Perfusion 2014-01

1 2014

Kreislauf- und Stoffwechselerkrankungen in Klinik und Praxis Jahrgang 27, Heft 1 Februar 2014

VERLAG

PERFUSION Offizielles Organ der Deutschen Gesellschaft für Arterioskleroseforschung Current Contents/ Clinical Medicine

ORIGINALARBEIT

Zur Terminologie des inflammatorischen Aortenaneurysmas FOREN

Forum cardiologicum: • B ioresorbierbares Gefäßgerüst revolutioniert die Gefäßmedizin • Symptomatische KHK: Update zu Ivabradin Forum antithromboticum: Vitamin-K-Antagonisten und Gerinnungsselbstmanagement – eine erfolgreiche Kombination ABSTRACTS Abstracts der wissenschaftlichen Beiträge zur 28. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung e. V. vom 13. bis 15. März 2014 im Schloss Rauischholzhausen REDAKTIONELLER TEIL

Kongressberichte, Mitteilungen

ISSN 0935-0020

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Ausführliche Informationen zum Thema Vorhofflimmern und Schlaganfallprävention finden Sie unter www.vorhofflimmern.de Pradaxa® 75 mg/110 mg/150 mg Hartkapseln, Wirkstoff: Dabigatranetexilat. Verschreibungspflichtig. Zusammensetzung: Jede Hartkapsel enthält 75 mg/110 mg/150 mg Dabigatranetexilat (als Mesilat); Sonstige Bestandteile: Weinsäure, Arabisches Gummi, Dimeticon 350, Talkum, Hyprolose, Carrageenan, Kaliumchlorid, Titandioxid, Indigokarmin (E 132), Gelborange S (E 110), Hypromellose, gereinigtes Wasser, Schellack, Butan-1-ol, 2-Propanol, Ethanol vergällt (mit Aceton, Methanol und Acetylacetat), Eisen(II,III)-oxid (E 172), Propylenglykol. Anwendungsgebiete: Primärprävention von venösen thromboembolischen Ereignissen bei erwachsenen Patienten nach elektivem chirurgischen Hüft- oder Kniegelenksersatz. Prävention von Schlaganfall und systemischer Embolie bei erwachsenen Patienten mit nicht valvulärem Vorhofflimmern mit einem oder mehreren der folgenden Risikofaktoren, wie zum Beispiel: Vorausgegangener Schlaganfall oder transitorische ischämische Attacke (TIA); Alter ≥ 75 Jahre; Herzinsuffizienz (New York Heart Association (NYHA) Klasse ≥ 2); Diabetes mellitus; arterielle Hypertonie. Gegenanzeigen: Überempfindlichkeit gegen den Wirkstoff oder einen der sonstigen Bestandteile; schwere Beeinträchtigung der Nierenfunktion (Kreatinin-Clearance < 30 ml/min); akute, klinisch relevante Blutung; Läsionen oder klinische Situationen, die als hohes Risiko einer schweren Blutung gewertet werden; spontane oder pharmakologisch bedingte Einschränkung der Hämostase; Beeinträchtigung der Leberfunktion oder Lebererkrankung, die Auswirkungen auf das Überleben erwarten lässt; gleichzeitige Behandlung mit systemisch verabreichtem Ketoconazol, Ciclosporin, Itraconazol und Dronedaron. Patienten mit künstlichen Herzklappen, die eine gerinnungshemmende Therapie benötigen.

Nebenwirkungen: Anämie, Hämoglobin vermindert, Nasenbluten, gastrointestinale Blutung, urogenitale Blutung, Bauchschmerzen, Diarrhoe, Dyspepsie, Übelkeit, Thrombozytopenie, Hämatokrit vermindert, ArzneimittelÜberempfindlichkeit, Hautausschlag, Pruritus, Anaphylaktische Reaktion, Angioödem, allergische Reaktion, intrakranielle Blutungen, Hämatom, Hämoptyse, Wundblutung, Blutung, rektale Blutung, hämorrhoidale Blutung, gastrointestinale Ulzera, Gastroösophagitis, gastroösophageale Refluxkrankheit, Erbrechen, Dysphagie, abnorme Leberfunktion/abnormer Leberfunktionstest, Leberenzyme erhöht, Transaminasen erhöht, Hyperbilirubinämie, Hautblutung, Hämarthrose, Hämaturie, blutige Absonderung, traumatische Blutung, postoperatives Hämatom, postoperative Blutung/Anämie/Absonderung, Wundsekretion, Wunddrainage, Urtikaria, Blutung an einer Injektionsstelle, Blutung an der Eintrittsstelle eines Katheters, Blutung am Inzisionsort, postoperative Drainage, Bronchospasmus. Weitere Einzelheiten enthalten die Fach- bzw. Gebrauchsinformationen, sowie die Ratgeber und Patientenausweise, deren aufmerksame Durchsicht wir empfehlen. Stand der Information: Januar 2014 Boehringer Ingelheim Pharma GmbH & Co. KG, Binger Str. 173, 55216 Ingelheim am Rhein, Tel.: 08 00 / 77 90 90 0, Fax: 0 61 32 / 72 99 99, E-Mail: info@boehringer-ingelheim.de

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EDITORIAL

Pseudo-systematic reviews Systematic reviews are generally considered to be the most reliable type of evidence regarding the effectiveness of therapeutic interventions. They have become essential tools, for instance, for busy clinicians who need quick and trustworthy information. Rather than tempting us to pick and chose from a usually mixed bag of findings, systematic reviews summarise the totality of the reliable evidence available for a given research question. In scientific terms, they are superior to other types of evidence because they minimise random and selection biases.

For conducting a systematic review, researchers need to first define a concise and well-focussed research question.

Subsequently, they must locate all the relevant primary studies. Finally, they need to critically evaluate and summarise the data either narratively or statistically; in the latter case we speak of a meta-analysis. In this complex development of a systematic review, there are numerous pitfalls which might impact on the quality and reliability of the final product.

Failure to define a focussed research question will automatically cause problems. Asking whether a given type of

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01/14 11

Prof. Dr. med. E. Ernst, Exeter, U.K.

treatment is superior to placebo, might be too broad a question to generate a sensible answer. Is Aspirin better than placebo? Yes, but for what condition!

Failure to locate all relevant studies might seriously distort the overall result. This means we need clearly defined inclusion and exclusion criteria as well as a well-designed search strategy. Depending on the research subject, literature searches can be complex and time-consuming. For instance, a systematic review of Traditional Chinese Medicine that does not include searches in the relevant Chinese data-bases will hardly produce the totality of the relevant clinical trials. Similarly imposing language restrictions will not necessarily yield all of the relevant primary studies.

Failure to include a transparent process of critical evaluation of all included clinical trials would open the door wide to bias. A simple example might explain: Imagine a systematic review that includes 100 clinical trials, of which 80 conclude that the experimental therapy is effective. Without any assessment of the methodological quality of the primary studies, the conclusion of the systematic review would have to be

positive as well â&#x20AC;&#x201C; simply because the majority of all relevant studies are positive. Imagine now that the vast majority of the 80 positive trials were seriously flawed and that all of the 20 negative trials were of high quality. Consequently, the conclusion of a rigorous systematic review ought to be negative.

In other words, if the systematic review failed to include a transparent quality assessment, it would dramatically mislead us.

Systematic reviews are meant to be objective â&#x20AC;&#x201C; and by and large they are.

But there are several steps in their development that either are based on somewhat subjective judgements rather than on objective science. Consequently, systematic reviews are open to bias and even to cheating. The final product may not readily reveal their defects, and particularly readers who are not experts in both the subject area and the methodology of systematic reviews can easily be misled.

My advice therefore is to always be cautious: Yes, systematic reviews are potentially the best type of evidence, but not everything that shines like gold is of value.

Edzard Ernst, Exeter

Š Verlag PERFUSION GmbH

Offizielles Organ der Deutschen Gesellschaft für Arterioskleroseforschung Current Contents/Clinical Medicine

Heft 1 Februar 2014

7, 8 Forum cardiologicum 10 Forum antithromboticum 6, 11 Kongressberichte 9, 14, 50 Mitteilungen

INHALT EDITORIAL 1 Pseudosystematische Reviews E. Ernst ORIGINALARBEIT 4 Zur Terminologie des inflammatorischen Aortenaneurysmas J. Janzen, R. Lützenberg ABSTRACTS 15 Abstracts der wissenschaftlichen Beiträge zur 28. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung e. V. vom 13. bis 15. März 2014 im Schloss Rauischholzhausen

7, 8 Forum cardiologicum 10 Forum antithromboticum 6, 11 Congress reports 9, 14, 50 Informations

CONTENTS EDITORIAL 1 Pseudo-systematic reviews E. Ernst

4

ORIGINAL PAPER Terminology of the inflammatory aortic aneurysm J. Janzen, R. Lützenberg

ABSTRACTS 15 Abstracts of the 28th Annual Meeting of the German Atherosclerosis Society, March 13–15 2014, Schloss Rauischholzhausen

BEI TYP-1UND TYP-2-DIABETES TYP-1 UND TYP-2 DIABETES LANTUS ® is wirtschat ftlich

h g durc n u t t a t Ers ge verträ t t a b a R ert * gesich

Das 24-Stunden-Original.

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NSULIN, DEM ICH VERTRAUE. 1FACH VON ANFANG AN – direkt nach Metformin möglich 1) Über 13 JAHRE LANGZEITERFAHRUNG in der täglichen Praxis 2)

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KARDIOVASKULÄRE LANGZEITSICHERHEIT über > 6 Jahre bestätigt3)

Das 24-Stunden-Original

INSULINPENS

RUNDUMRUNDUM SERVICE

BLUTZUCKERMESSGERÄTE

ÜBER 90 JAHRE INSULINERFAHRUNG.

*Für ca. 99,9% der GKV-Versicherten bestehen Verträge nach §130a Abs. 8 SGB V. Stand 10/2013 1) Bundesärztekammer (BÄK), Kassenärztliche Bundesvereinigung (KBV), Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften (AWMF). Nationale VersorgungsLeitlinie Therapie des Typ-2-Diabetes – Langfassung, 1. Auflage. Version 1. 2013. Available from: http://www.versorgungsleitlinien.de/themen/diabetes2/dm2_Therapie; [cited: 25.09.2013]; DOI: 10.6101/AZQ/000145; 2) Zulassung und Ausbietung von Lantus® im Juni 2000; 3) Gerstein HC. et al. N Engl J Med. 2012 Jul 26;367(4):319-28 Lantus® 100 Einheiten/ml Injektionslösung in einer Patrone · Lantus® SoloStar® 100 Einheiten/ml Injektionslösung in einem Fertigpen. · Lantus® 100 Einheiten/ml Injektionslösung in einer Durchstechflasche. Wirkstoff: Insulin glargin. Verschreibungspflichtig. Zusammensetzung: 1 ml enthält 100 Einheiten Insulin glargin (entsprechend 3,64 mg). Sonstige Bestandteile: Zinkchlorid, m-Cresol, Glycerol, Salzsäure, Natriumhydroxid, Wasser für Injektionszwecke, (Durchstechflasche 10 ml: Polysorbat 20). Anwendungsgebiete: Zur Behandlung von Diabetes mellitus bei Erwachsenen, Jugendlichen und Kindern im Alter von 2 Jahren und älter. Dosierung, Art und Dauer der Anwendung: Lantus® enthält Insulin glargin, ein Insulinanalogon mit einer lang anhaltenden Wirkdauer. Lantus® sollte einmal täglich zu einer beliebigen Zeit, jedoch jeden Tag zur gleichen Zeit, verabreicht werden. Dosierung und Zeitpunkt der Verabreichung von Lantus® sollten individuell festgelegt werden.

Bei Patienten mit Typ-2-Diabetes kann Lantus® auch zusammen mit oralen Antidiabetika gegeben werden. Lantus® wird subkutan verabreicht. Gegenanzeigen: Überempfindlichkeit gegenüber dem Wirkstoff oder einem der sonstigen Bestandteile. Warnhinweise/Vorsichtsmaßnahmen: Nicht das Insulin der Wahl bei diabetischer Ketoazidose. Umstellung auf anderen Insulintyp/-marke/ -ursprung nur unter strenger ärztlicher Überwachung. Bei Kombination mit Pioglitazon Herzinsuffizienz möglich, besonders bei Patienten mit Risikofaktoren. Bei Verschlechterung der kardialen Symptomatik Pioglitazon absetzen. Nebenwirkungen: Sehr häufig: Hypoglykämie. Häufig: Reaktionen an der Einstichstelle, Lipohypertrophie. Gelegentlich: Lipoatrophie. Selten: Allergische Reaktionen, Ödeme, Sehstörungen, Retinopathie. Sehr selten: Myalgie, Geschmacksstörung. Im Allgemeinen ist das Sicherheitsprofil bei Kindern und Jugendlichen (≤18 Jahre) ähnlich dem bei Erwachsenen. Gekürzte Angaben, vollständige Information siehe Fachinformation, die wir Ihnen auf Wunsch gern zur Verfügung stellen. Pharmazeutischer Unternehmer: Sanofi-Aventis Deutschland GmbH, D-65926 Frankfurt am Main, Postanschrift: Sanofi-Aventis Deutschland GmbH, Potsdamer Straße 8, 10785 Berlin. Stand: Juli 2013 (031682). AVS 210 13 027b-032310

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J. Janzen, R. Lützenberg: Zur Terminologie des inflammatorischen Aortenaneurysmas

ORIGINALARBEIT

Zur Terminologie des inflammatorischen Aortenaneurysmas PERFUSION 2014; 27: 4–6

Die Ätiologie des abdominalen Aorten­ aneurysmas (AAA) ist heterogen. So findet man bei mikroskopischen Un­ tersuchungen von Operationspräpara­ ten sowohl atherosklerotische als auch nicht atherosklerotische Ursachenkom­ plexe [1]. In der klinischen Terminolo­ gie hat sich der Begriff des inflamm­ atorischen Aortenaneurysmas etabliert [2]. Bei näherer Betrachtung fällt je­ doch auf, dass die Atherosklerose dabei nicht berücksichtigt wird.

Material und Methoden Grundlage für die vorliegende Arbeit ist das VascPath-Aortenregister. In diesem seit dem Jahre 1995 bestehen­ den Register sind Aortenpräparate im klinischen und histomorphologischen Kontext nach folgenden Parametern aufgeführt: • Aortendurchmesser, intramurales Hämatom, Dissektion • Plaquekonfiguration und -komposi­ tion (Schaumzellen, Cholesterin­ kristalle, Hämorrhagien, entzündli­ che Infiltrate, bindegewebige Kap­ pe etc.)

• Stary-Grad der Atherosklerose • Schlatmann-Becker-Score zur Me­ diabeurteilung • Intima-, Media- und Adventitiabrei­ te • Kalzifikationen und Ossifikationen • Vasa-vasorum-Morphologie und Zellkomposition der Periadventi­ tia Im VascPath-Aortenregister sind 92 Fälle mit einem AAA gelistet; der Perfusion 01/2014

27. Jahrgang

Jan Janzen1, Ronald Lützenberg2 1 VascPath, Bern, Schweiz 2 Universitätsklinikum Magdeburg

Zusammenfassung 92 abdominale Aortenaneurysmata (AAA) wurden mikroskopisch mit besonderem Blick auf die entzündliche Genese analysiert. In 33 Fällen (35,9 %) wurde eine Atherosklerose und in 15 Fällen (16,3 %) ein „reines“ inflammatorisches AAA nachgewiesen. 11 Fälle (12 %) wiesen eine degenerative Aortopathie und 2 Fälle (2,2 %) einen Morbus Erdheim-Gsell auf. In 31 Fällen (33,7 %) fanden wir heterogene mikroskopische Aspekte, die eine klare Zuordnung verhinderten. Die Atherosklerose sollte in der Terminologie des inflammatorischen Aortenaneurysmas berücksichtigt werden.

Schlüsselwörter: abdominales Aortenaneurysma, Atherosklerose, inflammato­ risches Aneurysma, Terminologie Summary In 92 cases of abdominal aortic aneurysms (AAA) microscopical analyses were performed with a special view on the inflammatory genesis. In 33 cases an atherosclerosis was seen and in 15 cases a „purely“ inflammatory AAA was shown. 11 cases displayed a degenerative aorthopathy und 2 cases a Morbus Erdheim-Gsell. Additionally we found 31 cases with heterogenous microscopic aspects which didn’t allow a clear attribution.

Since the atherosclerosis is an inflammatory disease, the terminology of the inflammatory aortic aneurysm should be specified.

Key words: abdominal aortic aneurysm, atherosclerosis, inflammatory aneu­ rysms, terminology

durchschnittliche AAA-Durchmesser liegt bei 6,5 cm (5,5–7 cm).

Resultate 33 Fälle (35,9 %) zeigten eine Athe­ rosklerose als Aneurysmaursache, wobei Früh- und Spätstadien gemäß der Stary-Klassifikation atheroskle­ rotischer Läsionen auftraten (Abb. 1

und 2). In 15 Fällen (16,3 %) kam das „reine“ inflammatorische AAA vor, gekennzeichnet durch eine zellarme Fibrose mit eingelagerten lympho­ plasmazellulären Infiltraten unter­ schiedlichen Ausmaßes (Abb. 3). Die Häufigkeitsrate dieser beiden Grup­ pen lag bei 52,2 %. Eine Besiede­ lung mit Mikroorganismen wurde in den konventionellen Färbungen nicht nachgewiesen. © Verlag PERFUSION GmbH

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J. Janzen, R. Lützenberg: Zur Terminologie des inflammatorischen Aortenaneurysmas

nicht, weil überlappende mikroskopi­ sche Befunde vorhanden waren. 2 Pa­ tienten (2,2 %) dieser Ursachengruppe hatten eine HIV-Erkrankung (Tab.1). Ätiologie Atherosklerose „Reines“ inflammatorisches AAA Degenerative Aortopathie Morbus ErdheimGsell Mischbilder

Anzahl

%

33 15

35,9 % 16,3 %

11

12,0 %

2

2,2 %

31

33,7 %

Tabelle 1: Ätiologie der abdominellen Aor­ tenaneurysmata (n=92)

Diskussion

Abbildung 1: Makroskopischer Aspekt eines atherosklerotischen AAA (Stary-Grad III)

Abbildung 2: Progrediente Atherosklerose (Stary-Grad IV) mit Cholesteringranulomen und Entzündungszeichen

Abbildung 3: Inflammatorisches AAA mit zellarmer Fibrose und lymphoplasmazellu­ lären Infiltraten

Nicht entzündliche AAA traten in 11 Fällen (12 %) auf, wobei es sich um degenerative Veränderungen handelte.

Mikroskopisch sind hier konfluierende Kalzifikationen im Sinne eines Morbus Mönckeberg und/oder Ossifikationen

der Aneurysmawand wegweisend. In 2 Fällen (2,2 %) lag ein Morbus ErdheimGsell vor.

In 31 Fällen (33,7 %) bestanden his­ tomorphologische Mischbilder; eine präzise ätiologische Zuordnung gelang

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Allgemein bekannt ist, dass es sich bei der Atherosklerose um eine entzünd­ liche Erkrankung der Tunica intima handelt. In den meisten Stadien der Atherogenese sind Entzündungszellen, Zytokine und deren Mediatoren betei­ ligt. Bereits mit einfachen mikroskopi­ schen Färbetechniken lassen sich Lym­ phozyten und Plasmazellen darstellen.

Immunhistochemische Markierungen (CD3, 8, 20 und 45) erlauben zudem eine weitere Infiltrattypisierung in der Aortenwand [3].

Die aktuelle klinische Terminologie des inflammatorischen Aneurysmas be­ zieht sich auf Formen, die intraoperativ durch eine verdickte Aneurysmawand ohne eigentliche Trennschichten sowie eine diffuse perianeurysmale (retrope­ ritoneale) Fibrose gekennzeichnet sind.

Die Inzidenz des inflammatorischen Aneurysmas – in unserer Studie als „reines“ inflammatorisches Aneurysma ohne Atherosklerose bezeichnet – wird in der Literatur mit 5–10 % angegeben [2]. Als Ursache werden unter anderem Autoimmunerkrankungen erwähnt, ein konkretes Antigen ist jedoch nach wie vor unbekannt. In mikrobiologischen Studien der AAA wurde unter anderem auf Zytomegalieviren und Chlamydia pneumoniae als mögliche Erreger hin­ gewiesen [4, 5]. Zudem gibt es Mittei­ lungen über die Rolle eines schlechten Zahnstatus (Eintrittspforte der Erreger) als Ursache kardiovaskulärer Erkran­ kungen [6]. © Verlag PERFUSION GmbH

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J. Janzen, R. Lützenberg: Zur Terminologie des inflammatorischen Aortenaneurysmas

Als mögliche Ursache eines inflam­ matorischen AAA ist zudem die HIVErkrankung zu erwähnen. Beispiels­ weise berichteten südafrikanische Gefäßchirurgen, dass bei 15 % der AAA eine HIV-Erkrankung im Spätstadium nachgewiesen wurde. Mikroskopische Leitkriterien der HIV-Vaskulopathie sind die leukozytoklastische Vasoritis und Kollagenveränderungen der Ad­ ventitia [7, 8].

Unsere mikroskopischen Untersuchun­ gen zeigten, dass die Ätiologie des AAA heterogen ist. Da die Atheroskle­ rose per se als eine entzündliche Ge­ fäßerkrankung angesehen wird, liegt die Häufigkeitsrate der entzündlich bedingten AAA im VascPath-Aortenre­ gister bei 52,2 %. Zudem konnten wir 3 histomorphologische Grundtypen des inflammatorischen AAA nachweisen: 1. Atherosklerotisches AAA 2. „Reines“ inflammatorisches AAA (nicht atherosklerotisches AAA) 3. Gemischtes AAA

KONGRESSE Edoxaban eine neue Option in der Schlaganfallprävention bei Vorhofflimmern Die Einführung der neuen oralen An­ tikoagulanzien (NOAC) bedeutet für die Behandlung von Patienten, die über lange Zeit antikoaguliert werden müs­ sen, einen deutlichen Fortschritt. Ein Vorteil besteht darin, dass anders als bei Vitamin-K-Antagonisten wie War­ farin die routinemäßige Überprüfung der Gerinnungshemmung nunmehr entfallen kann, führte Professor Micha­ el Böhm, Homburg/Saar, auf der PostAHA Pressekonferenz von Daiichi San­ kyo in München aus. Angriffspunkte für die Hemmung der Gerinnungskas­ kade sind bei den bisher zugelassenen NOAC der Faktor IIa (Thrombin) oder der Faktor Xa. Zur Gruppe der FaktorXa-Inhibitoren gehört auch Edoxaban, das sich derzeit noch in der klinischen Entwicklung befindet.

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Aufgrund unserer Ergebnisse möchten wir anregen, die Atherosklerose in der klinischen Terminologie des inflamm­ atorischen AAA zu berücksichtigen.

Danksagung Wir danken den Gefäßchirurgen Prof.

Dr. J. Schmidli, Bern, PD Dr. H. Scholz, Berlin, und Prof. Dr. M. Veller, Johannesburg, für die Bereitstellung des Operationsmaterials und die Mitar­ beit am VascPath-Aortenregister sowie Frau Tanja Schneider für die Hilfe bei der Manuskripterstellung.

Literatur 1 Janzen J, Schmidli J. Histopathologische Analysen komplettieren die Diagnostik der Aortenerkrankungen. Gefäßchirurgie 2012;17:203-208 2 Hellmann DB, Grand DJ, Freischlag JA. Inflammatory abdominal aortic aneurysm.

J Am Med Ass 2007;297:395-400

Edoxaban so effektiv wie Warfarin Edoxaban zeichnet sich durch eine hohe Bioverfügbarkeit von etwa 60 % aus, die Plasmahalbwertszeit beträgt 8–10 Stunden und etwa die Hälfte der im Plasma vorhandenen wirksamen Substanz wird renal eliminiert, erläu­ terte Professor Johannes Waltenberger, Münster.

Auf der Jahrestagung der American Heart Association (AHA) in Dallas wurden erstmals die Daten einer großen Vergleichsstudie von Edoxaban gegen Warfarin zur Schlaganfallprävention bei nicht valvulärem Vorhofflimmern (VHF) präsentiert. In die Phase-III-Stu­ die ENGAGE AF-TIMI 48 waren über 21.000 Patienten eingeschlossen, be­ richtete Professor Andreas Goette, Pa­ derborn. Edoxaban wurde in der dreiar­ migen Studie entweder mit 30 mg oder mit 60 mg täglich appliziert. Die Kon­ trollgruppe erhielt Warfarin mit einem Ziel-INR zwischen 2,0 und 3,0. Patien­ ten mit einer Niereninsuffizienz, einem Gewicht unter 60 kg oder gleichzeitiger Behandlung mit einem P-GlykoproteinInhibitor bekamen Edoxaban jeweils in halbierter Dosis.

3 Stary HC. Atlas of atherosclerosis: pro­ gression and regression. New York Parthe­ non Publishing Group; 1999:10-125 4 Fiehn NE, Larsen T, Christiansen N, Holmstrup P, Schroeder TV. Identification of periodontal pathogens in atherosclerotic vessels. J Periodontol 2005;76:731-736 5 Karlsson L, Gnarpe J, Nääs J, Olsson G, Lindholm J, Steen B, Gnarpe H. Detection of viable Chlamydia pneumoniae in ab­ dominal aortic aneurysms. Eur J Vasc En­ dovasc Surg 2000;19:630-635 6 Haheim LL (ed). Oral infections and car­ diovascular disease. Bentham e-books, Bentham Science Publishers; 2013 7 Veller M, Pillai J. Gefäßerkrankungen bei HAART-naiven HIV-Patienten. Gefäßchir­ urgie 2012;1:8-9 8 Robbs JV, Paruk N. Management of HIV vasculopathy – a South African experi­ ence. Eur J Vasc Endovasc Surg 2010; 39:25-31

Für die Verfasser: Dr. med. Jan Janzen, MPhil VascPath Schweiz Postfach 350 CH- 3000 BERN 22 E-Mail: info@janlab.ch

Der primäre Endpunkt in ENGAGE AF-TIMI 48 war die Nichtunterlegen­ heit von Edoxaban in der Prävention von Schlaganfällen und systemisch embolischen Ereignissen (SEE). Die jährliche Inzidenz in der Hochdosis­ gruppe mit 60 mg Edoxaban lag bei 1,18 % versus 1,50 % unter Warfarin (HR: 0,79), in der Niedrigdosisgruppe bei 1,61 % versus 1,50 % (HR: 1,07).

Somit war Edoxaban gleich wirksam und hatte den vorgegebenen Endpunkt erfüllt.

Deutlich weniger schwere Blutungen Bezüglich der Sicherheit stand die Häufigkeit schwerer Blutungen im Vor­ dergrund. Diese traten in der Hochdo­ sisgruppe im Vergleich zu Warfarin um 20 % seltener auf (2,75 % vs. 3,43 % pro Jahr; HR: 0,80) auf. Unter der niedrigeren Dosierung waren schwere Blutungen sogar um 53 % und damit signifikant seltener (1,61 % vs. 3,43 % pro Jahr; HR: 0,47).

Martin Bischoff, Planegg © Verlag PERFUSION GmbH

FORUM CARDIOLOGICUM

7

Bioresorbierbares Gefäßgerüst revolutioniert die Gefäßmedizin

Gefäßverengungen oder -verschlüsse behandeln zu können, ohne ein per­ manentes Implantat im Körper zu hin­ terlassen, ist ein Konzept über das in der Gefäßmedizin seit Jahren immer wieder diskutiert wird. Mit der Markt­ einführung von Absorb, dem weltweit ersten, Medikament freisetzenden, bio­ resorbierbaren Gefäßgerüst von Abbott Vascular ist dieses Konzept Realität geworden (Abb. 1).

Medizinprodukt der neuesten Generation Während das Design des neuen bio­ resorbierbaren Gefäßgerüsts dem des bekannten drug-eluting Stents (DES) entspricht, ist das Gerüstmaterial statt Edelmetall ein Polylactid. Polylactid ist eine Milchsäureverbindung, die schon in anderen Bereichen in der Me­ dizin erfolgreich zum Einsatz kommt, zum Beispiel bei selbstauflösendem Nahtmaterial.

In der Gefäßmedizin ist der Einsatz des Materials relativ neu – und kann dem Patienten im Vergleich zum bislang als Therapiestandard geltenden Medika­ ment freisetzenden Metallstent Vorteile bieten.

Milchsäure-„Scaffold“ wird rückstandslos ausgeschieden Absorb setzt das antiproliferative Me­ dikament Everolimus frei. Everolimus inhibiert das In-Stent-Neointimawachs­ tum im Bereich der Koronargefäße,

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Abbildung 1: Das Gerüst der Everolimus freisetzenden Gefäßstütze Absorb besteht aus Poly­ lactid, das sich im Verlauf von 2–3 Jahren vollständig auflöst. Übrig bleiben nur 2 Paar kleine Platinmarker, die bei der Platzierung des Systems helfen und es ermöglichen zu kontrollieren, wo das System platziert wurde.

in die ein Stent oder ein Gerüst implan­ tiert wurde.

Im Unterschied zum Stent, der in die Gefäßwand einwächst dort als Gefäß­ stütze das restliche Leben lang verbleibt und die Arterie unbeweglich hält, löst sich das neue, auch als „Scaffold“ (auf Deutsch „Gerüst“) bezeichnete Implan­ tat auf natürliche Weise auf. Die Milch­ säureverbindung wird zu Wasser und Kohlendioxid abgebaut und vom Kör­ per rückstandslos ausgeschieden. Dieser Prozess beginnt nach etwa 6 Monaten und ist nach 2–3 Jahren abgeschlossen.

Allein 2 Paar kleine Marker verbleiben im Gefäß. Durch sie ist es möglich zu kontrollieren, wo das Gerüst platziert wurde.

Damit hinterlässt der nur zeitlich be­ grenzt vorhandene Scaffold einen behandelten Bereich des Gefäßes, der sich frei von einem permanenten Implantat wieder aufdehnen und zu­ sammenziehen kann. Darüber hinaus werden durch die Implantation des bioresorbierbaren Scaffolds die Ge­ fäßabschnitte für Bypass-Grafts nicht dauerhaft durch Metallstents blockiert.

Daher sind, sollte dies der Krank­ heitsverlauf des Patienten notwendig machen, spätere Bypass-Operationen nach wie vor möglich.

Elisabeth Wilhelmi, München

© Verlag PERFUSION GmbH

FORUM CARDIOLOGICUM

8

Symptomatische KHK: Update zu Ivabradin

Wegweisende Ergebniss der BEAUTIfUL-Studie Bereits vor mehreren Jahren hatten die Ergebnisse der BEAUTIfUL-Studie [1] aufhorchen lassen und auf einen erheb­ lichen prognostischen Nutzen von Iva­ bradin (Procoralan®) bei KHK-Patien­ ten mit linksventrikulärer Dysfunktion hingewiesen. Zwar wurde für Ivabradin in Kombination mit einer Vortherapie einschließlich Betablocker im Gesamt­ kollektiv bereits mit geringer Fre­ quenz kein signifikanter Vorteil beim primären kombinierten Endpunkt aus kardiovaskulärem Tod und Hospitali­ sierung wegen eines Myokardinfarkts dokumentiert, eindeutig profitiert hat­ ten jedoch jene 1.507 Patienten einer Subgruppe mit limitierenden Anginapectoris-Beschwerden [1]. Im Ergebnis führte Ivabradin trotz der bereits opti­ malen Begleittherapie zu einer Reduk­ tion des primären zusammengesetzten Endpunkts um 24 % (p=0,05) und in­ farktbedingter Hospitalisierungen um 42 % (p=0,021). Bei gleichzeitig hoher Herzfrequenz über 70/min wurde das Risiko für einen Myokardinfarkt unter Perfusion 01/2014

27. Jahrgang

Ivabradin sogar um 73 % (p=0,002) ge­ senkt (Abb. 1) [2].

Genauere Erkenntnisse zum potenziel­ len prognostischen Nutzen von Ivabra­ din bei asymptomatischen KHK-Pati­ enten sind von den zum ESC-Kongress 2014 angekündigten Ergebnissen der randomisierten, kontrollierten SIG­ NIfY-Studie zu erwarten. In die groß angelegte Studie zur Erfassung von Mortalität und kardiovaskulären Er­ eignissen wurden für mindestens 18 Monate 19.102 Patienten mit KHK und einer Herzfrequenz über 70/min, aber ohne linksventrikuläre Dysfunktion oder Zeichen einer Herzinsuffizienz eingeschlossen, die auf Basis einer leit­

15

liniengerechten Standardtherapie mit täglich 2 × 7,5 mg Ivabradin auf eine Herzfrequenz von <60/min eingestellt werden sollten [3].

Neue ESC-Leitlinie 2013 im Fokus Wird in SIGNIfY endgültig der Nach­ weis einer signifikanten Reduktion der kardiovaskulären Mortalität und Mor­ bidität durch Ivabradin geführt, hat dies einschneidende Folgerungen für die künftige Prävention koronarer Er­ eignisse bei stabiler KHK. In der neuen ESC-Leitlinie 2013 wird derzeit Iva­ bradin im Hinblick auf eine wirksame

Hazard Ratio: 0,27 (0,11–0,66) p=0,002

–73%

10

Ereignisse (%)

Bei Patienten mit stabiler KHK schon lange etabliert ist die gute symptoma­ tische Wirksamkeit von Ivabradin mit und ohne Betablocker. Erneut unter­ mauert wird die bedeutende Rolle von Ivabradin durch die aktuelle ESC-Leit­ linie 2013 zum Therapiemanagement der stabilen KHK. Weitere Evidenzen an einer zugleich auch effektiven Re­ duktion von kardiovaskulären Ereig­ nissen bei nicht symptomatischer KHK sollen jetzt durch die SIGNIfY-Studie endgültig bewiesen werden.

Placebo* 5

Ivabradin*

0

0 0,5 1 1,5 2 Jahre * + Vortherapie inkl. Betablocker

Abbildung 1: Signifikante Reduktion von Myokardinfarkten durch Ivabradin in einer BEAUTIfUL-Subgruppenanalyse von symptomatischen KHK-Patienten mit einer HF≥70/min [2] © Verlag PERFUSION GmbH

MITTEILUNGEN

FORUM CARDIOLOGICUM

Ernährung bei Diabetes und Nieren­erkran­ kungen: Phosphathaltige Lebensmittel meiden

p<0,001

Anzahl der Angina-pectorisBeschwerden/Woche

5

≥ 100% der Betablocker-Zieldosis 50% bis 99% der Betablocker-Zieldosis < 50% der Betablocker-Zieldosis

4 3 2 1

12 Monate

8 Monate

4 Monate

1 Monat

Baseline

0

Abbildung 2: Signifikante Reduktion der wöchentlichen Angina-pectoris-Attacken durch Ivabra­ din in Kombination mit einem Betablocker (unabhängig von dessen Dosis) in der ADDITIONSStudie (n=2.330) [7]

Linderung von Angina-pectoris-Symp­ tomen zum Einsatz in Kombination mit einem Betablocker und/oder Kalzium­ antagonisten empfohlen [4]. Nicht zu­ letzt wird in diesem Zusammenhang eine noch konsequentere Einstellung der Herzfrequenz auf den Zielbereich von <60/min angemahnt [4]. Dass die­ se Forderung durchaus ihre Berech­ tigung hat, verdeutlicht das in zahl­ reichen KHK-Patientenregistern wie z.B. CLARIfY [5] selbst unter einer gut eingestellten Betablocker-Therapie ermittelte, weit verbreitete und oftmals sogar ausgeprägte Verfehlen dieses Zielfrequenzbereichs.

Dass sich die im Praxisalltag mit einem Betablocker allein oft nicht realisierba­ ren Zielwerte bei den meisten Patienten durch eine Kombination mit Ivabradin erreichen lassen, wird nicht nur durch klinische Studien wie ASSOCIATE [6] gestützt. Unter Anwendung einer solchen Kombinationsstrategie wurden mit der Gabe eines Betablockers und Ivabradin in der deutschen nicht inter­ ventionellen „Real-life“-Studie ADDI­ TIONS nach 12 Monaten eine effektive Einstellung der Herzfrequenz und eine Perfusion 01/2014

27. Jahrgang

9

rasche und vor allem nachhaltige Bes­ serung der Beschwerden unter Belas­ tung erreicht [7]. Unabhängig von der erreichten Betablocker-Dosis wurde durch die Kombination mit Ivabradin sowohl die wöchentliche Anzahl von Angina-pectoris-Beschwerden (Abb. 2) als auch der Verbrauch kurzwirksa­ mer Nitrate signifikant reduziert (je p<0,001). Bei zugleich guter Therapie­ sicherheit konnte überdies auch die mit dem EQ-5D-Index ermittelte Lebens­ qualität signifikant verbessert werden (p<0,001).

Dr. Michael Lohmann, Limburg Literatur 1 Fox K. et al. Lancet 2008;372:807-816 2 Fox K. et al. Eur Heart J 2009;30:23372345 3 Fox K. et al. Am Heart J 2013;166:654661 4 Montalescot G. et al. Eur Heart J 2013; 34:2949-3001 5 Steg PG et al. PLoS ONE 2012;7:e36284 6 Tardif JC et al. Eur Heart J 2009;30:540548 7 Werdan K et al., Eur Heart J 2013;34(Abs­ tr. Suppl.):175-176

Nierenkrankheiten sind nach Ampu­ tationen die zweithäufigste Kompli­ kation bei Diabetikern. Etwa 40–50 % aller Diabetiker entwickeln im Verlauf ihrer Stoffwechselerkrankung eine diabetische Nephropathie. Dadurch erhöht sich auch ihr Risiko für HerzKreislauf-Erkrankungen. Denn die „Klärleistung“ der Niere nimmt ab, Blutdruck und Blutfette steigen an. In Deutschland sind Menschen mit Dia­ betes mellitus die größte Gruppe der Patienten, die an die Dialyse müssen oder sogar eine neue Niere benötigen.

Gegenwärtig sind etwa 20.000 davon betroffen.

Diabetiker mit Nierenerkrankungen sollten auf ihre Ernährung achten. „In der Vergangenheit sollten Diabetiker Typ 1 und Typ 2 nach Auftreten einer diabetischen Nephropathie ihre Ei­ weißzufuhr beschränken“, erläutert Professor Eberhard Ritz vom Nieren­ zentrum in Heidelberg. „Dies wird heute wegen fehlender Effektivität und der Gefahr einer Fehlernährung nicht mehr empfohlen.“ Ein neu erkannter Risikofaktor bei chronisch Nierenkranken ist jedoch die Aufnahme von künstlichen Phosphaten über die Nahrung: „Hier gibt es Hin­ weise darauf, dass Phosphat zu Nie­ renversagen, Gefäß- und Herzerkran­ kungen führen kann“, warnt Professor Ritz. „Ein erhöhter Phosphatspiegel im Blut kann bei Menschen mit Diabetes auch die Wirkung von blutdrucksen­ kenden Medikamenten wie ACE-Hem­ mern verringern oder gar aufheben.“ Daher sollten Betroffene Lebensmittel mit hohem Phosphatgehalt möglichst meiden.

Phosphate sind unter anderem als Konservierungsmittel in Schmelzkäse, vielen Fleisch- und Wurstwaren, Fer­ tiggerichten wie Tiefkühlpizza oder Softdrinks enthalten.

DDG © Verlag PERFUSION GmbH

FORUM ANTITHROMBOTICUM

10

Vitamin-K-Antagonisten und Gerinnungsselbstmanagement – eine erfolgreiche Kombination

Zur Prophylaxe und Therapie von thromboembolischen Erkrankungen stehen seit vielen Jahrzehnten VitaminK-Antagonisten (VKA) zur Verfügung.

Derzeit werden etwa 1,1 Millionen Menschen dauerhaft damit behandelt.

VKA haben auch nach der Einführung der neuen oralen Antikoagulanzien (NOAK) weiterhin einen hohen Stel­ lenwert, denn diese decken bislang nicht das gesamte Spektrum der In­ dikationen mit ihren Zulassungen ab.

Dagegen sind bei künstlichen Herz­ klappen, valvulärem Vorhofflimmern, Kontraindikationen gegen NOAK oder Komorbiditäten wie schwerer Nieren­ insuffizienz ausschließlich VKA zur Langzeittherapie zugelassen. Außer­ dem ist eine Überlegenheit der NOAK gegenüber VKA durch Studien bei gut eingestellten Patienten nicht belegt [1, 2]. Eine optimierte Behandlungs­ qualität lässt sich laut aktuellem Leit­ faden der Arzneimittelkommission der deutschen Ärzteschaft [2] erzielen, wenn die VKA-Therapie mit Gerin­ nungsselbstmanagement (GSM) kom­ biniert wird. Die regelmäßige Messung des Gerinnungswertes durch den Pati­ enten lässt sich mit Messgeräten wie dem CoaguChek® XS System schnell und sicher durchführen.

Gerinnungsselbstmanagement optimiert VKA-Therapie Das konventionelle Monitoring der INR-Werte in der Praxis ist oftmals mit Hürden verbunden: Neben der venösen Blutentnahme verzögern die lange Dauer der Ergebnisgenerierung sowie eine mögliche Dosisanpassung Perfusion 01/2014

27. Jahrgang

durch den Arzt die sofortige Therapie­ entscheidung. Darüber hinaus sind die Testintervalle häufig zu lang, sodass kritische Situationen unbemerkt blei­ ben und zeitnahe Reaktionen daher nicht möglich sind. Aus diesem Grund sollte die VKA-Therapie in Verbindung mit dem leitliniengesicherten [3] und kostengünstigen [2] GSM durchge­ führt werden. Patienten im GSM wei­ sen eine deutlich erhöhte TTR (time in therapeutic range) von 78 % gegen­ über konventionell betreuten Patienten (61 %) auf [4]. Da bei Bedarf eine so­ fortige Dosisanpassung erfolgen kann, werden schwerwiegende thromboem­ bolische Komplikationen gegenüber der ärztlichen Routinekontrolle signifi­ kant um 58 % reduziert. Mortalität und schwerwiegende Blutungen wurden um 25 % respektive 14 % (nicht signi­

fikant) reduziert [5]. Zudem wird die Therapieadhärenz der Patienten durch das gestärkte Arzt-Patienten-Bündnis und die erlernte Eigenverantwortung deutlich gesteigert.

Sichere Messung durch Patientenschulungen Um eine selbstständige und korrekte Durchführung der wöchentlichen INRMessung durch die Patienten zu ge­ währleisten, müssen diese im Vorfeld der Therapie entsprechend geschult werden. Grundsätzlich kann jeder Pa­ tient auf das GSM eingestellt werden, der kognitiv und physisch dazu in der Lage ist. Im Falle einer körperlichen oder geistigen Beeinträchtigung kön­ nen Angehörige, Pflegekräfte oder Be­

Gerinnungsselbstmanagement mit dem CoaguChek® XS System Das Gerät misst die Zeit vom ersten Kontakt der Blutprobe (8 µl aus der Fingerbeere) mit dem Teststreifen bis zum Einsetzen der Gerinnung. Der Teststreifen enthält ein Reagenz in getrockneter Form, dessen reaktive Bestandteile aus dem Thromboplastin und einem Peptid-Substrat bestehen. Wenn die Probe aufgetragen wird, aktiviert das Thromboplastin die Gerinnungskaskade, was zur Bildung von Thrombin führt. Dieses Enzym spaltet das Peptid-Substrat, das ein elektrochemisches Signal erzeugt. Dieses Signal wird in Abhängigkeit vom Zeitpunkt seines Auftretens durch einen entsprechenden Algorithmus in die Gerinnungseinheiten INR, % Quick und Sekunden umgerechnet und nach 60 Sekunden auf dem Display angezeigt. Die letzten 100 Messwerte werden mit Datum und Uhrzeit gespeichert. © Verlag PERFUSION GmbH

FORUM ANTITHROMBOTICUM

treuer ganz einfach mitgeschult werden [6]. Das GSM wird mittlerweile von etwa 195.000 Menschen aller Alters­ klassen in Deutschland genutzt.

Ein von der Arbeitsgemeinschaft Selbstkontrolle der oralen Antiko­ agulation (ASA) e.V. zertifiziertes Schulungsprogramm ist SPOG (Schu­ lungsprogramm für Patienten mit ora­ ler Gerinnungshemmung). Es wird in Kleingruppen von 3–6 Personen durch­

geführt und beinhaltet 4 Unterrichts­ einheiten zu je 90–120 Minuten. Diese umfassen sowohl theoretische Themen wie die Grundlagen der Blutgerinnung als auch den praktischen Umgang mit dem Messgerät.

Weitere Informationen sind unter www.coaguchek.de/schulungsadressen erhältlich.

Brigitte Söllner, Erlangen

11

Literatur 1 Wallentin L et al. Lancet 2010;376(9745): 975-983 2 Leitfaden der Arzneimittelkommission der deutschen Ärzteschaft (AkdÄ) 2012; Ver­ sion 1.0 3 Camm A et al. Eur Heart J 2010;31:23892429 4 Koertke H, Koerfer R. Ann Thorac Surg 2001;72:44-48 5 Heneghan C et al. Lancet 2012;379:322334 6 Braun S et al. DMW 2009;134:695-700

KONGRESSE TAVI-System mit breitem Einsatzspektrum

JenaValve erfolgreich bei Aortenstenose und Aorteninsuffizienz Vor knapp 50 Jahren wurde die erste chirurgische Aortenklappe implantiert.

Seither hat sich eine rasante Entwick­ lung vollzogen. Mit der katheterba­ sierten Aortenklappen-Implantation (TAVI) gibt es seit 2002 auch eine Al­ ternative für inoperable Risikopatien­ ten mit schwerer Aortenstenose (AS).

Im September 2013 ist das erste TAVISystem zur Behandlung einer reinen Aorteninsuffizienz (AI) dazugekom­ men. JenaValve Technology bietet weltweit das einzige TAVI-System, das für beide Aortenvitien, Stenose und Insuffizienz, zugelassen ist. Dieser Fortschritt ist dem einzigartigen Stent­ design des JenaValve TAVI-Systems zuzuschreiben. Neue Indikationen und weniger TAVI-typische Komplikati­ onen sind der Entwicklungstrend in diesem Bereich. Sollte das Entwick­ lungstempo anhalten, könnten in 5–10 Jahren die typischen TAVI-Kompli­ kationen sogar gänzlich überwunden sein. Dies war das Fazit der Experten auf dem JenaValve-Symposium im Rahmen der Jahrestagung der Euro­ pean Association for Cardio-Thoracic Surgery in Wien.

Perfusion 01/2014

27. Jahrgang

Abbildung 1: Details des JenaValve TAVI-Systems (© JenaValve Technology GmbH).

Innovativer Clippingmechanismus ermöglicht Einsatz bei AI-Patienten Laut Dr. Hendrik Treede vom Uni­ versitären Herzzentrum Hamburg hat die Zulassung des JenaValve TAVISystems für die AI einen hohen Stel­ lenwert. Denn bisher fehlte gerade in diesem Bereich für Patienten, die nicht mehr konventionell operiert werden konnten, eine adäquate Therapiemög­ lichkeit. Daten von 10 am UKE Ham­ burg versorgten AI-Patienten zeigten vielversprechende Ergebnisse. Mit den Resultaten, so Treede, sei er äußerst zufrieden: „Das hämodynamische Er­ gebnis nach drei Monaten war bei allen Patienten sehr gut“. Ein Patient aus der Hochrisikogruppe starb nach 7 Mona­ ten infolge einer nicht kardialen Ursa­

che. Deutschlandweit erhobene Daten aus 7 Zentren werden derzeit in einer retrospektiven Analyse ausgewertet.

Das JenaValve System ist das einzige TAVI, das sich aufgrund des Designs für den Einsatz bei AI-Patienten eig­ net. Der Grund ist der einzigartige Me­ chanismus, mit dem die Prothese auf die nativen Klappensegel „geclippt“ wird (Abb. 1). Das Prinzip entspricht dem einer Büroklammer und macht die Fixierung der Prothese damit unab­ hängig vom Verkalkungsgrad der na­ tiven Aortenklappen. Zusätzlich kann der Implanteur die Prothese mithilfe von 3 Positionsfühlern anatomisch korrekt positionieren. Die optimale Lage und Positionshöhe bekommt er durch taktiles Feedback angezeigt.

Clippingmechanismus, anatomisch © Verlag PERFUSION GmbH

12

Kongresse

korrekte Positionierung und das nied­ rige Stentprofil sind mitverantwortlich dafür, dass kaum radialer Druck auf die Herzwand im linksventrikulären Ausflusstrakt ausgeübt wird. Somit ist das Risiko für eine postprozedurale Schrittmacherpflichtigkeit gering.

Für Treede sind zwei weitere Indikati­ onen vorstellbar, in denen das System künftig eingesetzt werden könnte: bei der Behandlung von Risikopatienten mit schwerer Hypertrophie im links­ ventrikulären Ausflusstrakt und wenn der Abstand zwischen Annulus und Koronarostien kurz ist.

Implantation waren es nur noch 26,7 % mit NYHA III, kein Patient hatte mehr NYHA IV. Die 30-Tage-Mortalität lag bei 12,4 %. „Hier muss aber das klei­ ne Patientenkollektiv (n=113) berück­ sichtig werden“, sagte Wendler. „Denn fasst man alle bisherigen Studien zu­ sammen, d.h. First-In-Man, CE-Zu­ lassung und JUPITER-Register, so er­ gibt sich eine Mortalitätsrate von 10 % (n=190).“ Die 30 Tage-Mortalität be­ trug im JUPITER-Register nur 6,2 %.

Erfolgreiche Implantation bei 94 Prozent der AS-Patienten

Durch die Einführung des neuen De­ livery-Systems „Cathlete plus“ Mitte November 2013 wurde der Freiset­ zungsmechanismus des JenaValve TAVI-Systems nochmals vereinfacht. „Die Klappenfreisetzung basiert nun ausnahmslos auf einer Rotationsbe­ wegung am Implantationskatheter.

Anstelle von drei Schritten in drei Dimensionen (drehen, schieben, zie­ hen), muss jetzt pro Arbeitsschritt nur noch eine Drehbewegung ausgeführt werden. Der Komfort für den Im­ planteur ist gestiegen. Und durch die bedienungsfreundliche Anwendung wird sich sicherlich die Lernkurve der Anwender, die das System noch nicht kennen, verkürzen“, beschrieb Dr. Ar­ dawan Rastan vom Herz- und Kreis­ laufzentrum Rotenburg a.d. Fulda die Vorteile des neuen Delivery-Systems.

Die technische Erfolgsrate der Im­ plantation mit dem JenaValve TAVISystem lag bei 94 %, so das aktuelle Interimergebnis des JUPITER-Regis­ ters, in dem Daten von 117 Hochrisi­ kopatienten mit AS und einem mittle­ ren logistischen EuroScore von 23,6 % und STS-Score von 7,1 ausgewertet wurden. Positiv zu vermerken ist vor allem die niedrige Rate an typischen TAVI-Komplikationen, die in erster Li­ nie dem spezifischen Design und dem innovativen Clippingmechanismus zu­ zurechnen ist: 72,5 % der AS-Patienten hatten bei Entlassung keine paravalvu­ lären Lecks. Lediglich bei 27,5 % trat eine milde PVL, bei keinem eine mo­ derate oder schwere PVL auf. „Das Outcome ist beeindruckend“, re­ sümierte Professor Olaf Wendler vom King’s College Hospital in London, und ergänzte: „Damit sind die para­ valvulären Lecks so gut wie überwun­ den“. Laut Wendler sind auch die hä­ modynamischen Ergebnisse exzellent: Der mittlere AV-Gradient lag nach der Operation bei 8,3 mmHg. Die Schritt­ macherrate betrug 2,6 % in den ersten 48 Stunden nach Implantation. Ein schwerwiegender Schlaganfall trat nicht auf, lediglich ein Patient erlitt ei­ nen Herzinfarkt. In keinem Fall wurden die Koronararterien verlegt. Der Funk­ tionsstatus verbesserte sich deutlich: Vor der Implantation hatten 85,4 % der Patienten eine Herzinsuffizienz im Sta­ dium NYHA III und höher. Nach der Perfusion 01/2014

27. Jahrgang

Prozedur durch neues DeliverySystem vereinfacht

Transfemorales System in Aussicht In Kürze soll es neben dem transapika­ len auch ein transfemorales TAVI Sys­ tem von JenaValve geben. In den letz­ ten Monaten wurde dieses von Prof. Dr. Stephan Baldus vom Herzzentrum der Uniklinik Köln im In-vivo-Modell ge­ testet. „Dabei zeigte sich, dass sich das transfemorale TAVI-System sehr gut steuern und die Prothese anatomisch korrekt positionieren lässt“, berichtete Baldus. Ende 2013 begann die erste klinische Studie, mit der CE-Zulassung wird 2014 gerechnet.

Fabian Sandner, Nürnberg

Schlaganfallprophylaxe bei nicht valvulärem Vorhofflimmern – effektiv und verträglich mit Apixaban Vitamin-K-Antagonisten (VKA) wur­ den lange Zeit vorwiegend zur Schlag­ anfallprophylaxe eingesetzt. „Sie wei­ sen jedoch mehrere Limitationen wie ein relativ enges therapeutisches Fens­ ter und ein aufwendiges Gerinnungs­ monitoring auf, die ihren Einsatz in der Praxis erschweren [1]“, gab Professor Johann Bauersachs, Hannover, auf einem Pressegespräch anlässlich der DGK-Herbsttagung 2013 zu bedenken.

Aufgrund des überlegenen NutzenRisiko-Profils sowie der einfacheren Handhabung gegenüber VKA werden zunehmend neue orale Antikoagulanzi­ en (NOACs) wie der direkte, orale Fak­ tor-Xa-Inhibitor Apixaban (Eliquis®) in der Praxis eingesetzt.

Apixaban ist zur Schlaganfallprophy­ laxe bei nicht valvulärem Vorhofflim­ mern (VHF) und mindestens einem weiteren Risikofaktor wie Schlagan­ fall oder transitorische ischämische Attacke (TIA) in der Anamnese, Al­ ter ≥75 Jahre, Hypertonie, Diabetes mellitus, symptomatische Herzin­ suffizienz (NYHA ≥II) in der fixen Standarddosierung von 5 mg zweimal täglich zugelassen; bei mindestens 2 der folgenden Kriterien Alter ≥80 Jahre, Körpergewicht ≤60 kg oder Se­ rumkreatinin ≥1,5 mg/dl (133 μmol/l) bzw. bei schwerer Nierenfunktions­ störung (Kreatinin-Clearance 15– 29 ml/min) sollte die Dosis auf 2,5 mg zweimal täglich reduziert werden.

Apixaban verfügt über ein breites the­ rapeutisches Fenster und regelmäßige Gerinnungsmonitorings sind ebenso wenig notwendig wie häufige Dosis­ anpassungen [2]. Der Zulassung lie­ gen 2 Studien – ARISTOTLE [3] und AVERROES [4] – mit insgesamt etwa 24.000 Patienten zugrunde, in denen Wirksamkeit und Verträglichkeit von Apixaban umfassend nachgewiesen wurden.

© Verlag PERFUSION GmbH

13

Kongresse

*

**

p=0,01 für Überlegenheit

***

p<0,001

p=0,047

Ereignisrate pro Jahr (%)

4 3 2 1

Apixaban

0

Warfarin INR-Zielwert: 2,0-3,0

(n/N****)

(265/9081)

(212/9120)

Primärer Wirksamkeitsendpunkt

(462/9052)

(327/9088)

(669/9081)

Sekundärer Endpunkt

(603/9120)

Sekundärer Endpunkt

Medianwert der Beobachtungszeit: 1,8 Jahre

Abbildung 1: In der ARISTOTLE-Studie war der direkte Faktor-Xa-Inhibitor Apixaban Warfarin in 3 wichtigen Endpunkten signifikant überlegen [3]. * HR 0,79; 95%-KI: 0,66–0,95; p<0,001 für Nicht-Unterlegenheit ** HR 0,69; 95%-KI: 0,60–0,80 *** HR 0,89; 95%-KI: 0,80–0,99 **** n = Anzahl der Patienten mit nicht valvulärem Vorhofflimmern mit einem Ereignis

N = Anzahl der Patienten mit nicht valvulärem Vorhofflimmern in der Apixaban- oder Warfarin-Gruppe

RRR = Relative Risikoreduktion

Studien belegen überzeugendes Nutzen-Risiko-Profil für Apixaban In der ARISTOTLE-Studie wurden 18.201 Patienten doppelblind im Me­ dian 1,8 Jahre entweder mit Apixaban 5 mg zweimal täglich (ausgewählte Patienten mit entsprechenden Kriterien erhielten 2,5 mg zweimal täglich) oder mit Warfarin (INR: 2,0–3,0) behandelt.

Das Ergebnis wies die signifikante Überlegenheit von Apixaban gegen­ über Warfarin in gleich 3 wichtigen Endpunkten nach (Abb. 1): Apixaban reduzierte signifikant das relative Ri­ siko für Schlaganfälle und systemische Embolien um 21 % (p=0,01 für Über­ legenheit) und für schwere Blutungen um 31 % (wichtiger sekundärer End­ punkt; p<0,001). Zudem wurde die Ge­ samtmortalität als weiterer wichtiger Endpunkt relativ um 11 % (p=0,047) gesenkt [3]. Bauersachs wies darauf hin, dass der Nutzen von Apixaban gegenüber Warfarin hinsichtlich der Reduktion von Schlaganfällen oder systemischen Embolien, schweren Blutungen und der Gesamtmortalität Perfusion 01/2014

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sich auch konsistent in allen präspezi­ fizierten Subgruppenanalysen gezeigt habe – so profitierten auch Patienten mit Schlaganfall/TIA in der Anamnese oder in hohem Alter sowie Patienten unabhängig von ihrem Risikoprofil von der Schlaganfallprophylaxe mit dem direkten Faktor-Xa-Inhibitor [5, 6, 7].

Auch in der AVERROES-Studie, die Wirksamkeit und Verträglichkeit von Apixaban gegenüber Acetylsalicyl­ säure (ASS) bei insgesamt 5.599 für eine Therapie mit VKA ungeeigne­ ten Patienten untersuchte, verringer­ te Apixaban das relative Risiko für Schlaganfälle und systemische Em­ bolien gegenüber ASS signifikant um 55 % (HR 0,45; 95%-KI: 0,32–0,62; p<0,001), bei vergleichbarem Risiko für schwere Blutungen (sekundärer Endpunkt) [4].

Aufgrund der klinischen Studienda­ ten und der darin nachgewiesenen Wirksamkeit, dem günstigen Verträg­ lichkeitsprofil und der einfacheren Anwendung im Vergleich zu Warfarin geben die aktuellen Leitlinien der Eu­ ropean Society of Cardiology (ESC)

der Substanzklasse der NOACs, zu der auch Apixaban zählt, einen gewis­ sen Vorrang gegenüber VKA [8]. Die Deutsche Gesellschaft für Neurologie (DGN) nennt die NOACs als Alternati­ ve zu den VKA in ihren Leitlinien und empfiehlt aufgrund des günstigeren Nutzen-Risiko-Profils deren Anwen­ dung in der Sekundärprävention [9].

Neue Daten vom ESC-Kongress – Subgruppe mit Herzklappen­ erkrankung Aktuelle Daten einer Post-hoc-Sub­ gruppenanalyse der ARISTOTLEStudie mit 4.808 Patienten (26,4 % der ARISTOTLE-Gesamtpopulation) mit nicht valvulärem VHF und einer Herzklappenerkrankung wurden auf dem ESC-Kongress 2013 vorgestellt.

Die Ergebnisse waren mit den Ge­ samtergebnissen konsistent. Der Nut­ zen von Apixaban gegenüber Warfarin war hinsichtlich der Reduktion von Schlaganfällen und systemischen Em­ bolien, schweren Blutungen und der © Verlag PERFUSION GmbH

14

Kongresse / Mitteilungen

Gesamtmortalität konsistent, unab­ hängig von dem Vorliegen einer Herz­ klappenerkrankung. Ausgeschlossen von der ARISTOTLE-Studie waren Patienten mit einer klinisch signifi­ kanten Mitralstenose oder einer me­ chanischen Herzklappenprothese [10]. „Die Ergebnisse dieser Subgruppen­ analyse liefern wichtige Erkenntnisse zur Verträglichkeit und Wirksamkeit von Apixaban bei dieser Patientenpo­ pulation, die häufig älter ist und ein höheres Risiko für klinische Ereignis­ se trägt als Patienten mit nicht valvu­ lärem Vorhofflimmern ohne Herzklap­ penerkrankung“, kommentierte Dr. Alvaro Avezum, São Paolo, Brasilien.

Nutzen für die Praxis „Apixaban reduzierte in fixer Dosie­ rung sowohl signifikant die Rate an Schlaganfällen/systemischen Embolien als auch an schweren Blutungen“, resü­ mierte Bauersachs und ergänzte: „Des Weiteren zeichnet sich Apixaban durch

MITTEILUNGEN Neu: Blutzuckermessgerät MyStar Extra® Ab sofort ist in Deutschland das neue Blutzuckermessgerät MyStar Extra® von Sanofi erhältlich. Als erstes und einziges Blutzuckermessgerät be­ rechnet MyStar Extra® einen HbA1cSchätzwert und einen HbA1c-Trend.

Mit seinen Extra-Funktionen und der einfachen, intuitiven 1-2-3-Klick-Be­ dienung ermöglicht MyStar Extra® eine frühzeitige und selbstständige Kontrol­ le des Therapieverlaufs und motiviert zu einer konsequenten Durchführung der Diabetestherapie.

eine signifikante Reduktion intrakrani­ eller Blutungen, keine Erhöhung schwe­ rer gastrointestinaler Blutungen und die Überlegenheit gegenüber dem VKA Warfarin unabhängig von der Güte der INR-Erstellung aus [11].“ Apixaban weist zudem auch bei alten und/oder niereninsuffizienten Patienten sowie bei Patienten nach Schlaganfall/TIA ein ge­ genüber Warfarin deutlich überlegenes Nutzen-Risiko-Profil auf und kann so einem breiten Spektrum von Patienten mit nicht valvulärem Vorhofflimmern eine effektive, verträgliche und einfach zu handhabende Schaganfallprophylaxe ermöglichen [6, 7, 12]. Als bislang ein­ ziges NOAC wurde Apixaban zudem im Vergleich zu ASS evaluiert – mit dem Ergebnis einer starken Reduktion von Schlaganfällen bei vergleichbarer Rate an schweren Blutungen.

Fabian Sandner, Nürnberg Literatur 1 Wilke T et al. Thromb Haemost 2012; 107:1053-1065

Gespräch beim Arzt – oft reicht die Blutzuckerselbstmessung allein nicht aus, um einen Zusammenhang zwi­ schen Ernährung, Bewegung, Thera­ pie, den Blutzuckerwerten und dem Einfluss auf den sog. Langzeitblut­ zuckerwert HbA1c herzustellen. Die­ ser wichtige Marker wird einmal im Quartal beim Arzt gemessen. Liegt er dauerhaft unter 7 %, wird das Risiko für Folgeerkrankungen nachweislich verringert. Zwischen den Arztbesuchen kann der von MyStar Extra® berechne­ te HbA1c-Wert und -Trend eine Orien­ tierungshilfe sein.

Der HbA1c-Schätzwert basiert auf ei­ nem Algorithmus, der von führenden Wissenschaftlern der University of Vir­ ginia entwickelt und zum Patent einge­ reicht wurde.

HbA1c-Schätzwert als Orientierungshilfe

Intuitive Bedienung und übersichtlicher Datenspeicher

Egal ob zur Selbstkontrolle, als Hilfe zur Lebensstiländerung oder für das

MyStar Extra® arbeitet mit der dy­ namischen Elektrochemie, verfügt

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2 Fachinformationen Eliquis® 2,5 mg, 5 mg, Stand September 2013 3 Granger CB et al. N Engl J Med 2011; 365:981-992 4 Connolly SJ et al. N Engl J Med 2011;3: 806-817 5 Lopes RD et al. Lancet 2012;380:17491758 6 Easton JD et al. Lancet Neurol 2012;11: 503-511 7 Halvorsen S et al. J Am Coll Cardiol 2013;61:5 8 Camm AJ et al. Europace 2012;14:13851413 9 Diener HC et al. Leitlinien für Diagnostik und Therapie in der Neurologie. Stuttgart: Thieme, 2012 10 Avezum A. Abstract 4384 presented at the annual congress of the European Society of Cardiology, August 31–September 4, 2013. Amsterdam, Netherlands 11 Wallentin L et al. Circulation 2013;127: 2166-2176 12 Hohnloser SH et al. Eur Heart J 2012;33: 2821-2830

Quelle: Pressegespräch anlässlich der DGKHerbsttagung am 11. Oktober 2013 in Dres­ den; Veranstalter: Bristol-Myers Squibb GmbH & Co. KGaA, München und Pfizer Pharma GmbH, Berlin

über ein großes, beleuchtetes Dis­ play, leicht lesbare Ziffern und intui­ tive Symbole. Angezeigt werden die Durchschnittswerte der letzten 3, 7 und 30 Tage – entweder für alle Mess­ werte oder für solche, die mit Symbo­ len für nüchtern, vor oder nach einer Mahlzeit markiert wurden. Zusätzlich zeigt das Gerät mit einem Trendpfeil die Entwicklung der Nüchternblutzu­ ckerwerte an. MyStar Extra® speichert alle Blutzuckermesswerte automatisch im integrierten Tagebuch und schlägt für die gemessenen Werte Mahlzeiten­ markierungen vor.

Das Blutzuckermessgerät verwendet die bewährten BGStar®-Teststreifen. Übergreifend für alle Blutzuckermess­ geräte von Sanofi werden ab 1. Febru­ ar 2014 die Teststreifen in die Preis­ gruppe B bzw. in die Preisklasse 2 des VdEK bzw. BARMER GEK Vertrags aufgenommen.

E. W.

© Verlag PERFUSION GmbH

ABSTRACTS

Abstracts der wissenschaftlichen Beiträge zur 28. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung e. V. 13. bis 15. März 2014 Schloss Rauischholzhausen I. Vorträge Hyperreactivity of junctional adhesion molecule A-deficient platelets accelerates atherosclerosis in hyperlipidemic mice E. Karshovska, Z. Zhao, M. M. M. Schmitt, X. Blanchet, K. Bidzhekov, O. Soehnlein, P. von Hundelshausen, N. Mattheij, J. W. M. Heemskerk, T. A. Koeppel, T. M. Hackeng, C. Weber, R. R. Koenen Maastricht University, CARIM, School for Cardio­ vascular Diseases, Maastricht, The Netherlands Background: Besides their essential role in haemostasis, platelets also have role in inflammation. Platelets may in­ duce endothelial dysfunction through direct interactions or through the re­ lease of pro-inflammatory cytokines.

Junctional adhesion molecule (JAM-) A is an IgG-superfamily transmem­ brane molecule abundantly expressed on endothelial cells and platelets. Re­ cent studies have implied endothelial JAM-A in the recruitment of mono­ cytes during atherogenesis and JAM-A on platelets was recently identified as an inhibitor of integrin α2bβ3-mediated outside-in signaling. Deficiency of JAM-A in platelets resulted in hyper­ reactivity. In this study, this gain-offunction of JAM-A deficient platelets was exploited to investigate the role of platelet hyperreactivity in plaque de­ velopment.

Methods and results: Mice with floxed JAM-A alleles were crossed with PF4-Cre apolipoprotein Edeficient (ApoE–/–) mice to obtain PF4-Cre+ or PF4-Cre– JAM-Aflox/flox Perfusion 01/2014

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ApoE–/– mice (trJAM-A–/– ApoE–/– or trJAM-A+/+ ApoE–/–, respectively) and were placed on a high-fat diet for 2, 6, and 12 weeks. Characterization of the response of trJAM-A–/– and trJAM-A+/+ to various platelet agonists confirmed the previously published hyperreactiv­ ity of trJAM-A–/– platelets. Under shear flow conditions, trJAM-A–/– showed increased adhesion to collagen and in­ creased activation of integrin α2bβ3.

After up to 12 weeks of high-fat die trJAM-A–/– ApoE–/– mice showed in­ creased plaque formation in the aorta compared with trJAM-A+/+ ApoE–/– controls. However in the aortic roots, plaque formation in trJAM-A–/– ApoE–/– mice was only enhanced after 2 weeks of diet and the differences were no longer present at 6 and 12 weeks. At 2 weeks but not at 12 weeks, the plaques of the trJAM-A–/– ApoE–/– animals showed increased macrophage, T cell and smooth muscle cell content and also increased mRNA expression of cytokines interferone γ and tumor ne­ crosis factor α, compared to controls.

Interestingly, the plasma levels of chemokines CCL5 and CXCL4 were increased in the trJAM-A–/– ApoE–/– mice and JAM-A-deficient platelets showed increased binding to mono­ cytes and leukocytes, both indicative for increased baseline activation or hyperreactivity. Finally, whole-blood perfusion experiments in vitro and in­ travital microscopy in vivo revealed increased recruitment of monocytes to the endothelium in blood of trJAM-A–/– ApoE–/– mice compared to controls.

Conclusions: Deletion of JAM-A causes a gain-of-function in platelets, with decreased activation thresholds

15

and increased inflammatory activities.

This leads to an increase of plaque for­ mation, particularly in early stages of the disease.

Expression of anaphylatoxin receptors on platelets in patients with coronary heart disease J. Patzelt1, 2, K. Mueller2, S. Breuning2, M. Gawaz2, H. F. Langer1, 2, T. Geisler2 1 Section for Cardioimmunology, Eberhard Karls University Tuebingen, Germany 2 University Hospital, Department of Cardiovascular Medicine, Eberhard Karls University Tuebingen, Germany Background: Inhibition of compo­ nents of the complement system or of its receptors has been postulated as a concept for secondary prevention in atherosclerosis and was applied in clin­ ical trials. Although the anaphylatoxinreceptors C3aR and C5aR are com­ monly associated with inflammatory cells, in vitro studies suggested their expression on platelets.

Patients and method: Here, we ana­ lyzed the expression levels of C3aR and C5aR in a collective of 405 pa­ tients with documented coronary artery disease.

Results: In comparison to healthy controls (n=21), patients with sympto­ matic coronary artery disease or acute myocardial infarction had significantly stronger expression of C5aR on plate­ lets (MFI 16.1 and 14.1, respectively, versus 7.0 and 10.9; p<0.001). Fur­ thermore, the expression of C3aR on platelets was significantly enhanced in patients with symptomatic coronary artery disease but not in patients with myocardial infarction. While there was a strong correlation between the solu­ ble ligands of these receptors C3a and C5a, we observed only a weak corre­ lation with their receptors on platelets.

Similarly, agonist-induced aggregation (MEA, ADP, and TRAP) showed only weak correlation with the expression level of anaphylatoxin-receptors on platelets. Interestingly, however, the © Verlag PERFUSION GmbH

ABSTRACTS

16

expression of both anaphylatoxin-re­ ceptors on platelets strongly correlated with platelet activation as assessed with the surface activation marker Pselectin (r=0.51, p>0.001 for C3aR, r=0.77 for C5aR, p<0.001). Likewise, we observed a positive correlation of C3aR with other molecules associated with platelet activation such as SDF-1.

Conclusion: In summary, we observed a clear correlation between the expres­ sion of anaphylatoxin-receptors C3aR and C5aR with platelet activation in patients with coronary artery disease.

Further investigations are needed to study the prognostic and mechanistic relevance of the unexpected expression of inflammatory receptors C3aR and C5aR on platelets.

Clonal restriction and predominance of regulatory T cells in coronary thrombi of patients with acute coronary syndromes R. Klingenberg1, 2, C. E. Brokopp3, A. Grivès4, A. Courtier4, M. Jaguszewski1, N. Pasqual4, E. Vlaskou Badra1, A. Lewandowski1, O. Gaemperli1, S. P. Hoerstrup3, W. Maier1, U. Landmesser1, 2, T. F. Lüscher1, 2, C. M. Matter1, 2 1 University Heart Center, Department of Cardiology, University Hospital Zurich, Switzerland 2 Cardiovascular Research, Zurich Center of Integrative Human Physiology (ZIHP), Institute of Physiology, University of Zurich, Switzerland 3 Regenerative Medicine Center, Department of Cardiothoracic Surgery, University Hospital Zurich, Switzerland 4 ImmunID Technologies, Grenoble, France Aims: Regulatory T cells (Treg) exert anti-inflammatory and atheroprotective effects in experimental atherosclerosis.

Treg can be induced against specific antigens using immunization strategies associated with clonal restriction. No data exist on Treg in combination with clonal restriction of T cells in patients with acute coronary syndromes (ACS).

Methods and results: Among T cell subsets characterized by flow cytome­ try, Treg (CD4+ CD25+ CD127low) were twice as frequent in coronary thrombi Perfusion 01/2014

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compared with peripheral blood. To evaluate clonal restriction, genomic DNA was extracted from coronary thrombi and peripheral blood in order to evaluate T cell receptor β chain di­ versity by means of Multi-N-plex PCR using a primer specific for all T cell re­ ceptor β V gene segments and a primer specific for T cell receptor β J gene segments. T cell receptor diversity was reduced in thrombi compared with pe­ ripheral blood (intra-individual com­ parisons in 16 patients) with 8 gene rearrangements in the T cell receptor common in at least 6 out of 16 analyzed coronary thrombi. Compared with agematched healthy controls (n=16), T cell receptor diversity was also reduced in peripheral blood of patients with ACS; these findings were independent of pe­ ripheral T cell numbers.

Conclusion: We provide novel evi­ dence for a perturbed T cell compart­ ment characterized by clonal restric­ tion in peripheral blood and coronary thrombi from patients with ACS. Treg prevailed among T cell subsets identi­ fied in coronary thrombi, suggesting Treg as a novel target for specific thera­ pies such as immunization to enhance the anti-inflammatory component of adaptive immunity in coronary athero­ thrombosis.

Macrophage accumulation during plaque progression depends on local macrophage proliferation rather then cell recruitment I. Hilgendorf, C. S. Robbins, F. K. Swirski University Heart Center Freiburg Bad Krozingen, Department of Cardiology and Angiology, University of Freiburg, Germany Rationale: A dominant paradigm states that macrophage accumulation in atherosclerotic lesions results from the continuous influx of circulating mono­ cytes. Here, we challenge this para­ digm and show that local macrophage proliferation rather than monocyte re­ cruitment drives rapid cell turnover in atheromata.

Methods and results: BrdU incorpora­ tion studies revealed that lesional mac­ rophage turnover is rapid; self renewal of the entire population occurs within 4 weeks. Surprisingly, short-term mono­ cyte depletion by clodronate liposomes did not affect aortic macrophage turn­ over and numbers, suggesting that macrophage renewal can occur in­ dependent of monocytes. Cell cycle analysis using 4’,6-diamidino-2-phe­ nylindole (DAPI), Ki-67 immuno-his­ tochemical staining, and image stream analysis showed that lesional aortic macrophages proliferate in situ in mice as well as in human disease. Indeed, parabiosis experiments confirmed that local proliferation accounted for 87 % of cell turnover in established athero­ mata. Increased macrophage prolif­ eration depended on the local plaque milieu and scavenger receptor A sign­ aling. Anti-proliferative treatment ef­ fectively reduced macrophage content and lesion size.

Conclusion: We propose a biphasic model of plaque development. Initially lesional macrophages directly derive from newly recruited monocytes. With disease progression, however, mac­ rophages predominantly accumulate by local proliferation. These studies enrich our understanding of atherogen­ esis and identify macrophage prolifera­ tion as a potentially important thera­ peutic target.

Regulation of macrophage polarization by extracellular RNA: the role of sialoadhesin-1 H. A. Cabrera-Fuentes1, S. Galuska1, S. Meiler2, Y. Baumer2, S. McCurdy2, S. Fischer1, K. T. Preissner1, W. A. Boisvert2 1 Institute for Biochemistry, Medical School, Justus Liebig University, Giessen, Germany 2 Center for Cardiovascular Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, U.S.A. Background: Macrophages respond to external stimuli with rapid changes in their expression of many inflammation© Verlag PERFUSION GmbH

ABSTRACTS

related genes to undergo polarization towards the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype.

This unique property of macrophages allows these cells to modulate chronic inflammatory processes such as athero­ genesis. We have previously shown that extracellular RNA (eRNA) can exert prothrombotic and inflammatory properties in the vasculature as a co­ factor in protease auto-activation and cytokine mobilization.

Aim: Given the association of eRNA with macrophages within atheroscle­ rotic lesions, we assessed whether eRNA generated by the macrophages themselves may induce inflammatory responses within macrophages, inde­ pendent of Toll-like receptor signaling.

Method and results: Recombinant mouse macrophage CSF-driven bone marrow-derived macrophage (BMDM) differentiation was skewed towards the M1 phenotype by exposure of cells to eRNA. This resulted in the overexpres­ sion of inflammatory markers such as TNF-α, Arg2, IL-1β, IL-6, or IfN-γ to­ gether with IL-12 and iNOS, whereas anti-inflammatory genes such as IL-10 and IL-4 together with Arg1 and mac­ rophage mannose receptor-2 (Cd206) were significantly down-regulated by eRNA. Accordingly, the release of TNF-α and IL-6 proteins into the cell supernatant was significantly elevated by eRNA stimulation. Moreover, the capacity of granulocyte macrophage CSF-driven BMDM differentiation (already representing M1 phenotype) towards further M1 polarization in re­ sponse to eRNA was moderate. Never­ theless, a significant down-regulation of M2 markers was found.

Sialoadhesin-1 (SN-1) is a membraneanchored basic protein, predominantly expressed on monocytes/macrophages and involved in cellular interactions via recognition of sialylated glycocon­ jugates. Interestingly, exposure of SN1–/– BMDM towards eRNA resulted in prominent down-regulation of proinflammatory cytokines together with M1 phenotype markers, while antiinflammatory cytokines together with Perfusion 01/2014

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M2 phenotype mediators were signifi­ cantly raised.

Conclusion: In accordance with our proposal that eRNA serves as proinflammatory “alarm signal” during physiologically distressed conditions, these data may shed light on the role of eRNA and macrophages in chronic inflammatory environment such as atherosclerosis. The self-perpetuating process of atherosclerosis as a largely inflammation-driven pathology may be controlled by SN-1 expression on lesional macrophages as well as the injury-mediated release of eRNA as a master trigger in inflammation.

Deficiency of the sialyltransferase St3Gal4 reduces Ccl5-mediated myeloid cell recruitment and arrest

17

lyltransferase ST3Gal-IV in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis.

Methods and results: St3Gal4-defi­ cient myeloid cells showed a reduced binding of Ccl5 and impaired Ccl5triggered integrin activation. Corre­ spondingly, Ccl5-induced arrest on TNF-α-stimulated endothelium was almost completely abrogated, as ob­ served in flow chamber adhesion as­ says and during ex vivo perfusion or intravital microscopy of carotid arter­ ies. Moreover, Ccl5-triggered neutro­ phil and monocyte extravasation into the peritoneal cavity was severely re­ duced in St3Gal4–/– mice. In contrast, St3Gal4-deficiency did not significant­ ly affect Ccl2 binding and only mar­ ginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accu­ mulation in atherogenesis, and the im­ portance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4deficiency drastically reduced the size, stage and inflammatory cell content of atherosclerotic lesions in ApoE–/– mice on high-fat diet.

Conclusion: In summary, these find­ ings identify ST3Gal-IV as a promis­ ing target to reduce inflammatory leu­ kocyte recruitment and arrest.

H. Noels1, Y. Döring2, M. Mandl2, B. Kramp2, C. Neideck2, D. Lievens2, M. Drechsler2, R. T. A. Megens2, 7, P. V. Tilstam1, M. Langer2, H. Hartwig2, W. Theelen1, J. D. Marth3, M. Sperandio4, 5, O. Soehnlein2, 5, 6, C. Weber2, 5, 7 1 IMCAR, RWTH Aachen University, Germany 2 IPEK, Ludwig Maximilians University Munich, Germany 3 Center for Nanomedicine, Sanford-Burnham Medical Research Institute, University of California Santa Barbara, USA 4 WBex, Ludwig Maximilians University Munich, Germany 5 DZHK (German Center for Cardiovascular Research), partner site Munich Heart Alliance, Germany 6 AMC, Amsterdam, The Netherlands 7 Cardiovascular Research Institute Maastricht, The Netherlands

Long-chain metabolites of α-tocopherol and a δ-tocotrienolrelated natural compound regulate inflammatory pathways in macrophages

Aims: Sialylation by α2-3 sialyltrans­ ferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor. As the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruit­ ment of neutrophils and monocytes, we here investigated the role of the sia­

M. Wallert1, L. Schmölz1, J. Heise1, V. Krauth2, O. Werz2, M. Birringer3, S. Lorkowski1 1 Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 Department of Pharmaceutical Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Germany 3 Department of Nutritional, Food, and Consumer Studies, University of Applied Sciences Fulda, Germany © Verlag PERFUSION GmbH

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18

Background: Inflammation is a key initiator and driving force for many age-associated diseases, such as ath­ erosclerosis. Macrophages regulate in­ flammation by producing chemotactic and inflammatory mediators. Vitamin E, a mixture of tocopherols and to­ cotrienols, is the most important lipid antioxidant but next to its anti-oxidant properties, vitamin E regulates inflam­ mation and signal transduction. Little effort has been spend so far on unrave­ ling the effects of structurally related compounds and metabolites of vitamin E on the regulation of inflammatory pathways.

Aim: We therefore investigated the in­ fluence of the physiologically relevant long-chain α-tocopherol metabolites α-13’-COOH and α-13’-OH, and the tocotrienol-related natural compound garcinoic acid on the LPS-induced in­ flammatory response of murine mac­ rophages.

Methods and results: We measured a selection of pro- and antiatherogen me­ diators, i.e. IL-6, IL-1β, TNF-α, iNos, Cox2, IL-12p40, TGF-β1 and IL-10, using RT-qPCR and Western blotting.

We further analyzed the subsequent ef­ fects on nitric oxide and prostaglandin E2 production by murine macrophag­ es. Lipopolysaccharide-induced iNos and Cox2 mRNA and protein levels were significantly reduced by all com­ pounds by a comparable extent. The LPS-induced production of nitric ox­ ide and prostaglandin E2, measured in the supernatant using Griess assay and prostaglandin E2 ELISA, showed sig­ nificant reductions in the presence of the compounds. To explain the immu­ nomodulatory properties of the com­ pounds we are currently investigating the underlying regulatory pathways.

Conclusion: Oxidized metabolites and isomers of α-tocopherol and δ-tocotrienol show high activity in inhibiting proinflammatory path­ ways and associated signal transduc­ tion. We also assume a new molecu­ lar mode of action of vitamin E via its metabolites.

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The histone deacetylase SIRT1 modulates STAT3 activity and prevents de-differentiation and proliferation of vascular smooth muscle cells J.-M. Daniel, J. Dutzmann, S. Vogel, D. G. Sedding Hannover Medical School (MHH), Hannover, Germany Background: De-differentiation and proliferation of vascular smooth mus­ cle cells (SMCs) are hallmarks of vasculo-proliferative diseases. Activa­ tion of the class III histone deacetylase SIRT1 has been shown to exert protec­ tive effects on vascular lesion progres­ sion; however, the cell-specific molec­ ular pathophysiological mechanisms remain incompletely understood. In this study, we tested the hypothesis that SIRT1 exerts specific effects on the dif­ ferentiation and proliferation of SMCs and investigated the underlying mecha­ nisms.

Methods and results: SIRT1 expres­ sion levels were significantly downregulated in proliferating human coronary SMCs in vitro and in SMCs following wire-induced neointimal le­ sion formation in mice in the mouse femoral artery in vivo. Over-expres­ sion and activation of SIRT1 using adenoviral vector systems or pharma­ cologic activation of SIRT1 attenuated proliferation of cultured SMCs in a dose dependent manner. In addition, SIRT1 activation induced a more dif­ ferentiated phenotype of SMCs, which was accompanied by higher expres­ sion levels of cytoskeletal and contrac­ tile proteins (sma, smmhc, calponin) as determined by proteomic analysis (MALDI-TOF) and subsequent im­ mune blotting as well as qPCRhigher expression levels of cytoskeletal and contractile proteins using qPCT, im­ munoblotting and MALDI-TOF analy­ sis. In a mouse model of wire-induced injury, over-expression of wild typeSIRT1 but not a deacetylase inactive SIRT1-H355A mutant prevented ne­ ointimal formation and significantly reduced SMC proliferation at 3 weeks

after dilation (neointima/media ratio: pAd control: 1.94±0.31, pAd-SIRT1H355A 2.04±0.24, pAd-SIRT1-WT 0.56±0.49, n=8, p<0.05). A gene mi­ croarray analysis identified STAT3 de­ pendent target genes to be strongly reg­ ulated in response to changes in SIRT1 expression levels and SIRT1 activity.

Using immunoprecipitation analysis, we confirmed a direct interaction of SIRT1 with the transcription factor STAT3. Mechanistically, deacetylation of STAT3 by SIRT1 significantly pre­ vented transcription of cyclinD1 and survivin in activated SMCs. In conclu­ sion, our data provide evidence that activation of SIRT1 prevents STAT3 signaling in SMCs and holds promise as an excellent therapeutic strategy to target vasculo-proliferative diseases.

Characterization of atherosclerotic plaques by Raman spectroscopy and optical coherence tomography C. Matthäus1, 2, S. Dochow1, 2, K. Egodage1, 2, C. Krafft1, B. R. Brehm3, J. Popp1, 2 1 Leipniz Institute of Photonic Technology, Jena, Germany 2 Institute of Physical Chemistry and Abbe Center of Photonics – Friedrich Schiller University Jena, Germany 3 Catholic Clinic – Koblenz, Internal Medicine & Cardiology, Koblenz, Germany Aims: Visualization and characteriza­ tion of inner arterial plaque depositions is of vital diagnostic interest, especial­ ly for the early recognition of vulner­ able plaques. Established clinical tech­ niques provide valuable morphological information, but can not deliver infor­ mation about the chemical composition of individual plaques. Raman spectro­ scopy is a well established methodol­ ogy in routine analytical chemistry.

The spectroscopic information can eas­ ily distinguish major plaque compo­ nents such as cholesterol, cholesterol esters, triglycerides or crystalline cal­ cium. Here, we employ Raman-probe spectroscopy to characterize the plaque compositions in arterial walls of a rab­ © Verlag PERFUSION GmbH

ABSTRACTS

bit model in vivo. Rabbits were fed with a cholesterol-enriched diet. In order to correlate the obtained spectral information, a combination of Raman spectroscopy with existing imaging techniques, such as optical coherence tomography (OCT) would offer great potential for future applications in a clinical setting.

Methods: For in vivo characterization a Raman probe, available from Emvi­ sion (Loxahatchee, FL, USA), coupled to a Raman spectrometer from Kai­ ser Optical Systems, Inc. (Ann Arbor, MI, USA) was employed and tested on rabbits (New Zealand White), fed with a cholesterol enriched diet. OCT measurements were performed using a Thorlabs (Newton, NJ, USA) TelestoII (1300 nm) imaging system.

Results: Atherosclerotic plaque depo­ sitions were successfully detected and characterized in vivo. Cholesterol and cholesterol esters were identified as main plaque components. OCT images of the same positions were acquired.

The correlation of the results is cru­ cial for the technical development of a combined instrument.

Conclusions: Vibrational spectroscopy in combination with non-linear imag­ ing techniques were successfully em­ ployed and showed great potential for clinical applications. The techniques offer the possibility to gain information about the biochemical composition of plaque depositions in a qualitative and quantitative manner.

Acknowledgments: Financial support from the BMBF (FKZ: 13N12525) is highly acknowledged.

Molecular basis for treatment of diabetes type 2 with inhibitors of sodium-D-glucose cotransporters H. Koepsell Institute of Plant Physiology and Biophysics, University of Wuerzburg, Germany For treatment of diabetes type 2 a con­ tinuous down-regulation of increased blood glucose is required to avoid Perfusion 01/2014

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disastrous follow-up diseases which include injury of vascular systems in different organs. To achieve this aim treatment of diabetes with combina­ tions of antidiabetic drugs employing different mechanisms of action are required, for example the combina­ tion of metformin with insulin and a glucagon-like-peptide 1 (GLP-1) analog. In recent years it has been recognized that inhibitors of the Na+D-glucose cotransporters SGLT2 and SGLT1 decrease postprandial blood glucose. Whereas SGLT1 expressed in intestine is pivotal for the absorption of D-glucose in small intestine, SGLT2 expressed in the first two thirds of the renal proximal tubule is responsible for bulk reabsorption of ultrafiltrated D-glucose. SGLT1 which is expressed in the distal part of the proximal tubule reabsorbs the remaining 3 % of D-glu­ cose from the glomerular ultrafiltrate.

Selective SGLT2 inhibitors have been developed. Five inhibitors including empagliflozin reached clinical phase 3 trials and one, dapagliflozin, has been approved for treatment of diabetes in Europe. Evidence has been provided in preclinical and clinical studies that selective SGLT2 inhibitors decrease glucose reabsorption in the kidney and thereby reduce postprandial blood glu­ cose.

Employing a genetic mouse model of diabetes it has been shown that em­ pagliflozin attenuated albuminuria, diabetic increase of kidney growth and inflammation secondary to lowering blood glucose. In addition, empagliflo­ zin decreased the diabetic increase of glomerular filtration rate independent­ ly of lowering blood glucose. The data suggest that SGLT2 inhibitors protect against diabetic nephropathy.

Preclinical studies showed that inhibi­ tion of SGLT1 in small intestine in­ creased the glucose induced secretion of GLP-1 in distal ileum and thereby decreased the postprandial elevation of blood glucose. These data suggest that the down-regulation of SGLT1 in small intestine represents an additional way to control blood glucose during diabe­

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tes type 2. In summary, inhibitors of SGLT2 and SGLT1 have the potential to improve treatment of diabetes type 2.

Clinical profile and therapeutic role of SGLT-2 inhibition J. Seufert Division of Endocrinology and Diabetology, University Hospital of Freiburg, Germany Despite a substantial variety of thera­ peutic options, a good part of type 2 diabetic patients is not able to achieve adequate glycemic control. Novel phar­ macological treatment modalities ide­ ally provide a mode of action that can be widely combined with other drugs, has optimal cardiovascular effects, has beneficial effects on increased body weight, and brings along a low intrin­ sic risk of hypoglycemia. The develop­ ment of SGLT-2 inhibitors is providing a class of drugs that comes close to these expectations. SGLT-2 inhibition is a novel treatment modality that acts directly at the kidney and is completely independent of insulin secretion, or ac­ tion.

The kidneys represent an important organ for the regulation of glucose homoeostasis. They are substantially contributing to gluconeogenesis, but importantly also regulate glucose ho­ moeostasis through glomerular filtra­ tion and tubular resorption. Glucose resorption occurs in the proximal tu­ bule of the nephron by active trans­ portation across the luminal cellular membrane via the transporter proteins sodium-glucose cotransporters type 2 (SGLT-2, ca. 90 % of glucose resorp­ tion) and SGLT-1 (ca. 10 % of glucose resorption). Experimentally, it could be demonstrated that diabetes mellitus is accompanied by a dysregulation of glucose resporption in the proximal tu­ bule through up-regulation of SGLT-2 expression. Inhibition of SGLT-2 and subsequent induction of „therapeutic glucosuria“, therefore, is an attractive approach to counteract the „paradoxi­ cal“ and pathologically increased glu­ © Verlag PERFUSION GmbH

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cose resorption in patients with type 2 diabetes. Drugs that are already licensed or are in development for SGLT-2 inhibition are dapagliflozin, canagliflozin and empagliflozin. Trial results consistently have demonstrated for all three drugs clinically relevant HbA1c-reductions of about 0.7–1.1 %.

With once daily dosing, based on their insulin independent mode of action, SGLT-2 inhibitors can be combined with all other antidiabetic drugs in­ cluding insulin in dual, but also triple combination therapy regimes with syn­ ergistic actions. SGLT-2 inhibitors per se only have a very low intrinsic risk of hypoglycemia that is merely dependent on the antihyperglycemic combination partner.

Based on the excretion of both glucose and sodium, SGLT-2 inhibitors provide additional beneficial effects by lower­ ing body weight and blood pressure for patients with type 2 diabetes mel­ litus. Indeed, trial results demonstrate consistent weight reduction by 2–3 kg, and a systolic blood pressure lowering propensity of 4–5 mmHg. These “by­ stander effects” are specifically inter­ esting to lower the cardiovascular risk of type 2 diabetic patients. SGLT2inhibitors are generally well tolerated.

Main side effects comprise genital mycotic infections and hypovolemic episodes that can be clinically handled very well. Use of SGLT2-inhibitors is not recommended in patients with ad­ vanced kidney failure as the effect is dependent on the glomerular filtration rate.

Summary: Therapy of type 2 diabetes requests a multifactorial approach that integrates individual treatment targets with the patients’ comorbidities. Novel principles of action such as SGLT-2 in­ hibition that can be used at all stages of the disease are welcome and needed.

Drugs represent dapagliflozin, cana­ gliflozin und empagliflozin. SGLT-2 inhibitors • significantly improve glycemic con­ trol in clinical studies (increased ef­ fectivity when compared to sulfonyl­ ureas and DPP-4 inhibitors) Perfusion 01/2014

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• a ct independently of insulin secre­ tion, or action and can, therefore, be combined with all other antidiabetic drugs including insulin • bear a very low intrinsic risk of hypo­ glycemia (80 % less when compared to sulfonylureas) • support weight reduction in over­ weight type 2 diabetic patients • act to reduce the cardiovascular risk factor blood pressure • are well tolerated, but can cause geni­ tal mycotic infections.

Cardiovascular endpoint studies are running.

Obesity-induced dysfunction of endothelial nitric oxide synthase in perivascular adipose tissue N. Xia, S. Horke, A. Habermeier, E. I. Closs, G. Reifenberg, Z. Wu, D. Siuda, U. Förstermann, Y. Mikhed, A. Daiber, H. Li Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany Aims: Perivascular adipose tissue (PVAT) has recently been recognized as a novel modulator of vascular func­ tion. The present study was designed to study the role of PVAT in obesityinduced vascular dysfunction.

Methods: Obesity was induced in C57BL/6J mice with high-fat diet (60 % energy from fat).

Results: In PVAT-free aortas from mice fed a high-fat diet (HFD) for 20 weeks, the endothelium-dependent, NO-mediated vasodilator response to acetylcholine remained normal. In con­ trast, a clear reduction in the vasodila­ tor response to acetylcholine was ob­ served in aortas from obese mice when the PVAT was left in place. In immu­ nohistochemistry analysis, staining of endothelial NO synthase (eNOS) could be clearly seen in the aortic PVAT as well as in the aortic endothelium. Ace­ tylcholine induced vasodilation in the mouse aorta (either with or without PVAT) was completely blocked by the NOS inhibitor L-NAME, indicating

that this response is NO-dependent.

Thus, the reduced vasomotor function in the aorta of obese mice was likely to result from a dysfunction of eNOS in PVAT, but not in the endothelium.

Indeed, PVAT NO production was significantly reduced in obese mice compared to lean mice, as detected by 4,5-diaminofluorescein diacetate (DAF2-DA) staining with a Zeiss Laser Scanning Microscope (LSM).

HFD had no effect on the expression of eNOS at mRNA or protein levels, neither in PVAT nor in endothelium.

However, obesity led to a reduction of eNOS phosphorylation at serine 1177 (and thus in eNOS activity) in aortic PVAT, but not in the aorta itself. Acetyl­ choline enhanced the serine 1177 phos­ phorylation of PVAT eNOS in control mice, but not in obese mice. A number of kinases are known to phosphoryl­ ate eNOS at serine 1177, with Akt and AMPK being the most important ones.

The reduced PVAT eNOS serine 1177 phosphorylation in obesity was associ­ ated with a reduced Akt phosphoryla­ tion at serine 473 (and thus reduced ac­ tivity), whereas the activity of AMPK remained unchanged. Therefore, the reduced eNOS activity was likely to be due to an inhibition of Akt. Another potential mechanism underlying the re­ duced NO production in PVAT seemed to be a deficiency of L-arginine due to an induction of arginases.

Conclusion: A reduction of PVAT NO production is likely to be implicated in the obesity-induced vascular dysfunc­ tion.

Identification of GFAT1 as novel physiological AMPK substrate in the endothelial cells D. Zibrova1, 2, F. Vandermoere2a, O. Göransson3, M. Peggie4, S. Lindenmüller1, K. Spengler1, B. Viollet5, N. A. Morrice2b, K. Sakamoto2c, R. Heller1 1 Institute for Molecular Cell Biology, University Hospital Jena, Germany 2 MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, UK © Verlag PERFUSION GmbH

ABSTRACTS

Department of Experimental Medical Sciences, Lund University, Sweden 4 Division of Signal Transduction Therapy, University of Dundee, UK 5 Institut Cochin, Université Paris Descartes, CNRS, France Present addresses: a Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Montpellier, France b Beatson Institute for Cancer Research, Glasgow, UK c Nestlé Institute of Health Sciences, Lausanne, Switzerland 3

Aims: Adenosine monophosphateactivated protein kinase (AMPK), a master sensor of the cellular energy levels contributes to an anti-inflamma­ tory and anti-atherogenic phenotype of endothelial cells as well as to the regulation of angiogenesis. However, the molecular mechanisms mediating these functions remain elusive. Using a phosphoproteomic approach in mouse embryonic fibroblasts (MEFs) lacking the catalytic subunits of AMPK, we identified glutamine:fructose-6-phos­ phate amidotransferase 1 (GFAT1) as a potential AMPK substrate with serine 243 as a phosphorylation site. GFAT1 is the rate-limiting enzyme of the hex­ osamine biosynthesis pathway (HBP), which converts glucose to UDP-Nacetylglucosamine (UDP-GlcNAc).

The latter mediates posttranslational modification (O-GlcNAcylation) of various cellular proteins and may contribute to hyperglycemia-induced endothelial dysfunction. The current study was aimed to identify GFAT1 as an AMPK substrate in endothelial cells and to characterize a possible func­ tional role of the AMPK-GFAT axis in these cells.

Material and methods: Experiments were performed in human umbilical vein endothelial cells (HUVEC), in which AMPK was down-regulated by specific siRNAs or stimulated with 5-aminoimidazole-4-carboxamide ri­ bonucleoside (AICAR) or vascular endothelial growth factor (VEGF). The phosphorylation states of AMPK and Perfusion 01/2014

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its substrates were determined by im­ munoblotting of total lysates or immu­ noprecipitates. To study the regulation of GFAT1 by AMPK at the biochemical level, a GFAT1 phospho-specific anti­ body was generated. Total O-GlcNAc levels were analyzed by immunoblot­ ting using an anti-O-GlcNAc antibody.

To investigate the involvement of HBP in angiogenesis, sprouting of capillarylike structures from the endothelial cell spheroids was evaluated under normal (5.5 mM) and high (25 mM) glucose conditions in the presence or absence of the GFAT antagonist 6-diazo-5-ox­ onorleucine (DON).

Results: We first confirmed our data from the global phosphoproteomic screen by demonstrating that GFAT1 is an AMPK target in human embryonic kidney 293 cells and in MEFs. Next we showed that the chemical AMPK activator AICAR and, importantly, also VEGF, which is a major physiologi­ cal AMPK agonist, increased GFAT1 phosphorylation in primary endothe­ lial cells. With both stimuli, GFAT1 phosphorylation was not observed when AMPKα catalytic subunits were down-regulated by siRNAs. To inves­ tigate whether the AMPK-GFAT1 axis is of functional importance, we com­ pared O-GlcNAcylation patterns in en­ dothelial cells with or without AMPKα double knockdown. Interestingly, O-GlcNAcylation was considerably increased in HUVEC with AMPKα double knockdown. Oppositely, OGlcNAc levels were decreased in HU­ VEC treated with AICAR in compari­ son with control cells suggesting that GFAT1 and the HBP were inhibited by AMPK. Since VEGF is a potent an­ giogenic factor and AMPK has been shown to be involved in its angiogenic effects, we wanted to know whether inhibition of GFAT belongs to the un­ derlying mechanisms. Interestingly, pharmacological inhibition of GFAT1

by DON increased basal and VEGFinduced angiogenesis indicating that the HBP has an antiangiogenic role which may be counteracted by AMPK. In line with this, hyperglycemia, which

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is known to trigger the HBP, reduced VEGF-induced angiogenesis and this was rescued by DON. Conclusions: In the present study we have shown for the first time that GFAT1 (i) is AMPK substrate in the endothe­ lium, (ii) is a component of VEGFAMPK signaling and (iii) might medi­ate the effects of AMPK on angiogenesis.

Via GFAT1 phosphorylation, AMPK might control O-GlcNAc levels and counteract the adverse effects of hyper­ glycemia on endothelial function.

A score to predict coronary plaque vulnerability in high risk patients with type 2 diabetes: an optical coherence tomography study M. Burgmaier1, M. Hellmich2, S. Battermann1, N. Marx1, S. Reith1 1 Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany 2 Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne, Germany Introduction: Optical coherence to­ mography (OCT) is a novel intravascu­ lar imaging modality which allows the exact visualization and quantification of several vulnerable plaque character­ istics such as plaque macrophage con­ tent. This study tested the diagnostic value of OCT-derived coronary plaque characteristics to predict vulnerable le­ sions both individually and when com­ bined in a score in high-risk patients with type 2 diabetes.

Methods: OCT has been performed in 102 patients with type 2 diabetes.

Plaques of patients with ACS (n=40) were defined as vulnerable, those of patients with stable angina (n=62) as stable plaques.

Results: Vulnerable lesions were characterized by a smaller minimal fibrous cap thickness (FCT) overlying a lesions’s lipid core (51.5±0.9 µm vs.

80.3±26.7 µm), more frequent plaque macrophage infiltration (80.0 % vs.

33.9 %) and a larger medium lipid arc (174±33.8° vs. 122.9±43.9°) compared to stable plaques, all p<0.001. © Verlag PERFUSION GmbH

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Multivariable logistic regression anal­ ysis demonstrated that minimal FCT (odds ratio [OR] 0.354, p<0.011), the medium lipid arc (OR 15.843, p=0.016) and the presence of macrophages (OR 23.527, p=0.005) all predicted plaque vulnerability independently.

Receiver operated curve statistics showed that among all parameters in­ vestigated the minimal FCT had the best diagnostic efficiency to predict plaque vulnerability (AUC 0.840, 95% CI 0.735–0.944). However, when combined in a score the minimal FCT, the mean lipid arc and the presence of macrophages predicted plaque vulner­ ability with an excellent diagnostic ef­ ficiency (AUC 0.936, 95% CI 0.876– 0.996).

Conclusion: We report for the first time a score to predict plaque vulner­ ability in patients with type 2 diabe­ tes using OCT-derived features of coronary plaque composition. Future studies are warranted to determine if this score may be useful to reduce car­ diovascular morbidity and mortality in this high-risk population.

Association of SFRP4 with diabetes in patients with stable coronary artery disease M. M. Hoffmann1, C. Werner2, M. Böhm2, U. Laufs2, K. Winkler1 1 Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Freiburg, Germany 2 Clinic for Internal Medicine III (Cardiology, Angiology and Internal Intensive Medicine), Saarland University Medical Center, Homburg/ Saar, Germany Background: Secreted frizzled-relat­ ed proteins (SFRP) are regulators of Wnt signaling and therefore play an important role in embryonic develop­ ment and carcinogenesis. Recently, SFRP1/2/4 have been described as adi­ pokines in humans and SFRP4 as a reg­ ulator of insulin exocytosis in murine islet cells. Moreover, preliminary data showed an association of SFRP4 serum Perfusion 01/2014

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levels with insulin resistance and type 2 diabetes mellitus.

To confirm this observation we meas­ ured SFRP4 in participants of the Homburg Cream & Sugar Study, a pro­ spective cohort study of patients with stable coronary artery disease.

Methods and results: Fasting SFRP4 concentrations were measured by ELISA in 504 consecutive patients with stable CAD confirmed by angio­ graphy. The median age was 68 years and 83 % of patients were male. Oral glucose tolerance tests were performed in all patients without known diabetes for metabolic characterization. 24.4 % of patients showed normal glucose tol­ erance, 29.4 % impaired glucose tol­ erance and 46.2 % diabetes mellitus.

SFRP4 concentrations correlated with insulin (R=0.119, p=0.007) and HbA1c (R=0.146, p=0.011). SFRP4 was as­ sociated with gender, type 2 diabetes, insulin therapy, metabolic syndrome, and severity of diabetes.

The primary outcome was the com­ posite of cardiovascular death and cardiovascular hospitalization within 48 months follow-up. Comparison of event-free survival between SFRP4 tertiles showed that SFRP4 concentra­ tions were not predictive for CV out­ come. In a subgroup analysis including only patients with impaired glucose tolerance or diabetes there was a trend for a higher risk associated with the third tertile, which did not reach sig­ nificance.

Conclusion: SFRP4 concentrations are associated with impaired glucose me­ tabolism but do not predict outcome in patients with stable coronary artery disease.

New drugs fort he treatment of diabetes R. A. Ritzel Division of Endocrinology and Diabetes, Klinikum Schwabing, Munich, Germany Type 2 diabetes is characterized by relative insulin deficiency due to low

functional beta-cell mass and insulin resistance. The human pancreatic betacell has exquisite functional character­ istics (e.g. biphasic insulin secretion, pulsatile insulin secretion) which are difficult to reproduce by exogenous therapies. Since each patient has typi­ cal pathophysiologic characteristics, therapy needs to be individualized to address this metabolic heterogeneity and support residual beta-cell function directly or indirectly. Recent evidence suggests that early intervention is more effective to reduce cardiovascular end­ points than late establishment of glu­ cose control. Furthermore, multiple therapeutic mechanisms (= combina­ tion therapy) provide better glycemic durability compared to monotherapies.

Hence, there is great interest in new mechanisms to treat hyperglycemia and other features of diabetes mellitus.

New treatment strategies would also expand the possibilities of personalized diabetes therapy.

For several years inhibitors of 11βhydroxysteroid dehydrogenase type 1 (11βHSD1) are in development to re­ duce endogenous glucocorticoid pro­ duction. Since there are similarities between obesity, type 2 diabetes and Cushing’s syndrome it is hypothesized that inhibition of 11βHSD1 exerts an­ tidiabetic actions. 11βHSD1 catalyzes the intracellular conversion of inactive cortisone into bioactive cortisol and is expressed most prominently in the liv­ er, adipose tissue, vascular tissue, brain and macrophages. 11βHSD does not participate in the intraadrenal biosyn­ thesis of cortisol. Clinical trials showed some glucose lowering activity, how­ ever safety concerns arise from sub­ stantial increases of ACTH, DHEAS and androstendione. In women there also was a dose-dependent increase in total testosterone concentrations.

Another approach to address multiple aspects of metabolic disease in diabe­ tes mellitus may be the coagonism at the glucagon and the glucagon-likepeptide 1 receptors. In preclinical stud­ ies this dual-hormone receptor agonism decreased energy intake, body weight © Verlag PERFUSION GmbH

ABSTRACTS

and adipose tissue mass, improved glucose tolerance and decreased blood glucose concentrations. There also was a reduction of cholesterol and trigly­ ceride levels and a reversal of hepatic steatosis. Since these compounds are largely developed in proprietary set­ tings, little is published about potential side-effects and safety.

Other ongoing activities to develop new antidiabetic treatments include research on glucokinase activation, free fatty acid receptors (FFAR) and the human circadian rhythm. However, besides being effective new treatments should also improve patient outcome.

Impaired adipose tissue function und endothelial dysfunction M. Blüher Department of Medicine, University of Leipzig The incidence of obesity has increased dramatically during recent decades.

Obesity increases the risk for meta­ bolic and cardiovascular diseases and may therefore contribute to premature death. With increasing fat mass, secre­ tion of adipose tissue derived bioactive molecules (adipokines) changes to­ wards a pro-inflammatory, diabetogen­ ic and atherogenic pattern. Adipokines are involved in the regulation of ap­ petite and satiety, energy expenditure, activity, endothelial function, hemosta­ sis, blood pressure, insulin sensitivity, energy metabolism in insulin sensitive tissues, adipogenesis, fat distribution and insulin secretion in pancreatic β-cells. Therefore, adipokines are clini­ cally relevant as biomarkers for fat dis­ tribution, adipose tissue function, liver fat content, insulin sensitivity, chronic inflammation and have the potential for future pharmacological treatment strategies for obesity and its related diseases.

Impaired adipose tissue function may either directly (via secretion of athero­ genic adipokines and cytokines) or indirectly (via factors contributing to hypertension, insulin resistance, oxida­ Perfusion 01/2014

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tive stress, impaired glucose and lipid metabolism) contribute to premature atherosclerosis. The clinical relevance of selected adipokines as markers or predictors of obesity related endothelial dysfunction and as potential therapeutic tools or targets in metabolic and cardio­ vascular diseases is discussed.

Early cardiovascular comorbidity in childhood obesity (KFO Leipzig Atherobesity Childhood Cohort) J. T. Schwartze1, K. Landgraf1, D. Friebe1, T. Ullrich1, J. Kratzsch2, K. Scheuermann1, G. Herberth3, V. Adams4, W. Kiess1, S. Erbs4, A. Körner1 1 Center for Pediatric Research, Department of Women and Child Health, University of Leipzig, Germany 2 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Germany 3 Helmholtz Center for Environmental Research – UFZ, Leipzig, Germany 4 Heart Center Leipzig, University Hospital Leipzig, Germany Aims: We aim to identify new factors linking obesity and early-onset meta­ bolic and vascular sequelae in children.

In this context we identified the asso­ ciation of several adipokines including chemerin, NAMPT, and vaspin with obesity, early cardiovascular dysfunc­ tion, and impaired insulin resistance in children.

Methods: We assessed anthropomet­ ric, metabolic, and cardiovascular pa­ rameters in 174 children (69 lean/105 obese) including age, sex, body mass index (BMI), blood serum parameters, oral glucose tolerance testing, 24 hour blood pressure, the reactive hyper­­emia index (RHI), carotid intima media thickness (IMT), as well as endothelial progenitor cell number and migration.

Results: Significantly increased insu­ lin resistance was already evident in obese children. Markers of cardiovas­ cular dysfunction including hyperten­ sion, thickening of the carotid intima and media, a severely impaired RHI, as

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well as decreased numbers of endothe­ lial progenitor cells were also detected in obese children. Furthermore, mark­ ers of endothelial activation includ­ ing intercellular adhesion molecule (ICAM)-1 and E-selectin as well as high sensitive C-reactive protein were increased in children suffering from obesity whereas interestingly, stromal cell-derived factor (SDF)-1α as well as vascular endothelial growth factor (VEGF) serum levels were significant­ ly decreased compared to lean controls.

Conclusions: Our results suggest that well-known complications of obesity including insulin resistance and car­ diovascular dysfunction can already be detected in obese children demonstrat­ ing that pathogenic alterations caused by obesity already occur during child­ hood and adolescence.

Vaspin and glucose homeostasis J. T. Heiker1, N. Klöting2, P. Kovacs2, E. B. Küttner3, N. Sträter3, S. Schultz1, M. Kern2, M. Stumvoll4, M. Blüher4, A. G. Beck-Sickinger1 1 Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany 2 IFB Adiposity Diseases, University of Leipzig, Germany 3 Center for Biotechnology and Biomedicine, Institute for Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany 4 Department of Medicine, University of Leipzig, Germany Aims: Obesity significantly increases the risk of developing type 2 diabetes, hypertension, coronary heart disease, stroke and several types of cancer. The expression of human vaspin (serpin A12) is positively correlated to BMI and insulin resistance and increases glucose tolerance in vivo, suggesting a compensatory role in response to di­ minished insulin sensitivity in obesity.

We have investigated structural and mechanistical aspects of vaspin ac­ tion to elucidate the molecular basis of vaspin action in vitro and in vivo. © Verlag PERFUSION GmbH

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Methods: We have recombinantly expressed human vaspin for crystalli­ zation and protease target screenings.

We used immunohistochemistry and Western blot analysis to study protein expression in pancreatic islets and ad­ dressed vaspin effects on isolated mu­ rine islets. Furthermore, we have in­ vestigated in vivo effects of vaspin and generated vaspin mutants on glucose tolerance and insulin sensitivity in the db/db mouse strain. In addition, we in­ vestigated central effects of vaspin on blood glucose levels and food intake.

Results: We have solved the vaspin X-ray structure confirming the typical serpin fold suggesting an inhibitory ser­ pin. We identified kallikrein 7 (KLK7) as the first target protease of vaspin and characterized inhibition mechanism and kinetics. We detect vaspin-KLK7 complexes in human plasma and find co-expression of both proteins in mu­ rine pancreatic β-cells. We further demonstrate that KLK7 cleaves human insulin in the A- and B-chain. Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion.

After application of vaspin and inac­ tive vaspin mutants, we find the sig­ nificantly improved glucose tolerance mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspin-me­ diated changes in insulin sensitivity as determined by euglycemic-hyperinsu­ linemic clamp studies. In addition, we could demonstrate that central vaspin administration leads to reduced food intake and has sustained blood glucoselowering effects in mice.

Conclusions: Here, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissuederived serpin vaspin, and our findings indicate KLK7 inhibition by vaspin as an underlying physiological mecha­ nism for its compensatory actions on obesity-induced insulin resistance. Our results suggest the vaspin-kallikrein system as a potential novel target for anti-diabetic treatment strategies.

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Metabolic consequences of genetic variation in the vaspin gene J. Breitfeld1, A. Tönjes1, 2, D. Schleinitz1, Y. Böttcher1, C. Marzi3, 4, C. Brockhaus5, 6, W. Rathmann7, C. Huth8, H. Grallert3, 4, T. Illig3, 9, M. Stumvoll2, M. Blüher2, P. Kovacs1 1 IFB Adiposity Diseases, University of Leipzig, Germany 2 Department of Medicine, University of Leipzig, Germany 3 Research Unit of Molecular Epidemiology, Helmholtz Center Munich, Neuherberg, Germany 4 German Center for Diabetes Research (DZD e. V.), Neuherberg, Germany 5 Department of Medicine I, University Hospital Grosshadern, Ludwig Maximilians University Munich, Germany 6 Institute of Genetic Epidemiology, Helmholtz Center Munich, Neuherberg, Germany 7 Institute of Biometrics and Epidemiology, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany 8 Institute of Epidemiology II, Helmholtz Center Munich, Neuherberg, Germany 9 Medical School Hannover, Hannover Unified Biobank, Hannover, Germany Aims: The adipokine vaspin (visceral adipose tissue derived serine protease inhibitor) is suggested to link obe­ sity, insulin resistance (IR) and type 2 diabetes (T2D), but so far its patho­ physiological role remains largely un­ known. In human studies we analyzed the relationship between serum vaspin concentrations and metabolic traits in­ cluding eating behavior. Furthermore, we investigated whether variability in vaspin serum concentrations might be explained by its genetic variants.

Methods: Vaspin was sequenced in DNA samples from 48 unrelated Cau­ casian subjects. Twenty-eight single nucleotide polymorphisms (SNPs) rep­ resentative for their linkage disequilib­ rium groups (r2>0.8 and minor allele frequencies >0.05) were genotyped in 1046 clinically well-characterized Sorbs from Germany for subsequent association studies on metabolic traits including IR and insulin secretion in­ dices. In addition, we assessed eat­

ing behavior by using the three-factor eating questionnaire in a subgroup of 618 Sorbs. Serum vaspin concentra­ tions were determined by ELISA in 2665 samples (Sorbs and KORA co­ horts). We performed a meta genomewide association study (GWAS) on serum vaspin concentrations, correla­ tion analyses between eating behavior and serum vaspin as well as associa­ tion analyses with genetic variants and quantitative traits.

Results: Serum vaspin levels corre­ lated with gender, waist-to-hip-ratio (WHR), 2-hr glucose, insulin (fasting, 30 min, 2 hr), HOMA-IR and QUICKI (all p<0.05) in the Sorbs. Additionally there was a strong association of vaspin SNPs with serum vaspin concentra­ tions (with 7 SNPs reaching P-values between 10–8 and 10–35) in a metaGWAS including Sorbs and KORA. Also, genetic variants were nominally associated with WHR, 30-min glucose, 2-hr insulin, AUCglucose and IR indices (adj. for age, sex and BMI). We found positive correlations between serum vaspin concentrations and factors rep­ resenting restraint (p=0.006), disinhi­ bition (p=0.004) and hunger (p=0.01; all Pearson correlations). These data have been independently supported by genetic association analyses in which two SNPs (rs2236242, rs3736803) were significantly associated with both serum vaspin (p<10–8) and measures of eating behavior (p<0.05; all in regres­ sion analyses employing additive mode of inheritance).

Conclusions: Our data suggest the role of vaspin and its genetic variation in the pathophysiology of IR. Correla­ tions between serum vaspin and eating behavior along with associations of genetic variants with both vaspin con­ centrations and with eating behavior further suggest the role of vaspin in the regulation of eating behavior.

© Verlag PERFUSION GmbH

ABSTRACTS

Influence of CCL19 and CCL21 on the intimal inflammatory milieu during atherogenesis in Ldlr–/– mice M. Akhavanpoor1, C. A. Gleissner1, S. Gorbatsch1, E. Blessing1, S. Wangler1, T. J. Dengler1, F. Lasitschka2, H. A. Katus1, C. Erbel1 1 Department of Cardiology, University of Heidelberg, Germany 2 Institute of Pathology, University of Heidelberg, Germany Background: Atherosclerosis is a chronic inflammatory disease. CCL19 and CCL21 are homeostatic chemokines that induce chemotaxis of T, B and den­ dritic cells. In the murine genome, there are two copies of CCL21 that differ from each other by one amino acid at position 65 (either a serine – CCL21-Ser – or leucine – CCL21-Leu – residue).

CCL21-Ser is expressed predominantly in lymphoid organs while CCL21-Leu is expressed in nonlymphoid organs without significant functional differ­ ences. Elevated expressions of CCL19 and CCL21 have been observed in ruptured lesions of coronary arteries of patients with myocardial infarction and carotid plaques of patients with ischem­ ic symptoms as well as in plasma of CAD patients. However, the exact role of CCL19 and CCL21 in atherogenesis remains unknown.

Aim and methods: In the present study, we investigated the role of both chemokines in the inflammatory pro­ cess of atherogenesis in vivo. 16 male Ldlr–/– mice were irradiated with 9.5 Gy, followed by bone marrow transplanta­ tion (1×106 cells, plt/plt mice, lacking CCL19 and CCL21-Ser) for the therapy group (n=8) and C57BL/6 for controls (n=8) within 24 hours after irradia­ tion. 4 weeks later a western type diet was started. After additional 14 weeks aortic root was dissected and embed­ ded in OCT for immunohistochemistry, the thoracic aorta snap-frozen for gene expression and blood serum stored at –20°C for ELISA analysis.

Results: The study demonstrates that in thoracic aorta of plt/plt/Ldlr–/– mice CCL19 was significantly down-regulat­ Perfusion 01/2014

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ed, whereas CCL21-Leu in the thoracic aorta as well as CCL5 and CCL3 in the serum were markedly up-regulated compared to controls. This led to sig­ nificantly increased inflammatory cel­ lular infiltration including macrophag­ es, T cells, B cells and dendritic cells into the lesions of plt/plt/Ldlr–/– mice vs. controls. Although the level of che­ moattractants was increased, mRNA and protein levels in thoracic aorta and serum of several proinflammatory cytokines (TNF-α, IFN-γ, IL-6, IL-12, IL-17 and IL-23) were significantly re­ duced in plt/plt/Ldlr–/– vs. control mice.

Increased influx, accompanied by reduced activation of leukocytes in atherosclerotic lesions was not ac­ companied by significant changes in atherosclerotic lesion development and plaque stability between the groups.

However, a significant reduction of the lipid content and thus the size of mac­ rophages within atherosclerotic lesions were observed in plt/plt/Ldlr–/–mice vs. controls. This is due to an increased oxLDL uptake in monocyte derived mac­ rophages as well as a foam cell forma­ tion through an up-regulation of CD36 on the surface of these cells by CCL19, as demonstrated in vitro data.

Conclusion: In conclusion, our data reveal that CCL21-Leu regulates che­ moattraction of leukocytes into athero­ sclerotic lesions, whereas CCL19 in­ fluences the activation of leukocytes, lipid uptake of macrophages and foam cell formation. Further studies are needed to investigate the long-term in­ fluence of the chemokine modification on plaque progression and stability.

CD40-TRAF6 signaling is a novel therapeutic target in obesity-related metabolic dysregulation A. Chatzigeorgiou1, 2, 3, T. Seijkens4, B. Zarzycka5, D. Engel6, M. Poggi6, R. Garcia-Martin1, K.-J. Chung1, E. Lutgens1, 8, T. Chavakis1, 2, 3, 8 1 Department of Clinical Pathobiochemistry, Technical University of Dresden, Germany 2 Department of Medicine, Technical University of Dresden, Germany

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Paul-Langerhans Institute Dresden, German Center for Diabetes Research, Dresden, Germany 4 Department of Medical Biochemistry, Subdivision Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, The Netherlands 5 Department of Biochemistry, University of Maastricht, The Netherlands 6 Department of Pathology, University of Maastricht, The Netherlands 7 Institute for Cardiovascular Prevention, Ludwig Maximilians University Munich, Germany 8 Institute for Clinical Chemistry and Laboratory Medicine, Technical University of Dresden, Germany 3

Aims: Inflammation is a critical con­ tributor to the pathogenesis of obesityassociated metabolic dysregulation and atherosclerosis and co-stimulatory molecules, including CD40, are crucial mediators of this process. Aim of this study was to reveal the role of the costimulatory molecule, CD40, and its signaling intermediates, TNF-receptorassociated factors (TRAFs), in dietinduced obesity (DIO).

Methods: We performed the DIO model in CD40-deficient or -sufficient mice as well as in mice with deficient CD40-TRAF2/3/5 signaling or CD40TRAF6 signaling in MHCII+ cells.

Body weight was measured weekly and metabolic functions were assessed by measuring blood glucose, cholesterol, triglycerides, insulin and performing insulin tolerance tests, body composi­ tion analysis by using MRI and indirect calorimetry/metabolic cage analysis.

After the experimental period, subcu­ taneous and gonadal adipose tissues and liver were isolated. FACS analysis of the stromal vascular fraction from adipose tissues as well as qPCR analy­ sis and histochemistry of the isolated tissues were performed. Virtual ligand screening (VLS), TRAF6 C-domain expression, purification and binding analyses were also performed to con­ struct a CD40-TRAF6 signaling in­ hibitor that was used as therapy in DIO mice.

Results: CD40ko mice in DIO dis­ played worsened insulin resistance © Verlag PERFUSION GmbH

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associated with excessive adipose tis­ sue (AT) inflammation, characterized by increased accumulation of CD8+ T cells and M1 macrophages, and en­ hanced hepatosteatosis. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance, and showed a reduc­ tion in both AT inflammation and hepa­ tosteatosis in DIO, whereas the mice with deficient CD40-TRAF2/3/5 sign­ aling exhibited a phenotype similar to the CD40ko mice. DIO mice that were treated with a small-molecule inhibitor that specifically blocks CD40-TRAF6 interactions displayed improved insu­ lin sensitivity, reduced AT inflamma­ tion and decreased hepatosteatosis.

Conclusions: The CD40-TRAF2/3/5 signaling pathway protects against obesity-associated metabolic dys­ function, whereas the CD40-TRAF6 pathway rather contributes to the det­ rimental consequences of obesity. The amelioration of insulin resistance by blocking the CD40-TRAF6 pathway via a newly developed inhibitor that we constructed could represent a thera­ peutic breakthrough in the field of im­ munometabolism.

PDE4 inhibition prevents neointima formation and inhibits VCAM-1 expression and histone methylation in an Epac-dependent manner F. Kahles, M. Lehrke, A. Makowska, J. Marx, K. Hess, D. Bruemmer, N. Marx, H. M. Findeisen Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany Purpose: Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation at sites of vascular inflammation and tissue remodeling. In this approach we have investigated the effects of specific PDE4 inhibition on SMC activation and neointima formation after vascular injury.

Methods: C57BL/6 mice treated with the new selective PDE4 inhibitor roflu­ milast (added to the diet, 21 mg/kg) Perfusion 01/2014

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underwent guide wire-induced en­ dothelial denudation injury of the left femoral artery. Neointima formation was quantified after 4 weeks. In vitro, we analyzed the effects of roflumilast on SMC proliferation and inflamma­ tory activation.

Results: Roflumilast treatment attenu­ ated neointima formation and femoral artery intima-media ratio by more than 50 % (p<0.01). In vitro analysis of cell growth, BrdU incorporation, cell cy­ cle distribution and cyclin D1 expres­ sion exhibited no difference in mito­ gen-induced vascular smooth muscle cell (VSMC) proliferation following stimulation with roflumilast or con­ trol. However, roflumilast treatment significantly diminished expression of the inflammatory marker vascular cell adhesion molecule 1 (VCAM-1) by 60 % (p<0.05) in TNF-α treated VSMCs, while other cell adhesion molecules were affected less. Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 ex­ pression. Consistently, the reduction of VCAM-1 expression was rescued in the presence of the novel specific Epac inhibitor ESI-09 and following siRNA mediated knockdown of Epac1. Unex­ pectedly, TNF-α induced NF-κB p65 translocation and VCAM-1 promotor activity were not altered by roflumi­ last in VSMCs. Indeed, roflumilast treatment and Epac activation directly repressed the induction of the activat­ ing epigenetic histone mark H3K4me2 at the VCAM-1 promoter, while PKA activation showed no effect. In accord­ ance with the reduction of VCAM-1 in VSMC, roflumilast treated mice displayed decreased expression of VCAM-1 and the macrophage mark­ ers F4/80 and CD68 during neointima formation. Consistently, immunohis­ tochemical analysis of the vessel wall showed reduced Mac2 staining, indi­ cating decreased macrophage accumu­ lation after vascular injury.

Conclusion: Inhibition of PDE4 re­ duces neointima formation and attenu­ ates VSMC inflammatory activation in

vitro. Roflumilast regulates VCAM1 through a novel Epac-dependent mechanism, which modulates specific histone methylation patterns. All in all, PDE4 inhibition might represent a nov­ el approach for the treatment of vascu­ lar diseases, including atherosclerosis and in-stent restenosis.

P2Y6 deficiency limits vascular inflammation and atherosclerosis in mice A. Peikert1, P. Stachon1, N. Anto Michel1, S. Hergeth1, D. Wolf1, B. Dufner1, C. Bode1, M. Idzko2, A. Zirlik1 1 University Heart Center Freiburg Bad Krozingen, Department of Cardiology and Angiology I, University of Freiburg, Germany 2 University Hospital Freiburg, Department of Pneumology, University of Freiburg, Germany Aim: Nucleotides such as ATP, ADP, UTP, and UDP serve as pro-inflam­ matory danger signals via purinergic receptors upon their release to the ex­ tracellular space by activated or dying cells. UDP binds to the P2Y6 recep­ tor and propagates vascular inflam­ mation by inducing the expression of chemokines such as MCP-1, IL-8, and adhesion molecules such as VCAM-1 and ICAM-1. Thus, P2Y6 contributes to leukocyte recruitment and inflam­ mation in conditions such as allergic asthma or sepsis. Since atherosclero­ sis is a chronic inflammatory disease driven by leukocyte recruitment to the vessel wall we hypothesized a role of P2Y6 in atherogenesis.

Approach and results: Intraperito­ neal stimulation of wild-type mice with UDP induced rolling and adhe­ sion of leukocytes to the vessel wall as assessed by intravital microscopy.

This effect was not present in P2Y6deficient mice. Moreover, subcutane­ ous injection of Matrigel containing UDP increases migration of F4/80-pos­ itive cells compared to Matrigel alone.

However, migration of F4/80 cells was reduced in P2Y6-deficient mice after UDP stimulation. Atherosclerotic aor­ © Verlag PERFUSION GmbH

ABSTRACTS

tas of LDLR–/– mice consuming high cholesterol diet for 16 weeks expressed significantly more transcripts and pro­ tein of P2Y6 than respective controls.

Finally, P2Y6–/–/LDLR–/– mice con­ suming high cholesterol diet for 16 weeks developed significantly smaller atherosclerotic lesions compared with P2Y6+/+/LDLR–/– mice. Atherosclerotic lesions of P2Y6-deficient mice con­ tained fewer macrophages and fewer lipids as determined by immunohis­ tochemistry. The uptake of modified LDL was reduced in P2Y6-deficient PBMCs in contrast to P2Y6-competent PBMCs. Mechanistically, RNA ex­ pression of VCAM-1 and IL-6 was de­ creased in these lesions. Furthermore, the intima of atherosclerotic lesions of P2Y6-deficient mice expressed signifi­ cantly lower levels of VCAM-1 than that of P2Y6+/+/LDLR–/– mice.

Conclusion: We show for the first time that P2Y6 deficiency limits atheroscle­ rosis and plaque inflammation in mice.

The present study suggests the UDPP2Y6 axis as promising potential target for prevention or treatment of vascular inflammation as seen when atheroscle­ rosis is present.

Extracellular ATP induces atherosclerosis and vascular inflammation via purinergic receptor 2 (P2Y2) in mice P. Stachon1, A. Peikert1, N. Anto Michel1, D. Wolf1, N. Hoppe1, B. Dufner1, C. Bode1, M. Idzko2, A. Zirlik1 1 University Heart Center Freiburg Bad Krozingen, Department of Cardiology and Angiology I, University of Freiburg, Germany 2 University Hospital Freiburg, Department of Pneumology, University of Freiburg, Germany Aim: A solid body of basic evidence supports a role for extracellular ATP and its receptors as danger signals in various chronic inflammatory diseases including asthma, chronic obstructive pulmonary disease (COPD), arthritis, allergic dermatitis, graft versus host disease and other entities. The ATP binding P2Y2 receptor is involved in inflammatory cell recruitment and Perfusion 01/2014

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chronic inflammatory diseases like COPD. Hypothesis: Atherosclerosis repre­ sents a chronic inflammatory disease and several lines of evidence suggest that extracellular ATP and the P2Y2 re­ ceptor may contribute to atherogenesis.

Methods: The expression of P2Y2 was determined by realtime PCR and im­ mune histology in aortas after feeding LDLR- deficient mice (LDLR–/–) a high (HCD) or low (LCD) cholesterol diet for 16 weeks. To gain into the function­ al role of extracellular ATP in athero­ genesis, LDLR–/– mice on HCD for 18 weeks received either ATP or PBS intra­ peritoneally 3 times per week. Further­ more, P2Y2 knockout mice (P2Y2–/–) were crossed with LDLR–/– and fed a HCD for 18 weeks. LDLR–/– served as control group. Atherosclerosis was determined histologically. To evaluate the role of ATP and P2Y2 in vascular inflammation, intravital microscopy was performed in P2Y2–/– and LDLR–/– mice stimulated with ATP or vehicle.

Results: P2Y2 is overexpressed in mu­ rine atherosclerotic aortas compared to healthy vessels indicating a relevant role in atherosclerosis. Stimulation of the P2Y2 receptor by ATP increases atherosclerotic lesions in aortic arches (n=15, control: 0.30 mm², ATP group: 0.35 mm², p=0.04) and abdominal aortas (n=15, control: 0, 0.34 mm², ATP group: 0.18 mm², p<0.01) sig­ nificantly. Accordingly, LDLR–/– mice treated with ATP showed an increased inflammatory status characterized by more adherent leukocytes after TNF-α stimulation and increased Interleukin-6 expression in peripheral blood after HCD. However, the knockout of the ATP binding P2Y2 receptor reduces atherosclerosis in aortic arches com­ pared to the LDLR–/– control group (n=10, LDLR–/–: 0.26 mm², P2Y2–/–/ LDLR–/–: 0.14 mm², p=0.03).

Since leukocyte recruitment to the ves­ sel wall is the crucial step in atherogen­ esis, we examined ATP and its P2Y2 receptor in vascular inflammation.

Stimulating mice with ATP intraperito­ neally induces leukocyte adhesion and

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rolling in wildtype mice. In P2Y2–/– mice this effect of ATP is reduced indi­ cating a relevant role of P2Y2 in ATPdependent vascular inflammation.

Conclusion: Extracellular ATP acts as a danger signal in vascular inflam­ mation and induces atherosclerosis in mice via purinergic receptor 2. The ATP-P2Y2 axis represents a potential new pathway in pathogenesis of ath­ erosclerosis.

First worldwide sterol and oxysterol survey D. Lütjohann1, H. Schött1, R. Kruse2, W. J. Geilenkeuser2 1 Institute for Clinical Chemistry and Clinical Pharmacology, University Clinics Bonn, Germany 2 Reference Institute for Bioanalytics, Bonn, Germany Aims: Non-cholesterol sterols (NCS), which encompass endogenous cholesterol precursors (lanosterol, lathosterol, desmosterol) and exogenous phytosterols (campesterol, sitosterol, 5α-campestanol, 5α-sitostanol) as well as cholesterol metabolites (5α-cholestanol; bile acid precursors/oxysterols: 7α-, 24S-, and 27-hydroxycholesterol; other oxysterols: 7β- and 25-hydroxy­ cholesterol, 7-ketocholesterol), are widely used in biomedical research as surrogate markers for estimating cho­ lesterol synthesis, cholesterol absorp­ tion efficiency, bile acid synthesis and radical oxygen species (ROS)-status.

Furthermore, NCS and oxysterols are used as prognostic and diagnostic tools for the description of dyslipidemic and neurodegenerative states in mamma­ lian subjects. These NCS and oxys­ terols show even stronger correlation with cholesterol absorption or synthesis or bile acid synthesis when expressed as ratios to total cholesterol, which standardizes for variations in ster­ ol transport protein concentrations.

Specifically, when reporting NCS in a ratio to cholesterol, the cholesterol measurement should ideally be from the same sample preparation as the NCS, © Verlag PERFUSION GmbH

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and measured by the same chromato­ graphic method.

Methods: Twenty laboratories special­ ized in chromatographic lipid analysis, either gas or liquid chromatography, agreed to participate in a first world­ wide survey under the expertise of the reference institute for bioanalytics (RfB) located in Bonn, Germany. A set of two different lyophilized pool sera (A and B) was sent to each participant and the results were sent back from 18 participants and evaluated by RfB. The different sterols, stanols and oxysterols were quantified either by GC-flameionization detection, GC-mass selec­ tive detection or LC-mass selective de­ tection. Additionally, different internal standards were used: 5α-cholestane, epicoprostanol, deuterium labeled ster­ ols/oxysterols (isotope dilution mass spectrometry, GC- and LC-MS) and in one case 19-OH-cholesterol for oxy­ sterols. The data were given in the in­ dividual units used by the participant and converted into standard units for comparison (cholesterol and NCS as mg/dL; oxysterols as ng/mL). Each participant received a clear overview of his position in form of Youden-Plots and basic statistical evaluation (mean, median, SD, min, max).

Results: Unfortunately, cholesterol was autoxidized as proofed by ex­ tremely high concentrations of 7α- and 7β-hydroxycholesterol as well as by increased 7-ketocholesterol. Those oxysterols, which are exclusively en­ zymatically produced (24S-, 25- and 27-hydroxycholesterol) were in the range as known from the literature and are integrated in the evaluation process.

The lowest coefficient of variation is presented for cholesterol (A: 25.1 % and B: 21.5 %). For non-cholesterol sterols there are coefficients of vari­ ation between 54 % and 189 %. For oxysterols, except 7-oxygenated cho­ lesterol, coefficients of variation vary between 36 % and 125 %.

Conclusions: We need further careful considerations and proposals to harmo­ nize the methods for GC- and LC-an­ alytics of cholesterol, non-cholesterol Perfusion 01/2014

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sterols/stanols and oxysterols. This includes the work-up procedure, safety parameters, use of standards and inter­ nal standards, chromatographic separa­ tion and detection mode and finally the unit to present each parameter.

Genetic deficiency of TRAF-1 attenuates diet-induced adipose tissue inflammation by limiting the pool of circulating and splenic monocytes in mice D. Wolf, N. Anto Michel, P. Stachon, I. Hilgendorf, C. Bode, A. Zirlik Atherogenesis Research Group, University Heart Center Freiburg-Bad Krozingen, University of Freiburg, Germany Background: Accumulation of inflam­ matory leukocytes is a prerequisite of adipose tissue inflammation during the metabolic syndrome. We recently reported that genetic deficiency of Tumor necrosis receptor–associated factor (TRAF)-1 attenuated inflam­ matory cell recruitment in atheroscle­ rosis. Here, we tested whether genetic deficiency of TRAF-1 modulates dietinduced obesity (DIO) in mice.

Methods and results: To test the as­ sociation of TRAFs and obesity we screened for expression of different TRAFs in mouse adipose tissue after 20 weeks of feeding with a high fat diet (HFD). HFD induced up-regulation of TRAF-1, -3, -5, -6, and -7 mRNA. Inter­ estingly, the amplitude of gene regula­ tion was highest for TRAF-1 (4.9-fold, p=0.002). To test functional relevance of our findings, WT or TRAF-1–/– mice consumed HFD for 20 weeks (n≥10 mice per group). Interestingly, TRAF1–/– mice gained less weight during DIO (119±7.5 % vs. 41±3.7 % for WT and TRAF-1–/–, respectively). Accord­ ingly, total body weight and weight of fat pads was decreased in TRAF-1–/– mice. Moreover, TRAF-1–/– mice dem­ onstrated lowered glucose levels after intraperitoneal glucose and insulin tol­ erance tests. Finally, inflammatory cell recruitment was impaired in TRAF-1–/–

mice with reduced numbers of adipose tissue macrophages. Functionally, cir­ culating and splenic monocytes were lowered in TRAF-1–/– mice proposing that TRAF-1 modulates monocyte mo­ bilization during inflammation.

Conclusion: We present the novel finding that TRAF-1 is regulated in obese adipose tissue. Genetic defi­ ciency of TRAF-1 attenuates adipose tissue inflammation in mice by limit­ ing monocyte recruitment. These find­ ings identify TRAF-1 as a mediator of cardio-metabolic disease.

Influence of immune cells on local collagen production, collagen pattern and cardiac remodeling after myocardial infarction A. Schuh1, S. Simsekyilmaz2, O. Bucur5, A. Curaj2, 3, I. Kanzler2, 4, X. Li2, N. Marx N1, 2, E. A. Liehn2 1 Department of Cardiology, University Hospital of the RWTH Aachen, Germany 2 Institute for Molecular Cardiovascular Research (IMCAR), University Hospital of the RWTH Aachen, Germany 3 Department of Experimental Molecular Imaging, University Hospital of the RWTH Aachen, Germany 4 Department of Biochemistry and Molecular Cell Biology, University Hospital of the RWTH Aachen, Germany 5 Department of Pathology, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA, USA Aim: Despite the significant progress in the treatment of cardiovascular dis­ eases in the last decades, it is some­ times impossible to prevent fibrosis and scar formation in the heart. How­ ever, this process belongs to the repara­ tory mechanisms and often represents a protection and an adaptation of the heart to sustained mechanical forces.

Recent studies suggest that contrary to previous belief, increased collagen content in the infarct scar does not nec­ essarily lead to a deterioration of myo­ cardial function. The aim of this study was to perform a complete characteri­ zation of scar formation, particularly © Verlag PERFUSION GmbH

ABSTRACTS

collagen, at different time points after myocardial infarction in the infarcted area and to investigate the influence of different inflammatory cells on the col­ lagen pattern and the functional stabil­ ity/elasticity of the scar.

Methods: We first quantified in vitro the production of all types of collagen by cultured myofibroblasts, which we subsequently co-cultivated with differ­ ent inflammatory cells. For the “deple­ tion” of various inflammatory cells, we subsequently conducted in vivo infarct surgery series in four different “knock out” mice strains which lack specific leukocyte subsets (CCR1–/–, CCR2–/–, CCR5–/–, CX3CR1–/–) and a wild type control group. A quantitative represen­ tation of the expressed collagen types by mRNA detections at different time points (until 4 weeks) after myocar­ dial infarction as well as in the various “knock out” mice strains was carried out. To analyze the origin of the myo­ fibroblasts involved in scar forma­ tion, we performed transplantation of GFP-labeled bone marrow in irradi­ ated mice. To illustrate the elasticity of scar areas, we performed in all groups atomic force microscopy (AFM) meas­ urements in addition to the represen­ tation of left ventricular function by echocardiography at different time points after myocardial infarction.

Results: Under normoxia, in vitro iso­ lated myofibroblasts exhibited mainly a production of type collagen I, IV, VI, XII, XIV and XVIII, under hypoxic conditions, a reduction of collagen I, IV and VI was observed. Co-incuba­ tion with mononuclear cells reduced mainly the amount of collagen IV and VI, while co-incubation with neutro­ phils reduced collagen type I, VI and XII. In vivo, collagen changes its pat­ tern during the scar formation, which was mostly produced by the local pro­ liferated myofibroblasts, and not from the blood recruited cells, as the trans­ plantation studies demonstrated. Four weeks after myocardial infarction, we detected different collagen subtypes in the various “knock out” strains of mice. For example: CX3CR1–/– mice Perfusion 01/2014

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showed an increased amount of col­ lagen types III , VIII , XII and XIV, while CCR2–/– mice presented a highly significant increase of collagen XIII. Consequently, AFM measurements showed significant differences in tissue elasticity between the scar tissues of all groups. Especially in the CCR1–/– and CCR2–/– groups higher grain elasticity correlated with a better left ventricular function by echocardiography.

Conclusions: Both in vitro and in vivo experiments suggest that inflammatory cells can take a significant influence on the expression of different collagen ex­ pression patterns in the infarct scar tis­ sue, with important effects on stability/ elasticity of the scar areas, and on func­ tion of the heart. An intervention in the processes of migration of inflammatory cells after myocardial infarction could therefore influence therapeutic cardiac repair processes in the future and could complete the current treatment, which is aimed to sustain the cardiomyocytes survival and function.

Down-regulation of MMP8 by 15-deoxy-∆12,14-prostaglandin J2 involves PPARγ S. Becher, M. Abhari, M. Schubert, S. Lorkowski Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany Background: 15-Deoxy-∆12,14-prosta­ glandin J2 (15dPGJ2) is a natural lipid metabolite produced in macrophages, which is an anti-inflammatory and negative mediator of macrophage ac­ tivation. This compound acts either as PPARγ ligand or independently of PPARγ. Since weakening of the fibrous cap by macrophage-derived matrix metalloproteinases (MMPs) is an im­ portant event in atherogenesis, we are interested in the influence of 15dPGJ2 on the expression of MMPs, in particu­ lar MMP8, which are known to con­ tribute to plaque stability.

Aim: We are investigating the effect of 15dPGJ2 on the expression of MMPs,

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such as MMP8, in human macrophages and aim at unraveling involved signal­ ing pathways.

Methods and results: Using THP-1 macrophages we investigated the ef­ fects of 15dPGJ2 on MMP8 mRNA by RT-qPCR and MMP8 protein by West­ ern blotting. Since MMP8 is secreted by macrophages after glycosylation, expression of MMP8 protein was ana­ lyzed in cell lysates and cell culture su­ pernatants after precipitation with am­ monium sulfate. Further, we analyzed MMP8 proteolytic activity in the su­ pernatant by gelatin zymography cou­ pled with magnetic immunoprecipita­ tion for specific enrichment of MMP8 protein. For investigating PPARγ de­ pendency, we arranged combination experiments of 15dPGJ2 or the syn­ thetic PPARγ ligand rosiglitazone with the obligate RXR partner ligand 9-cis retinoic acid or blocking experiments with the PPARγ antagonist GW9662.

15-Deoxy-∆12,14-prostaglandin J2 downregulates MMP8 dose- and time-de­ pendently at the mRNA and protein level in THP-1 macrophages. Downregulation of MMP8 by 15dPGJ2 was also confirmed in primary human mac­ rophages. The effect of 15dPGJ2 on MMP8 was blocked in part by GW9662 and augmented by 9-cis retinoic acid.

In comparison with other natural and synthetic PPARγ ligands, 15dPGJ2 showed most efficient down-regulation of MMP8 suggesting that PPARγindependent effects also contribute the regulatory effect of 15dPGJ2. We are currently analyzing several signaling pathways that may mediate the downregulation of MMP8 independently of PPARγ in macrophages using different inhibitors. But, no inhibitor tested so far was able to block the down-regula­ tion of MMP8 by 15dPGJ2. Thus, the metabolite itself may act as kinase in­ hibitor or inhibitor of kinase signaling pathways in our macrophage model.

Conclusions: Down-regulation of MMP8 by 15dPGJ2 takes place at least in part via PPARγ but also involves PPARγ-independent signaling path­ ways which remain to be identified. © Verlag PERFUSION GmbH

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Effects of maternal PETN treatment of spontaneously hypertensive rats on blood pressure in the offspring Z. Wu*, D. Siuda*, A. Habermeier, E. I. Closs, N. Xia, G. Reifenberg, A. Daiber, U. Förstermann, H. Li Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany (*both authors contributed equally to this study) Aims: Pentaerithrityl tetranitrate (PETN) is an organic nitrate used for prevention of angina pectoris in clinic.

PETN has little effect on blood pres­ sure when administrated directly. The present study was designed to test the “perinatal programming” effect of PETN in spontaneously hypertensive rats, a rat model of genetic hyperten­ sion.

Methods: The F0 parent SHR animals were treated with PETN (50 mg/kg/ day) during pregnancy and lactation periods; the offspring received stand­ ard chow without PETN after weaning.

Blood pressure was measured with the tail-cuff method.

Results: Maternal PETN treatment had little effect on blood pressure in male offspring. In the female F1 SHR ani­ mals, however, a persistent reduction in blood pressure was observed. This long-lasting effect of maternal PETN treatment on blood pressure was ac­ companied by a substantial change in gene expression even evident at the age of 8 months. Maternal PETN treat­ ment led to an up-regulation of the endothelial NO synthase (eNOS), the mitochondrial superoxide dismutase (SOD2), the glutathione peroxidase I (GPx1) and the heme oxygenase-1 (HO-1). This was associated with epi­ genetic changes (enhanced histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation) and transcrip­ tional activation (enhanced binding of RNA poly­merase 2a to the promoter region of the abovementioned genes).

In organ chamber experiment, the en­ dothelium-dependent, NO-mediated vasodilation to acetylcholine was en­ hanced in aorta from female F1 SHR animal of the PETN group.

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Conclusion: The improved endothe­ lial function, which may result from expression changes of the above-men­ tioned genes, is likely to represent a key mechanism for the blood pressure reduction in the offspring.

II. Poster AnxA5 decreases plaque inflammation of advanced atherosclerotic lesions in ApoE–/– mice M. Burgmaier1, K. Schutters2, B. Willems2, D. Kusters2, M. Chatrou2, E. A. L. Biessen3, J. Cleutjens3, L. Schurgers2, C. Reutelingsperger2 1 Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany 2 Department of Biochemistry, University of Maastricht, Cardiovascular Research Institute Maastricht, The Netherlands 3 Department of Pathology, University of Maastricht, Cardiovascular Research Institute Maastricht, The Netherlands Introduction: Annexin A5 (anxA5) has been demonstrated to exert antiinflammatory, anti-coagulant and antiapoptotic effects through binding cell surface expressed phosphatidylserine.

The actions of anxA5 on the pathogen­ esis of atherosclerosis are incompletely understood. We investigated effects of exogenous anxA5 on plaque morphol­ ogy and phenotype of advanced lesions in a murine model of atherosclerosis.

Methods and results: Advanced ath­ erosclerotic lesions were induced in 12 weeks-old ApoE–/– mice using a collar placement around the right ca­ rotid artery and a high-fat diet. Af­ ter 5 weeks mice were injected either with anxA5 (1 mg/kg i.p., 3 times a week, n=8) or with vehicle for anoth­ er 4 weeks. No change in lesion size was observed between mice injected with anxA5 (0.175±0.026 mm3) and vehicle (0.175±0.042 mm3) in the ca­ rotid artery. However, anxA5 reduced plaque macrophage content both in the intima (59 % reduction; 6.7±5.6 % vs. 16.2±10.2 % for anxA5 vs. con­

trols, p<0.05) and the media (72.9 % reduction; 2.3±2.4 % vs. 8.5±4.1 % for anxA5 vs. controls, p<0.01) in ad­ vanced atherosclerotic lesions as deter­ mined by MAC3 immunohistochem­ istry. These findings were confirmed in advanced lesions of the aortic arch, where a 66.7 % reduction in plaque macrophage content was observed with anxA5 compared to controls (2.6±3.2 % vs. 7.8±2.5 %, p<0.01).

AnxA5 did not change plaque apopto­ sis, collagen content, smooth muscle cell content or acellular plaque com­ position after 4 weeks of treatment as determined by immunohistochemistry in advanced carotid lesions (p=ns). An in vitro flow chamber based assay dem­ onstrated that anxA5 inhibited adhe­ sion of monocytes to TNF-α-activated endothelial cells.

Conclusion: Short term treatment with anxA5 decreases plaque inflammation of advanced lesions in a mouse model of atherosclerosis likely through inter­ fering with recruitment of monocytes to the inflamed lesion site. Suppressing chronic inflammation by targeting ex­ posed phosphatidylserine may become a viable strategy to treat patients suffer­ ing from advanced atherosclerosis.

Non-invasive up-regulation of heart preconditioning program in vivo with minimal side effects A. Curaj1, 2, 3, S. Simsekyilmaz1, M. Staudt1, X. Li2, A. Götzenich4, A. Urs3, M. Leabu3, E. A Liehn1 1 Institute for Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Germany 2 Department of Experimental Molecular Imaging, RWTH Aachen University, Germany 3 Victor Babes National Institute of Pathology, Bucharest, Romania, Department for Thorax Surgery, University Hospital of the RWTH Aachen, Germany Aims: Despite considerable progress over the last period, acute myocardial infarction continues to remain the ma­ jor cause of morbidity and mortality worldwide. Phosphatidylserine (PS) is a phospholipid component of the inner © Verlag PERFUSION GmbH

ABSTRACTS

leaflet of cell membranes and seems to be a messenger for the cellular death and an important destruction signal for the macrophages. On the other hand, PS has demonstrated some usefulness in treating cognitive impairment but also speeded up recovery, prevented muscle soreness, improved well-being, and might possess ergogenic proper­ ties. In our studies, we also found an up-regulation of the PS in cxcr4+/– de­ ficient hearts, as an adaptation to the poor blood supply present in these mice. The best known mechanism which is described to protect the heart in the absence of oxygen is the ischem­ ic preconditioning. Ischemic precon­ ditioning is an experimental technique gaining resistance against the loss of blood supply in many tissues. By a short (under 5 minutes) and repeated (2 or 3 times) impairment of the blood supply, the tissue or the organ becomes robustly protected from a predicted se­ vere ischemic insult. In this regard, we believe that PS can play an important role in myocardial preconditioning and protection of the heart from ischemia.

Methods and results: The effect of PS was tested in vitro on isolated cardio­ myocytes undergoing hypoxia. After 3 hours of preincubation the cardio­ myocytes increased significantly their protection, indicating no stress sing­ nals as analyzed by Alamar blue stain­ ing after 5 hours of ischemia. Using a mouse model of myocardial infarction, we tested the effect of oral adminis­ tration of PS on myocardial function and biology. The mice pre-treated with PS one week before myocardial infarction induction showed a signifi­ cantly preserved heart function and reduced infarction size. Starting the administration after myocardial infarc­ tion induction also reduced infarction size, however the hearts remain with a significant diastolic dysfunction. No differences in apoptosis were detect­ ed, as measured by TUNEL staining in infarcted myocardium. Inflamma­ tion was also reduced, due to probably reduced tissue damages after PS ad­ ministration. mRNA extraction from Perfusion 01/2014

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isolated cardiomyocytes after hypoxia and myocardium after myocardial in­ farction showed a significant up-reg­ ulation of main players of precondi­ tioning program such as protein kinase C type ε, cyclooxygenase-2, aldose reductase, Mn superoxide dismutase.

As already known, PS side effects are rare and include only mild gastrointes­ tinal discomfort. When taken together with other blood thinning drugs (such as warfarin, aspirin, pentoxifylline, clopidogrel, ticlopidine, garlic and vi­ tamin E), thinning the blood was also reported. However, to exclude any ma­ jor changes in lipid composition, lipids were extracted from heart, kidney, liver, lung after PS administration and fatty acids were analyzed. We did not detect any significant changes in fatty acids or lipid composition after one week of PS administration in any analyzed organs.

Moreover, the blood analysis showed normal and unchanged parameters in all groups.

Conclusions: We demonstrated that PS can be used to activate preconditioning program of the heart, to assure a signif­ icant protection from ischemia in vitro and in vivo. We strongly believe that PS can be used in the treatment of heart failure to protect the cardiomyocytes and to adapt them to chronical hypoxic conditions thus preserving their func­ tion. Comparing with other methods used in present, as remote ischemia or opioide administration, PS adminis­ tration seems to be easy to apply with minimal side effects, no professional supervision being neccesary. Since PS is already in clinical use for some cognitive diseases, we believe that our findings would be of high relevance for the cardiovascular community, with immediate clinical implication.

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Augmented atherosclerotic lesion formation in ApoE-FcγRIII-deficient hyperlipidemic mouse model J. Köhncke1, Y. Asare1, 2, J. Selle1, S. Simsekyilmaz1, J. E. Gessner3, E. Shagdarsuren1 1 Institute for Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Germany 2 Institute of Biochemistry and Molecular Cell Biology, University Hospital of the RWTH Aachen, Germany 3 Molecular Immunology Research Unit, Clinical Department of Immunology and Rheumatology, Hannover Medical School, Germany Objective: Accumulating data suggest that oxidized low density lipoproteins (oxLDL) induce autoimmune response as evidenced by the presence of antioxLDL antibodies in mice and human atherosclerotic lesions. Immunoglobu­ lin-Fc gamma receptors (FcγRs) play an important role in inflammatory cell activation, clearance of immune com­ plexes and also in maintaining immu­ noglobulin (Ig) homeostasis. They are expressed as surface receptors on the hematopoietic cells and specifically bind to IgG. This binding of immune complexes to FcγRs on immune cells, such as macrophages, can induce a variety of proinflammatory immune responses, including an activation of proinflammatory complement ana­ phylatoxin C5a/C5a receptor-axis, which can accelerate the local inflam­ mation in tissues. Therefore, we hy­ pothesized that activating Fcγ receptor type III (FcγRIII)-deficiency would be protective against atherosclerotic vas­ cular inflammation.

Methods and results: The role of the activating FcγRIII in atherosclero­ sis was analyzed using ApoE/FcγRIII double-knockout (DKO) and control apolipoprotein E-deficient (ApoE–/–) mice at different interval, 12 weeks and 24 weeks, of atherogenic diet. Analysis of Oil-red-O stained whole aortas and sections of aortic roots revealed sig­ nificantly increased plaque size along the aorta and in aortic roots in DKO mice after 12 weeks when compared to control mice. There was no differ­ © Verlag PERFUSION GmbH

ABSTRACTS

32

ence in lesion size between the strains after 24 weeks of atherogenic diet. To determine the cellular composition of the plaque, serial sections of the aortic roots were analyzed by quantitative immunohistochemistry. A significant reduction of α-SMA positive cells in the DKO mice was revealed after 12 weeks of atherogenic diet when com­ pared to control mice, whereas no dif­ ference was observed after 24 weeks.

In parallel, macrophage content was decreased in DKO mice after 12 and 24 weeks when compared to control mice. Importantly, as determined by staining in aortic root lesions, FcγRIIIdeficiency resulted in an increase in whole fraction IgG antibodies after 12 and 24 weeks on high-fat diet com­ pared to control. Of note, analysis of anti-oxidized low density lipoprotein (oxLDL) and anti-malondialdehydeoxidized (MDA-LDL) immunoglobu­ lins in serum using ELISA revealed a higher IgG2a, IgG2b and IgG3 titer against oxLDL in DKO mice when compared to control mice. However, the IgA and IgM titer did not change at both intervals of atherogenic diet.

Differences in IgG and IgG1 antibod­ ies were observed against MDA-LDL. Whereas serum CRP was reduced after 12 weeks, plasma C5a was elevated af­ ter 24 weeks as measured by ELISA. Conclusions: FcγRIII-deficiency in hypercholesterolemic mice leads to increased lesion formation with de­ creased inflammatory cells and accu­ mulation of IgG antibodies in plaques.

This was paralleled with higher serum titers of IgG subtypes against oxi­ dized LDL. These findings reveal an increased lesion formation in DKO mice which is associated with defec­ tive clearance of immune complexes through the immunoglobulin-receptor deficiency.

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Deficiency of endothelial Cxcr4 reduces reendothelialization and enhances neointimal hyperplasia after vascular injury in mice B. Zhou1, H. Noels1, P. V. Tilstam1, W. Theelen1, X. Li1, S. Akhtar1, S. Simsekyilmaz1, E. E. Liehn1, A. Schober2, R. H. Adams3, J. Bernhagen4, 5, Y. Döring2, C. Weber2, 6, 7 1 Institute of Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Germany 2 Institute for Cardiovascular Prevention, Ludwig Maximilians University Munich, Germany 3 Max Planck Institute for Molecular Biomedicine, University of Muenster, Germany 4 Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Germany 5 August-Lenz-Stiftung, Institute for Cardiovascular Research, Ludwig Maximilians University Munich, Germany 6 Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands 7 German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Germany Objectives: As the underlying pathol­ ogy of post-angioplasty restenosis, the processes driving neointima formation need to be clarified. Interestingly, the CXCR4/CXCL12 chemokine receptor/ ligand axis has been shown to medi­ ate the recruitment of smooth muscle cell progenitors to the neointima, driv­ ing neointimal hyperplasia. This study aims to investigate whether and how CXCR4 signaling in endothelial cells (ECs) is involved in neointima forma­ tion.

Methods and results: Neointima for­ mation was triggered by wire-mediated endothelial denudation of the common carotid artery of Cxcr4EC-KOApoE–/– mice, which show an inducible genetic deficiency of Cxcr4 in arterial ECs after tamoxifen treatment. Cxcr4ECWTApoE–/– mice were used as negative controls. After injury, mice received a high-fat diet for 3 weeks before being sacrificed. A tamoxifen-induced genet­ ic deficiency of Cxcr4 in arterial ECs significantly reduces reendothelializa­ tion and endothelial proliferating cells after arterial injury, followed by an in­

crease of neointimal lesion sizes. The lesions of EC-specific Cxcr4-deficient mice displayed a higher macrophage content and lower smooth muscle cell content. Endothelial outgrowth cells (EOCs) in the peripheral blood were characterized by flow cytometry as Sca1+Flk1+CD31+ cells before injury (baseline), 5 days after injury and 3 weeks after injury. Interestingly, pe­ ripheral blood EOCs were significantly decreased after injury upon endothelial Cxcr4 deletion, whereas there was no change of surface expression of Cxcr4 on EOCs in Cxcr4EC-KOApoE–/– mice compared to controls. Moreover, stim­ ulation of Cxcl12 on human aortic EC (HAoEC) lines showed a faster EC mi­ gration and endothelial Cxcl12 expres­ sion in neointima was also reduced in Cxcr4EC-KOApoE–/– mice.

Conclusions: Endothelial Cxcr4 defi­ ciency contributes not only to increased inflammation after injury but also to a reduced reendothelialization. Fur­ thermore, CXCR4 is involved in EOC homeostasis and endothelial Cxcl12 reduction induced less recruitment of peripheral EOC after injury also under­ lies a defective reendothelialization in endothelial Cxcr4-deficient mice. En­ dothelial Cxcr4 deficiency mice have also less wound healing or less reen­ dothelialization through less EC migra­ tion after carotid artery injury.

Characterization of atherosclerotic plaques in blood vessels carrying blood with low oxygen content (pulmonary trunk): role of GDF-15 N. Struck, G. A. Bonaterra, H. Schwarzbach, S. Zügel, J. Strelau, R. Kinscherf Institute for Anatomy and Cell Biology, Philipps University of Marburg, Germany Introduction: Growth differentiation factor-15 (GDF-15) is a member of the TGF-β superfamily. Increased GDF15 serum levels are suggested as a risk factor for cardiovascular diseases.

Recently we have shown that GDF-15 deficiency inhibits atherosclerosis pro­ © Verlag PERFUSION GmbH

ABSTRACTS

gression in the brachiocephalic trunk (BT) in hypercholesterolemic ApoE–/– mice. The aim of the present study was to investigate the effect of hypercholes­ terolemia on the development of ath­ erosclerotic plaques in blood vessels carrying blood with low oxygen con­ tent as the pulmonary trunk (PT) and to elucidate the possible role of GDF-15, both in comparison with the BT. Methods: 10 weeks old GDF-15+/+/ ApoE–/– and GDF-15–/–/ApoE–/– mice received a cholesterol-enriched diet (CED) for 20 weeks. After 20 weeks of CED the pulmonary trunks were dissected, fixed in 4 % PFA and em­ bedded in paraffin. 5 μm thick paraf­ fin sections were processed to perform immunohistomorphometrical analyses of APG5L/ATG5 (autophagy), α-actin (smooth muscle cells), CD68 (mac­ rophages), Ki67 (proliferation), lumen stenosis and TUNEL (apoptosis).

Results: After 20 weeks of CED, in the PT of GDF-15+/+/ApoE–/–, as well as in the PT of GDF-15–/–/ApoE–/– mice, we observed a development of atheroscle­ rotic plaques. However, we found an increase of 40 % apoptotic (TUNEL+) cells in the PT of GDF-15–/–/ApoE–/– mice compared to the GDF-15+/+/ ApoE–/– mice. Furthermore α-actin+ area was 70 % higher in the PT of GDF-15–/–/ApoE–/– than in GDF-15+/+/ ApoE–/– mice. However, no signifi­ cant differences were observed in PT concerning APG5L/ATG5, CD68 and Ki67 immunoreactivity or lumen ste­ nosis between GDF-15+/+/ApoE–/– and GDF-15–/–/ApoE–/– mice after 20 weeks of CED. Conclusion: Our present data demon­ strate that hypercholesterolemia leads to a development of atherosclerotic plaques in blood vessels carrying blood with low oxygen content like the PT. The plaque morphology in PT seems to be different from the BT. This differ­ ence may be related to the blood oxy­ gen concentration or the morphology of the vessels. The influence of GDF15 on plaque development seems to be more obvious in vessels carrying blood with high oxygen content.

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Influence of growth differentiation factor-15 (GDF-15) on the gene and protein expression in gastrocnemius muscle of hypercholesterolemic ApoE–/– mice M. Wagner1, G. A. Bonaterra1, H. Schwarzbach1, S. Zügel1, J. Strelau2, R. Kinscherf1 1 Institute for Anatomy and Cell Biology, Philipps University of Marburg, Germany 2 Institute for Anatomy and Cell Biology, University of Heidelberg, Germany Introduction: Growth differentiation factor-15 (GDF-15) is a distant mem­ ber of the TGF-β superfamily and is induced under inflammatory condi­ tions. Increased GDF-15 serum levels are suggested as a risk factor for car­ diovascular diseases. Recently we have shown that GDF-15 deficiency inhibits atherosclerosis progression in hyper­ cholesterolemic ApoE–/– mice. The aim of the present study was to investigate the role of GDF-15 on possible muscle dysfunctions using GDF-15 deficient (GDF-15–/–) mice in a mouse model for experimental atherosclerosis (ApoE–/–).

Methods: 10 weeks old GDF-15+/+/ ApoE+/+ (WT), GDF-15–/–/ApoE+/+, GDF-15+/+/ApoE–/– and GDF-15–/–/ ApoE–/– mice received a cholesterolenriched diet (CED) for 12 or 20 weeks.

After CED the gastrocnemius mus­ cles were dissected and shock-frozen.

RNA and proteins were isolated and the mRNA expression of relevant genes for apoptosis and inflammation were analyzed by real-time RT-PCR. Proteins were analyzed for pro-/anti-inflammato­ ry cytokines using a Proteome Profiler Mouse Cytokine Array Kit. Results: We found in gastrocnemius muscle after 12 weeks CED a signifi­ cant (p<0.05) increase in the mRNA expression of IL-6 and IL-1β in all groups. But after 20 weeks CED, IL-6 expression was inhibited in all groups compared with WT mice. However, in gastrocnemius muscle of GDF-15+/+/ ApoE–/– and GDF-15–/–/ApoE–/– mice, we found a high expression of IL-1β (10- and 15-fold) after 20 weeks CED compared with the WT group. Pro-

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apoptotic caspase-3 expression was increased after 12 weeks CED in GDF15–/–/ApoE+/+, GDF-15+/+/ApoE–/– and GDF-15–/–/ApoE–/– mice (5-, 3.1- and 2.9-fold) compared with WT. Pro­ tein expression array-analysis after 20 weeks CED showed an increase (>4fold) of the IL-1α and IL-17 cytokines in GDF-15+/+/ApoE–/– mice compared with WT. IL-1β and IL-6 protein ex­ pressions were increased in GDF-15+/+/ ApoE–/– mice (3.5- and 1.9-fold) com­ pared with WT. Conclusion: In gastrocnemius muscle of GDF-15+/+/ApoE–/– mice, GDF-15 increases the expression (at the protein level) of pro-inflammatory cytokines af­ ter 20 weeks CED compared to the oth­ er three groups. Thus, in gastrocnemius muscle GDF-15 seems to potentiate the pro-inflammatory condition induced by the CED and the lack of ApoE.

The role of IKKα in atherosclerosis P. V. Tilstam1, M. J. Gijbels2, 3, M. Habbeddine4, C. Cudejko4, Y. Asare1, 5, W. Theelen1, B. Zhou1, Y. Döring6, M. Drechsler6, L. Pawig1, S. Simsekyilmaz1, R. R. Koenen2, M. P. J. de Winther2, 3, T. Lawrence4, J. Bernhagen5, 7, A. Zernecke8, 9, C. Weber2, 6, 9, H. Noels1 1 Institute of Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Germany 2 Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands 3 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands 4 Centre d’Immunologie de Marseille-Luminy, Aix-Marseille Université, France 5 Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Germany 6 Institute for Cardiovascular Prevention, Ludwig Maximilians University Munich, Germany 8 Department of Vascular Surgery, Technical University Munich, Germany Aims: Most mortal cardio- and cer­ ebrovascular incidents are caused by atherosclerosis, a chronic inflamma­ tory disease of the blood vessel wall in which the transcription factor NFκB has been shown to be involved. © Verlag PERFUSION GmbH

ABSTRACTS

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Two main pathways activate NF-κB: while inflammatory signals induce “canonical” NF-κB activation through the IKKβ/IKKγ kinase complex, “al­ ternative” NF-κB activation, triggered by e.g. RANKL and CD40L, depends on the IKKα kinase. In addition, IKKα terminates canonical NF-κB activation in macrophages and thereby contrib­ utes to the resolution of inflammation.

Furthermore, IKKα exerts multiple NF-κB-independent functions, e.g. modulating gene expression by phos­ phorylating Histone H3. Despite these known functions of IKKα, the contri­ bution of IKKα to atherogenesis re­ mains unexplored. Therefore, the aim of this study is to identify the role of IKKα activation in hematopoietic ver­ sus resident vascular cells in athero­ sclerosis.

Methods: First, ApoE–/– mice received radiation and were subsequently trans­ planted with ApoE–/– bone marrow car­ rying a mutated, inactivation-resistant IKKα gene (IkkαAA/AAApoE–/–), or with ApoE–/– bone marrow as control. The mice received a high-fat diet for ei­ ther 8 or 13 weeks. Secondly, IkkαAA/ ApoE–/– and control ApoE–/– mice were fed a high-fat diet for 13 weeks. In both studies, the atherosclerotic progression was analyzed in the aortic root and the aorta. Lesion classification, cellular composition (T cells, macrophages and smooth muscle cells) and intracel­ lular lipid accumulation was analyzed in the aortic root. Blood lipid levels were measured in the blood serum by ELISA, cholesterol profiles were deter­ mined after HPLC-based fractionation and serum cytokine levels were meas­ ured with a cytokine bead array. The hematopoietic profile was studied by flow cytometric analysis of leukocytes isolated from bone marrow, blood, spleen and lymph nodes.

Results and conclusions: The hemat­ opoietic profiling revealed a significant decrease in B-cells, regulatory T cells and effector memory T cells in IkkαAA/ AA ApoE–/– bone marrow chimeras, whereas the naive T-cell population was increased. Surprisingly, no differences Perfusion 01/2014

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were observed in the size, lesion stage or cellular composition of atherosclerot­ ic lesions in the aorta and aortic root of IkkαAA/AAApoE–/– versus Ikkα+/+ApoE–/– bone marrow-transplanted mice, as shown by histological and immunofluo­ rescent stainings. Necrotic core sizes, cellular and macrophage apoptosis, and intracellular lipid accumulation in macrophages in aortic root lesions were unaltered. Furthermore, serum levels of inflammatory proteins as measured with a cytokine bead array by flow cytometry were comparable. In contrast, a total Ikkα knock-in did influence atheroscle­ rosis. IkkαAA/AAApoe–/– mice showed a significantly increased plaque forma­ tion in the aorta, but surprisingly the le­ sions in the aortic root – although more advanced – were significantly reduced compared to the control group. These results indicate a complex role of Ikkα in atherogenesis and future studies aim to identify the underlying mechanism of the differential effect of IkkαAA/AA on atherosclerosis in aorta vs aortic root.

Garcinoic acid, a tocotrienol-related natural product, reduces the inflammatory response of macrophages J. Heise1, M. Wallert1, L. Schmölz1, V. Krauth2, O. Werz2, M. Birringer3, S. Lorkowski1 1 Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 Department of Pharmaceutical Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Germany 3 Department of Nutritional, Food, and Consumer Studies, University of Applied Sciences Fulda, Germany Background: Garcinia kola is an Af­ rican plant which is widely used in African ethno-medicine. Beside its tra­ ditional use pharmacological effects of compounds from this plant have been shown. Its seed, the Garcinia kola nut, is rich in phytochemicals and second­ ary plant products. One of these com­ pounds is garcinoic acid which is a

δ-tocotrienol metabolite. Tocotrienols are well known antioxidants with antiinflammatory properties but only few studies have investigated the immu­ nomodulatory functions of garcinoic acid.

Aim: We are investigating the effect of isolated garcinoic acid on inflam­ matory markers which are released by activated macrophages.

Methods and results: We isolated gar­ cinoic acid from the nut by extraction and examined the effect of garcinoic acid and the crude nut extract on proand anti-atherogenic mediators, such as IL-1β, IL-6, IL-10, IL-12p40, Tgfβ1, TNF-α, iNos and Cox2, in LPS-stimu­ lated murine RAW 264.7 macrophages using RT-qPCR. In addition, we meas­ ured iNos and Cox2 protein by West­ ern blotting as well as release of nitric oxide using Griess reagent and pro­ duction of prostaglandin E2 (PGE2) by ELISA. Garcinoic acid reduced LPSinduced expression of iNos and Cox2 at the mRNA and protein level. Further, LPS-stimulated release of nitric oxide and PGE2 was reduced by garcinoic acid and the crude nut extract. We are currently investigating the influence of garcinoic acid on NF-κB signaling to explain its immunomodulatory effects on activated macrophages.

Conclusion: Garcinoic acid is a po­ tent anti-inflammatory compound with strong biological activity that can serve as a lead structure for developing new anti-inflammatory agents.

Long-chain metabolites of vitamin E protect human macrophages against lipotoxicity L. Schmölz1, M. Wallert1, M. Birringer2, S. Lorkowski1 1 Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 Department of Nutritional, Food, and Consumer Studies, University of Applied Sciences Fulda, Germany Introduction: Lipotoxicity describes the toxic effects of pathologically high © Verlag PERFUSION GmbH

ABSTRACTS

levels of lipids and free fatty acids. With respect to atherosclerosis lipotoxicity plays an important role in foam cell formation and particularly in plaque formation. Following endothelial dys­ function the arterial wall is perfused with lipoproteins which are assimilated by macrophages. These cells initially store excessive lipids in lipid droplets but if lipid levels overwhelm the stor­ age capacity of macrophages lipotox­ icity causes apoptosis and cell death, thus contributing to plaque formation.

For several years vitamin E, mostly in form of α-tocopherol (α-TOH), has been widely used as an antioxidant and protective agent to prevent cardiovas­ cular disease. Tocopherols are metabo­ lized via oxidative modification of the side-chain to 13’-hydroxychromanols (13’-OH) and 13’-carboxy-chromanols (13’-COOH) whose biological func­ tions remain unclear.

Aim: Previous studies of our group provided evidence for the physiologi­ cal relevance of the long-chain metab­ olites of vitamin E. We therefore inves­ tigated the mechanisms by which the metabolites affect macrophage foam cell formation.

Methods and results: In the present study we focused on stearic acidinduced lipotoxicity in human THP1 macrophages. We found that the long-chain metabolite α-13’-COOH reduced the lipotoxic effect of stearic acid up to 20 % as assayed by MTT. α-Tocopherol in contrast does not af­ fect stearic acid-induced lipotoxicity.

We also investigated the effects of α-TOH and long-chain metabolites on the expression of the lipid droplet-as­ sociated protein adipophilin (adipose tissue related protein, ADRP). While α-TOH reduced adipophilin expres­ sion on mRNA and protein level as measured by RT-qPCR and Western blotting respectively, the long-chain metabolite α-13’-COOH induced adi­ pophilin at both levels. Using adipo­ philin-specific siRNA, we are current­ ly investigating the hypothesis that the up-regulation of adipophilin by the long-chain metabolite prevents mac­ Perfusion 01/2014

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rophages from stearic acid-induced lipotoxicity.

Conclusion: Our investigations shed new light on the effects of the longchain metabolites of vitamin E in the context of atherosclerosis. We once again provide evidence for the diverse effects of α-TOH and its metabolites indicating a physiological role of the long-chain metabolites as signaling molecules.

α-Tocopherol long-chain-metabolites regulate the expression of ABCA1 in primary fibroblasts S. Kluge1, L. Schmölz1, M. Wallert1, M. Birringer2, S. Lorkowski1 1 Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 Department of Nutritional, Food, and Consumer Studies, University of Applied Sciences Fulda, Germany Background: α-Tocopherol (α-TOH) is metabolized in the liver by side-chain truncation initiated by CYP3A4-de­ pendent ω-hydroxylation which results in the formation of the long-chain-me­ tabolite (α-LCM) hydroxychromanol (α-13‘-OH). Subsequent α-oxidation in peroxisomes forms the α-LCM car­ boxychromanol (α-13‘-COOH) which also occurs in human plasma. Vitamin E is known to modulate cholesterol ho­ meostasis but almost nothing is known on the effects of the α-LCM α-13’-OH and α-13’-COOH on cholesterol ho­ meostasis.

Aim: We investigated the regulatory effects of α-LCM on the cholesterol export.

Methods and results: We have recent­ ly shown that the α-LCM up-regulate the expression of the scavenger recep­ tor CD36 but prevent oxLDL-mediated foam cell formation. We were therefore interested whether the α-LCM modu­ late cholesterol export and focused our interest on the cholesterol exporters ABCA1 and ABCG1. In primary fi­ broblasts α-13‘-COOH and α‑13‘-OH

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significantly down-regulated ABCG1 mRNA as well as ABCA1 mRNA and protein. We investigated the signaling pathways mediating these effects at protein level using Western blotting.

First we focused on blocking experi­ ments of the MAPK signaling cascade, PI3K and different protein kinases such as PKC and PKA which are known to regulate ABCA1 expression. Involve­ ment of these pathways in α-LCM signaling was excluded. We are there­ fore currently analyzing whether the metabolites act themselves as kinase inhibitors.

Conclusion: The cellular signaling of the α-LCM responsible in the reg­ ulation of ABCA1 has not yet been unraveled. Further experiments are required to elucidate the regulatory pathways.

Signaling pathways involved in PMA-induced up-regulation of CCR7 A.-A. Keller1, M. B. Maeß1, F. Dehm2, V. Krauth2, O. Werz2, S. Lorkowski1 1 Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 Department of Pharmaceutical Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Germany Background: Classical activation of macrophages (MΦ) plays a central role in the pathogenesis of atherosclerosis.

Macrophage activation is character­ ized by, for example, increased surface expression of CCR7. This chemokine receptor is involved in homing of MΦ and other immune cells into lymph nodes as well as their remaining in ath­ erosclerotic vessels. CCR7 is dose- and time-dependently induced by phor­ bol-12-myristate-13-acetate (PMA) in THP-1 MΦ. By now, no signaling pathways have been identified which explain this finding.

Aim: Unraveling the signaling path­ ways involved in PMA-induced up-regulation of CCR7 in mature hu­ man MΦ is our objective. © Verlag PERFUSION GmbH

ABSTRACTS

36

Methods and results: In order to achieve mature but unpolarized THP-1 MΦ we performed differentiation with low-doses of 10 ng/ml PMA. Up-reg­ ulation of CCR7 mRNA expression in mature MΦ followed stimulation with 100 ng/ml PMA not before 48 hours.

Therefore, we hypothesized an auto­ crine, positive feedback signaling loop inducing CCR7 via PKC and an extra­ cellular mediator. We assumed involve­ ment of prostaglandin E2 (PGE2) but al­ though release of PGE2 was increased in response to PMA as measured by ELISA blocking of PGE2 production by indometacin and celecoxib did not affect up-regulation of CCR7. Like­ wise, we determined inhibition of the 5-lipoxgenase pathway using BWA4C to be not involved on mRNA level. Us­ ing specific inhibitors we performed systematic investigations of potentially involved signaling pathways. We con­ firmed involvement of ERK, PKC and PI3K in PMA-induced up-regulation of CCR7. In contrast to p38 MAPK and mTOR, activation of JNK is likely involved in down-regulation of CCR7 mRNA expression. However, further studies are required for completely un­ raveling the PMA-induced MΦ activa­ tion pathway.

Conclusions: PMA-induced up-reg­ ulation of CCR7 involves several signaling proteins as well as macro­ phage-derived autocrine mediators that are potent modulators of classical acti­ vation of MΦ.

The effect of oxLDL on Jab1/COPS5 expression and foam cell formation in human macrophages A. Schwarz, G. Bonaterra, B. Wilhelm, R. Kinscherf Institute for Anatomy and Cell Biology, Philipps University of Marburg, Germany Aim: Atherosclerosis is an inflamma­ tory disease involving recruitment of macrophages (MΦ), which are gener­ ally the most abundant cell type in ath­ erosclerotic plaques. Previous studies of Jab1/COPS5 (c-Jun activation do­ Perfusion 01/2014

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main binding protein-1); the subunit 5 of the COP9 signalosome (COPS5) have shown a co-expression of MΦ migration inhibitory factor (MIF) in all stages of human plaques. MΦ in­ ternalize oxLDL (oxidized low density lipoprotein), develop into foam cells and release diverse pro-inflammatory cytokines, phenomena which are con­ stituents of vulnerable plaques. Thus, we were interested to investigate the role of Jab1/COPS5 during foam cell formation and release of pro-inflamma­ tory cytokines.

Methods: U937 cells were differenti­ ated (24 hours; phorbol 12-myristate 13-acetate [PMA, 20 nM]) to MΦ and afterwards incubated (4 hours or 24 hours) with human oxLDL (50 µg/ml, 100 µg/ml) to induce foam cell forma­ tion (confirmed with OilredO stain­ ing). The viability of unstimulated and stimulated MΦ were analyzed by Prestoblue. Using real time RT-PCR (qRT-PCR) we measured the expres­ sion of Jab1/COPS5 and TNF-α. JAB1/ COPS5 and IkBα protein expression were analyzed and quantified by West­ ern blot. Additionally, we quantified the release of TNF-α by ELISA. Treat­ ment with 0.1 µg/ml LPS was used as positive control.

Results: The viability of human MΦ was unchanged after 4 hours and 24 hours treatment with oxLDL. In ox­ LDL-treated MΦ we observed a con­ centration- and time-dependent foam cell formation. Moreover, in oxLDL (50 µg/ml, 100 µg/ml) stimulated MΦ we found a significant 34- and 50-fold increase of TNF-α release in comparison with the negative control.

Additionally, we found a significantly enhanced of TNF-α mRNA expression by concentration of 50 µg/ml oxLDL. The JAB1/COPS5 mRNA expression was unchanged after 4 hours oxLDL treatment, but the relative protein ex­ pression was significantly increased (27–34 %). Furthermore, we found a significant 33–43 % decrease of Jab1/ COPS5 mRNA expression after 24 hours oxLDL treatment in comparison with the control, whereas the relative

JAB1/COPS5 protein expression was unchanged. In addition, we found a significant 2-fold increase of relative IkBα protein expression after 4 hours and a significant 5-fold decrease of rel­ ative IkBα protein expression after 24 hours L treatment in comparison with the negative control.

Conclusion: Our results suggest an oxLDL-mediated inverse regulation of Jab1/COPS5 expression and inflamma­ tory markers (TNF-α) in human mac­ rophages, which may be related to an enhanced internalization of oxLDL in macrophages.

Induction of myocardial ischemic injury by extracellular RNA: a role for tumor necrosis factor (TNF-α) shedding H. A. Cabrera-Fuentes1, 4, M. Ruiz-Meana2, O. N. Ilinskaya4, D. Garcia-Dorado2, K. D. Schlüter3, K. T. Preissner1 1 Institute for Biochemistry, Justus Liebig University, Giessen, Germany 2 Hospital Universitari Vall d´Hebron, Servicio de Cardiologia, Barcelona, Spain 3 Institute of Physiology, Medical Faculty, Justus Liebig University, Giessen, Germany 4 Kazan Federal University, Department of Microbiology, Kazan, Russia Aim: Extracellular RNA (eRNA), ex­ posed after tissue trauma, ischemia or tissue damage, has been shown to exert prothrombotic and hyperpermeabilityinducing functions. Following myocar­ dial ischemia, the presence of eRNA (as cofactor for cytokines and coagula­ tion proteases) is expected to potentiate cell injury. Due to the causative role of the mitochondrial permeability transi­ tion pore (mPTP) in cell death, the aim of this study was to investigate whether eRNA contributes to mPTP opening and necrosis during hypoxia.

Methods and results: eRNA was re­ leased from cardiac myocytes under hypoxic conditions in a time-dependent manner. Addition of eRNA (1–10 µg/ ml) under normoxia did not promote cell death but induced the release of © Verlag PERFUSION GmbH

ABSTRACTS

TNF-α and IL-6 in a dose-dependent manner. During hypoxia, cell death (as quantified by lactate dehydrogenase, LDH) was increased in the presence of both eRNA (1-10 µg/ml) or TNF-α (10 ng/ml). The effect of eRNA on cell death could be reverted in the pres­ ence of RNase1 or TAPI (an inhibitor of TNF-α converting enzyme, TACE). eRNA (10 µg/ml) significantly accel­ erated cell energy exhaustion (rigor contracture) in cardiomyocytes as well as the time of mPTP opening upon oxidative damage (tetramethylrhoda­ mine-ethylester-loaded cardiomoy­ cytes submitted to intermittent 568 nm laser illumination). Moreover, eRNAinduced TNF-α release was effectively reduced by the TACE inhibitor TAPI (10 µM) and was fully prevented by anti-TNF-α. Hydrolyzed eRNA did not show any effect under normoxia or hypoxia.

Conclusion: These results demonstrate that eRNA is released from cardio­ myocytes under hypoxic conditions, promoting cell death by mechanisms related to TNF-α release and mPTP opening. The use of RNAse-1 to re­ duce the deleterious effects of eRNA may serve as a new potential therapeu­ tic approach to prevent ischemic myo­ cardial damage.

Platelets induce apoptosis via membrane-bound FasL R. I. Schleicher1, 2, F. Reichenbach3, 4, 5, P. Kraft6, A. Kumar7, M. Lescan8, F. Todt3, 4, 5, K. Goebel9, T. Geisler2, A. Bauer2, S. Wesselborg10, L. O’Reilly11, S. G. Meuth9, K. Schulze-Osthoff12, M. Gawaz2, X. Li13, C. Kleinschnitz6, F. Edlich3, 4, 5, 14, H. F. Langer1, 2 1 Section for Cardioimmunology, Eberhard Karls University Tuebingen, Germany 2 University Hospital, Department of Cardiovascular Medicine, Eberhard Karls University Tuebingen, Germany 3 Institute for Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, Germany 4 Spemann Graduate School of Biology and Medicine, SGBM, Freiburg, Germany 5 Faculty of Biology, University of Freiburg, Germany Perfusion 01/2014

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Department of Neurology, University Hospital Wuerzburg, Germany 7 National Eye Institute, Bethesda, USA 8 University Hospital, Department of Cardiovascular Surgery, Eberhard Karls University Tuebingen, Germany 9 Department of Neurology and Institute of Physiology I – Neuropathophysiology, University of Muenster, Germany 10 Institute for Molecular Medicine, Heinrich Heine University Duesseldorf, Germany 11 The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia 12 Interfaculty Institute of Biochemistry, University of Tuebingen, and German Cancer Consortium (DKTK) and German Research Cancer Center (DKFZ), Heidelberg, Germany 13 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Guangdong, P.R. China 14 BIOSS, Center for Biological Signaling Studies, University of Freiburg, Germany 6

Background: After tissue injury, both wound sealing and apoptosis contrib­ ute to restoration of tissue integrity and functionality. Although the role of platelets for wound closure and induc­ tion of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete.

Results: Here, we show that platelets present the death receptor Fas ligand (FasL) on their surface after activation.

Activated platelets as well as the iso­ lated membrane fraction of activated platelets but not of resting platelets induced apoptosis in a dose-dependent and cell-specific manner. In contrast, membrane protein from platelets lack­ ing membrane-bound FasL (FasLΔm/ Δm ) failed to induce apoptosis. Mito­ chondrial apoptosis signaling via Bax and Bak activation was not required for platelet-induced cell death, but increased the apoptotic response to platelet-induced Fas signaling. In vivo, platelet depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-D-aspartic acid (NMDA)induced retinal apoptosis. Further­ more, reduction of apoptosis in FasLΔm/ Δm mice compared to wild type mice

37

was abolished in the absence of plate­ lets demonstrating a role of plateletderived FasL for tissue apoptosis.

Conclusion: Since apoptosis second­ ary to injury prevents inflammation, our findings describe a novel mecha­ nism how platelets could contribute to tissue homeostasis.

Regulation of endothelial cell PARP by oxidized lipoprotein in atherosclerosis L. Mey, A. Hildenberg, M. Fahrleitner, H. Langer, N. Al-Fakhri Center of Transfusion Medicine and Hemotherapy, Faculty of Medicine, Philipps University Marburg, Germany, and Department of Internal Medicine III, AG Cardioimmunology, University Clinic Tuebingen, Germany Introduction: Poly-ADP-ribose poly­ merase (PARP) is the main intracel­ lular target for the antiapoptotic effect of vascular endothelial growth factor (VEGF) in vascular endothelial cells (EC), as demonstrated previously.

Here we show that PARP is regulated in vitro by oxLDL and could play a role in endothelial apoptosis of the ApoE–/– mouse model.

Methods: HUVEC stimulated with oxidized and native LDL and incu­ bated with or without VEGF-A were analyzed by means of Western blot, q-RT-PCR and activity assay for the expression of PARP-1 and Caspase-3.

Aortae of ApoE–/– mice that were fed normal chow or high fat diet (HFD) were analyzed by immunohistochemis­ try for PARP, VEGF receptors, SOD-1, catalase and activated caspase-3 ex­ pression. Controls were aortae of wild type (WT) animals fed HFD or normal chow. Additionally, PARP-1 expres­ sion was quantified in protein and RNA isolated from the aortae by Western blot and q-RT-PCR (LightCycler).

Results: PARP expression was induced in HUVEC by stimulation with oxLDL, but not with nLDL, and increased by VEGF incubation. Arteriosclerotic aor­ tae of ApoE–/– mice, on HFD or normal © Verlag PERFUSION GmbH

ABSTRACTS

38

chow, showed an increased expression of PARP, SOD-1 and caspases com­ pared to WT animals.

Conclusion: PARP, an anti-apoptotic intracellular mediator and substrate of caspases, is regulated by VEGF on en­ dothelial cells. The effect of oxidized lipoproteins on PARP expression in vitro and the arteriosclerotic animal model indicate a role for PARP in arte­ riosclerosis development.

The endothelial expression of neuropilin-1 is regulated by oxidized lipoprotein L. Mey, A. Hildenberg, M. Fahrleitner, H. Langer, N. Al-Fakhri Center of Transfusion Medicine and Hemotherapy, Faculty of Medicine, Philipps University Marburg, Germany, and Department of Internal Medicine III, AG Cardioimmunology, University Clinic Tuebingen, Germany Introduction: Vascular endothelial growth factor (VEGF) is the most po­ tent anti-apoptotic growth factor for endothelial cells (EC). The effect of VEGF is transduced via the VEGF receptor 2 (VEGF-R2) and the coreceptor neuropilin-1 (NP-1). In our previous work we have shown that endothelial NP-1 plays an important role in the regulation of VEGF effects in vascular endothelial apoptosis and arteriosclerosis development. The aim of this study was to identify a possible relationship between oxLDL-induced oxidative stress of the endothelium and neuropilin-1 in vitro and in an ApoE–/– animal model.

Methods: HUVEC stimulated with oxidized and native LDL and incubated with or without VEGF were analyzed by means of Western blot, q-RT-PCR and activity assay for the expression of NP-1 and Caspase-3. Aortae of ApoE–/– mice that were fed normal chow or high fat diet (HFD) were analyzed by im­ munohistochemistry for NP-1, SOD-1, catalase and activated caspase-3 ex­ pression. Controls were aortae of wild type (WT) animals fed HFD or normal Perfusion 01/2014

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chow. Additionally, NP-1 expression was quantified in protein and RNA iso­ lated from the aortae by Western blot and q-RT-PCR (LightCycler).

Results: NP-1 expression was induced by oxLDL stimulation in HUVEC, this effect was slightly increased by VEGF incubation. Arteriosclerotic aortae of ApoE–/– mice, on HFD or normal chow, showed an increased expression of NP1, SOD-1 and caspases compared to WT animals.

Conclusion: NP-1, an important regu­ latory receptor for VEGF that inhib­ its apoptosis, is induced by oxidized lipoproteins, most likely to increase protection of the vascular endothelium against apoptosis.

The role of 5’-AMP-activated kinase (AMPK) in the regulation of endothelial cell dysfunction induced by inflammatory stress S. Lindenmüller1, 2, K. Spengler1, B. Viollet3, R. Heller1, 2 1 Institute for Molecular Cell Biology, Center for Molecular Biomedicine (CMB), University Hospital Jena, Germany 2 Center for Sepsis Control and Care (CSCC), University Hospital Jena, Germany 3 Institut Cochin, Université Paris Descartes, CNRS, France Aim: Sepsis and systemic inflamma­ tory response syndrome (SIRS) are characterized by a generalized dys­ regulated inflammatory response. One hallmark is microvascular dysfunction which is linked to endothelial dysfunc­ tion and which results in decreased organ perfusion and subsequent devel­ opment of organ failure. Thus, strate­ gies to stabilize the microvasculature may help to prevent or ameliorate or­ gan dysfunction. In this context, the energy-sensing enzyme AMP-activat­ ed kinase (AMPK) may represent an interesting target. It is expressed in endothelial cells and involved in the regulation of energy supply and stress protection. The goal of this study was to understand whether AMPK is able to

protect endothelial cells from dysfunc­ tion induced by inflammatory stimuli thereby contributing to the prevention of microvascular dysfunction.

Methods: Experiments were per­ formed in mice, in which the cata­ lytic subunits AMPKα1 or AMPKα2 were knocked out, as well as in human umbilical vein endothelial cells (HU­ VEC), in which these subunits were down-regulated by specific siRNAs. In addition, two AMPK activators were applied (5-aminoimidazole-4-carbox­ amide ribonucleoside [AICAR] and A769662). Cytokine stimulation of cells was carried out with tumor ne­ crosis factor α (TNF-α), interleukin 1β (IL-1β) and lipopolysaccharide (LPS) alone or in combination. Expression of adhesion molecules ICAM-1 and VCAM-1 was detected by flow cy­ tometry using specific antibodies and endothelial barrier function was meas­ ured by means of the electrical cellsubstrate impedance sensing method (ECIS). To characterize the influence of AMPK on endothelial permeability in vivo, a vascular leakage assay was established, in which the dye Evan’s Blue, which binds to plasma albumin, is injected and its distribution in organs after application of permeability-in­ creasing compounds is evaluated.

Results: AMPK activation leads to a decrease of cytokine-induced VCAM1 and ICAM-1 expression suggesting an aniinflammatory effect of the en­ zyme. Importantly, AMPK affects en­ dothelial barrier function. ECIS meas­ urements in AMPKα-depleted HUVEC showed an amplification of cytokine/ LPS-induced permeability suggesting a barrier-protecting role of AMPK. To characterize the influence of AMPK on endothelial permeability in vivo, vas­ cular leakage assays after i.p. injection of LPS were performed. We observed an early and more intense increase in vascular permeability in the liver in re­ sponse to LPS in AMPKα1 knockout mice as compared to wild type control mice suggesting a protective effect of AMPK early after an initiation of an inflammatory response. © Verlag PERFUSION GmbH

ABSTRACTS

Conclusion: Our results indicate that AMPK may protect endothelial cells from cytokine- or LPS-induced bar­ rier dysfunction, especially in liver. To­ gether, our data raise the possibility that AMPK may be a target through which vascular function can be maintained under inflammatory conditions, which may help to attenuate organ failure and decrease the severity of disease.

C5a receptor deficiency enhances cardiac repair after myocardial infarction by regulating angiogenesis and myofibroblast transdifferentiation S. Simsekyilmaz1, E. A. Liehn1, Y. Asare1, 2, G. Shagdarsuren3, A. Togtokh3, A. Klos4, E. Medina5, J. E. Gessner6, E. Shagdarsuren1 1 Institute for Molecular Cardiovascular Research (IMCAR), University Hospital of the RWTH Aachen, Germany 2 Institute of Biochemistry and Molecular Cell Biology, University Hospital of the RWTH Aachen, Germany 3 Department of Nephrology, Health Sciences University of Mongolia, Ulaanbaatar, Mongolia 4 Department for Medical Microbiology, Hannover Medical School, Germany 5 Department of Medical Microbiology, Helmholtz Center for Infection Research, Braunschweig, Germany 6 Molecular Immunology Research Unit, Clinical Department of Immunology and Rheumatology, Hannover Medical School, Germany Objective: The complement anaphyla­ toxin C5a functions through its two functional receptors, the C5aR (CD88) and C5a receptor-like 2 (C5L2). Sever­ al studies have shown the protective ef­ fect of C5aR-inhibition in myocardial infarction (MI). However, the underly­ ing mechanisms of C5a/C5aR-mediat­ ed action remain elusive and the role of the second receptor of C5a, C5L2, is completely unexplored. Therefore, we analyzed the role of these receptors in cardiac remodeling after MI in C5aRand C5L2-deficient mice.

Methods and results: MI was induced by temporary ligation of the left ante­ Perfusion 01/2014

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rior descending coronary artery. Echo­ cardiography, histology, quantitative immunohistochemistry, quantitative RT-PCR and in vitro angiogenesis and myofibroblast transdifferentiation as­ says were performed. Neutrophil re­ cruitment was significantly reduced 1 day after MI in C5aR-deficient mice compared to wild-type (WT) mice, while no changes were observed on monocyte infiltration 1 week after MI. Four weeks after MI, the infarct size was reduced by 58 % in C5aR-deficient mice compared to WT mice, but not in C5L2-deficient mice. Cardiac ejection fraction was improved in C5aR-defi­ cient mice accompanied by a reduced left ventricular diastolic volume com­ pared to WT mice, whereas the cardiac output was unchanged. The reduction in infarct size in C5aR-deficient mice 4 weeks after MI was associated with a reduced neutrophil and monocyte infil­ tration, as well as a decreased content of collagen and myofibroblasts. Fur­ thermore, an increased amount of proangiogenic CD31+ endothelial cells was detected in infarcted areas, which was confirmed by an improved tube formation in vitro. In addition, while the number of apoptotic cells was sig­ nificantly reduced, the amount of pro­ liferating cells was increased. Notably, C5a mRNA and protein expression were elevated in C5aR- and C5L2-de­ ficient mice in infarcted cardiac tissue one week after MI compared to WT mice, whereas TGF-β1 and VEGF-A mRNA and protein expression were reduced. In the presence of C5a, the fibroblast-to-myofibroblast transition was reduced, whereas neutralizing C5a with an antibody promoted the trans­ differentiation in vitro.

Conclusions: C5aR plays a crucial role in endogenous remodeling pro­ cesses after MI, while the gene deletion positively affects cardiac remodeling by contributing to the inhibition of in­ flammatory cell recruitment, apopto­ sis, myofibroblast transdifferentiation and promoting the neovascularization and proliferation. In contrast, C5L2 showed no effect.

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Re-occurrence of ED-A+ fibronectin after human cardiac transplantation: pivotal role as diagnostic marker and therapeutic target?

B. Ziffels1, M. Matusiak-Brückner1, H. Maschek2, P. Richter3, U. Schulz4, J. Gummert4, H. R. Figulla1, A. Renner4, A. Berndt3, M. Franz1 1 Department of Internal Medicine I, University Hospital Jena, Germany 2 Institute of Pathology, “Pathologie am Tiergarten”, Hannover, Germany 3 Institute of Pathology, University Hospital Jena, Germany 4 Clinic for Thoracic and Cardiovascular Surgery, Heart Center North Rhine-Westphalia, Ruhr University of Bochum, Bad Oeynhausen, Germany Background and aims: Clinical man­ agement of acute and chronic rejec­ tion in cardiac transplant recipients is still challenging. While acute rejection can be sufficiently controlled with ef­ ficient immunosuppressive drugs, its early detection requires invasive endomyocardial biopsy, which is stressful for the patients. For chronic rejection with allograft vasculopathy (CAV) and interstitial fibrosis (CIF), there are no therapeutic strategies available until now to prevent or treat the disease.

Rejection associated tissue-remodeling entails the re-occurrence of fetal vari­ ants of fibronectin (Fn) and tenascin-C (Tn-C), which are virtually absent in healthy adults. In a rat heart transplan­ tation model, an extensive rejectionassociated re-expression could be dem­ onstrated for ED-A+ Fn with spatial association to CAV and CIF. A human antibody specific to this molecule is ca­ pable for targeted delivery of bioactive payloads as well as diagnostic agents like radionuclides or magnetic beads.

Thus, it is of great interest to analyze ED-A+ Fn deposition in human cardiac biopsies and to establish a method to specifically assess serum levels of the molecule.

Methods: From 48 heart transplanted patients, tissue specimens derived from right ventricular biopsies were available. Histopathological analysis was performed according to the ISHLT © Verlag PERFUSION GmbH

ABSTRACTS

40

guidelines for the detection of acute rejection. By immunohistochemistry, protein expression of ED-A+ Fn and B+ Tn-C was assessed and analyzed semiquantitatively. Co-localization studies to show the spatial relation of both pro­ teins to CAV and CIF were performed by means of immunofluorescence dou­ ble labeling. ED-A+ Fn protein expres­ sion in tissue extracts was also ana­ lyzed by Western blot. Furthermore, an ELISA method for the quantification of ED-A+ Fn concentration in human and rat serum was developed.

Results: Histopathological analysis of biopsy samples revealed different ISH­ LT grades (0R, 1R and 2R). There was a distinct and quantitatively relevant reoccurrence of ED-A+ Fn and B+ Tn-C in most samples. Semi-quantitative evaluation did not show any correla­ tion to rejection grade for all markers.

Furthermore, there was a significant correlation between ED-A+ Fn and B+ Tn-C protein deposition (p=0.000). A spatial association of ED-A+ Fn and B+ Tn-C to CAV and CIF could be shown by means of immunofluorescence dou­ ble labeling. By Western blot analysis, ED-A+ Fn could be detected in diseased human cardiac tissue. Using a newly established ELISA protocol, ED-A+ Fn could be specifically detected both in human and rat serum samples.

Conclusions: A relevant re-occurrence of ED-A+ Fn and B+ Tn-C following human heart transplantation could be demonstrated as an early step of tissue remodeling. Since a correlation of EDA+ Fn expression to chronic rejection in animal models could be proven re­ cently, it can be postulated that an early re-occurrence of the molecule identi­ fies patients at increased risk to develop CAV and CIF. The spatial association of ED-A+ Fn and B+ Tn-C deposition to signs of chronic rejection and the avail­ ability of antibodies usable for targeted delivery of drugs or diagnostic agents might pave the way for novel strategies to image, prevent or treat the disease.

Moreover, the specific quantitative as­ sessment of ED-A+ Fn in serum using ELISA technique facilitates further re­ Perfusion 01/2014

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search on its possible value as a novel biomarker in heart failure patients.

The role of p53 in transcriptional regulation of Nox4 S. Göbel, D. Siuda, G. Reifenberg, N. Xia, U. Förstermann, H. Li Department of Pharmacology, University Medical Center Mainz, Germany Introduction: NADPH oxidases (Nox) represent an important source of reactive oxygen species (ROS) in the vasculature and Nox4 is the major Nox isoform in endothelial cells. Un­ like other Nox isoforms, Nox4 does not require the regulatory subunits for acti­ vation. In other words, Nox4 is consti­ tutively active and its activity mainly depends on the level of expression.

Therefore, it is of importance to study the molecular mechanisms controlling Nox4 transcription.

Aims: The aim of the study was to identify regulation mechanisms of Nox4 expression under conditions of ischemia or starvation.

Methods: Human umbilical vein en­ dothelial cells (HUVEC) and HUVECderived EA.hy 926 cells were starved in diluted culture media for 24 hours to produce ischemia-like conditions.

Nox4 mRNA and protein expression was analyzed with quantitative realtime PCR (qPCR) and Western blot, respectively.

Results: Starvation of HUVEC and EA.hy 926 cells led to an up-regulation of Nox4 at both mRNA and protein levels which was associated with an induction of the tumor suppressor p53. p53 functions as a transcription regula­ tor in cells undergoing stressful events.

Consistently, multiple p53 binding sites could be identified in Nox4 pro­ moter region by in silico analyses.

Importantly, siRNA-mediated knockdown of p53 reduced the basal Nox4 mRNA expression and prevented the starvation-induced Nox4 up-regula­ tion, indicating a crucial role of p53 in Nox4 transcriptional regulation.

Conclusions: The present study has identified a new regulation mecha­ nism of Nox4 expression. p53 is likely to play an essential role in Nox4 up­ regulation in response to starvation/ ischemia.

Effects of Pandanus conoideus Lam on endothelial nitric oxide synthase C. Schirra, N. Xia, G. Reifenberg, D. Siuda, U. Förstermann, E. Thines, H. Li Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany Aims: Red fruit (Pandanus conoideus Lam; PCL) is used in traditional Indo­ nesian medicine and believed to possess multiple actions including protective effects on the cardiovascular system.

The underlying molecular mechanisms, however, are poorly understood. Nitric oxide (NO) produced by endothelial cells is a protective principle with anti­ hypertensive, antithrombotic and antiatherosclerotic properties. Therefore, we hypothesized that PCL may enhance NO production by stimulating endothe­ lial NO synthase (eNOS).

Methods: Human EA.hy 926 endothe­ lial cells were treated with PCL juice and PCL fractions. Phosphorylation of eNOS was studied with Western blot analyses. NO production was deter­ mined with electron paramagnetic spin resonance (ESR) and rat fetal lung fi­ broblasts (RFL-6) reporter cell assay.

Results: Treatment of EA.hy 926 en­ dothelial cells with PCL juice for 5 min led to an enhanced eNOS phospho­ rylation at serine 1177 (an indicator of eNOS activation) and NO production.

These effects were evident even at a dilution of 1:1.000.000, indicating the PCL contains eNOS-stimulating com­ pounds with high-potency. In order to identify these active compounds, PCL juice was separated into fractions. Five (3A, 3B, 4B, 4C and 5B) out of the 12 fractions showed significant effects on eNOS phosphorylation and NO pro­ duction. The constituents of these frac­ © Verlag PERFUSION GmbH

ABSTRACTS

tions are currently being analyzed to find out the compounds of interest.

Conclusions: Pandanus conoideus Lam contains eNOS-stimulating com­ pounds with therapeutic potential for cardiovascular disease.

Premature senescence and proteolytic activities in endothelial cells O. Meçe1, T. Grune2, R. Heller1 Institute for Molecular Cell Biology, Center for Molecular Biomedicine (CMB), University Hospital Jena, Germany 2 Department of Nutritional Toxicology, Institute of Nutrition, Jena, Germany

1

Aim: Endothelial cells underlie se­ nescence in vivo, which is believed to contribute to endothelial dysfunction and vascular diseases. Senescence in endothelial cells may be triggered by stress-induced pathways and may es­ pecially take place in atherosclerotic plaques where inflammatory factors and oxidants are released from adja­ cent macrophages. One way through which inflammatory or oxidative stress may elicit or aggravate senescence of endothelial cells is the induction of a functional decline in proteasomal pro­ tein degradation systems. As a con­ sequence protein aggregates may ac­ cumulate, which in turn may promote senescence and cellular dysfunction.

The current project aims to character­ ize protein degradation pathways in endothelial cells undergoing oxidative stress-induced premature senescence and to investigate the effect of protea­ somal inhibition on endothelial senes­ cence.

Methods: The study was performed in human umbilical vein endothelial cells (HUVEC). To obtain chronic oxidative stress, 100 µM or 200 µM H2O2 were supplemented to growth medium con­ taining 10 % fetal calf serum on a daily basis. Several parameters of oxidative stress (protein carbonylation in West­ ern blots) or senescence (staining of senescence-associated-β-galactosidase Perfusion 01/2014

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(SA-β-gal) and the proliferation mark­ er Ki-67) were assessed up to 8 days.

To characterize proteolytic capacity of cells, fluorigenic substrates specific for peptidyl-glutamyl-like (β1 subunit), trypsin-like (β2 subunit) and chymo­ trypsin-like (β5 subunit) activities of the 20S proteasome were employed.

The expression of proteasomal sub­ units was analyzed by Western blot­ ting. To investigate long-term effects of proteasome inhibition on endothe­ lial senescence, the compound MG132 was added to growth medium on a dai­ ly basis for 4 consecutive days and se­ nescence markers were analyzed after 6 additional inhibitor-free days.

Results: Our results show that H2O2 treatment led to major intracellular oxidative stress as monitored by pro­ tein carbonylation. In parallel, a time and dose-dependent increase of SAβ-gal positive cells (45 % of total cell numbers at 8 days after treatment with 200 µM H2O2) as well as growth arrest (reduction of the percentage of Ki67-positive cells) were observed. At the same time, proteasomal activities, especially the chymotrypsin-like activ­ ity, declined while the expression levels of the proteasomal subunits remained unchanged. To understand whether the development of senescence and protea­ somal decline in response to chronic oxidative stress may be causally relat­ ed we investigated the effect of a direct proteasomal inhibition on endothelial senescence. Our preliminary data dem­ onstrate that long-term incubation (4 days) of cells with MG132 followed by an inhibitor-free period (6 days) led to a considerable increase of SA-β-gal positive cells.

Conclusion: Taken together, our data show that chronic oxidative stress leads to an induction of premature senescence in endothelial cells and to a decline of proteolytic activities. Proteasomal inhi­ bition alone is able to aggravate senes­ cence suggesting a causal relationship between catabolic insufficiency and senescence. Future studies will be re­ quired to understand whether and how premature senescence and proteolytic

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decline are linked and to characterize responsible mechanisms.

Regulation of endothelial cell function by the AMPK/autophagy axis K. Spengler, S. Lindenmüller, N. Kryeziu, R. Heller Institute for Molecular Cell Biology, Center for Molecular Biomedicine (CMB), University Hospital Jena, Germany Aims: 5’AMP-activated protein kinase (AMPK) is a ubiquitously expressed in­ tracellular energy sensor, which main­ tains the balance between ATP produc­ tion and ATP consumption. In addition, AMPK is involved in the regulation of cellular homeostasis and signaling.

Recent studies have shown that AMPK may also be involved in the regula­ tion of an autophagosomal/lysosomal pathway, which plays a role in the degradation of long-lived or damaged proteins and organelles and contributes to stress adaptation and cell survival in an adverse environment. Autophagy is regulated by protein complexes that are formed during the assembly of au­ tophagosomes, which finally fuse with lysosomes to allow proteolysis. The present study was aimed at investigat­ ing the role of AMPK in the regulation of autophagy in endothelial cells and at characterizing the functional signifi­ cance of the AMPK/autophagy path­ way in these cells.

Methods: Experiments were per­ formed in human umbilical vein en­ dothelial cells (HUVEC). AMPK was activated by three different approaches (i) metabolic stress induced by 2-de­ oxyglucose (2-DG), (ii) 5-aminoimi­ dazole-4-carboxamide ribonucleoside (AICAR), which mimics AMP and (ii) A769662, a novel chemical AMPK activator. Autophagy was monitored by analyzing phosphorylation or con­ jugation of autophagy-regulating pro­ teins (ULK1, p70S6K or LC3B, re­ spectively) in immunoblots. Inhibition of autophagy was achieved by down­ regulation of ULK1 and Beclin1 us­ © Verlag PERFUSION GmbH

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42

ing specific siRNAs. Cellular ATP was measured with the ATP Kit SL. Cell cycle analysis was performed by flow cytometry and apoptosis was quanti­ fied according to the proportion of cells in the subG1 fraction. Angiogenesis was monitored in spheroid assays by quantifying sprouting from endothelial spheroids under basal and VEGF-stim­ ulated conditions.

Results: Our data show that AMPK is involved in autophagy induction in endothelial cells. This was confirmed by phosphorylation of the autophagy initiator ULK1 in response to AMPK activators, while the phosphorylation of p70S6K, a downstream target of mTOR, was decreased. The latter indi­ cates mTOR inhibition by AMPK and an indirect stimulation of autophagy by releasing the repressive effect of mTOR on autophagy. AMPK activa­ tors also induce conjugation of LCB3, which indicates the formation of au­ tophagosomes. We also show that au­ tophagy is essential for endothelial function. Impairment of autophagy by siRNA-mediated down-regulation of ULK1 and Beclin1 led to a clear in­ hibition of VEGF-induced angiogen­ esis. In parallel, cell proliferation was decreased and the amount of apoptotic cells was increased. The effect of au­ tophagy on endothelial function may be related to its role in providing en­ ergy sources for cellular metabolism in conditions of cellular activation and/or stress. In line with this we show that autophagy is necessary for the recov­ ery of intracellular ATP levels in re­ sponse to metabolic stress.

Conclusions: Taken together, our re­ sults demonstrate that AMPK activa­ tion induces autophagy. Autophagy is essential for VEGF-induced angiogen­ esis, which may be related to effects on cell survival and energy supply under these conditions. Previous results of our group have shown that AMPK is activated by VEGF and required for VEGF-induced angiogenesis but the underlying mechanism had not been clarified. The data of this study sug­ gest that AMPK may support VEGFPerfusion 01/2014

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induced angiogenesis via promoting autophagy.

15-deoxy-∆12,14-prostaglandin J2 is a mediator of arterial remodeling by controlling proteolytic activity of macrophages M. Abhari, M. Schubert, S. Becher, S. Lorkowski Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany Background: The complications of atherosclerosis are still the leading cause of death worldwide. Macrophag­ es play an important role in arterial remodeling during atherogenesis. On the one hand macrophages contribute to the preservation and build-up of the extracellular matrix (ECM) of the fibrous cap covering atherosclerotic plaques, and on the other hand mac­ rophages especially in the shoulder re­ gion of atherosclerotic plaques produce large amounts of proteinases, such as matrix metalloproteinases (MMPs).

Extensive release of MMPs leads to the destruction of the ECM of the fi­ brous caps, thus resulting in plaque rupture. 15-Deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) is a multifunctional antiinflammatory lipid metabolite which is abundantly produced by macrophages.

We have shown that 15dPGJ2 downregulates the expression of MMP8 and MMP14 by macrophages.

Aim: We aim to unravel the signaling pathways involved in balancing the proteolytic activity of macrophages by 15dPGJ2.

Methods and results: Studies of our group revealed that 15dPGJ2 decreases dose- and time-dependently the ex­ pression of MMP8 and MMP14 at the mRNA and protein level in human THP-1 macrophages as well as in hu­ man primary macrophages. Whereas regulation of MMP14 by 15dPGJ2 takes place independently of PPARγ, expression of MMP8 is regulated via PPARγ and in addition to some extent

by PPARγ-independent signaling path­

ways. PPARγ-independent signaling pathways were investigated by treat­ ment of mature macrophages with vari­ ous inhibitors of key signaling proteins, such as PI3K, JAK, JNK and MAPKs, in combination with 15-deoxy-∆12,14prostaglandin J2 for 24 hours. Effects of the inhibitors were examined at the mRNA and protein level of MMP8 and MMP14 using RT-qPCR and Western blotting, respectively. While investi­ gating PPARγ-independent signaling pathways, none of the inhibitors tested so far was able to block the downregulation of MMP8 by 15-deoxy∆12,14-prostaglandin J2, thus providing evidence that the metabolite itself acts as kinase inhibitor or inhibitor of ki­ nase signaling pathways. In contrast to MMP8, the expression of MMP11 has been up-regulated by 15dPGJ2. So far, we have no data providing evidence which pathways are responsible for this up-regulation.

Conclusions: The regulation of the proteolytic activity of macrophages by 15dPGJ2 involves several pathways and the functional consequences of the regu­ lation of MMP expression are not clear yet. However, our research may help for identifying new approaches in prevent­ ing myocardial infarction by preventing atherosclerotic plaque rupture.

Regulation of matrix metallo­pro­teinase 14 by 15-deoxy-∆12,14prostaglandin J2 in human macrophages M. Schubert, M. Abhari, S. Becher, S. Lorkowski Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany Background: Matrix metalloprotein­ ases (MMPs) are a group of zinc-de­ pendent enzymes that degrade several components of the extracellular matrix.

Hence they play a pivotal role in the stability of atherosclerotic plaques, a critical factor for the outcome of vas­cular disease. The lipid mediator 15deoxy-∆12,14-prostaglandin J2 (15dPGJ2), © Verlag PERFUSION GmbH

ABSTRACTS

known for promoting the resolution of inflammation, down-regulates expres­ sion of MMP14 in human macrophag­ es. 15-deoxy-∆12,14-prostaglandin J2 exerts its diverse effects via peroxi­ some proliferator-activated receptor γ (PPARγ)-dependent and -independent pathways. Unraveling the molecular modes of action of 15dPGJ2 on MMP expression may help to identify key regulators of plaque stability.

Aim: Our aim is to elucidate the sign­ aling pathways involved in the regula­ tion of MMP14 by 15dPGJ2 in human macrophages.

Methods and results: For our investi­ gations we used macrophages obtained from the monocytic cell line THP-1 and primary macrophages. THP-1 macrophages were generated from monocytes using phorbol 12-myristate 13-acetate. For analyzing PPARγdependent effects of 15dPGJ2 we used combinations of 15dPGJ2 or rosiglita­ zone with either the PPARγ antagonist GW9662 for blocking experiments or the retinoid X receptor ligand 9-cis retinoic acid for co-stimulatory ex­ periments. No convincing effects on MMP14 mRNA and protein levels were found in these experiments. PPARγindependent signaling pathways were investigated by treatment of mature macrophages with various inhibitors of key signaling proteins, such as phos­ phoinositide 3-kinase, janus kinases, c-Jun N-terminal kinases and mito­ gen-activated protein kinases in com­ bination with 15dPGJ2. Effects of the inhibitors were examined at MMP14 mRNA and protein level using RT-qP­ CR and Western blotting, respectively.

No inhibitor tested so far was able to block the down-regulation of MMP14 by 15dPGJ2. Thus, the metabolite itself may act as kinase inhibitor or inhibitor of kinase signaling pathways.

Conclusions: PPARγ is not involved in the down-regulation of MMP14 by 15dPGJ2. Signaling pathways in­ dependent of PPARγ are likely in­ volved but the exact mode of action of 15dPGJ2 on MMP14 expression re­ mains unclear.

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GLP-1 (7-37) and the GLP-1 metabolite (9-37) improve myocardial function and reduce infarction size after LAD ligation J. Möllmannn, E. A. Liehn, S. Simsekyilmaz, K. Hess, H. Findeisen, C. Lebherz, N. Marx, M. Lehrke Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany Introduction: GLP-1 (7-37) is an in­ cretin hormone which is released in response to nutritional stimuli from the gut and improves glucose metabo­ lism by increasing glucose-dependent insulin secretion. Furthermore, GLP1 (7-37) has cardioprotective effects and was found to reduce myocardial infarction size and improve contractile function following ischemia. Similar cardio­protective effects have been re­ ported for the GLP-1 metabolite (9-37) under in vitro conditions.

Methods and results: To evaluate whether the GLP-1 metabolite (9-37) has cardioprotective functions also under in vivo conditions we overex­ pressed GLP-1 (9-37) in direct com­ parison to GLP-1 (7-37) and LacZ (control) in 6 week old C57BL/6J mice using an adeno-associated viral vector system (n=7/group). Myocar­ dial infarction was induced by LADligation. Overexpression of GLP-1 (7-37) led to the expected improve­ ment of glucose metabolism (p<0.01 in oGTT) while GLP-1 (9-37) had no effect. Nevertheless, both peptides caused a comparable reduction of myocardial infarction size (p<0.05) and improved echocardiographic and in Langendorff assessed myocardial function in rest as well as under dobu­ tamin stress (all p<0.05). In addition GLP-1 (7-37) and GLP-1 (9-37) sig­ nificantly improved coronary blood (in rest and under dobutamin stress; all p<0.05) while also reducing the in­ filtration of leucocytes to the infarcted tissue on day 1 following LAD liga­ tion. This suggests anti-inflammatory effects as a possible mechanism of cardioprotection.

43

Conclusion: GLP-1 (7-37) and its metabolite GLP-1 (9-37), both reduce myocardial infarction size and improve left ventricular function following LAD ligation which might be mediated by anti-inflammatory effects of both peptides.

The GLP-1 metabolite (9-37) improves myocardial function in the TAC model by reducing myocardial hypertrophy and improving glucose uptake J. Möllmannn , R. Stöhr, W. A. Coumans, O. Winz, A. T. Vogg, H. J. Kaiser, C. Lebherz, F. M. Mottaghy, J. J. Luiken, J. F. Glatz, N. Marx, M. Lehrke Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany Introduction: Diastolic dysfunction and myocardial hypertrophy are early signs of hypertensive cardiomyopa­ thy. GLP-1 (7-37) is an incretin hor­ mone which is released in response to nutritional stimuli from the gut and improves glucose metabolism by in­ creasing glucose-dependent insulin secretion from the pancreas. GLP-1 (7-37) is rapidly cleaved to its inac­ tive metabolite GLP-1 (9-37) which is unable to bind to the GLP-1 receptor and does not cause insulin secretion.

Nevertheless both peptides have been found to hold cardioprotective actions.

Methods and results: To investigate the effects of GLP-1 on hypertensive cardiomyopathy we injected 6 week old C57BL/6J mice with an adenoassociated viral vector system over­ expressing GLP-1 (7-37), GLP-1 (937) or LacZ (control) (n=15/group).

Cardiac hypertrophy was induced by transversal aortic constriction (TAC).

Overexpression of GLP-1 (7-37) led to the expected improvement of glucose metabolism (p<0.01) while GLP-1 (9-37) had no effect. Despite, both peptides similarly reduced myo­ cardial hypertrophy (p<0.01; n=5-10/ group) and reduced myocardial col­ lagen content (gomori stain and PCR © Verlag PERFUSION GmbH

ABSTRACTS

44

all p<0.05; n=6-8/group) and apopto­ sis (caspase-3 p<0.05). Interestingly however, only GLP-1 (9-37) led to a significant improvement of diastolic myocardial function (dp/dt-min) while reducing LVEDP (all p<0.05; n=13/ group after 4 weeks under dobutaminstress by millar catheter).This was ac­ companied by a significant reduction of myocardial F18-FDG-glucose uptake (p<0.05; n=11/group in the PET) in GLP-1 (9-37) expressing mice and re­ duced expression of the glucose trans­ porters GLUT1 (p<0.01) and GLUT4 (p<0.01). In addition, GLP-1 (9-37) expressing mice were found to hold in­ creased ACC phosphorylation (p<0.05) pointing to activation of AMPK as a possible mechanism.

Conclusion: The GLP-1metabolite (937) improves hypertrophic cardiomyo­ pathy in the TAC model by reducing myocardial glucose uptake, ventricular hypertrophy, myocardial collagen con­ tent and apoptosis.

GLP-1 regulates the metabolic response during acute inflammation and predicts outcome in critically ill patients: central role of IL-6 F. Kahles, C. Meyer, J. Möllmann, C. Lebherz, H. M. Findeisen, S. Diebold, A. Koch, F. Tacke, N. Marx, M. Lehrke Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany Purpose: Hypoglycemia predicts ad­ verse outcome in critically ill patients including those with sepsis or myocar­ dial infarction. The underlying mech­ anisms for inflammation-mediated hypoglycemia are incompletely un­ derstood. Experimental data suggest that hyperinsulinemia might play a role here and that LPS induces insulin se­ cretion in a glucose-dependent manner.

Since the incretin hormone glucagonlike-peptide 1 (GLP-1) causes insulin secretion also in a glucose-dependent manner, we speculated that GLP-1 may be of relevance for inflammationdependent hypoglycemia.

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Methods and results: GLP-1 serum levels are significantly increased (6.9fold) in critically ill patients (n=155) in comparison to healthy controls (p<0.001) and independently predict unfavorable short- (p=0.001) and long­ er term (p=0.01) mortality. To examine the underlying molecular mechanisms we injected C57Bl6 mice with endo­ toxin as an inflammatory stimulus.

LPS increased GLP-1 secretion in a dose and time-dependent manner with a maximal 3.4-fold increase (p<0,001) 120 minutes post LPS injection. This was paralleled by a time and dosedependent increase of serum insulin (p<0,001) and a drop of blood glucose (p<0,001). A similar increase of serum GLP-1 and insulin was found after IL1β or IL-6 administration. Experiments in IL-1-receptor- and IL-6 knockout mice demonstrated that LPS-depend­ ent GLP-1 secretion was selectively dependent on IL-6 but not on IL-1 secretion. Consistently IL-6 (p<0,05) but not IL-1β or LPS (all 100 ng/ml) stimulated GLP-1 secretion from intes­ tinal L cells under in vitro conditions.

Interestingly, the antiinflammatory properties of GLP-1 remain present during endotoxemia. Administration of the GLP-1 receptor antagonist exendin (9-39) (100 nM/kg) augmented LPS in­ duced IL-1, IL-6 and MCP-1, whereas DPP4 inhibition (sitagliptin 40 mg/kg) lowered IL-6 and increased antiinflam­ matory cytokine IL-10 serum levels (all p<0,05). To assess the functional relevance of GLP-1 for glucose home­ ostasis in response to endotoxemia we administered sitagliptin, which aug­ mented LPS-induced insulin secretion (p<0,05) and blood glucose lowering (p<0,05) in C57Bl6 mice. Conversely, injection of exendin (9-39) markedly blunted LPS-dependent increase of se­ rum insulin and prevented endotoxic hypoglycemia.

Conclusion: GLP-1 provides a new metabolic cross talk between the gut and the immune system with LPS-de­ pendent hypoglycemia being depend­ ent on an inflammatory cascade includ­ ing IL-6, GLP-1 and insulin. Increased

GLP-1 serum levels are associated with unfavorable prognosis in critically ill patients.

The effect of platelet derived DPP4 inhibition on cardiovascular disease W. Theelen1, N. Klein1, M. Nelson1, R. Soltan1, M. Garbe1, J. W. M. Heemskerk2, H. Noels1 1 Institute for Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Germany 2 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Center, The Netherlands For approximately the last decade, in­ hibition of serine protease dipeptidyl peptidase 4 (DPP4) receives a lot of interest as anti-hyperglycemic therapy in type 2 diabetes. Simultaneously, it has been found that this treatment also positively influences cardiovas­ cular outcome. This has mostly been attributed to glucagon-like peptide 1 (GLP1) since this is the most described substrate for DPP4. However, DPP4 is known to have many substrates and several of them (i.e. CXCL12, CCL5) have been described to play a role in cardiovascular disease, indicating that this positive outcome may also be due to one of these other substrates.

Platelets play a crucial role in the onset of atherosclerosis through the recruit­ ment of leukocytes by direct interac­ tions or by the secretion of inflamma­ tory cytokines and chemokines. Also, platelets are essential in thrombus for­ mation following rupture of vulnerable plaques. The effect of DPP4 inhibitors on platelet functions in the context of cardiovascular disease remains unin­ vestigated, with only one recent study (Gupta et al. 2012) demonstrating the DPP4 inhibitor sitagliptin to reduce platelet aggregation and intracellular calcium signaling in vitro.

Immunohistochemical staining showed that DPP4 is present in platelet gran­ ules and that these granules are most likely the lysosomes since there is no colocalization with CXCL12, which is localized in the α-granules, or P© Verlag PERFUSION GmbH

ABSTRACTS

selectin, which is localized in both the α- and δ-granules. Using flow cyto­metry the presence of DPP4 in platelets could not be shown both extracellular and intracellular, whereas both smooth muscle cells and endothelial cells did show positive signals for DPP4. This indicates that either the amount of DPP4 in platelets is extremely low or that the DPP4 present in these granules is only the soluble form of DPP4. Fur­ thermore, DPP4 activity studies show that platelet activation leads to an in­ crease in DPP4 activity.

However, despite this seemingly low thrombocytic DPP4 expression, treat­ ment of platelets with DPP4 inhibitors was previously shown to reduce plate­ let aggregation in vitro, which indicates that DPP4 activity plays an important role in platelet activation. In our future studies, we aim to unravel the impor­ tance of thrombocytic DPP4 activity on platelet function and atherogenesis.

Zonulin, a potential biomarker of metabolic inflammation, in an out-patient cohort at very high cardiovascular risk T. Dschietzig1, 2, R. Klüsener2, F. P. Armbruster1, C. Melzer2 1 Immundiagnostik AG, Bensheim, Germany 2 Charité University Medicine Berlin, Campus Mitte, Department of Cardiology and Angiology Background: In obesity and metabolic syndrome, disturbed intestinal perme­ ability and low-grade chronic systemic inflammation appear to act in a vicious circle called metabolic inflammation.

Zonulin, a tight junctions modulator and key regulator of intestinal permea­ bility, has been shown to be up-regulat­ ed in individuals with type-1 diabetes and to play a role in gut-related dys­ functional auto-immunity. In addition, there are some preliminary reports in­ dicating a possible association between zonulin and metabolic inflammation or type-2 diabetes as well. Moreover, zonulin is implicated in the regula­ tion of general endothelial/epithelial Perfusion 01/2014

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permeability, and its association with increased pulmonary permeability has been demonstrated in animal experi­ ments.

Aim: This study aimed at investigating plasma zonulin and its dependence on various clinical and biochemical fac­ tors in 225 patients carrying automatic implantable cardioverters/defibrillators (AICD), with 75 % of them suffering from systolic heart failure, 69 % from coronary artery disease (CAD), and 27 % from type-2 diabetes (T2D).

Results: Simple correlation analysis showed that zonulin levels were as­ sociated with plasma creatinine, plas­ ma nitrotyrosine, severity of CAD, left ventricular ejection fraction, and NYHA functional class, but not with high-sensitivity C-reactive protein (hsCRP), body mass index, weight, height, sex, or age. After multiple lin­ ear regression analysis, the negative association with creatinine (p=0.006) and the positive one with NYHA class (p=0,013) remained significant. In the subgroup of individuals with T2D, multiple regression revealed a signifi­ cant positive affection of zonulin by hsCRP only (p=0.025).

Conclusion: These findings may sup­ port reports on zonulin’s involvement in the phenomenon of metabolic in­ flammation in T2D patients. The as­ sociation of zonulin with NYHA may reflect its newly established role in altering endothelial/pulmonary perme­ ability in heart failure. The robust nega­ tive correlation with creatinine is unex­ pected and needs further clarification in experimental and clinical studies.

Stable and instable coronary artery disease and clot lysis: are patients with diabetes mellitus different?

M. Berger, K. Lysaja, M. Lehrke, N. Marx, K. Hess Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany Aims: Diabetes mellitus is associated with increased cardiovascular risk and fibrin clot lysis is a critical factor de­

45

termine predisposition to atherothrom­ botic disease. A previous study dem­ onstrated prolonged clot lysis in acute coronary syndrome. However, it is un­ known if this holds true in patients with diabetes and whether patient character­ istics or medication have any impact in patients with and without diabetes.

Therefore, the present study investi­ gates clot lysis in stable and instable coronary artery disease (SCAD and ICAD respectively) in patients with and without diabetes.

Methods: Fibrin clot lysis was de­ termined by validated turbidimetric assay in 123 patients, 57 without dia­ betes (SCAD n=33, ICAD n=24) and 56 with diabetes (SCAD n=33, ICAD n=23). ICAD was defined as unsta­ ble angina, non-ST elevation MI and STEMI. Clot lysis data were corrected by a multivariate analysis for potential confounding by medical therapy and patient characteristics (sex, age, BMI, height, weight). Plasminogen activator inhibitor (PAI)-1 plasma levels were determined by ELISA. Results: Clot lysis was significant­ ly increased in ICAD compared to SCAD in patients without diabetes (1872±419 s and 850±9 s respectively; p<0.01). PAI-1, a classical inhibitor of fibrinolysis, had no impact as plasma levels did not differ between the groups (1688±220 µg/ml and 1536±224 µg/ml respectively; p=0.63). Using a mul­ tivariate analysis including SCAD/ ICAD and the use of anti-platelets or anti-coagulation the effect was only as­ sociated with instable coronary artery disease (p=0.016). Similarly, none of the investigated patient characteristics influenced clot lysis. SCAD patients with diabetes exhibited a significantly longer clot lysis compared to subjects without diabetes (1597±181 s and 850±79 s respectively; p<0.001). In­ terestingly, in patients with DM clot lysis did not significantly differ be­ tween SCAD and ICAD (1597±181 s and 2361±585 s respectively; p=0.51) as did PAI-1 plasma levels (2424±325 and 2408±378 µg/ml respectively; p=0.4). Furthermore, stable patients © Verlag PERFUSION GmbH

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46

with diabetes had a similar clot ly­ sis time compared to patients without diabetes but instable coronary artery disease (1597±181 s and 1872±419 s; p=0.51). Using a multivariate analysis including SCAD/ICAD and the use of anti-platelets, anti-coagulation and an­ ti-diabetic agents, a significant correla­ tion was found for anti-diabetic therapy (p=0.002), with a use of combined oral anti-diabetic therapy being associated with prolongation of clot lysis. Neither ICAD nor anti-platelet or anti-coag­ ulation was associated with clot lysis time. Similarly, patient characteristics had no influence on clot lysis time in patients with diabetes.

Conclusions: In unstable coronary ar­ tery disease clot lysis time is increased in patients without diabetes. In con­ trast, in patients with diabetes clot lysis time is no further elevated in the unsta­ ble situation. However, in patients with diabetes clot lysis is already in the sta­ ble situation as impaired as in non-dia­ betic patients with ICAD. Interestingly, in both groups neither the classical an­ tifibrinolytic protein PAI-1 nor therapy with anti-platelets or anti-coagulation did influence clot lysis time suggesting other mechanism to be involved. Fur­ ther studies are needed to evaluate the underlying mechanism and evaluate potential treatment strategies.

Plant sterol and oxyphytosterol levels in plasma and aortic valves in patients with aortic stenosis H.-F. Schött1, A. Luister2, C. Husche1, H.-J. Schäfers3, M. Böhm3, J. Plat4, U. Laufs2, D. Lütjohann1, O. Weingärtner2 1 Institute for Clinical Chemistry and Clinical Pharmacology, University Clinic Bonn, Germany 2 Clinic for Internal Medicine III (Cardiology, Angiology and Internal Intensive Medicine), Saarland University Medical Center, Homburg/ Saar, Germany 3 Clinic for Thoracis and cardiovascular Surgery, Saarland University Medical Center, Homburg/ Saar, Germany 4 Department of Human Biology, Maastricht University, The Netherlands Perfusion 01/2014

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Objective: We hypothesize that pa­ tients with severe aortic stenosis pre­ sent correlation between serum and aortic valve plant sterols and oxyphy­ tosterols. This should be independent from the use of statins.

Method: We included 104 consecutive patients (65/39; m/f) between 41 and 86 years (mean 68.5 yrs.) of age who were admitted to the University Clin­ ics of the Saarland, (Homburg/Saar, Germany) for elective aortic valve re­ placement owing to severe aortic ste­ nosis. During a structured interview, study participants were assessed for established cardiovascular risk fac­ tors and concomitant statin treatment (no statins: 33, statin user: 71). Ve­ nous blood samples were drawn on the day before the scheduled valve re­ placement. Resected valve cusps were dried in a speed-vac-concentrator.

After drying and mechanical separa­ tion of the calcified part of the resect­ ed valve cusps the non-cholesterols (cholestanol, lathosterol, lanosterol and desmosterol) and oxyphytosterols (7α-, 7β-, 7-ketocampesterol, -sitoster­ ol) and cholesterol within the residual tissue fragments were extracted with chloroform/methanol. The organic ex­ traction was followed by alkaline hy­ drolysis, extraction and separation of free sterols from oxyphytosterols by solid-phase extraction and derivatiza­ tion to the corresponding trimethyl­ silyl-ethers in n-decane. Finally the samples were analyzed by gas chroma­ tography-flame ionization (cholesterol, cholestanol) or mass spectrometry se­ lected ion-monitoring (non-cholesterol sterols and oxyphytosterols).

Results: As expected, our results show significantly lower absolute and cho­ lesterol corrected serum concentrations of the cholesterol precursors (lathoster­ ol, lanosterol and desmosterol) in the statin treated group compared with the non-users. Furthermore the absolute serum level of sitosterol is lower in the statin-treated group. Therefore we decided to split up the ongoing analy­ sis into statin users and non-users.

The concentrations of the two most

common plant sterols (sitosterol and campesterol) strongly correlate to each other in plasma and aortic valve cusps as well as between the two groups. In­ terestingly, only in the valve tissue a highly significant correlation between campesterol and sitosterol and their respective corresponding oxidized forms is observed. This correlation is independent from statin treatment. Ad­ ditionally, our results indicate that 7α-, 7β-, 7-ketocampesterol and -sitosterol highly significantly correlate with each other in the valve tissues of both groups (untreated and treated with statins).

These results suggest that oxidized phytosterols are locally produced and their oxidation is not affected by sta­ tin treatment. Moreover, we observe a highly significant correlation between age and cholesterol, cholestanol, camp­ esterol and sitosterol in the valve tis­ sues from patients treated with statins.

In conclusion, a highly significant correlation between phytosterols and oxyphytosterols and between the oxy­ phytosterols with themselves is clearly shown in the valve tissue. This effect is not apparent in the serum.

Conclusion: All these results com­ bined together lead us to the assump­ tion that serum values of oxyphytoster­ ols do not reflect the condition in the aortic valve.

Genetic variation at chromosome 9p21.3 and atherosclerosis: the Ludwigshafen Risk and Cardiovascular Health Study M. B. Maeß1, M. E. Kleber2, 3, M. M. Hoffmann4, H. Scharnagl5, T. B. Grammer6, W. März3, 6, 7, S. Lorkowski1 1 Department of Nutritional Biochemistry, Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 Department of Internal Medicine II, Cardiology, University of Ulm Medical Center, Germany 3 Mannheim Institute of Public Health, Social and Preventive Medicine, Medical Faculty Mannheim, Ruprechts Karls University, Heidelberg, Germany 4 Clinical Chemistry, University Medical Center, Freiburg, Germany © Verlag PERFUSION GmbH

ABSTRACTS

Department of Internal Medicine, Division of Cardiology, Medical University of Graz, Austria 6 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria 7 Synlab Academy, Mannheim, Germany 5

Background: Large-scale genomewide association studies (GWAS) have consistently associated chromosomal locus 9p21.3 with cardiovascular dis­ ease. However, the underlying mo­ lecular mechanisms have not yet been elucidated.

Aim: The rs1333049 genotype is locat­ ed in a haplotype block at the 9p21.3 locus; therefore its association with the severity of coronary artery obstruction and mortality was investigated.

Methods and results: Participants of the LURIC study were genotyped us­ ing Affymetrix Human SNP Array 6.0.

Measured outcomes were total mortal­ ity and mortality caused by cardiovas­ cular and associated diseases. Study subjects were clinically stable except for acute coronary syndrome and coro­ nary angiograms were available. Ad­ ditionally common clinical parameters were measured (fasting glucose, gly­ cosylated hemoglobin, total, LDL and HDL cholesterol, triglycerides, C-reac­ tive protein, serum amyloid A, fibrino­ gen, IL-6, adhesion molecules, platelet activating factor). Data were analyzed

by ANOVA. No significant correlation was found for rs1333049 genotype and analyzed parameters. However the G/G genotype is characterized by reduced severity and later onset of myocardial infarctions and peripheral vascular dis­ ease in comparison to the C/C and G/C genotypes.

Conclusions: Severity of atherosclero­ sis and onset of its complications are affected by rs1333049 genotype and are independent from established risk factors.

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The impact of uric acid on long-term mortality in patients with asymptomatic carotid atherosclerotic disease F. J. Mayer1, 2, 3, C. Mannhalter1, M. Schillinger2, E. Minar2, T. Chavakis3, 4, R. Koppensteiner2, M. Hoke2 1 Department of Laboratory Medicine, Medical University of Vienna, Austria 2 Department of Internal Medicine II, Division of Angiology, Medical University of Vienna, Austria 3 Department of Clinical Pathobiochemistry, University of Dresden, Germany 4 Institute of Clinical Chemistry and Laboratory Medicine, University of Dresden, Germany Background: Serum uric acid (SUA) levels are thought to be related to car­ diovascular disease and outcome. We investigated whether levels of SUA predicts long-term mortality in neuro­ logically asymptomatic patients with carotid atherosclerotic disease.

Methods: We prospectively studied 959 consecutive patients with carotid atherosclerosis as evaluated by duplex Doppler sonography for all-cause and cardiovascular death, respectively.

Results: During a median follow-up time of 6.3 years (IQR 5.4–7.1 years) 246 deaths (25.7 %), including 160 cardiovascular deaths (16.7 %), were recorded. Median baseline serum uric acid (SUA) levels were 5.9 mg/dl (IQR, 5.0–7.0 mg/dl). SUA was signifi­ cantly associated with all-cause death and cardiovascular death, respectively.

Adjusted hazard ratios (HR) for an in­ crease of 1 mg/dl of SUA levels were 1.12 (95% CI 1.04–1.21; p=0.003) and 1.20 (95% CI 1.11–1.30; p<0.001) for all-cause and CV-death, respectively.

Quartiles of SUA levels showed a sig­ nificant association with cardiovascu­ lar mortality (log rank p=0.002). For cardiovascular death, adjusted HRs for increasing quartiles of SUA levels were 1.45 (95% CI 0.87–2.43), 1.44 (95% CI 0.85–2.46), and 2.26 (95% CI 1.36– 3.76), compared to the lowest quartile, respectively (p=0.011 for trend). Pa­ tients with baseline carotid stenosis of more than 50 % and/or increased levels of SUA (≥ median), had a ~2-fold in­

47

crease in risk of (CV-) death, compared to patients with carotid narrowing of less than 50 % and/or SUA levels less than the median (p<0.001).

Conclusion: Levels of SUA were found to be independent predictors for all-cause and cardiovascular mortality, respectively, among a cohort of pa­ tients with asymptomatic carotid ath­ erosclerosis.

Atherosclerosis-associated mixed testicular atrophy: novel hormonal and microvascular aspects in ApoE–/–/LDL receptor–/– double knockout mouse model K. Steinfeld1, R. Middendorff2, M. Kampschulte3, A. Mietens2, A. C. Langheinrich3, G. Krombach3, T. Linn4, C. Mühlfeld5, S. Wudy6, M. Hartmann6, A. Paradowska-Dogan1, B. Altinkilic1, M. Bergmann7, W. Weidner1 1 Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Germany 2 Department of Anatomy and Cell Biology, Justus Liebig University Giessen, Germany 3 Department of Radiology, Justus Liebig University Giessen, Germany 4 Department of Internal Medicine, Justus Liebig University Giessen, Germany 5 Department of Functional and Applied Anatomy, Hannover Medical School, Germany 6 Department of General Pediatrics and Neonatology, Justus Liebig University Giessen, Germany 7 Department of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Giessen, Germany Aims: Age-related testicular changes are linked with a reduced spermato­ genesis and low testosterone levels.

A relationship to atherosclerosis has never been investigated systemati­ cally. We want to analyze different parameters of arterial supply of the tubules in context with spermatoge­ netic alterations in an animal model providing a remarkable homology to human atherosclerosis to investigate the possible relationship between ath­ erosclerotic lesions and unexplained infertility in men. © Verlag PERFUSION GmbH

ABSTRACTS

48

Methods: The ApoE–/–/LDL recep­ tor–/– double knockout (KO) mouse de­ veloping atherosclerotic plaques is the optimal animal model to investigate atherosclerosis-related spermatogenet­ ic alterations similar to humans. KOand wild-type (WT) mice at the age of 20, 40, 60 and 80 weeks were system­ atically perfused with glutaraldehyde and with contrast agent. The contrast agent perfused testes were scanned by micro-CT to quantify total testis vol­ ume and vascular volume. Semithin sections of the glutaraldehyde perfused testes were analyzed by newCAST to estimate total length, volume and surface area of capillaries. Spermato­ genesis was analyzed by spermatoge­ netic scores in histological sections and sperm counts were quantified in the epididymis. Testosterone levels were determined by gas chromatographymass spectrometry (GC-MS). Testis gene expression levels were measured

by RT-PCR using GAPD as the house­ keeping gene.

Results: KO mice show a decrease of total testis volume and vascular volume compared to controls. There are sig­ nificant declines of length (p<0.0001), volume (p=0.0409) and surface area (p=0.000246) of capillaries in the tes­ tes of KO mice compared to those of controls. The testosterone serum levels are reduced significantly (p=0.0315) in KO mice. The differences between KO- and WT mice reach their peaks in older ages in all of these parameters.

KO mice exhibit mixed atrophy in vari­ ous seminiferous tubules and a reduc­ tion of epididymal sperm counts. Ex­ pression of inflammatory (IL-1, IL-6, VEGF) and ER-stress (ATF-3, ERdj4) genes was elevated in testicular tissue of KO as compared to WT mice.

Conclusions: Mixed testicular atrophy in KO mice is linked to reduced testis volume and vascular volume fraction as well as a decrease of length, vol­ ume and surface area of capillaries in­ cluding low testosterone serum levels, suggesting a direct relation between atherosclerosis and disturbed spermat­ ogenesis. Further evaluation of testicu­ Perfusion 01/2014

27. Jahrgang

lar perfusion in human tissue is neces­ sary to find possible associations with testicular sperm extraction (TESE) out­ comes.

Differentiation of endothelial-like cells from adult murine germlinederived pluripotent stem cells R. Klocke1, J. Kim2, S. Eligehausen1, M. Stehling2, S. Nikol1, K. Ko2, J. Waltenberger1 1 Department of Cardiovascular Medicine, University Hospital of Muenster, Germany 2 Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Muenster, Germany Aims: Functional endothelial cells and their progenitors are required for in vitro basic physiological and pharma­ cological vascular research. Moreover, they are useful for cell-based therapies of ischemic cardiovascular diseases in­ cluding approaches based on tissue en­ gineered vascular grafts or single cell preparations, respectively. Currently, such applications are limited by the low abundance and functionality of subject/ patient-derived endothelial progenitor cells. Furthermore, their purification from peripheral blood or bone marrow is laborious. Accordingly, the evalua­ tion of alternative sources of functional endothelial-like cells (ECs) is required.

We have recently obtained germlinederived pluripotent stem (gPS) cells from adult mouse unipotent germline stem cells. The objective of our study is to reveal whether functional ECs can be derived from pluripotent stem cells.

Methods and results: CD31-positive cells were isolated from single cell suspensions of gPS-derived embryoid bodies (EBs) by fluorescence-activat­ ed cell sorting (FACS) and subculti­ vated on OP9 stromal cells. Mechani­ cally isolated EC-like colonies were cultivated on collagen IV to obtain monolayers displaying a cobblestone EC-like morphology. FACS, immu­ nofluorescence analysis and real-time RT-PCR analyses demonstrated that the cells expressed various endothelial

cell-specific markers and/or their mR­ NAs including CD31, von Willebrand Factor, Tie2, Flk1, and vascular en­ dothelial-cadherin. Furthermore, a sig­ nificantly higher level of expression of the mRNA of ephrin B2 as compared to those of ephrin B4 and VEGFR3 ar­ gues for an arterial rather than a venous or lymphatic character of the cells. The gPS-derived ECs were capable to form capillary-like structures on Matrigel and to take up Dil-conjugated acety­ lated low-density lipoprotein thereby confirming their functionality in vitro.

Conclusions: Functional ECs poten­ tially suitable for basic research and for therapeutic applications can be derived from gPS cells.

Raman microscopic investigation of the lipid metabolism in macrophages C. Stiebing1, 2, C. Matthäus1, 2, C. Krafft1, 2, S. Lorkowski3, J. Popp1, 2 1 Leibniz Institute of Photonic Technology (IPHT), Jena, Germany 2 Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena, Germany 3 Institute of Nutrition and Abbe Center of Photonics, Friedrich Schiller University Jena, Germany Aims: Recently, Raman microscopy has become a popular tool to charac­ terize and image cellular processes.

As a non-invasive technique, it com­ bines optical imaging with vibrational spectroscopy. The distinct spectro­ scopic signals of cellular components allow the investigation of the compo­ sition and structure of a cell, without the introduction of external labels or dyes. The aim of this study is the in­ vestigation of the uptake, metabolism and storage of different types of fatty acids by macrophages using Raman microscopy. Macrophages play an im­ portant role during the development of atherosclerotic plaques. Often located in the subendothelial layer of arterial walls, macrophages take up lipids from the blood stream. In case of high lipid content in the blood, toxic levels can be © Verlag PERFUSION GmbH

ABSTRACTS

reached within the cell, which leads to apoptosis or necrosis. Consequently, a lipid-rich plaque forms within the arte­ rial walls, which can rupture and dis­ turb the blood flow.

Methods: Macrophages were differ­ entiated from THP-1 monocytes and afterwards treated with deuterated fatty acids, which were complexed to bovine serum albumin. Investigated fatty acids were for example the saturated palmit­ ic acid and the unsaturated arachidonic acid. Incubation times between 30 min­ utes and 32 hours were chosen. After incubation, the cells were fixed with formalin. Raman measurements were performed at an excitation wavelength of 785 nm. Raman images were re­ corded at a spatial resolution of 0.5 μm using a 60×/NA=1 water immersion objective.

Results: The labeling with stable iso­ topes such as deuterium allows dis­ tinguishing spectroscopically between the fatty acid of interest and naturally occurring lipids within a cell. The deu­ terium label exhibits a specific Raman signal in a spectroscopic region where cellular components usually do not show any characteristic signals, thus providing a high specificity and sensi­ tivity. The investigated fatty acids were stored in lipid droplets within the cells, but had different effects on the viability of the cells. While d-palmitic acid was stored in high amounts as triglycerides in lipid droplets, which led to foam cell formation, d-arachidonic acid was stored less efficiently in lipid droplets

by THP-1 macrophages. Even after 32 hours there was no foam cell formation detectable.

Conclusions: Raman microscopy is a powerful tool to assess cellular infor­ mation. With this technique it is pos­ sible to selectively detect the uptake of individual fatty acids by macrophages and to localize them within the cell.

Through the analysis of the obtained spectra, it is possible to investigate metabolic changes of the fatty acids.

Acknowledgments: The financial sup­ port by the Carl-Zeiss-Stiftung is high­ ly acknowledged.

Perfusion 01/2014

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Differences in microcirculation of exercising skeletal muscle between middle-aged and young males detected by a new contrastenhanced ultrasound method H. Schwarzbach1, A. Pardun1, L. Hannemann1, B. Bogs1, P. Mayr2, O. Hildebrandt2, U. Koehler2, R. Kinscherf1, W. Hildebrandt1 1 Department of Medical Cell Biology, Institute of Anatomy and Cell Biology, Philipps University Marburg, Germany 2 Department of Sleep Medicine, Division of Pneumology, Internal Medicine, University Clinics of Giessen and Marburg UKGM, Germany Aims: Impaired skeletal muscle micro­ circulation is considered a novel factor of insulin resistance as an important factor in aging and metabolic syn­ drome. Contrast-enhanced ultrasound (CEUS) is a novel in-vivo method for real-time quantification of skeletal muscle microvascular blood volume (MBV indicating capillary recruit­ ment), flow velocity (MFV) and blood flow (MBF) in response to adequate stimuli like exercise or hyperinsulin­ emia. As the present state-of-the art, CEUS with low mechanical index (MI) allows to detect circulating microbub­ bles (e.g. Sonovue™, Bracco, Milan, Italy) at low-background conditions and thus to assess replenishment kinet­ ics of microbubbles immediately after their destruction with a high MI USpulse (flash) within a region of interest.

Replenishment curves can be modelled according to the non-linear regression equation y = A (1 – e –β ( t – B1)) where y = acoustic intensity (AI) at any given time t, A = plateau (saturation) of re­ plenishment representing MBV, β = flow rate constant (1 s –1) and repre­ senting MFV, t = time (s), and B1 = time used for background subtraction.

The product of MBV (A) and MFV (β) represents MBF i.e. the initial slope of replenishment.

Methods: Using CEUS as presently suggested we evaluated possible agerelated changes in the microvascular response of skeletal muscle to light ex­ ercise, which should increase recruit­

49

ment of fiber-adjacent capillaries and therefore MBV and MBV. Using the Logiq E9 system with the linear 9L-D scanner (General Electric, Connecticut, USA) we applied CEUS to the vastus lateralis/ intermedius muscle in a group of 12 middle-aged (MA, age 41.2±1.8 years, BMI 27.1±1.0 kg m-2) as com­ pared to a group of 7 young (YG, age 25.3±0.7 years, BMI 24.0±1.0) fasting healthy male subjects. Knee extension peak torque and maximal oxygen up­ take were not significantly different between MA and YG. Following 190 seconds of Sonovue™ equilibration at an antecubital-venous infusion of 1 ml/ min (viewject, Bracco), CEUS replen­ ishment curves covering 25 seconds were obtained in duplicate at rest and after 70 seconds of 2 minutes of iso­ metric knee extension (at 15 % of peak torque). Femoral artery Doppler-flow was monitored in parallel.

Results: Upon exercise, femoral ar­ terial Doppler-flow was lower in MA as compared to YG (779±86 vs.

1072±84 ml/min, p<0.05). CEUS anal­ ysis during exercise revealed lower lev­ els (relative to baseline rest) of MBV 1.12±0.10 vs. 1.63±0.21, p<0.02) and MFV (1.60±0.27 vs. 4.13±1.03, p<0.01) in MA compared to YG, re­ sulting in lower MBF (0.01±0.30 vs.1.83±1.11, p<0.06). These sig­ nificant differences were undetectable when applying replenishment curve analysis, as recently reported by others.

Conclusion: Improved CEUS-anal­ ysis reveals an important age-related impairment of capillary recruitment which might limit ‘local’ muscle en­ durance as well as insulin and nutrition availability. Analyses of capillarization in biopsies will provide further infor­ mation.

© Verlag PERFUSION GmbH

ABSTRACTS

50

Improving contrast-enhanced ultrasound (CEUS) of muscular microcirculation in humans H. Schwarzbach1, A. Pardun1, L. Hannemann1, B. Bogs1, O. Hildebrandt2, U. Koehler2, R. Kinscherf1, W. Hildebrandt1 1 Department of Medical Cell Biology, Institute of Anatomy and Cell Biology, Philipps University Marburg, Germany 2 Department of Sleep Medicine, Division of Pneumology, Internal Medicine, University Clinics of Giessen and Marburg UKGM, Marburg, Germany Aims: Compromised skeletal muscle microcirculation is considered to con­ tribute to peripheral insulin resistance as implicated in aging or metabolic syndrome. In vivo, contrast-enhanced ultrasound (CEUS) has been proposed for real-time quantification of skeletal muscle microvascular blood volume (MBV indicating capillary recruit­ ment), flow velocity (MFV) and blood flow (MBF) at rest and upon challeng­ es like hyperinsulinemia or exercise.

CEUS allows to detect circulating inert gas microbubble contrast agents (e.g.

Sonovue™, Bracco, Milan, Italy) at a low mechanical index (MI) i.e. lowbackground conditions, and to assess replenishment kinetics of microbub­ bles immediately after their destruc­

MITTEILUNGEN Cholesterinsenker sind nicht schädlich für das Gedächtnis Die Sorge, dass die häufig verschrie­ bene Medikamentenklasse der Cho­ lesterinsenker (Statine) das Gedächtnis schädigen könnte, scheint unbegrün­ det. In einer Übersichtsarbeit, bei der Studien mit mehr als 23.000 Männern und Frauen ausgewertet worden wa­ ren, fanden US-Forscher bei kurzfris­ tiger Einnahme kein erhöhtes Risiko und bei längerer sogar einen Schutz­ Perfusion 01/2014

27. Jahrgang

tion with a high MI US-pulse (flash) within a region of interest. Replenish­ ment curves are modelled according to the non-linear regression equation y = A (1 – e –β ( t – B1)) where y = acous­ tic intensity (AI) at any given time t, A = plateau (saturation) of replenish­ ment (MBV), β = flow rate constant 1 s –1 (MFV), t = time (s), and B1 = time used for background subtraction and origin correction. The product of MBV and MFV yields MBF (A × β), i.e. the initial slope of replenishment.

As a critical point these calculation may vary massively with the definition of background-subtracted AI at origin, e.g. the usual averaging of post-flash losses in microbubble signal (cover­ ing 0.5 seconds), which range between 74 % and 112 %.

Methods and results: Such AI-bias may massively impact A (MBV) as well as β (MBV) and their product (MBF), e.g. a seemingly negligible AIbias of 10 % or even of 20 % results in a ~20 % or even ~40 % over-/underesti­ mation of MBF, respectively. We there­ fore evaluated a new approach, which uses a least-squares minimization to the parameterized model to fit CEUS replenishment data, i.e. the model: A × (1 – e (–β × (t ± 0.25 s bias))) ± AI bias) instead of the above-mentioned fixed pre­ sumptions. This parameterized model

improves the median of variances by nearly 50 % (4.33e-6 vs. 2.22e-6, n=333 flashes) and furthermore the fraction of post-flash losses of the preflash microbubble signal from median 74 % to 97 % (p<0.0001) at rest and from 69 % to 84 % (p=0.0012) during exercise. The data base for these cal­ culations were representative replen­ ishment curves of Sonovue™ (antecu­ bital-venous infusion of 1 ml/min by viewject, Bracco, with 190 seconds for equilibration) obtained by the Logiq E9 system with the linear 9L-D scanner (General Electric, Connecticut, USA) from vastus lateralis/intermedius mus­ cle of a group of 12 middle-aged (MA, age 41.2±1.8 years) and a group of 7 young (YG, age 25.3±0.7 years) fasted healthy male subjects. In fact, we show that there is a significance difference in MBV and MBV between MA and YG during exercise, which is only detect­ able by the present new model.

Conclusions: The present parameter­ ized model works without fixed pre­ sumptions about AI averaging and fill­ ing time intervals, improves curve fit as well as the bias of origin, and therefore the determination of the initial slope (MBF). Biopsies will allow to further validate this CEUS method via capil­ lary histomorphometry.

effekt. „Unterm Strich könnten laut dieser Studie Statine das Risiko einer Demenz um 29 % senken, wenn sie ein Jahr oder länger eingenommen wer­ den“, fasst Professor Matthias Endres, zweiter Vorsitzender der Deutschen Schlaganfall-Gesellschaft (DSG) und Direktor der Klinik für Neurologie an der Berliner Charité, das Ergebnis der Meta-Analyse zusammen. Im Um­ kehrschluss sollten Statine aber nicht entgegen ihrer Zulassung als Anti-De­ menzmittel eingesetzt werden, betont Professor Hans-Christoph Diener von der Deutschen Gesellschaft für Neuro­ logie (DGN). Regelmäßiger moderater Sport zeige sich als Vorsorgemaßnah­ me gegen Demenz ähnlich wirksam.

Verunsicherung durch wider­ sprüchliche Studienergebnisse Mit einem jährlichen Gesamtumsatz von etwa 20 Milliarden Euro bis zum Ablauf ihres Patentschutzes waren die Statine zumindest aus wirtschaftlicher Sicht die erfolgreichste Arzneimittel­ klasse der vergangenen 30 Jahre. Etwa 220 Millionen Menschen weltweit neh­ men diese Medikamente ein, um sich vor Herzinfarkten und Schlaganfällen zu schützen.

Mehrere Studien berichteten in der Vergangenheit, dass Menschen, die Statine einnehmen, seltener von einer Demenz wie zum Beispiel die Alzhei­ mer-Krankheit betroffen sind. Andere © Verlag PERFUSION GmbH

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Untersuchungen fanden aber keinen Effekt. Im Gegenteil wurden nach der Einnahme vereinzelt sogar Gedächt­ nisstörungen und Vergesslichkeit be­ obachtet, bis hin zum vorübergehenden Gedächtnisverlust, was heute in den Beipackzetteln dieser Präparate auch nachzulesen ist. „Zusammen mit ent­ sprechenden Presseberichten hat dies auch zur Verunsicherung der Patienten beigetragen“, so Endres. „Deshalb war es enorm wichtig, die Studien dazu ge­ nau unter die Lupe zu nehmen.“ Statine über kurze Zeit – kein Unterschied erkennbar Forscher um Kristopher J. Swiger von der Johns Hopkins University in Bal­ timore hatten diese Lupe herausgeholt, die Publikationen systematisch ana­ lysiert und unter den 41 Studien zum Thema die 16 methodisch besten aus­ gewählt. Für den ersten Teil der Unter­ suchung wurden 8 Studien erfasst, die einen kürzeren Gebrauch von Statinen untersucht hatten. Drei dieser Studien ermöglichten eine quantitative Aus­ wertung, weil hier die Teilnehmer bei einem Denktest unter Zeitdruck Zah­ len durch Symbole ersetzen mussten.

Dabei fanden sich keine eindeutigen Unterschiede zwischen denjenigen, die Statine bekommen hatten, und je­ nen Teilnehmern, die stattdessen ein Scheinmedikament einnahmen. Swiger fand „keinen Zusammenhang zwischen dem kurzfristigen Gebrauch von Stati­ nen und Gedächtnisverlust oder ande­ ren kognitiven Beeinträchtigungen“.

Statine über längere Zeit – Demenzrisiko scheint sogar zu sinken Teil 2 der Analyse sollte die langfris­ tigen Folgen der Statin-Einnahme klären. Hier standen die Daten aus 8 Studien mit mehr als 23.000 Menschen zur Verfügung, bei denen es anfänglich keine Hinweise auf Gedächtnisstörun­ gen gab. Im Durchschnitt waren diese Patienten zwischen 3 bis maximal 25 Jahre lang untersucht worden. In 3 der 8 Studien fand sich kein Zusam­ Perfusion 01/2014

27. Jahrgang

menhang zwischen der Einnahme von Statinen und dem Demenzrisiko. In 5 Studien aber fanden die Wissenschaft­ ler einen positiven Einfluss der Cho­ lesterinsenker. Zusammengenommen errechneten sie für alle 8 Langzeitstu­ dien ein um 29 % geringeres Risiko, an einer Demenz zu erkranken, gegenüber Patienten, die lediglich ein Scheinme­ dikament erhalten hatten.

Endres kommentiert dies kritisch: „Einige große Statin-Studien, wie die PROSPER-Studie (Prospective Study of Pravastatin in the Elderly at Risk) oder die Heart Protection Stu­ dy, ergaben keinen Hinweis auf einen schützenden Effekt der Statine. Diese wurden aber in der aktuellen Über­ sichtsstudie nicht mit eingerechnet.“ Vorsichtiger Optimismus „Auch aufgrund früherer Berichte zu möglichen Nebenwirkungen der Sta­ tine auf das Gedächtnis ist auch diese neue Studie mit Vorsicht zu betrach­ ten“, kommentiert Professor HansChristoph Diener, Direktor der Klinik für Neurologie am Universitätsklini­ kum Essen. „Es stimmt mich aber opti­ mistisch, zu sehen, dass die Einnahme von Cholesterinsenkern über einen län­ geren Zeitraum womöglich das Risiko verringert, an einer Demenz zu erkran­ ken“. Dass Statine einen solchen Ef­ fekt haben, scheint plausibel. Schließ­ lich verringern oder stabilisieren die Cholesterinsenker jene Ablagerungen (Plaques) in den Gefäßen, die Veren­ gungen verursachen, den Blutfluss ins Gehirn verringern und durch Verstop­ fungen Schlaganfälle auslösen können. „Was die neurokognitiven Effekte der Statin-Therapie angeht, so könnten Ärzte und Patienten nun beruhigt sein“, folgert Diener aus der neuen Studie.

Nutzen und Risiken mit dem Arzt besprechen Millionen von Menschen nehmen Sta­ tine ein, erinnert Endres. „Der mögli­ che Nutzen muss gegen die Gefahr von unerwünschten Nebenwirkungen wie Schädigungen der Muskulatur abge­

wogen werden. Eine Beratung mit dem Arzt ist deshalb vor der Einnahme un­ bedingt erforderlich.“ DGN und DSG Quelle: Swiger KJ et al. Statins and cognition: a systematic review and meta-analysis of short- and long-term cognitive effects. Mayo Clin Proc 2013;88:1213-1221

Zulassung für Levosimendan erteilt Seit Januar 2014 ist Levosimendan (Simdax®) auch in Deutschland zur Behandlung der akut dekompensier­ ten Herzinsuffizienz verfügbar. Der Wirkstoff Levosimendan zeichnet sich durch einen dreifachen Wirkmechanis­ mus aus: Neben der positiven Inotropie sind die Vasodilatation und die Kardio­ protektion wesentliche pharmakologi­ sche Eigenschaften, die das Wirkprofil der Substanz einzigartig machen.

Levosimendan erhöht als Kalziumsen­ sitizer die Empfindlichkeit der kontrak­ tilen Elemente für Kalzium und steigert auf diesem Weg die kardiale Kontrakti­ onskraft. Im Gegensatz zu anderen in­ otropen Substanzen erhöht Levosimen­ dan die Kontraktilität des Myokards ohne einen Anstieg der intrazellulären Kalziumkonzentration, was sich in ei­ ner ausgewogenen Sauerstoffbilanz der myokardialen Filamente widerspiegelt.

Als Vasodilatator aktiviert Levosimen­ dan ATP-sensitive Kaliumkanäle in der glatten Gefäßmuskulatur. Die dadurch induzierte Gefäßerweiterung führt zu einer Senkung der Vor- und Nachlast sowie zu einer verbesserten Organ­ durchblutung.

Der molekulare Mechanismus, der den kardioprotektiven Eigenschaften von Levosimendan zugrunde liegt, findet sich in einer Aktivierung der ATP-ab­ hängigen Kaliumkanäle der myokardi­ alen Mitochondrien. Über die Öffnung mitochondrialer KATP-Kanäle werden bei kritischen Zuständen wie Ischä­ mie, oxidativem Stress oder Reperfu­ sion zellprotektive Signalwege ange­ schaltet, die das Ausmaß potenzieller Zellschädigungen eindämmen und die Myokardzellen schützen.

E. W. © Verlag PERFUSION GmbH

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Nachruf auf den Kardiologen Professor Rolf Schröder Der Berliner Kardiologe Herr Prof. Dr. Rolf Schröder verstarb am 25.12.2013 im Alter von 85 Jahren.

Geboren am 16. Februar 1928 in Osterode am Harz, erhielt er seine klinische und wissenschaftliche Ausbildung bei dem großen Internisten Prof. Dr. Rudolf Schoen in Göttingen, wo er 1962 mit der Arbeit „Untersuchungen über das Verhalten der Nebennierenrindenhormone bei hydropischer Herzinsuffizienz“ habilitiert wurde.

Nach seinem Wechsel in das damalige Westberlin arbeitete der junge Privatdozent bei Prof. Dr. M. Schwab in der 1. Medizinischen Universitätsklinik der Freien Universität Berlin im Klinikum Westend. Hier wurde eine der ersten kardiologischen Intensivstationen aufgebaut, parallel dazu gedieh das differenzialdiagnostische EKGWerk von R. Schröder bis zur 4. Auflage.

Am 3. März 1969 erfolgte der Umzug in das neu errichtete Klinikum Steglitz der Freien Universität Berlin, wo Schröder zum Professor und zum Abteilungsleiter für Innere Medizin mit Schwerpunkt Kardiologie und Pulmologie ernannt wurde. Dort wurden vielfältige Forschungsaktivitäten entwickelt, von der Digitalisforschung über den Dopamineinsatz bis hin zum Cor pulmonale. Dies waren zum Teil auch wesentliche Themen der unter dem gemeinsamen Tagungsvorsitz von R. Schröder und H. Schmutzler abgehaltenen Herbsttagung der DGK 1978 in Berlin. Die Ende der siebziger Jahre aufgekommene Rekanalisationstherapie des akuten Myokardinfarktes veranlasste Schröder zur Entwicklung der systemischen Lyse mit Streptokinase und zur Einführung der Prähospitallyse. Hierzu erfolgte in den achtziger Jahren von ihm die Konzeption und Durchführung großer multizentrischer Studien.

Kombination aus Dapagliflozin und Metformin zur Behandlung des Typ-2Diabetes zugelassen Am 24. Januar hat 2014 hat die Euro­ päische Kommission Xigduo®, die ers­ te Fixkombination aus einem Inhibitor des Natrium-Glukose-Cotransporters-2 (SGLT-2) und Metforminhydrochlorid in der EU für die Behandlung des Typ2-Diabetes zugelassen. Xigduo® kom­ biniert die beiden Wirkstoffe Dapagli­ flozin und Metformin in einer Tablette (5 mg/850 mg und 5 mg/1000 mg) zur Perfusion 01/2014

27. Jahrgang

Unmittelbar nach der Grenzöffnung gehörte R. Schröder 1990 zum Gründungsvorstand für eine gemeinsame Berlin-Brandenburgische Gesellschaft für Kardiologie (BBGK) und wurde im November 1990 zu deren erstem Vorsitzenden gewählt.

Mit großer Initiative begleitete er die ersten zwölf Tagungen der Gesellschaft. Am 30.9.1993 wurde er emeritiert und trat auch nicht mehr zum Gesellschaftsvorsitz an. Professor Schröder war noch für weitere zwanzig Jahre zusammen mit einigen seiner Schüler in einer kardiologischen Gemeinschaftspraxis tätig.

1997 wurde er zum Ehrenmitglied der Berlin-Brandenburgischen Kardiologengesellschaft ernannt und 2003 erhielt er die Carl-Ludwig-Ehrenmedaille der Deutschen Gesellschaft für Kardiologie. 2010 war er Ehrenpräsident zum 20-jährigen Jubiläum der Berliner Kardiologen-Gesellschaft.

Unter seiner klinischen und wissenschaftlichen Leitung wurde eine große Zahl von Schülern und Habilitierten herangebildet, die heute die Mehrzahl der kardiologischen Kliniken Berlins und viele auswärtige Kliniken leiten.

Prof. Dr. Rolf Schröder hat über fünf Jahrzehnte die Berliner Kardiologie maßgeblich mitbestimmt, wir werden ihm ein ehrendes Andenken bewahren.

Prof. Dr. H. Eichstädt, Geschäftsführer der BBGK

zweimal täglichen Einnahme. Die beiden Wirkstoffe bieten ergänzende Wirkeigenschaften, um die glykämi­ sche Kontrolle zu verbessern.

Indikationen Xigduo® ist zugelassen zur Behand­ lung von erwachsenen Patienten mit Typ-2-Diabetes im Alter ab 18 Jahren, ergänzend zu Diät und Bewegung zur Verbesserung der Blutzuckerkontrol­ le. Das Kombinationspräparat ist in­ diziert, wenn bei Patienten der Blut­ zucker mit der maximal verträglichen Dosis von Metformin allein nicht aus­

reichend kontrolliert wird – auch in Kombination mit anderen blutzucker­ senkenden Arzneimitteln einschließ­ lich Insulin – oder wenn Patienten bereits die Kombination von Dapa­ gliflozin und Metformin in separaten Tabletten erhalten.

Forxiga® war der erste Vertreter der SGLT-2-Substanzklasse. Derzeit ist das orale Antidiabetikum in 40 Län­ dern weltweit zugelassen. In Deutsch­ land wurde Forxiga® am 15. Dezember 2013 aus wirtschaftlichen Gründen au­ ßer Vertrieb genommen. Mit der Zulas­ sung von Xigduo können die Patienten nun weiter von Dapagliflozin profitie­ ren. © Verlag PERFUSION GmbH

53

Mitteilungen

Wirkprinzip der SGLT-2-Inhibition An der Regulation des Glukosestoff­ wechsels sind maßgeblich auch die Nieren beteiligt. Diese filtern üblicher­ weise 180 Gramm Glukose täglich und resorbieren diese aus dem Primärharn zurück in den Blutkreislauf. SGLT-2 ist ein wichtiger Natrium-GlukoseCotransporter in der Niere und ein in­ sulinunabhängiger Weg für die Rück­ resorption der Glukose ins Blut. Durch eine selektive Inhibition von SGLT-2 lässt sich die Rückresorption reduzie­ ren und somit die Ausscheidung über­ schüssiger Glukose mit dem Harn for­ cieren. Dadurch wird der Blutzucker gesenkt.

F. S.

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E. W. Perfusion 01/2014

27. Jahrgang

PERFUSION

IMPRESSUM

OFFIZIELLES ORGAN DER DEUTSCHEN GESELLSCHAFT FÜR ARTERIOSKLEROSEFORSCHUNG

Herausgeber: Univ.-Prof. Dr. Dr. Edzard Ernst, Emeritus Professor of Complementary Medicine, University of Exeter, Peninsula Medical School,Salmon Pool Lane, Exeter EX2 4SG, UK Prof. Dr. med. W. Koenig, Abt. Innere Medizin II, Med. Univ.-Klinik, Robert-Koch-Str. 8, 89070 Ulm Wissenschaftlicher Beirat: Prof. Dr. med. T. von Arnim (Kardiologie), München Prof. Dr. med. G. V. R. Born (Arterioskleroseforschung), London Prof. Dr. med. C. Diehm (Angiologie), Karlsbad Priv.-Doz. Dr. med. Dr. phil. C. Drosde (Kardiologie), Freiburg Dr. med. J. Dyerberg MD, Ph. D. (Klin. Chemie), Aalborg Sygehus, Dänemark Univ.-Prof. Dr. med. H. W. Eichstädt, (Kardiologie), Berlin Doz. Dr. rer. nat. F.-D. Ernst (Hämorheologie), Dresden Dr. med. J. Gehring (Kardiologie, Rehabilitation), München Prof. Dr. med. J. D. Gruß (Gefäßchirurgie), Kassel Prof. Dr. J. Harenberg (Hämostaseologie), Mannheim Prof. Dr. med. L. Heilmann (Gynäkologie), Rüsselsheim Prof. Dr. med. H. M. Hoffmeister (Kardiologie), Solingen Prof. Dr. med. H. U. Janka (Diabetologie), München Dr. med. J. Janzen MPhil (Pathologie), Bern, Schweiz Prof. Dr. med. L. Kollár M. D., PhD (Gefäßchirurgie), Universität Pécs, Ungarn Prof. Dr. med. M. Marshall (Phlebologie), Rottach Egern Prof Dr. med. J. Matsubara (Chirurgie), Ishikawa, Japan Prof. Dr. med. G. Mchedlishvilli (Mikrozirculation), Tbilisi, Georgien Prof. Dr. med. V. Mitrovic (Kardiologie, Klinische Pharmakologie), Bad Nauheim Prof. Dr. med. H. Mörl (Angiologie), Mannheim Prof. Dr. med. F. J. Neumann (Kardiologie), Bad Krozingen Prof. Dr. med. K. L. Resch (Medizin-Statistik), Bad Elster Prof. Dr. med. G. Rettig (Kardiologie), Homburg Prof. Dr. med. G. Schmid-Schönbein (Biomechanik), La Jolla, USA Prof. Dr. med. H. Schmid-Schönbein (Physiologie), Aachen Prof. Dr. med. A. Schrey (Pharmakologie), Düsseldorf Prof. Dr. med. H. Sinzinger (Nuklearmedizin), Wien, Österreich Prof. Dr. med. T. Störk (Kardiologie, Angiologie), Göppingen Prof. Dr. med. I. Szirmai M. D. (Neurologie), Universität Budapest, Ungarn Prof. Dr. med. G. Trübestein (Angiologie), Bonn Prof. Dr. med. B. Tsinamdzvrishvili (Kardiologie, Hypertonie), Tbilisi, Georgien Prof. Dr. med. W. Vanscheidt (Dermatologie), Freiburg Prof. Dr. med. H. Weidemann (Kardiologie, Sozialmedizin), Bad Krozingen

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