Perfusion 1 2015 by Sibylle Michna

1 2015

Kreislauf- und Stoffwechselerkrankungen in Klinik und Praxis Jahrgang 28, Heft 1 März 2015

VERLAG

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

ORIGINAL PAPER

Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system FOREN

Forum antithromboticum: • E ffektive Schlaganfallprophylaxe bei nicht valvulärem Vorhofflimmern: Überzeugendes Nutzen-Risiko-Profil von Apixaban • C ATCH-Studie bestätigt Wirksamkeit von Tinzaparin zur VTE-Rezidivprophylaxe bei Tumorpatienten Forum cardiologicum: Evolut™ R – eine innovative Aortenklappe für inoperable Patienten ABSTRACTS Abstracts der wissenschaftlichen Beiträge zur 29. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung e. V. vom 26. bis 28. März 2015, Schloss Rauischholzhausen, Tagungsstätte der Universität Gießen REDAKTIONELLER TEIL

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EDITORIAL

01/15

A Scientist in Wonderland Prof. Dr. med. E. Ernst, Exeter, U.K.

Earlier this year, my memoir was published [1]; the original version is in English and has the title ‘A Scientist in Wonderland’, while the German edition to be released shortly will be entitled ‘Nazis, Nadeln und Intrigen’ [2]. Even though it was published by a small publisher, it has already received much attention and numerous amazing reviews; perhaps most significantly, the journal ‘Nature’ described my book as ‘a ferociously frank autobiography’ and a ‘clarion call for medical ethics’ [3]. It is true that, looking back on to several decades of my professional life, most of the things that really mattered can, in the final analysis, be boiled down to ethical issues. Fairly early on in my career, I developed an interest in the significant role played by the German medical profession in the atrocities committed during the Third Reich. Few would doubt that this is a subject which is very much related to medical ethics. Later, when it became my job to conduct research into alternative medicine, the links to ethics were perhaps a little less clear. But that does not mean that they were absent. The longer I researched alternative medicine, the stronger I felt that these two subjects were inextricably and intimately connected. By far the best way to prove my point might be to provide a short, abbreviated excerpt from the book:

just be switched off at will. No branch of health care, including alternative medicine, can be considered exempt from it …

It should be axiomatic that ethics is indispensable to the practice of medicine, and is not something that can

Some might criticize me here for claiming the moral high ground. But if I do so, it is for a good reason. Medical

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Medical ethics are violated, for example: when homeopaths prescribe or recommend homeopathic vaccinations for which there is not a shred of evidence; when chiropractors or other alternative practitioners happily promote bogus treatments for children with asthma or other serious conditions; when practitioners fail to obtain informed consent before commencing their treatments; when Prince Charles sells his “detox tincture” which is unable to eliminate poisons from your body, merely cash from your purse; when quacks inveigle desperate cancer patients by pretending they have found a cure; when pharmacists sell Bach Flower Remedies or other gloriﬁed placebos; when applied kinesiologists, iridologists, etc. claim that their baseless diagnostic tests are able to identify serious diseases; when pseudoscientists claim that certain alternative therapies are evidence-based because they managed to generate a false positive result purely by cherrypicking or massaging their data; when politicians who lack even the most basic understanding of science publicly support quackery, proclaiming that it is evidence-based. And so on, and so on.

consultations are intrinsically unequal, with the clinician occupying a position of considerable power over often highly vulnerable patients. This places an important ethical onus on the caregiver to assist patients in making informed choices – an imperative and a trust that is breached each and every time that unproven nostrums born of ideology and wishful thinking are offered to people with assertions that they are an effective, valid approach to the treatment of disease. When science is abused, hijacked or distorted in order to serve political or ideological belief systems, ethical standards will inevitably slip. The resulting pseudoscience is a deceit perpetrated on the weak and the vulnerable. We owe it to our-selves, and to those who come after us, to stand up for the truth, no matter how much trouble this might bring. I sincerely hope that this short excerpt will stimulate a wider reﬂection on medical ethics – perhaps it might even motivate some readers to study my memoir in more detail. Edzard Ernst, Exeter References 1 Ernst E: A Scientist in Wonderland. A memoir of searching for truth and ﬁnding trouble. Exeter: Imprint Academic; 2015 2 Ernst E: Nazis, Nadeln und Intrigen. Eine Autobiographie. Hannover: JMB Verlag; 2015 3 Books in brief: A Scientist in Wonderland. Nature 2015;518:33

Heft 1 März 2015

14, 16 Forum antithromboticum 18 Forum cardiologicum 19 Kongressberichte 22 Mitteilungen

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

INHALT EDITORIAL 1 Ein Wissenschaftler im Wunderland E. Ernst ORIGINALARBEIT 4 Evaluation des FreeStyle Precision Neo Messsystems für Blutzucker und Blutketone C. Brannan ABSTRACTS 24 Abstracts der wissenschaftlichen Beiträge zur 29. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung e.V. vom 26. bis 28. März 2015, Schloss Rauischholzhausen, Tagungsstätte der Universität Gießen

14, 16 Forum antithrom boticum 18 Forum cardiologicum 19 Congress reports 22 Informations

CONTENTS EDITORIAL 1 A scientist in wonderland E. Ernst ORIGINAL PAPER 4 Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system C. Brannan ABSTRACTS 24 Abstracts of the 29th Annual Meeting of the German Atherosclerosis Society, March 26–28 2015, Conference Venue of the University Giessen, Rauischholzhausen

C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

ORIGINAL PAPER

Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system PERFUSION 2015; 28: 4–13

A new version of the international standard governing blood glucose monitoring systems for self-testing (ISO 15197) was published in 2013 [1]. The updated standard includes more stringent accuracy criteria and additional requirements around the limit to which systems may be influenced by haematocrit and interferences from substances potentially in a patient’s blood. The FreeStyle Precision Neo blood glucose and ketone monitoring system (herein referred to as the FreeStyle Precision Neo system) has been designed to meet the performance requirements introduced in the ISO 15197:2013 standard [1] and to simplify patient testing. It also provides features that can enhance diabetes management, including blood glucose trend indicators and insulin dosing guide. These additional features will not be discussed herein since the focus of this paper is the performance of the FreeStyle Precision Neo system in relation to ISO 15175:2013. This paper details a comprehensive evaluation of the FreeStyle Precision Neo system against the requirements for glucose monitoring in ISO 15175:2013. A multicentre clinical study was conducted to evaluate performance with fresh capillary whole blood. Additional laboratory studies were performed at Abbott Diabetes Care to verify performance claims under various testing conditions.

Claire Brannan Abbott Diabetes Care, Witney, UK

Summary Objectives: To evaluate performance of the Abbott Diabetes Care FreeStyle Precision Neo blood glucose and ketone monitoring system against the performance requirements for blood glucose monitoring in ISO 15197:2013 [1]. Methods: Accuracy and user performance in testing fresh capillary whole blood samples were assessed at two diabetes clinics. Results obtained with 3 lots of test strips were compared to plasma equivalent glucose values from the YSI analyser. Lay users were also asked to rate ease of use of the system via a questionnaire. Laboratory studies were performed at Abbott Diabetes Care to verify system performance under varied test conditions. Results: Clinical accuracy of the FreeStyle Precision Neo system was demonstrated by comparing results from 186 blood samples (from 165 subjects), tested across 3 test strip lots by trained operators, to results obtained with the YSI analyser. Results met the system accuracy requirements of ISO 15197:2013: • For the 3 test strip lots, 100 %, 98.5 % and 98.8 % (99.1 % overall) of results agreed within ±15 mg/dL of the reference values at glucose concentrations <100 mg/dL and within at ±15% of the reference values at glucose concentrations ≥100 mg/dL. • 100 % of results were in zones A and B of the consensus error grid. Similarly, accuracy requirements of the standard were met in the lay user testing, with 97.9 % of results within the accuracy criteria. The overall mean rating for the 174 subjects completing the ease of use questionnaire was 5.5 (out of 6), demonstrating that these users found the FreeStyle Precision Neo system easy to use. Repeatability evaluation yielded standard deviations (SD) ≤2.7 mg/dL at glucose concentrations <100 mg/dL and coefficients of variation (CV) ≤3.5 % at glucose concentrations ≥100 mg/dL. Intermediate precision assessment demonstrated SDs ≤3.3 mg/dL at glucose concentrations <100 mg/dL and CV of 3.1 % at glucose concentrations ≥100 mg/dL. Results were not affected by high altitude (3,048 m). The FreeStyle Precision Neo system provided accurate results across the claimed haematocrit range (30 to 60 %) and with 30 potentially interfering substances at high concentrations – biases observed were less than the criteria which trigger inclusion of the results in the product labelling per ISO 15197:2013. Conclusions: The clinical studies verify accuracy of the FreeStyle Precision Neo system for fingerstick capillary testing when compared to laboratory method results – accuracy in lay user and trained operator testing met the requirements in ISO 15197:2013. The FreeStyle Precision Neo system had a high ease of use rating by first time users. Laboratory studies demonstrated that the system maintained accuracy in various challenging conditions that may be encountered in everyday home testing. These also demonstrated that influence conditions identified in ISO 15197:2013 (haematocrit & interfering substances) did not influence the FreeStyle Precision Neo system to such an extent that would require description of their effects in the product labelling.

Keywords: FreeStyle Precision Neo, ISO 15197:2013, accuracy, haematocrit, blood glucose monitoring system Perfusion 01/2015

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C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

Zusammenfassung Ziele: Evaluation der Systemgenauigkeit des FreeStyle Precision Neo Blutzucker-Messsystems von Abbott Diabetes Care hinsichtlich der Genauigkeitsanforderungen für Blutzucker-Messsysteme nach DIN ISO-Norm 15197:2013 [1]. Methoden: Die Systemgenauigkeit durch den Anwender bei der Messung von frischen Kapillarvollblutproben wurde in 2 Diabeteskliniken untersucht. Die mittels 3 Teststreifenchargen erzielten Ergebnisse wurden mit plasmaäquivalenten Glukosewerten aus dem YSI-Analysegerät verglichen. Die Gruppe der Laien-Anwender wurde außerdem gebeten, die Benutzerfreundlichkeit anhand eines Fragebogens zu bewerten. Die Laboruntersuchungen zur Verifizierung der Systemgenauigkeit unter verschiedenen Testbedingungen wurden bei Abbott Diabetes Care durchgeführt. Ergebnisse: Die klinische Genauigkeit des FreeStyle Precision Neo Blutzucker-Messsystems wurde nachgewiesen, indem die Ergebnisse aus 186 Blutproben (von 165 Probanden), die von geschultem Personal anhand von 3 Teststreifenchargen untersucht wurden, mit den Ergebnissen aus dem YSI-Analysegerät verglichen wurden. Die Ergebnisse entsprachen den Genauigkeitsanforderungen der DIN ISO-Norm 15197:2013: • Für die 3 Teststreifenchargen lagen 100 %, 98,5 % und 98,8 % (übergreifend 99,1 %) der Ergebnisse innerhalb von ±15 mg/dL der Referenzwerte bei Glukosekonzentrationen <100 mg/dL sowie innerhalb von ±15 % der Referenzwerte bei Glukosekonzentrationen ≥100 mg/dL. • 100 % der Ergebnisse lagen in den Zonen A und B des Consensus Error Grid. Auch bezüglich der Systemgenauigkeit bei Anwendung durch Laien wurden die Genauigkeitsanforderungen der Norm erfüllt, wobei 97,9 % der Ergebnisse innerhalb der Genauigkeitskriterien lagen. Die durchschnittliche Gesamtbeurteilung der 174 Probanden, die den Fragebogen zur Benutzerfreundlichkeit ausfüllten, betrug 5,5 (von 6). Dies zeigt, dass diese Anwendergruppe das System als benutzerfreundlich einstufte. Die Untersuchung der Wiederholbarkeit ergab eine Standardabweichung (SD) von ≤2,7 mg/dL bei Glukosekonzentrationen <100 mg/dL und einen Variationskoeffizienten (CV) von ≤3,5 % bei Glukosekonzentrationen ≥100 mg/dL. Die Zwischenbeurteilung der Präzision ergab Standardabweichungen von ≤3,3 mg/dL bei Glukosekonzentrationen <100 mg/dL und einen Variationskoeffizienten von 3,1 % bei Glukosekonzentrationen ≥100 mg/dL. Die Ergebnisse zur Systemgenauigkeit wurden durch große Höhen (3048 m) nicht beeinträchtigt. Das FreeStyle Precision Neo Blutzucker-Messsystem lieferte über die gesamte angegebene Hämatokrit-Bandbreite (30–60 %) und auch in Anwesenheit von 30 potenziellen Störsubstanzen in hohen Konzentrationen genaue Ergebnisse – die beobachteten Effekte lagen dabei unterhalb der Kriterien, die eine Aufnahme in die Produktkennzeichnung gemäß der DIN ISO-Norm 15197:2013 verlangen. Schlussfolgerungen: Die klinischen Untersuchungen bestätigen die Systemgenauigkeit des FreeStyle Precision Neo Blutzucker-Messsystems für die Kapillarblutmessung verglichen mit der Laborreferenzmethode. Die Messgenauigkeit bei der Anwendung durch Laien und durch geschultes Personal entsprachen den Anforderungen der DIN ISO-Norm 15197:2013. Das FreeStyle Precision Neo Blutzucker-Messsystem erhielt von Erstbenutzern eine gute Bewertung der Benutzerfreundlichkeit. In Laboruntersuchungen wurde nachgewiesen, dass das System in verschiedenen herausfordernden Situationen, die auch beim täglichen Messen zu Hause auftreten können, seine Genauigkeit beibehielt. Weiterhin wurde gezeigt, dass störende Bedingungen, wie sie in der DIN ISO-Norm 15197:2013 genannt werden (Hämatokrit und Störsubstanzen), das FreeStyle Precision Neo Blutzucker-Messsystem nicht in dem Maße beeinflussten, dass eine Beschreibung ihrer Effekte in der Produktkennzeichnung aufgeführt werden müsste.

Schlüsselwörter: FreeStyle Precision Neo, ISO 15197:2013, Genauigkeit, Hämatokrit, Blutglukose-Messsystem

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Materials and methods The FreeStyle Precision Neo system Measurement principle: Glucose dehydrogenase (GDH-NAD), coenzyme nicotinamide adenine dinucleotide (NAD) and an electron mediator (phenanthroline quinone, PQ) are present on the working electrode of the test strip. Glucose in the blood sample is oxidised to gluconolactone by reaction with NAD, this oxidation is catalysed by GDH (Figure 1). The PQ reacts with the reduced coenzyme (NADH), thus reducing the mediator and returning the coenzyme to its oxidised state (NAD). The reduced mediator is oxidised at the working electrode, this produces a small electric current which is proportional to the concentration of glucose in the sample and is measured by the meter. Lack of interference: GDH can be used to perform electrochemical glucose measurements without direct interference by oxygen in the blood sample, thus reducing interfering effects caused by oxygen. Glucose oxidase (GOX), the enzyme used in some other blood glucose monitoring systems (BGMS), may react with oxygen to cause measurement errors. Low potential measurements (Figure 2) can minimize interference by substances present in the blood sample. For an electrochemical reaction to occur, a potential (voltage) is applied between the working and reference electrodes. The larger the applied potential, the greater the number of interfering substances that can be oxidized at the working electrode and produce a false signal. The electron mediator (PQ) used in these test strips allows the electrochemical reaction to occur at low potential, so there is minimal interference with the test strip results from other substances. Dual fill: The test strip retains the dualfill feature of previous Abbott Diabetes Care test strips that allows the user to apply blood to either the top or the end of the test strip (Figure 3). The blood is automatically drawn into the reaction area. © Verlag PERFUSION GmbH

C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

Fill trigger electrode: The test strip contains 3 electrodes (working, reference and fill trigger; see Figure 4). The circuit between the fill trigger and the reference electrodes must be detected by the meter before the test will start. The completed circuit is only detected when the applied sample flows beyond the reference and working electrodes to contact the fill trigger electrode (Figure 5). This feature minimizes errors due to insufficient sampling and reduces test strip waste. Upon application of sufficient sample, the test is automatically initiated. Summary of features: The combination of the fill trigger electrode, the GDH-NAD chemistry with low applied electric potential and the dual fill design with visual confirmation of fill is the basis of TrueMeasure technology, designed to minimise errors from insufficient blood samples and interfering substances, allow easy sample application and thus protect the integrity of glucose testing data from preventable errors. Test strips are individually wrapped in foil packets to protect from exposure to moisture, chemicals or contamination. In addition to the features described above, the FreeStyle Precision Neo system incorporates a number of enhancements to provide minimal sensitivity to haematocrit and high accuracy with the short (5 second) test time and no requirement for coding or calibration by the user. These features help to ensure compliance to performance requirements introduced in ISO 15197:2013 and also enhance the reliability of patient testing. Comparative methods The YSI 2300 Stat Plus glucose analyser served as the comparative method in the clinical and laboratory studies. The YSI whole blood glucose results were multiplied by 1.12 to obtain plasma equivalent glucose values for comparison with the test strip results. The YSI glucose analyser has metrological traceability to NIST certified reference material [2]. Perfusion 01/2015

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Figure 1: Reaction scheme

Lower applied potential

Enzyme very specific for glucose

New mediator

GLUCOSE SPECIFIC READING

Figure 2: Measurement principle of the FreeStyle Precision Neo system

Top Fill

End Fill

Visual Confirmation

Figure 3: End fill or top fill test strip

Statistical analysis All statistical analyses for the clinical studies were performed using SAS® version 9.2 (SAS Institute Inc., Cary, NC). Passing and Bablok regression [3] was used to correlate meter results with comparative method values in the capillary clinical evaluation. Passing and Bablok regression analysis is recommended by the American Association of Bioanalysts [4] for method comparison (accuracy) studies. Mean absolute relative difference (MARD) between meter results and comparative method values was calculated to assess the mean absolute bias. Data

were excluded from statistical analysis if (1) the drift between the first and second measurements of the comparative method was >4 mg/dL (0.22 mmol/L) at glucose ≤100 mg/dL (5.55 mmol/L) or >4 % at glucose >100 mg/dL (5.55 mmol/L); (2) time exceeded the interval specified in the protocol (e.g. the BGMS and YSI tests on each sample must be completed within 20 minutes of sample collection); or (3) the data set was not complete (e.g. missing haematocrit level or YSI value). Laboratory study results were evaluated using JMP version 5.1 statistical software (SAS Institute) or SAS® version 9.2. © Verlag PERFUSION GmbH

C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

Three test strip lots were used in the study; each sample was tested on 2 strip lots. Each strip lot tested per sample was tested once by the lay user (for user accuracy evaluation and ease of use survey) and in duplicate by the trained operator (for system accuracy evaluation). The FreeStyle Precision Neo system results were compared to results obtained on the YSI analyser. Each of the 174 lay users completed a questionnaire rating ease of use topics after reading the instructions for use and performing a glucose test on their own. A scale of 1 to 6 was used, with 6 being the highest rating. An overall ease of use rating was obtained by averaging all responses. The age of the users ranged from 13 to 84 years (mean 61 years). 51 % were male and 49 % were female. 56 % had college or higher level of education. 28 % had type 1 diabetes and 72 % had type 2 diabetes. The methods used in the user performance evaluation and the system accuracy evaluation are based on those outlined in ISO 15197:2013.

Figure 4: Test strip architecture

Precision evaluation

Figure 5: Test only starts when sufficient sample is applied. The completed circuit is only detected when sample reaches the trigger electrode

Clinical study – capillary (ﬁngerstick) Accuracy of the FreeStyle Precision Neo system was evaluated at two medical centres in the United States, who conducted the study on behalf of Abbott Diabetes Care. 174 subjects were enrolled in the study. 9 subjects were excluded from the analysis due to protocol deviations, yielding 165 subjects. Samples from 21 of these subjects were modified to provide additional samples (186 samples in total) at low and high glucose concentrations for Perfusion 01/2015

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system accuracy analysis. Blood samples collected with an appropriate anticoagulant were spiked with a 0.9 % saline solution containing a high concentration of glucose to prepare high glucose samples; the spiked samples were allowed to stand for at least 15 minutes before use to allow the added glucose to equilibrate between the plasma and red blood cells. To prepare low glucose samples, blood samples collected with an appropriate anticoagulant were incubated at 27 to 37 °C to allow glycolysis to occur.

Repeatability was evaluated using 10 meters, 3 test strip lots, and 1 venous blood sample with glucose concentrations adjusted to five concentration ranges. 10 measurements were made with each combination of meter, test strip lot and sample. Testing was completed in 1 day. Intermediate precision was evaluated using 10 meters, 3 test strip lots and 3 levels of control solution, representing hyperglycaemic, euglycaemic and hypoglycaemic conditions. Each sample was tested in duplicate on 3 test strip lots and 10 meters on each of 20 days. The methods used in the precision studies are based on those outlined in ISO 15197:2013. Haematocrit evaluation The effect of haematocrit on performance of the FreeStyle Precision Neo system was evaluated using 5 haematocrit levels, 3 glucose concentrations, © Verlag PERFUSION GmbH

C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

3 venous blood samples (from different subjects) and 3 test strip lots. Each venous blood sample was adjusted to the 5 haematocrit levels (30, 35, 42 [control sample], 50, and 60 %) by separating the plasma from the cells, then adding or removing aliquots of plasma in different proportions. The samples at each haematocrit level were divided into 3 portions and the glucose level of each sample was adjusted to the desired concentration. 30 tests (10 tests per strip lot) were performed for each of the 45 samples. Each sample was also tested on the YSI analyser and the results were used to calculate the bias of the meter results from the mean YSI reference value for each sample. To determine haematocrit effects, the difference between the average bias (from reference value) of each test sample and the average bias (from reference value) of the control sample (42 % haematocrit) was determined. The methods used in the haematocrit study are based on those outlined in ISO 15197:2013. Interference substances evaluation 30 substances have been evaluated across paired difference (25), dose response (3) and anticoagulant (2) testing. Paired difference: 25 substances (including reducing substances, common medications and non-glucose sugars) were tested for interference using venous blood in two glucose concentration ranges (50–100 mg/dL [2.78–5.55 mmol/L] and 250–350 mg/ dL [13.88–19.43 mmol/L]), 3 test strip lots and a paired-sample experimental design. The glucose level of the venous blood was adjusted to the desired concentrations. Paired samples were then spiked with a concentrated solution of the substance (test sample) and an equal volume of the solvent used to dissolve the substance (control sample). This was repeated for each potentially interfering substance. 30 tests were made per sample. The YSI analyser was used to Perfusion 01/2015

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assign glucose reference values to the samples. For each sample, the bias of the average measured values (test strip results) from the mean YSI reference value was determined. The difference in bias between test and control samples was then calculated for each substance. Dose response: An additional 3 substances were tested for interference using venous blood in two glucose concentration ranges (50–100 mg/dL [2.78–5.55 mmol/L] and 250–350 mg/ dL [13.88–19.43 mmol/L]), 3 test strip lots and a dose response experimental design. The glucose level of the venous blood was adjusted to the desired concentrations. For each glucose concentration, the sample was divided into 6 aliquots, 4 test samples and 2 control samples. The test samples were then spiked with different concentrations of the substance and an equal volume of the solvent used to dissolve the substance was added to each of the control samples. This was repeated for the 3 potentially interfering substances. 30 tests were made per sample. The YSI analyser was used to assign glucose reference values to the samples. A regression model was fit (across lots) between the individual test responses and the interferent concentration. The regression model was used to calculate the interferent concentration at which the effect on performance is considered clinically significant (10 % change in the response at the control [zero] concentration). The methods used in the interference studies are based on those outlined in ISO 15197:2013 and CLSI EP-7A [5]. Anticoagulants and pH: Potential interfering effects from common anticoagulants (heparin and EDTA, including short fill of tubes) were evaluated by comparing average measured values (test strip results) to the mean YSI reference value and effects of pH were evaluated by comparing difference in bias from reference for control and test pH levels, covering the pH range 7.01 to 7.74.

Altitude evaluation The effect of altitude on the performance of the FreeStyle Precision Neo system was evaluated at 2 altitudes (sea level and 10,000 feet; 3,048 meters), using 3 venous blood samples (from different subjects), 3 glucose concentrations and 3 test strip lots. 30 tests (10 tests per strip lot) were performed for each of the 18 samples. Each sample was also tested on the YSI analyser and the results were used to calculate the bias of the meter results from the mean YSI value for each sample. To determine altitude effects, the difference between the average bias (from reference) of the testing performed at high altitude and the average bias of the control samples (sea level) was determined. Results User accuracy evaluation and ease of use (lay user) The haematocrit range of the samples in this study was 25–51 %, and the range of glucose concentrations was 43–358 mg/dL (2.4–19.9 mmol/L). Good correlation was found between the FreeStyle Precision Neo system and the YSI analyser by regression analysis (r = 0.98, slope = 0.99, intercept = 0.9 mg/dL [0.05 mmol/L]) (Figure 6). Overall the mean absolute relative difference (MARD) was 5.2 %. Of the 330 test results (from 165 subjects), 328 (99.4 %) were in Zone A (clinically accurate) and 2 (0.6 %) were in Zone B (clinically acceptable) of the consensus error grid [6] (Figure 6). This study evaluating glucose values from fingertip capillary blood samples obtained by 165 lay users showed: • 97.7 % (43/44) of results were within ±15 mg/dL (0.83 mmol/L) of the reference values at glucose concentration <100 mg/dL (5.55 mmol/L). • 97.9 % (280/286) of results were within ±15 % of the reference values at glucose concentrations ≥100 mg/dL (5.55 mmol/L). In total, 97.9 % (323/330) of results met the accuracy criteria described in © Verlag PERFUSION GmbH

C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

ISO 15197:2013 (Section 8.2) for the user performance evaluation (Figure 7), thus meeting the requirement that 95 % of results should be within the accuracy criteria. An overall ease of use rating of 5.5 (out of 6) was obtained when all responses were averaged, indicating that the lay users found the FreeStyle Precision Neo system easy to use (Table 1). The ease of use survey confirmed that the instructions for use and the messages displayed on the meter are adequate, as required by ISO 15197:2013. System accuracy (trained operator)

Figure 6: Fingertip accuracy – consensus error grid and regression analysis

The haematocrit range of the samples in this study was 24–56 %, and the range of glucose concentrations was 29–438 mg/dL (1.6–24.3 mmol/L). Good correlation was found between the FreeStyle Precision Neo system and the YSI analyser by regression analysis (r = 0.99, slope = 1.00, intercept = –0.3 mg/dL [–0.02 mmol/L]). Overall the MARD was 5.3 % and the mean CV between the paired tests for the 186 samples was 3.4 %. Of the 786 test results, 784 (99.7 %) were in zone A (clinically accurate) and 2 (0.3 %) were in zone B (clinically acceptable) of the consensus error grid. System accuracy analysis for the 3 lots combined showed: 99.1% of results agreed within ±15 mg/dL (0.83 mmol/L) or ±15 % (for glucose concentrations ≥100 mg/dL [5.55 mmol/L]) of the reference value (Tables 2–4). As required by ISO 15197:2013, each lot showed >95 % of results agreed within ±15 mg/dL (0.83 mmol/L) or ±15% of the reference value: 100 %, 98.5 % and 98.8 % for lots A, B and C, respectively. These results, in combination with the results above confirming that 100 % of test strip results were in zones A and B of the consensus error grid, illustrate that the FreeStyle Precision Neo system meets the accuracy criteria in ISO 15197:2013 (Section 6.3.3).

Figure 7: Fingertip accuracy – system accuracy analysis Perfusion 01/2015

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C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

Statement

Precision

Mean rating*

The test instructions contain sufficient information for me to do a test The test instructions are easy to follow The meter was easy to learn The meter is easy to use The meter is easy to hold The meter looks attractive It was easy to insert the test strip into the meter The test strip is easy to use It was easy to read the meter display

5.7

Repeatability: Precision was pooled for 300 tests performed across 3 test strip lots using fresh venous blood, at each of 5 glucose concentrations (Table 5). The pooled SD was ≤2.7 mg/ dL (0.15 mmol/L) at glucose concentrations <100 mg/dL (5.55 mmol/L) and the pooled CV was ≤3.5 % at glucose concentrations ≥100 mg/dL (5.55 mmol/L).

5.6 5.7 5.6 5.5 4.7 5.6 5.4 5.9

Mean over all statements

5.5

* The rating scale is 1 to 6 for each statement; 6 = strongly agree, 1 = strongly disagree. Table 1: Ease of use rating by 174 lay users for the FreeStyle Precision Neo system

Accuracy criteria

Within ±5 mg/dL (0.28 mmol/L) of reference

Within ±10 mg/dL (0.56 mmol/L) of reference

Within ±15 mg/dL (0.83 mmol/L) of reference

Percent (n/n) within criteria

68.2 % (105/154)

96.8 % (149/154)

100.0 % (154/154)

Table 2: System accuracy results for glucose concentrations <100 mg/dL (5.55 mmol/L) Within ±5 % of reference

Within ±10 % of reference

Within ±15 % of reference

64.9 % (410/632)

91.9 % (581/632)

98.9 % (625/632)

Accuracy criteria Percent (n/n) within criteria

Table 3: System accuracy results for glucose concentrations ≥100 mg/dL (5.55 mmol/L)

Accuracy criteria

Within ±5 mg/dL (0.28 mmol/L) or 5 % of reference

Within ±10 mg/dL (0.56 mmol/L) or 10 % of reference

Within ±15 mg/dL (0.83 mmol/L) or 15 % of reference

Percent (n/n) within criteria

65.5 % (515/786)

92.9 % (730/786)

99.1 % (779/786)

Table 4: System accuracy results for all data

Mean test strip response Pooled SD Pooled CV (%)

Glucose level

Low

Low-mid 86.9

145.8

Mid

Mid-high 206.4

332.9

mmol/L

2.6

4.8

8.1

11.5

18.5

mg/dL mmol/L

1.9 0.10 4.0

2.7 0.15 3.1

4.5 0.25 3.1

7.3 0.41 3.5

10.0 0.55 3.0

mg/dL

47.3

High

Table 5: Repeatability (adjusted venous samples)

Mean test strip response Pooled SD

Glucose level

Low

Low-mid

High

mg/dL

43.2

91.7

292.5

mg/dL

1.9

3.3

9.0

mmol/L mmol/L

Pooled CV (%)

2.4

0.11 4.4

Table 6: Intermediate precision (quality control solution samples) Perfusion 01/2015

28. Jahrgang

5.1

0.18 3.6

16.2

0.50 3.1

Intermediate: Precision was pooled for 1200 tests performed across 3 test strip lots over 20 days, at each of 3 glucose concentrations (Table 6). The pooled SD was ≤3.3 mg/dL (0.18 mmol/L) at glucose concentrations <100 mg/ dL (5.55 mmol/L) and the pooled CV was 3.1 % at glucose concentrations ≥100 mg/dL (5.55 mmol/L). Haematocrit For each test strip lot, the average difference in bias between test samples and control samples were less than the criteria detailed in ISO 15197:2013 (Section 6.4.3.2), therefore results for each lot have been combined for presentation here (in line with the guidance in ISO 15197:2013) (Table 7). Differences in the haematocrit level of the blood sample affect results by ≤1.2 mg/ dL (0.07 mmol/L) at low glucose concentrations and by ≤3.5 % at higher glucose concentrations – these were less than the criteria in ISO 15197:2013, which trigger inclusion of the results in the product labelling. Interfering substances Paired difference: The 25 potentially interfering substances undergoing paired difference testing were evaluated at concentrations above the upper limit of therapeutic or normal concentration. Results for each test strip lot have been combined for presentation here (Table 8). Presence of the 25 substances in the blood sample affected results by ≤6 mg/dL (0.36 mmol/L) at low glucose concentrations and by less © Verlag PERFUSION GmbH

C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

Mean YSI reference value

Mean difference in bias from control (42 % haematocrit)

mg/dL

mmol/L

2.5

111

Haematocrit mg/dL

6.2

400

–1.2

–0.8

–0.4

–0.8

mmol/L

–0.07

–0.04

–0.02

–0.04

–2.1

–3.3

–0.8

–1.9

22.2

–1.5

–0.7

–2.3

–3.5

Table 7: Effect of haematocrit Mean difference in bias from control Upper limit of therapeutic or normal concentration [5, 7], mg/dL (mmol/L)

Substance

Test concentration, mg/dL (mmol/L)

Mean YSI reference = 83 mg/dL (4.6 mmol/L)

Mean YSI reference = 316 mg/dL (17.5 mmol/L)

mg/dL

mmol/L

Acetaminophen (Tylenol, Paracetamol)

3 (0.20)

20 (1.32)

0.02

–2

Beta-hydroxybutyrate

<7.6 (0.73)

265 (25.46)

–1

–0.07

–1

Bilirubin (unconjugated)

1.2 (0.02)

20 (0.34)

0.05

–2

Cholesterol

<200 (5.18)

503 (13.01)

0.36

Creatinine

1.3 (0.115)

5 (0.442)

0.29

Dopamine

0.03 (1.96 µmol/L)

0.10 (6.53 µmol/L)

0.07

–1

Ethanol

200 (43.38)

400 (86.77)

0.10

–1

Galactose

5.05 (0.28)

15 (0.83)

0.04

–1

Gentisic acid

0.6 (0.039)

1.8 (0.117)

0.04

Haemoglobin

200 (0.031)

0.04

–3

5 (0.24)

50 (2.42)

0.00

460

0.04

–1

L-Dopa

0.2 (0.010)

0.6 (0.030)

–0.01

Lactate

20 (2.22)

59 (6.55)

0.22

–1

Maltose

–

110 (3.21)

0.08

–2

Maltotetraose

–

60 (0.90)

–1

–0.06

–4

Maltotriose

–

120 (2.38)

–0.02

–2

0.75 (0.036)

1.5 (0.071)

0.06

Pralidoxime iodide

205 (7.76)

0.12

Salicylic acid (from Aspirin)

30 (2.17)

60 (4.34)

0.17

–4

Tetracycline

0.5 (0.011)

1.5 (0.034)

-2

–0.10

Tolazamide (Tolinase)

2.8 (0.09)

15 (0.48)

0.09

Tolbutamide (Orinase)

10.8 (0.40)

64 (2.37)

0.23

Triglycerides

< 150 (1.7)

1500 (17)

–1

–0.03

–2

Uric acid

7.2 (0.43)

24 (1.43)

0.10

Ibuprofen (Montril, Advil) Icodextrin

Methyl-Dopa (Aldomet)

Table 8: Paired difference interference testing Perfusion 01/2015

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C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

Substance Ascorbate (vitamin C) Glutathione Xylose

Upper limit of therapeutic or normal concentration [5, 7], mg/dL (mmol/L)

Maximum test concentration, mg/dL (mmol/L)

1.5 (0.085)

6.0 (0.341)

50 (3.33)

100 (6.66)

0.18 (0.006)*

92.1 (3.00)

Clinically significant concentration Mean YSI reference = 87 mg/dL (4.8 mmol/L)

Mean YSI reference = 308 mg/dL (17.1 mmol/L)

mg/dL

mmol/L

mg/dL

mmol/L

2.6

0.15

7.9

0.45

82.4

5.49

374.2

24.9

22.5

0.73

73.3

2.39

* G lutathione exists mainly within cells, the extracellular concentration is significantly lower than the intracellular concentration. Since FreeStyle Precision Neo does not measure intracellular concentrations, the observed plasma glutathione concentration (2–6 µmol/L observed across studies [8–11]) has been used as the normal physiological concentration. Table 9: Dose response interference testing Altitude

Mean YSI reference value, mg/dL (mmol/L)

Sea level

49 (2.72)

10,000 ft (3,048 m) Sea level

10,000 ft (3,048 m)

41 (2.28)

110 (6.11)

104 (5.77)

391 (21.70)

390 (21.65)

Difference in bias from control (sea level) mg/dL

2.3

mmol/L

0.13

–3.5

0.1

Average bias for glucose <100 mg/dL (5.55 mmol/L), mg/dL (mmol/L)

2.3 (0.13)

Average bias for glucose ≥100 mg/dL (5.55 mmol/L), %

–1.8

Table 10: Effect of altitude

than 4 % at higher glucose concentrations – these were less than the criteria in ISO 15197:2013 (Section 6.4.4.2), which trigger inclusion of the results in the product labelling. Therefore, at the specified test concentrations, none of these substances had an interferent effect on the FreeStyle Precision Neo system. Dose response: The 3 potentially interfering substances undergoing dose response testing were evaluated over a range of concentrations, with the maximum test concentration being above the upper limit of therapeutic or normal concentration. The concentration at which each substance was determined to show a clinically significant effect on performance is shown in Table 9. The clinically significant concentration was above the therapeutic or normal concentration in each case, therefore these substances were not considered to have an effect on the FreeStyle Precision Neo system. However, a limitation is included in the test strip insert that the Perfusion 01/2015

28. Jahrgang

product should not be used during a xylose absorption test for malabsorption, where high concentrations of xylose can be present. Anticoagulant and pH: Presence of lithium heparin and EDTA in the blood sample affected results by ≤6 mg/dL (0.33 mmol/L) at low glucose concentrations and by ≤10 % at higher glucose concentrations – these were less than the criteria in ISO 15197:2013 (Section 6.4.4.2), which trigger inclusion of the results in the product labelling. This evaluation included half filling and quarter filling the tubes, in order to evaluate concentrations at 2× and 4× the concentration expected from full tubes. Blood samples with pH across the range 7.01 to 7.74 affected results by ≤2.2 mg/dL (0.12 mmol/L) at low glucose concentrations and by ≤3.8 % at higher glucose concentrations.

Altitude High altitudes of up to 10,000 feet (3,048 meters) affect results by ≤2.3 mg/dL (0.13 mmol/L) at low glucose concentrations and by ≤3.5 % at higher glucose concentrations. This magnitude of change associated with extreme altitudes is clinically acceptable. Discussion The clinical, user and laboratory studies illustrate that the FreeStyle Precision Neo system is accurate, easy to use and meets the performance requirements of ISO 15197:2013. Reliable and accurate results for home monitoring. Accuracy was verified with capillary blood samples across the measurement range of the FreeStyle Precision Neo system, the haematocrit range of 30 to 60 %, at high altitude (10,000 feet; 3,048 meters), and in the presence of 30 potentially interfering substances. © Verlag PERFUSION GmbH

C. Brannan: Evaluation of the FreeStyle Precision Neo blood glucose and ketone monitoring system

The FreeStyle Precision Neo system delivers accurate results from fingertip capillary samples in testing by trained operators and lay users; >99 % of results were in the “clinically accurate” zone A of the consensus error grid for both user groups. The system accuracy evaluation (performed by trained operators, in line with ISO 15197:2013) showed 99.1 % of results agreed within ±15 mg/dL (0.83 mmol/L) or ±15 % (for glucose concentrations ≥100 mg/ dL [5.55 mmol/L]) of the reference value. Thus the FreeStyle Precision Neo system meets the accuracy criteria introduced in ISO 15197:2013. The FreeStyle Precision Neo system maintains accuracy across the haematocrit range. Extreme haematocrit levels affect the FreeStyle Precision Neo system by ≤1.2 mg/dL (0.07 mmol/L) at low glucose concentrations and by ≤3.5 % at higher glucose concentrations, the effects observed were less than the criteria introduced in ISO 15197:2013, which trigger reporting the haematocrit sensitivity in the product labelling. The FreeStyle Precision Neo system minimises the potential for interference. Use of GDH-NAD ensures high specificity of the test to glucose. Consequently, there is no interference from other sugars such as galactose and maltose in dialysis patients that use icodextrin-containing solution for dialysis. The FreeStyle Precision Neo system should not be used during xylose absorption testing. Use of a low potential in the electrochemical reaction also minimises interference from reducing substances commonly found in the blood, such as acetaminophen (paracetamol) and uric acid. Each of the substances tested had no clinically significant effect on the FreeStyle Precision Neo system results, the effects observed were less than the criteria introduced in ISO 15197:2013, which trigger reporting the substance as an interferent in the product labelling.

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Conclusions

References

In conclusion, the studies described in this paper show that the FreeStyle Precision Neo system delivers accurate, reliable glucose results whilst providing safeguards to ensure the integrity of the testing process. Specifically, the clinical studies demonstrated the accuracy of the FreeStyle Precision Neo system for capillary self-testing – accuracy of the system in the hands of the lay user and trained operators meets the accuracy criteria introduced in ISO 15197:2013. In user performance testing, the FreeStyle Precision Neo system had a high acceptance and ease of use rating among first-time users. Laboratory studies showed that the test strip performs well in the presence of interfering substances and across the claimed haematocrit range, the magnitude of the effect of these influence quantities was less than the criteria introduced in ISO 15197:2013, which trigger inclusion in the product labelling. Combined with features that can minimise short sampling and test strip contamination, the short test time and no requirement for coding or calibration by the user, these results show that the FreeStyle Precision Neo system is uniquely designed to provide accurate and reliable results in self-testing by people with diabetes.

1 International Organisation for Standardisation. In vitro diagnostic test systems – Requirements for blood-glucose monitoring systems for self-testing in managing diabetes mellitus. ISO 15197:2013(E) 2 NIST Standard Reference Materials. http://ts.nist.gov/MeasurementServices/ ReferenceMaterials/232.cfm 3 Passing H, Bablok W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Applications of linear regression procedures for method comparison studies in clinical chemistry, Part I. J Clin Chem Clin Biochem 1983;21:709-720 4 American Association of Bioanalysts. Letter to the FDA; September 13 2000; www. fda.gov/ohrms/dockets/dailys/00/ Sep00/091400/c000018.pdf (accessed 04 February 2013) 5 CLSI. Interference testing in clinical chemistry; approved guideline – 2nd edition. CLSI document number EP7-A2 6 Parkes JL, Slatin SL, Pardo S, Ginsberg BH. A new consensus error grid to evaluate the clinical significance of inaccuracies in the measurement of blood glucose. Diabetes Care 2000;23:1143-1148 7 Roberts WL, McMillin GA, Burtis CA, Bruns DE. Reference information for the clinical laboratory. In: Burtis CA et al., eds. Tietz Textbook of Clinical Chemistry, 4th ed. Philadelphia: W.B. Saunders; 2006: 2251-2318 8 Michelet F, Gueguen R, Leroy P, Wellman M, Nicolas A, Siest G. Blood and plasma glutathione measured in healthy subjects by HPLC: relation to sex, aging, biological variables, and life habits. Clin Chem 1995;41:1509-1517 9 Jones DP, Carlson JL, Samiec PS, Sternberg P Jr, Mody VC Jr, Reed RL, Brown LA. Glutathione measurement in human plasma evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin Chim Acta 1998;275:175-184 10 Adams JD, Johannessen JN, Dacon JP. Quantification of glutathione and glutathione disulfide in human plasma. Clin Chem 1987;33:1675-1676 11 Mansoor, MA, Svardal AM, Ueland PM. Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal Biochem 1992;200:218-219

Acknowledgements This study was funded by Abbott Diabetes Care. We thank the principal investigators, Leonard Chuck, PhD, MD at Diablo Clinical Research, Walnut Creek, CA and Elizabeth Taylor, MS, RD, CDE, CES, CCRC at MassResearch, Waltham, MA, and the study staff for their invaluable assistance in the execution of the clinical study. FreeStyle and related brand marks are trademarks of Abbott Diabetes Care Inc. in various jurisdictions.

Address for correspondence: Dr Claire Brannan Abbott Diabetes Care Range Road Witney, Oxon United Kingdom OX29 0YL Email: claire.brannan@abbott.com

FORUM ANTITHROMBOTICUM

Für Patienten mit nicht valvulärem Vorhofflimmern (VHF) geben die aktuellen ESC-Leitlinien zur Schlaganfallprophylaxe den neuen oralen Antikoagulanzien (NOAC) den Vorrang gegenüber der bisherigen Standardtherapie mit Vitamin-K-Antagonisten (VKA) [1]. Ein in Klinik und Praxis sehr gutes Nutzen-Risiko-Profil weist der orale, direkte Faktor-Xa-Inhibitor Apixaban (Eliquis®) auf. Apixaban ist sowohl in komplexen Therapiesituationen als auch bei unterschiedlichen Risikoprofilen geeignet und steht nach der neuesten Zulassungserweiterung nun auch für ein breites Patientenkollektiv sowie Indikationsspektrum* zur Verfügung. Relevanz für eine effektive Schlaganfallprophylaxe VHF ist eine der häufigsten Formen von Herzrhythmusstörungen und geht mit einem – im Vergleich zu altersgleichen Menschen ohne Vorhofflimmern – 5-fach höheren Schlaganfallrisiko einher [2]. Rund 1,8 Millionen Menschen sind in Deutschland von VHF betroffen [3] und die Verhinderung von Schlaganfällen bei ihnen ist ein zentrales Therapieziel. Aufgrund des überlegenen Nutzen-Risiko-Profils sowie der einfacheren Handhabung gegenüber VKA werden in der Praxis zunehmend NOAC eingesetzt. Der direkte, orale Faktor-Xa-Inhibitor Apixaban (Eliquis®) ist zur Prophylaxe von Schlaganfällen oder systemischen Embolien bei erwachsenen Patienten mit nicht valvulärem VHF und mindestens einem weiteren Schlaganfallrisikofaktor* zugelassen [4]. Überlegenes Nutzen-Risiko-Profil gegenüber Warfarin belegt Basis der Zulassung waren die Phase-IIIStudien ARISTOTLE und AVERROES. * Detaillierte Informationen zu den Indikationen und Dosierungen entnehmen Sie bitte den Fachinformationen. Perfusion 01/2015

28. Jahrgang

Effektive Schlaganfallprophylaxe bei nicht valvulärem Vorhofflimmern:

Überzeugendes Nutzen-Risiko-Profil von Apixaban

In die ARISTOTLE-Studie [5] wurden insgesamt 18.201 Patienten mit nicht valvulärem VHF und einem oder mehreren Risikofaktoren* eingeschlossen, um doppelblind die Wirksamkeit und Verträglichkeit von Apixaban mit der Standardtherapie Warfarin über einen Beobachtungszeitraum von 1,8 Jahren (im Median) zu vergleichen. Gegenüber Warfarin reduzierte Apixaban das Risiko für Schlaganfälle oder systemische Embolien – den primären Wirksamkeitsendpunkt – signifikant um relativ 21 % (Hazard Ratio [HR] 0,79; p<0,001 für Nicht-Unterlegenheit; p=0,01 für Überlegenheit). Das Risiko für einen hämorrhagischen Schlaganfall wurde unter dem direkten Faktor-Xa-Inhibitor versus dem VKA Warfarin signifikant um relativ 49 % gesenkt und damit nahezu halbiert (HR 0,51; p<0,001) [5]. Die guten Daten zur Wirksamkeit gingen zugleich mit einem überzeugenden Sicherheitsprofil einher. So verringerte Apixaban verglichen mit Warfarin die Rate an schweren Blutungen signifikant um relativ 31 % (wichtiger sekundärer Endpunkt; HR 0,69; p<0,001; Abb. 1). Das Risiko für intrakranielle Blutungen wurde unter Apixaban gegenüber Warfarin signifikant um relativ 58 % gesenkt (HR 0,42; p<0,001), die Rate schwerer gastrointestinaler Blutungen war vergleichbar [5]. Das sehr gute Nutzen-Risiko-Profil von Apixaban zeigte sich in diversen vordefinierten Subgruppenanalysen der ARISTOTLEStudie: So profitierten Patienten unabhängig von ihrem Risikoprofil für Schlaganfälle oder für Blutungen [6], von ihrem Alter [7], vom Grad der Nierenfunktionseinschränkung [8] oder von einer KHK in der Anamnese [9]. Eine weitere präspezifizierte Subgrup-

penanalyse zeigte, dass Apixaban auch bei Patienten mit nicht valvulärem VHF im Rahmen einer Kardioversion ohne Unterbrechung weiter angewendet werden kann [10]. Wirksamer bei vergleichbarer Verträglichkeit gegenüber ASS Jene Patienten mit nicht valvulärem VHF, die für eine VKA-Therapie ungeeignet sind und daher mit Acetylsalicylsäure (ASS) behandelt werden, haben ebenfalls ein hohes Risiko, einen Schlaganfall zu erleiden. In der AVERROES-Studie [11] mit 5.599 Patienten mit nicht valvulärem VHF, die für eine Antikoagulation mit VKA nach Beurteilung des behandelnden Arztes nicht in Frage kamen, wurde Apixaban mit ASS über einen Beobachtungszeitraum von 1,1 Jahren (im Mittel) verglichen [11]. Unter Apixaban konnte das Risiko für Schlaganfälle oder systemische Embolien im Vergleich zu ASS signifikant um relativ 55 % gesenkt werden (primärer Endpunkt; HR 0,45; p<0,001) [11]. Der Nutzen von Apixaban erstreckte sich konsistent über zahlreiche vordefinierte Subgruppen, wobei die mit Apixaban assoziierte Risikoreduktion für einen Schlaganfall oder eine systemische Embolie bei Risikopatienten mit vorangegangenem Schlaganfall oder TIA noch stärker ausgeprägt war [12]. Auch das gute Sicherheitsprofil von Apixaban zeigte sich in der AVER ROES-Studie: So war die Rate an schweren Blutungen (wichtiger sekundärer Endpunkt) vergleichbar zu ASS (Abb. 2) – ebenso wie die Inzidenz tödlicher Blutungen und intrakranieller Blutungen [11]. © Verlag PERFUSION GmbH

Signifikante Reduktion von Schlaganfällen/systemischen Embolien

Signifikante Reduktion schwerer Blutungen

21 % RRR

31 % RRR

p=0,01

p<0,001

4 3 2 1 0 (n/N)

(265/9081) (212/9120) Schlaganfälle oder systemische Embolien (pimärer Wirksamkeitsendpunkt) Apixaban

Warfarin

Nicht nur in der Schlaganfallprophylaxe bei nicht valvulärem VHF*, sondern auch in der Behandlung und Rezidivprophylaxe venöser Thromboembolien* (VTE) profitieren Patienten von dem überzeugenden Nutzen-RisikoProfil von Apixaban. Dass der FaktorXa-Inhibitor in der Behandlung von tiefen Venenthrombosen (TVT) und Lungenembolien (LE) sowie in der Prophylaxe rezidivierender TVT und LE bei Erwachsenen* der konventionellen Therapie (initial Enoxaparin, überlappend und Weiterbehandlung mit Warfarin) in Hinblick auf die Wirksamkeit ebenbürtig war, ergab die Phase-III-Studie AMPLIFY [13] mit 5.395 Patienten mit nachgewiesener, symptomatischer TVT und/oder LE. Durch die vergleichbare Rate rezidivierender, symptomatischer VTE oder VTE-bedingtem Tod belegte Apixaban im primären Endpunkt gegenüber der konventionellen Therapie eine signifikante Nicht-Unterlegenheit, unabhängig davon, ob Patienten eine TVT oder LE aufwiesen (relatives Risiko [RR] 0,84; p<0,001). Zugleich reduzierte der Faktor-Xa-Inhibitor signifikant die Perfusion 01/2015

28. Jahrgang

+13 % RR

p<0,001

p=0,57

4 3 2 1 0 (n/N)

Mediane Beobachtungszeit: 1,8 Jahre

Wirksam und verträglich auch bei venösen Thromboembolien

Keine signifikante Zunahme des Risikos für schwere Blutungen

–55 % RRR

(462/9052) (327/9088) Schwere Blutungen (wichtiger sekundärer Endpunkt)

Abbildung 1: ARISTOTLE-Studie: Apixaban (Eliquis®) ist Warfarin in zweifacher Hinsicht signifikant überlegen: Reduktion von Schlaganfällen oder systemischen Embolien sowie schweren Blutungen [5]. RRR = relative Risikoreduktion, n = Anzahl der Patienten mit einem Ereignis, N = Anzahl der Patienten in der Apixaban- oder Warfarin-Gruppe

Signifikante Reduktion des Risikos für Schlaganfälle/ systemische Embolien

Ereignisrate pro Jahr (%)

FORUM ANTITHROMBOTICUM

(113/2791) (51/2808) Schlaganfälle oder systemische Embolien (pimärer Wirksamkeitsendpunkt) Apixaban

ASS

(39/2791)

(44/2808)

Schwere Blutungen (wichtiger sekundärer Endpunkt)

Mittlere Beobachtungszeit: 1,1 Jahre

Abbildung 2: Die AVERROES-Studie zeigte die überlegene Wirksamkeit von Apixaban (Eliquis®) gegenüber ASS bei Patienten mit nicht valvulärem VHF bei vergleichbarem Risiko für schwere Blutungen [11]. RRR = relative Risikoreduktion, RR = relatives Risiko, n = Anzahl der Patienten mit einem Ereignis, N = Anzahl der Patienten in der Apixaban- oder ASS-Gruppe

Rate an schweren Blutungen um relativ 69 % (primärer Sicherheitsendpunkt; RR 0,31; p<0,001) [13]. Nutzen im Therapiealltag Apixaban bietet neben seinem überlegenen Nutzen-Risiko-Profil, das sich sowohl in der Schlaganfallprophylaxe bei nicht valvulärem VHF als auch in der Behandlung und Rezidivprophylaxe venöser Thromboembolien gegenüber Warfarin gezeigt hat, im Praxis alltag eine einfache Handhabung für Arzt und Patient. Mit der im Vergleich zu VKA relativ kurzen Halbwertszeit von etwa 12 Stunden ist bei kleineren Eingriffen – abhängig vom Blutungs-/Schlaganfallrisiko – eine vergleichsweise kurze Therapieunterbrechung ohne Bridging möglich [4]. Darüber hinaus wird die einfache praktische Handhabung von Apixaban unter anderem durch die orale Einnahme unabhängig von den Mahlzeiten gestützt. Zudem muss die Ernährung nicht eingeschränkt werden und es gibt im Vergleich zu VKA weniger Wechselwirkungen mit anderen Arzneimitteln [4]. Fabian Sandner, Nürnberg

Literatur 1 Camm AJ et al. Eur Heart J 2012;33:27192747 2 Camm AJ et al. Europace 2012;14:13851413 3 Informationen des Kompetenznetz Vorhofflimmern (http://www.kompetenznetz-vorhofflimmern.de/de/vorhofflimmern/patienteninformation-vorhofflimmern/volkskrankheit-vorhofflimmern) 4 Fachinformationen Eliquis® 5 mg, 2,5 mg; Stand Juli 2014 5 Granger CB et al. N Engl J Med 2011; 365:981-992 6 Lopes RD et al. Lancet 2012;380:17491759 7 Halvorsen S et al. Eur Heart J 2014;35: 1864-1872 8 Hohnloser S et al. Eur Heart J 2012;33: 2821-2830 9 Bahit MC et al. Int J Cardiol 2013; 170:215-220 10 Flaker G et al. J Am Coll Cardiol 2014; 63:1082-1087 11 Connolly SJ et al. N Engl J Med 2011; 364:806-817 12 Diener HC et al. Lancet Neurol 2012;11: 225-231 13 Agnelli G et al. N Engl J Med 2013;369: 799-808

Mit freundlicher Unterstützung von BristolMyers Squibb GmbH & Co. KGaA, München, und Pfizer Pharma GmbH, Berlin. © Verlag PERFUSION GmbH

FORUM ANTITHROMBOTICUM

CATCH-Studie bestätigt Wirksamkeit von Tinzaparin zur VTERezidivprophylaxe bei Tumorpatienten

Tumorpatienten haben ein insgesamt etwa 7-fach erhöhtes Risiko, im Laufe ihrer Erkrankung eine akute venöse Thromboembolie (VTE) zu erleiden [1]. Niedermolekulares Heparin (NMH, z.B. Tinzaparin/Innohep®) reduziert dieses Risiko im Vergleich zu VitaminK-Antagonisten (VKA) und wird in nationalen sowie internationalen Leitlinien als Antikoagulation der Wahl empfohlen [2, 3]. Diese Evidenz beruht jedoch im Wesentlichen auf einer einzigen, randomisierten Open-Label-Studie (CLOT-Studie) [4]. Angesichts der noch limitierten Datenlage zum Einsatz von NMH in dieser Patientenpopulation bietet die vor Kurzem abgeschlossene CATCH-Studie (Comparison of Acute Treatments in Cancer Haemostasis) wichtige Zusatzinformationen. Erste Ergebnisse wurden im Rahmen des Kongresses der American Society of Hematolgy (ASH) im Dezember 2014 in San Francisco präsentiert [5]. Tinzaparin senkt VTE-Risiko gegenüber Warfarin um 35 Prozent Mit 900 Patienten aus 165 Studienzentren in 32 Ländern handelt es sich bei der CATCH-Studie um die weltweit größte Untersuchung zur Behandlung tumorassoziierter Thrombosen [5]. Hauptziel dieser randomisierten, offenen, multizentrischen Phase-III-Studie war die Untersuchung der Wirksamkeit von Tinzaparin zur Vorbeugung von VTE-Rezidiven bei Patienten mit aktiver Tumorerkrankung und einer akuten symptomatischen, proximalen tiefen Venenthrombose (TVT) und/oder Lungenembolie (LE). Gynäkologische Karzinome, Kolorektal-, Lungen- soPerfusion 01/2015

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wie Mammakarzinome waren die häufigsten Primärtumoren. Die Studienteilnehmer erhielten über einen Zeitraum von 6 Monaten randomisiert entweder einmal täglich Tinzaparin oder initial über 5–10 Tage einmal täglich Tinzaparin mit überlappender und anschließender Gabe von dosisadjustiertem Warfarin. Im Tinzaparin-Arm traten nach 6 Monaten Studiendauer bei 31 Patienten (6,9 %) VTE-Rezidive auf verglichen mit 45 Patienten (10 %) im WarfarinArm (Hazard Ratio 0,65; 95%-KI 0,41– 1,03; p=0,07). Damit erreichten die mit Tinzaparin behandelten Tumorpatienten im Hinblick auf ein VTE-Rezidiv eine Risikoreduktion um 35 %. Symptomatische, nicht tödliche TVT wurden bei 12 Patienten (2,7 %) im TinzaparinArm und bei 24 Patienten (5,3 %) im Warfarin-Arm diagnostiziert (HR 0,48; 95%-KI 0,24-0,96]; p=0,04). Dies bedeutet eine signifikante Reduktion um relative 52 %. Die Inzidenz schwerwiegender Blutungsereignisse war mit 2,9 % im Tinzaparin-Arm und mit 2,7 % im Warfarin-Arm vergleichbar. Wie die Auswertung der Sicherheitsdaten zeigte, traten klinisch relevante nicht schwerwiegende Blutungen signifikant seltener unter Tinzaparin auf als unter Warfarin (bei 50 [11 %] bzw. 73 [16 %] Patienten; p=0,03). Bei der Mortalität wurden keine Unterschiede beobachtet, die 6-Monats-Überlebensraten betrugen 59 % bzw. 60 %. Fazit für die Praxis Die CATCH-Studie repräsentiert die bislang größte, randomisierte und

kontrollierte Studie, die sich mit der Behandlung von tumorassoziierten Thrombosen befasst. Ihre Ergebnisse bestätigen die Überlegenheit der Rezidivprophylaxe mit einem NMH (Tinzaparin) gegenüber Warfarin bei Patienten mit aktiver Tumorerkrankung und akuter, symptomatischer VTE: Das kumulative Risiko für ein VTERezidiv verringerte sich unter Tinzaparin von 10,5 % auf 7,2 %, wobei die Patienten von einer signifikanten Risikoreduktion für symptomatische TVT um mehr als 50 % profitierten. Besonders wichtig für die Praxis ist, dass die Behandlung mit Tinzaparin in voller therapeutischer Dosierung das Risiko für schwere Blutungen nicht erhöhte und gleichzeitig das Risiko für klinisch relevante nicht schwerwiegende Blutungen signifikant verringerte. Fabian Sandner, Nürnberg

Literatur 1 Blom JW et al. J Am Med Ass 2005;293: 715-722 2 Lyman GH et al. J Clin Oncol 2013; 31:2189-204 3 Deutsche Gesellschaft für Angiologie – Gesellschaft für Gefäßmedizin e.V. (2010) S2-AWMF-Leitlinie Diagnostik und Therapie der Venenthrombose und der Lungenembolie. Stand 2010 4 Lee AY et al. N Engl J Med 2003;349: 146-153 5 Lee AY et al. A randomized trial of longterm tinzaparin, a low molecular weight heparin (LMWH), versus warfarin for treatment of acute venous thromboembolism (VTE) in cancer patients – the CATCH study. ASH, 6.–9.12.2014, San Francisco, USA © Verlag PERFUSION GmbH

ER FA HRUNG. ZU V E R LÄ SSIGKEI T. VE R T RA U EN . DIE WELT VON CLEXANE ®

25 140 >

Erfahrung geben JAHRE Sicherheit

Weltweit zugelassen in mehr als L Ä NDE R N M E H R

A L S

17.000 500.000.000 Indikationsspektrum Ü B E R

wissenschaftliche Publikationen mit Enoxaparin1 behandelte Patienten

B R E I T E S T E S

www.thromboseportal.eu

Prophylaxe, Therapie, Kardiologie, Hämodialyse 2

Clexane® 20 mg / Clexane® 40 mg / Clexane® 20 mg Duo / Clexane® 40 mg Duo / Clexane® 60 mg / Clexane® 80 mg / Clexane® 100 mg / Clexane® multidose 100 mg/ml / Clexane® 20 mg Praxis / Clexane® 40 mg Praxis / Clexane® 20 mg Klinik / Clexane® 40 mg Klinik / Clexane® multidose akut / Clexane® multidose Praxis. Wirkstoff: Enoxaparin-Natrium Zusammens.: 1 Fertigspritze 20/-40/-60/-80/-100/- enth.: 20/40/60/80/100 mg Enoxaparin-Na (entspr. 2.000/4.000/6.000/8.000/10.000 I.E. anti-Xa. - multidose® 100mg/ml enth.: Arzneil. wirks. Bestandt.: 100 mg Enoxaparin (entspr. 10.000 I.E. anti-Xa) pro 1 ml Injekt.-lsg. Sonst. Bestandt.: Wasser f. Injekt.-zwecke. Zusätzl. –multidose: 15 mg/ ml Benzylalkohol. Anw.-geb.: Thromboseprophyl. u. Gerinnungshemmung b. extrakorpor. Kreislauf währ. d. Hämodialyse. -20 mg/-20 mg Duo/-20 mg Praxis/-Klinik/-multidose: Peri- u. postoperat. Primärprophyl. d. TVT b. Pat. m. niedrig. od. mittlerem thromboembol. Risiko (z. B. Allgem.chir.). -40 mg/-40 mg Duo/-40 mg Praxis/-Klinik/-multidose: Peri- u. postoperat. Primärprophyl. d. TVT b. Pat. m. hohem thromboembol. Risiko (z. B. orthopäd. Chir.). Primärprophyl. d. TVT b. nicht chir. Pat. m. mittlerem od. hohem thromboembol. Risiko b. akuten schweren intern. Erkrank. (Herzinsuff. NYHA III bzw. IV, Infektionen, respirator. Erkrank.), die weitgeh. Immobilisation z. Folge haben. - 60 mg/- 80 mg/-100 mg /-multidose: Ther. d. TVT m./oh. Lungenembolie; Ther. d. instab. A. pectoris u. des NSTEMI; Prävent. erneut auftretender schw. A. pectoris od. e. drohenden MI bei instab. A. pectoris od. NSTEMI. Ther. d. akuten STEMI b. Pat. die konservativ od. konservativ m. nachfolg. PCI versorgt werden. Thromboseprophyl. u. Gerinnungshemmung b. extra-korpor. Kreislauf währ. d. Hämodialyse. Gegenanz.: Überempfindl. gg. Enoxaparin-Na, Heparin od. Heparin-Abkömml., einschl. anderer niedermolek. Heparine. Kürzl. zurücklieg. Verletzungen od. OPs a. ZNS, Auge, Ohr. Kürzl. zurücklieg. klin. relev. Blutung. Akuter od. <6 Mo. zurücklieg. (od. Verdacht auf) hämorrhag. Schlaganfall od. and. intrakran. Blutungen. Akute od. anamnest. bek. intrakraniale Erkrank. Klin. relev. Gerinnungsstör. (auch i. d. Anamnese, Mangel an Gerinnungsfakt., Thrombozytopenie). Magen- od. Darmulzera. Abortus imminens. Schw. Leber- od. Pankreaserkrank. Unkontrollierb. schw. Hypertonie. Endokarditis. Aktuelle od. anamnest. bek. HIT Typ II. Verdacht a. vaskul. Retinopathie, Glaskörperblut. od. and. intraokul. Blutung. Zusätzl. - 60/- 80/- 100 mg /-multidose: Instabile A. pectoris, wenn vor Therapiebeg. orale Antikoagulanzien m. INR >1,4 bzw. aPTT >41 Sekunden. Gleichzeit. Anw. e. Spinal-, Peridural-, Epiduralanästh. od. Lumbalpunktion. Zusätzl. - multidose: Säugl. u. Kleinkind. bis 3 J. (Gehalt an Benzylalkohol, cave Gasping-Syndrom!). CABG-OP frühestens 12 h nach letzter Gabe v. Enoxaparin! Warnhinw. u. Vorsichtsmaßn.: Nicht i.m. verabreichen. Bes. Vorsicht b. erhöh. Blutungsrisiko (Stör. d. Thrombozytenfkt., gering bis mäßig eingeschr. Leber- od. Pankreasfkt., pept. Ulzera i. d. Vorgesch., Verdacht a. Malignome m. Blutungsneig., Nieren- /Harnleitersteine, gleichzeit. Behandl. m. OAK. In Abhängigk. v. Dos. vermehrt offene od. okkulten Blutungskomplik. Auslösung HIT Typ II mögl. Vorsicht b. gleichzeit. Anw. v. AM, die den Serum-K-Spiegel erhöhen. Vor Behandl. mögliche vorbesteh. Blutgerinnungsanomalien abklären, währ. d. Behandl. entspr. Laborkontr. durchführen. Auch niedermolek. Heparine können HIT-Typ II auslösen. Anw. b. Pat. m. künstl. Herzklappen nur nach strenger Indikation. B. älteren Pat. m. besond. Vorsicht anw. (eingeschr. Nierenfkt., erhöht. Blutungsrisiko). B. stark eingeschränkt. Nierenfkt. Blutungsrisiko erhöht (Anti-Xa-Spitzenspiegel überwachen). Anw. b. Kdr. keine Erfahr. Pat. m. geringem KG (Frauen <45 kg; Männer <57 kg): erhöht. Blutungsrisiko, Dos.anpassung erwägen. Adipöse Pat. (BMI >30 kg/m2) haben erhöhtes thromboembolisches Risiko, Sicherheit u. Wirksamk. nicht abschließend bestimmt, sorgf. Überwachung empf. B. Pat., die eine rückenmarknahe Regionalanästhesie (Peridual-/Spinalanästhesie) bzw. Lumbalpunktion erhalten u. mit Clexane antikoaguliert werden, kann es in seltenen Fällen zu einem epiduralen o. spinalen Hämatom kommen, welches zu neurologischen Komplikationen führen kann. Zusätzl. – 60/80/100 /-multidose: Keine Erfahr. b. koronarer Bypass-OP. Zur gleichzeit. Durchführung e. PTCA, kombin. Anw. m. Gp-IIb/IIIa Inhibit., STEMI u. Gefäßpunktion siehe FI. Schwangersch. u. Stillz.: Während d. Schwangersch. strenge Indikationsstellung. Währ. Stillz. gerinnungshemm. Effekt auf Sgl. unwahrscheinlich. Nebenw.: Klin. Studien: Gefäße: Sehr häufig Blutung (sehr häufig b. Chir. Prophyl., häufig b. internist Prophyl.). Gelegentl. intrakranielle Blutung (gelegentl. b. chir. Prophyl., Behandl. d. STEMI. Selten b. Behandl. d. AP u. NSETMI). Selten retroperitonoeale Blutung (internist. Prophyl.). Blut, Lymphsyst.: Sehr häufig Thrombozytose (Chir. Prophyl.); Thrombozytopenie (häufig b. Chir. Prophyl. Gelegentl. b. internist. Prophyl.). Weitere NW aus klin. Studien: Blut u. Lymphsystem: Selten asymptomat. Leukopenien. Immunsyst.: Häufig allerg. Reakt. Selten anaphylakt., anaphylaktoide Reakt. inkl. Schock. Nerven: Häufig Kopfschmerzen. Leber, Galle: Sehr häufig erhöh. Leberenzyme. Haut, Unterhautzellgew.: Häufig Urtikaria, Pruritus, Erythem. Gelegentl. bullöse Dermatitis. Allgemein: Häufig Hämatom, Schmerzen, sonstige Reakt. (Ödem, Blutung, Überempfindl., Entzündung, Verhärtung) a. d. Injektionsst. Gelegentl. lokale Irritation, Hautnekrose a. d. Injekt.-stelle. Untersuchungen: Selten Hyperkaliämie. Nach Markteinführ. (alle Häufigkeiten nicht bekannt): Gefäße: spin./neuraxiale Hämatome (gleichzeit. Perid.-/Spinal-Anästh. o. Lumbalpunkt.) mit. neurolog. Folgeschäden. Blut u. Lymphsyst.: Blutungsanämie, immuno-allerg. Thrombozytopenie m. Thrombosebildung (i. einig. Fällen m. Organinfarkt od. Ischämie d. Gliedmaßen), Eosinophilie. Haut, Unterhautzellgew.: Kutane Vaskulitis, Hautnekrose (dann Ther. beenden!) od. Knötchen a. d. Injekt.-stelle, Alopezie. Leber u. Galle: hepatozelluläre od. cholestat. Leberschäd. Skelettmskl, Bindegew., Knochen: Osteoporose nach Langzeitbehandl. Einzelfälle Azidose, Hautnekrosen, Priapismus, Hypotonie, Bradykardie, Hypoaldosteronismus, Überempf.-reakt. durch Benzylalkohol sind mögl. Klin.-chem. Untersuch.-ergebnisse können verfälscht werden. Verschreibungspflichtig. Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt am Main. Stand: Mai 2014 (037807). AVS 801 14 085-038015

1 EMBASE Datenbank online, Stand: 01.10.2014 2 Gilt für in Deutschland zugelassene NMH; Anwendungsgebiete gemäß jeweiliger Fachinformation, Stand: 01.10.2014 Leistungsstark gegen Thrombosen.

FORUM CARDIOLOGICUM

Evolut™ R – eine innovative Aortenklappe für inoperable Patienten

Ist die Herzklappe zwischen Hauptschlagader und linker Herzkammer verkalkt, wird mehr Druck benötigt, um Blut in die Hauptschlagader zu pumpen. Als Folge vergrößert sich der Herzmuskel krankhaft und die Patienten leiden an zunehmender Luftnot sowie einem Engegefühl in der Brust. Ohne eine neue Herzklappe stirbt die Hälfte der schwer Betroffenen innerhalb von 2 Jahren. Die herkömmliche Operation am offenen Herzen ist jedoch insbesondere bei schwerkranken Patienten ist nicht möglich, jeder Dritte ist auf alternative Behandlungsmethoden angewiesen. Eine lebensrettende Alternative ist der Transkatheter-Aortenklappenersatz, der mit dem innovativen CoreValve® Evolut™ R System von Medtronic jetzt noch schonender und präziser erfolgen kann. Besserer Zugang und optimale Platzierung Mit der Evolut™ R-Aortenklappe können auch Patienten mit verkalkten und engen Zugangsgefäßen behandelt werden, da der Einführkatheter einen Duchmesser von nur 4,5 mm hat. Damit sinkt auch die Gefahr, dass die Zugangsgefäße beim Einführen verletzt werden. Komplikationen wie Infektionen oder Blutungen nach der Operation werden so maßgeblich verringert. Eine bedeutende Verbesserung beim CoreValve® Evolut™ R System ist, dass die Klappe bei der Implantation bei Bedarf wieder auf den Katheter zurückgezogen werden kann, sodass

Perfusion 01/2015

28. Jahrgang

TAVI-System Core Valve® Evolut™ R

Die Evolut™R-Aortenklappe aus biologischem Material (Schweineherzbeutel) ist in ein selbstentfaltendes Geflecht aus Metall (Nitinol) integriert. Sie wird schmal zusammengefaltet auf einen Katheter geladen, der durch das Gefäßsystem navigiert werden kann. Im Herzen wird die Aortenklappe aus dem Katheter geschoben. Durch den Rückzug der Schutzhülle und unter Einfluss der Körperwärme entfaltet sich die Metallstütze und mit ihr die neue Herzklappe. Die Segel der nicht mehr funktionstüchtigen natürlichen Aortenklappe werden dabei seitlich an die Gefäßwand gedrückt. Das metallene Stützgeflecht passt sich mit seiner Spannkraft der Anatomie an der Mündung der Aorta in die linke Herzkammer an und verankert sich so selbst. Die neue Herzklappe fängt sofort an zu arbeiten. Sollte nicht gleich die perfekte Position erreicht werden, ermöglicht das Nitinol den Wiedereinzug in den Katheter und die Repositionierung (© Medtronic).

sie sich an der optimalen Position platzieren lässt. Die selbstexpandierende Evolut™ R-Aortenklappe gibt es in verschiedenen Durchmessern (23, 26, 29 bis zu 31 mm). Sie ist anatomisch geformt und ihr flexibles Metallgerüst passt sich perfekt an die individuelle

Anatomie des Patienten an. Dadurch wird auch ein nicht exakt runder Anulus perfekt abgedichtet und die bestmögliche hämodynamische Performance gewährleistet. Brigitte Söllner, Erlangen

Kongresse

KONGRESSE Insulin glargin: Bessere Stoffwechsel einstellung und weniger Hypoglykämien Für Menschen mit Typ-2-Diabetes, die mit Lebensstiländerungen und oralen Antidiabetika allein ihre Blutzuckerwerte nicht ausreichend kontrollieren können, gibt es zahlreiche Möglichkeiten der Therapieintensivierung. Anlässlich einer Pressekonferenz in Berlin wurden die Vorteile einer basalunterstützten oralen Therapie (BOT) mit Insulin glargin (Lantus®) diskutiert. BOT mit Insulin glargin senkt HbA1c-Wert stärker In einer gepoolten Analyse wurden Patientendaten aus 9 randomisierten, kontrollierten Studien zusammengefasst, in denen jeweils die BOT mit Insulin glargin mit anderen Therapieregimen verglichen worden war. Zu den Vergleichstherapien gehörten Lebensstiländerungen, die Zugabe weiterer oraler Antidiabetika oder Mischinsulin, der Beginn einer Insulintherapie mit NPH-Insulin sowie der Start einer supplementären Insulintherapie (SIT) mit Insulin lispro. Alle eingeschlossenen Patienten wiesen kardiovaskuläre Risikofaktoren wie Hypertonie, Dyslipidämie oder/und eine koronare Herzkrankheit auf. Sowohl hinsichtlich der Effektivität der Behandlung als auch hinsichtlich der Vermeidung von Hypoglykämien schnitten die Patienten unter Insulin glargin besser ab, betonte Professor Markolf Hanefeld, Dresden: „Eine HbA1c-Senkung auf Werte ≤7 % und/ oder um mindestens einen Prozentpunkt erzielten 57,7 % der Patienten unter Insulin glargin, aber nur 51,4 % in den Vergleichsgruppen. Es wurden 5,04 vs. 7,01 bestätigte Hypoglykämien pro Patientenjahr beobachtet.“ Der Vorteil der BOT mit Insulin glargin gegenüber den Vergleichstherapien war jeweils signifikant (p<0,001). Perfusion 01/2015

28. Jahrgang

Seltener Hypoglykämien als unter NPH-Insulin

Insulin glargin steht für hohe Therapiepersistenz

Die Vermeidung von Hypoglykämien spielt eine wichtige Rolle in der Insulintherapie, nicht nur bei kardial vorgeschädigten Patienten. Dass Insulin glargin hier sicherer ist als NPHInsulin, bestätigen weitere Studien. So wurde die Outcome-Analyse einer 5-Jahres-Studie von Rosenstock et al. vorgestellt, in der dieser seine Beobachtung aus einer früheren eigenen Studie, die Häufung von Hypoglykämien unter NPH-Insulin, noch einmal genauer betrachtete. Es bestätigte sich, dass die Zahl symptomatischer Hypoglykämien pro Patientenjahr unter Insulin glargin deutlich niedriger war als unter NPH-Insulin. Die Rate Ratio betrug 0,72 zugunsten von Insulin glargin, dieser Vorteil war signifikant (p=0,001). Dabei spielte es keine Rolle, welchen HbA1c-Wert die Patienten erreicht hatten. Die Überlegenheit von Glargin war besonders ausgeprägt bei der Vermeidung nächtlicher Unterzuckerungen.

Dr. Franz-Werner Dippel, Berlin, stellte Daten zum Einsatz von Insulin glargin unter realen Versorgungsbedingungen vor. In einer retrospektiven Datenbankanalyse mit Daten von 17.472 US-amerikanischen Patienten wurden die medizinischen und ökonomischen Konsequenzen eines Therapiewechsels zwischen Insulin glargin und Insulin detemir untersucht. Die 13.882 Patienten der Kohorte 1 wurden seit mindestens 6 Monaten mit Insulin glargin behandelt. Wurde diese Therapie fortgesetzt, gehörten sie zur Gruppe der Glargin-Continuer (GLAC). Wurden die Patienten auf Insulin detemir umgestellt, wurden sie als Detemir-Switcher (DET-S) erfasst. Die Kohorte 2 umfasste 3.590 Patienten, die ihre Therapie mit Insulin detemir begonnen hatten. Sie setzten die Behandlung entweder mit Insulin detemir fort (Detemir-Continuer, DET-C) oder wechselten zu Insulin glargin (GlarginSwitcher, GLA-S). Strukturelle Unterschiede zwischen den Gruppen wurden mittels Propensity Score Matching ausgeglichen. Letztlich gingen die Daten von 5.921 Patienten in die Auswertung ein (GLA-C: n=3.930; DET-S: n=792; DET-C: n=780; GLA-S: n=419). Die Verweildauer (Persistenz) der Glargin-Continuer bei ihrer Therapie war nach 1 Jahr Follow-up signifikant höher als die Persistenz der DetemirSwitcher; sie betrug im Mittel 281 vs. 250 Tage (p<0,0001). Zudem waren die HbA1c-Werte der Glargin-Continuer zum Ende der Beobachtungszeit deutlich niedriger (8,35 vs. 8,64 %, p<0,0165). 40 % der Detemir-Switcher wechselten zurück zu Insulin glargin. Die Patienten der Kohorte 2 zeigten dagegen in beiden Gruppen eine ähnliche Persistenz mit einer mittleren Verweildauer von 262 vs. 268 Tagen. Nur 20 % der Patienten der Glargin-Switcher wechselten zu Insulin detemir zurück. Fazit: In der Versorgungsrealität profitieren Menschen mit Typ-2-Diabetes, die mit Insulin detemir behandelt werden (DET-C), von einem Wechsel auf Insulin glargin (GLA-S). Elisabeth Wilhelmi, München

Therapiesicherheit in ORIGINStudie bestätigt Hanefeld erinnerte an die ORIGINStudie: Sie schloss 12.537 Patienten ein, die entweder einen frühen Typ2-Diabetes hatten (89 %) oder eine erhöhte Nüchternglukose bzw. gestörte Glukosetoleranz (11 %). Insulin glargin ist zur Therapie von Patienten mit manifestem Diabetes mellitus zugelassen. Über die Beobachtungsdauer von mehr als 6 Jahren war Insulin glargin neutral in Bezug auf kardiovaskuläre Risiken, darüber hinaus wurde die onkologische Sicherheit bestätigt. „Der mediane HbA1c-Wert der Patienten blieb über die gesamte Zeit ≤6,5 %“, konstatierte Hanefeld. Dabei war es in der Glargin-Gruppe seltener als in der Kontrollgruppe notwendig, 2 oder gar 3 orale Antidiabetika einzusetzen. Schwere Hypoglykämien traten in beiden Studienarmen selten auf; zudem waren sie in der Gruppe mit Insulin glargin seltener mit kardiovaskulären Komplikationen verbunden.

Kongresse

Orale Antikoagulation durch Faktor-XaInhibition: Ausblick und Einblick 2015 Zwischen Nicht-VKA oralen Antikoagulanzien (NOAKs) bestehen relevante substanzspezifische Unterschiede, die es bei der Anwendung im klinischen Alltag zu berücksichtigen gilt. Aktuelle Aspekte der oralen Antikoagulation durch NOAKs diskutierten Experten auf einem Symposium, das im Rahmen der 32. Arbeitstagung NeuroIntensivMedizin (ANIM) stattfand. Vorteile bei der Therapie und Sekundärprophylaxe venöser Thromboembolien Der Umgang mit den zahlreichen Limitationen der VKA gehörte über Jahrzehnte hinweg zum Praxisalltag. Professor Ulrich Hoffmann, München, erinnerte an das enge therapeutische Fenster, die individuelle Variabilität des antikoagulatorischen Effektes, die zahlreichen Arzneimittel- und Nahrungsmittelwechselwirkungen sowie den langsamen Wirkungseintritt bzw. Beendigung der Wirkung. Bei der Therapie mit NOAKs spielen diese Faktoren im klinischen Alltag kaum eine Rolle. Dies vereinfacht die orale Antikoagulation erheblich. Hoffmann verdeutlichte dies am Beispiel der Entwicklung des Faktor-Xa-Inhibitors Edoxaban, der in einem großen PhaseIII-Studienprogramm mit rund 30.000 Patienten untersucht wurde, unter anderem in der Therapie und Sekundärprophylaxe venöser Thromboembolien (VTE). Die Phase-3-Studie HOKUSAI-VTE untersuchte die Wirksamkeit und Sicherheit von Edoxaban bei 8.292 Patienten mit akuter, symptomatischer tiefer Venenthrombose und/oder Lungenembolie. Die Patienten wurden wie in der Praxis allgemein üblich zunächst initial mit Heparin behandelt und anschließend je nach klinischem Bild für 3–12 Monate oral antikoaguliert. Die Auswertung nach 12 Monaten bePerfusion 01/2015

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stätigte die Nicht-Unterlegenheit von Edoxaban (60 mg einmal täglich) versus Warfarin und dokumentierte, dass klinisch relevante Blutungen unter Edoxaban seltener auftraten als unter der Vergleichstherapie. Antikoagulation bei nicht valvulärem Vorhofflimmern: Einblicke in die Studie ENGAGE AF-TIMI 48 Bei Patienten mit nicht valvulärem Vorhofflimmern (non valvular atrial fibrillation, NVAF) belegte die Phase-III-Studie ENGAGE AF-TIMI 48 (Effective aNticoaGulation with Factor XA Next GEneration in Atrial Fibrillation) die Wirksamkeit und Sicherheit von Edoxaban zur Schlaganfallprophylaxe. Die 21.105 Studienteilnehmer erhielten randomisiert jeweils einmal täglich entweder eine hohe EdoxabanDosis (60 mg), eine niedrige EdoxabanDosis (30 mg) oder eine Vergleichstherapie mit dem VKA Warfarin (Ziel-INR 2,0–3,0). Wie Professor Wilhelm Haverkamp, Berlin, ausführte, wurde in beiden Regimen die Dosis von Edoxaban halbiert, wenn eine leichte Niereninsuffizienz (Kreatinin-Clearance 30– 50 ml/min) vorlag, das Körpergewicht unter 60 kg lag oder wenn die Patienten gleichzeitig mit starken P-Glykoprotein-Inhibitoren wie Verapamil, Chinidin oder Dronedaron behandelt wurden, da diese Faktoren die Wirkstoffexposition und damit das Blutungsrisiko erhöhen. Bei den Risikopatienten, die eine reduzierte Dosis erhielten (30 bzw. 15 mg), zeigten sich im Plasma erniedrigte Edoxaban-Talspiegel und eine herabgesetzte Anti-Faktor-Xa-Aktivität. Dies hatte jedoch keinen Einfluss auf die Wirksamkeit von einmal täglich Edoxaban: Die Risikopatienten waren in einem vergleichbaren Ausmaß vor Schlaganfällen und systemischen embolischen Ereignissen (SEE) geschützt wie die Patienten, bei denen die Dosis nicht reduziert worden war (jeweils im Vergleich zur VKA-Gruppe). Gleichzeitig war das Blutungsrisiko in beiden dosisreduzierten Gruppen deutlich geringer als unter dem VKA.

Besonderheiten und Unterschiede der NOAKs Professo Dietmar Trenk, Bad Krozingen, wies in diesem Zusammenhang darauf hin, dass zwischen den verschiedenen NOAKs wichtige Unterschiede in der Pharmakokinetik bestehen. Therapierelevant sind insbesondere die Unterschiede bei renaler Clearance und CYP-Metabolismus, die das Nutzen-Risiko-Verhältnis des einzelnen Patienten beeinflussen und eine Dosis anpassung erfordern. Für Edoxaban ist eine individuelle Dosisanpassung anhand weniger klinischer Kriterien möglich, nämlich einer eingeschränkten Nierenfunktion, einem Körpergewicht ≤60 kg oder einer Komedikation mit P-Glykoprotein-Inhibitoren. Für alle NOAKs gilt, dass sie aufgrund ihres schnellen Wirkungseintritts und der kürzeren Halbwertszeit ein vereinfachtes Therapiemanagement erlauben, z.B. ist kein Bridging bei Operationen notwendig. Therapieadhärenz ist bei alten und neuen oralen Antikoagulanzien ein „Muss“. Trenk erinnerte in diesem Zusammenhang daran, dass die Einstellung der VKA-Wirkung bzw. der INR aufgrund zahlreicher Nahrungsmittelund Arzneimittelinteraktionen trotz eines engmaschigen Monitorings ein erhebliches Problem darstellen kann. Selbst in klinischen Studien ist die INR-Einstellung der Patienten oft nicht zufriedenstellend. Aktivitäts- und Spiegel bestimmungen im klinischen Alltag Die Therapie mit NOAKs erfordert im Gegensatz zur VKA-Therapie kein therapiebegleitendes Monitoring. Dies vereinfacht die Anwendung für Arzt und Patient. Ein spezifisches Monitoring kann jedoch in bestimmten Situationen wünschenswert sein. Dr. Robert Klamroth, Berlin, nannte hier unter anderem die Abschätzung der Eliminationsdauer der Substanz in einer Notfallsituation, wenn bei Blutungen Unklarheit über eine NOAK-Einnahme besteht oder die Bestimmung © Verlag PERFUSION GmbH

Kongresse

des Talspiegels zum Ausschluss einer Wirkstoffkumulation z.B. bei Niereninsuffizienz. Konventionelle Gerinnungstests werden durch NOAKs unterschiedlich beeinflusst. Während Fibrinogen und die Thrombinzeit nicht von Faktor-XaInhibitoren beeinflusst werden, kann schon ein leicht erniedrigter QuickWert auf eine deutlich erhöhte Blutungsneigung hinweisen. Wenn der Quick (Reagens: Neoplastin) und die partielle Thromboplastinzeit normal sind, ist nach den Worten von Klamroth eine relevante Gerinnungshemmung durch NOAKs unwahrscheinlich. Zur direkten Messung der Plasmakonzentrationen von Faktor-Xa-Inhibitoren kann der Anti-Faktor-Xa herangezogen werden. Da der Test eine Kalibrierung erfordert, ist das beauftragte Labor darüber zu informieren, welcher FaktorXa-Hemmer eingenommen wurde. Spezifische Antidote für Faktor-XaInhibitoren sind (noch) nicht verfügbar; probatorisch können ProthrombinKomplex-Präparate und Faktor VIIa erwogen werden. Fabian Sandner, Nürnberg

Gerinnungsmanagement in Notfallsituationen Wie lassen sich antikoagulierte Patienten im Notfall behandeln? 250 Intensiv- und Notfallmediziner diskutierten am 4. Dezember 2014 auf dem Symposium von Boehringer Ingelheim anlässlich des 14. Deutschen Interdisziplinären Kongresses für Intensiv- und Notfallmedizin (DIVI) in Hamburg bestehende und künftige Strategien zur Aufhebung des gerinnungshemmenden Effekts. Für antikoagulierte Patienten steht im klinischen Alltag eine Reihe an Optionen zum Gerinnungsmanagement in der Notfall- und Intensivmedizin zur Verfügung. Diese Optionen kommen auch bei der Aufhebung der gerinnungshemmenden Wirkung von Dabigatran (Pradaxa®) zum Einsatz. Mit der Entwicklung eines spezifischen Antidots will Boehringer Ingelheim künftig diese Möglichkeiten erweitern. Perfusion 01/2015

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Neue Daten zu Idarucizumab Das in Entwicklung befindliche, spezifische Antidot Idarucizumab, ein humanisiertes Antikörperfragment (Fab), wurde im Rahmen des RE-VERSE AD™Programms bei 46 gesunden männlichen und weiblichen Erwachsenen mittleren Alters (45–64 Jahre), bei älteren Menschen (65–80 Jahre) und bei Personen mit leichten bis mittelgradigen Nierenfunktionsstörungen untersucht. Die Freiwilligen erhielten 3 ½ Tage lang Dabigatran, bevor das Antidot verabreicht wurde. Bei einigen Teilnehmern wurde die Dabigatran-Therapie 24 Stunden nach Gabe des Antidots wieder aufgenommen. Die Ergebnisse der Studie zeigten Folgendes: • Eine 5-minütige Infusion mit Idarucizumab bewirkte die sofortige, vollständige und anhaltende Aufhebung der Dabigatran-induzierten Gerinnungshemmung in beiden untersuchten Altersgruppen sowie bei Personen mit eingeschränkter Nierenfunktion. • Die Behandlung mit Dabigatran konnte 24 Stunden nach der Gabe von Idarucizumab wieder aufgenommen werden und führte zur erneuten effektiven Gerinnungshemmung. • Eine zweite Gabe von Idarucizumab 2 Monate nach der ersten führte gleichermaßen zur vollständigen Aufhebung der Dabigatran-induzierten Antikoagulation. • Idarucizumab wurde gut vertragen – es wurden keine klinisch relevanten unerwünschten Ereignisse berichtet. • Die 5-g-Dosis Idarucizumab hob die Antikoagulation unabhängig von Alter und Nierenfunktion gleichermaßen auf. Diese Dosis wird derzeit im klinischen Umfeld untersucht. (Quelle: Glund S et al. Idarucizumab, a specific antidote for dabigatran: immediate, complete and sustained reversal of dabigatran induced anticoagulation in elderly and renally impaired subjects. Presented on 8th December at the 56th American Society of Hematology Annual Meeting & Exposition, San Francisco, USA)

Optionen bei der Behandlung mit NOAK In der Schlaganfallprävention seien die Nicht-Vitamin-K Oralen Antikoagulanzien (NOAK) eine wirksame und zum Teil sicherere Alternative zu Vitamin-K-Antagonisten, betonte Professor Georg Nickenig, Bonn. Im Fall von schwerwiegenden Blutungskomplikationen hätten Patienten beispielsweise unter Dabigatran eine bessere Überlebensprognose und verbrachten weniger Zeit auf der Intensivstation als mit Warfarin behandelte Patienten. NOAKs erfordern im Regelfall kein routinemäßiges Gerinnungsmonitoring. Dennoch fühle sich mancher Behandler bei akuten Interventionen sicherer, wenn er wisse, wie viel Antikoagulanz noch im Blut sei, erklärte Professor Hanno Riess, Charité Berlin. Er zeigte auf, welche Messmethoden bei welcher Substanz aussagekräftige

Ergebnisse lieferten. So stehe bei Dabigatran neben der aktivierten partiellen Thromboplastinzeit (aPTT) auch die Thrombinzeit als laborchemischer Gerinnungsparameter sowie die verdünnte Thrombinzeit (dTT) mittels Hemoclot®-Test zur Verfügung. Um Ärzten für die seltenen klinischen Notfallsituationen eine zusätzliche Behandlungsoption an die Hand zu geben, entwickelt Boehringer Ingelheim mit Idarucizumab* ein hochspezifisches Antidot zu Dabigatran. Die aktuell weltweit laufende RE-VERSE AD™-Patientenstudie zum Einsatz von Idarucizumab ist die erste Studie, die ein Antidot für ein NOAK an Patienten untersucht. Auch erste Kliniken in Deutschland nehmen als Studienzentren an RE-VERSE AD™ teil. Aktuelle Ergebnisse wurden auf der ASHJahreskonferenz 2014 in San Francisco vorgestellt (siehe Insert). Elisabeth Wilhelmi, München © Verlag PERFUSION GmbH

Mitteilungen

MITTEILUNGEN Neuer Pen vereinfacht Anwendung des DepotExenatide Bydureon® Ab sofort ist das Depot-Exenatide Bydureon® in einem Fertigpen verfügbar. Der praktische und anwenderfreundliche Pen erleichtert die Vorbereitung und Applikation des einmal wöchentlich zu injizierenden GLP-1-Analogons. So entfällt das bisher notwendige Übertragen der Wirkstoffsuspension aus der Ampulle in eine Spritze und auch die subkutane Selbstinjektion wird dadurch einfacher. Bydureon® ist bei erwachsenen Typ-2-Diabetikern in Kombination mit Metformin, Sulfonylharnstoff, Thiazolidindionen, Metformin und Sulfonylharnstoff oder Metformin und Thiazolidindionen indiziert, wenn mit der maximal verträglichen Dosis dieser Therapien keine angemessene Blutzuckerkontrolle erreicht werden kann. Hierbei ermöglicht die jeweils injizierte Dosis von 2 mg neben einer starken und anhaltenden HbA1c-Senkung auch eine Gewichtsreduktion. Vorteile der Depotform Bydureon® ist eine Depotformulierung von Exenatide, einem GLP-1-Rezeptor-Agonisten, der den Blutzucker bei Typ-2-Diabetes über den Inkretin-

Effekt senkt. Während kurzwirksames Exenatide zweimal täglich injiziert werden muss, ist dies bei Bydureon® ohne Titration nur einmal pro Woche der Fall, da der Wirkstoff nach der Injektion verzögert aus Mikrosphären freigesetzt wird. Die gleichmäßige Verfügbarkeit des langwirksamen DepotExenatide ermöglicht im Vergleich zur kurzwirksamen, täglich zu applizierenden Formulierung von Exenatide eine bessere glykämische Kontrolle mit weniger Hypoglykämien und gastrointestinalen Nebenwirkungen. Geeignet für eine Therapie mit langwirksamem Depot-Exenatide können zudem Patienten sein, die sich um Gewichtszunahme oder Hypoglykämien sorgen oder eine tägliche Injektion vermeiden möchten. Einziges GLP-1-Analogon mit 6-Jahres-Daten In den klinischen DURATION-Studien senkte Bydureon® den HbA1c nach 24–30 Wochen um 1,3–1,9 % und bewirkte eine Gewichtsreduktion um 2,3–3,7 kg. Es ist zudem der einzige langwirksame GLP-1-Rezeptor-Agonist, dessen Wirksamkeit und Verträglichkeit über 6 Jahre hinweg nachgewiesen wurde. Hierbei ergab die offene Verlängerung der DURATION-1-Studie mit 127 Patienten, dass die erreichte signifikante HbA1c-Absenkung unter Bydureon® über diesen Zeitraum aufrechterhalten werden konnte (–1,6 %; 95%-KI –1,9 bis –1,4), was auch für die Gewichts-

Bydureon®-Pen: Einfache Anwendung in 3 Schritten: 1. Vorbereiten des Pens durch Anbringen der Nadel und Drehen des Griffs bis zum Klick 2. Mischen der Suspension durch Klopfen, bis eine gleichmäßig getrübte Lösung ohne Klumpen entsteht 3. Aktivieren des Injektionsknopfs durch Drehen am Griff, Abziehen der Schutzkappe von der Nadel und Injektion Perfusion 01/2015

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reduktion (–4,3 kg; 95%-KI –6,0 bis –2,6) galt (p jeweils <0,05). Die Langzeitstudie zeigte zudem keine unerwarteten Beobachtungen hinsichtlich der Verträglichkeit, wobei die Inzidenz von Übelkeit und Reaktionen an der Injektionsstelle im Laufe der Zeit abnahm. E. W.

Stabile symptomatische KHK: EMA attestiert Ivabradin weiterhin positive Nutzen-RisikoBilanz Nach Abschluss eines Review-Verfahrens folgte die Europäische Arzneimittel-Agentur (EMA) den Vorschlägen des Ausschusses für Pharmakovigilanz/Risikobewertung (PRAC) vom 7. November und veröffentlichte ihre abschließenden Empfehlungen zur Verordnung von Ivabradin bei stabiler symptomatischer KHK. Ivabradin (Procoralan®) wurde unverändert mit einem positiven Nutzen-Risiko-Verhältnis bewertet. Die EMA-Empfehlungen sehen geringfügige Änderungen der Fachinformation vor, die dazu beitragen sollen die Therapiesicherheit weiter zu verbessern. Der Ausschuss für Humanarzneimittel (CHMP) der EMA kommt zu dem Schluss, dass das Nutzen-Risiko-Profil von Ivabradin in den beiden zugelassenen Indikationen stabile symptomatische KHK und chronische Herzinsuffizienz unverändert als positiv zu bewerten ist. Wichtige Empfehlungen zum Einsatz von Ivabradin bei stabiler symptomatischer KHK • Eine Behandlung mit Ivabradin sollte bei stabiler symptomatischer KHK ab einer Ruhe-Herzfrequenz (HF) ≥70/min begonnen werden. • Die symptomatische Therapie der Beschwerden steht im Vordergrund der Therapie. Sollte nach 3-monati© Verlag PERFUSION GmbH

Mitteilungen

ger Therapie keine wesentliche Besserung der Angina-pectorisSymptomatik eintreten, ist die Therapie neu zu evaluieren. • Es wird empfohlen, unter der Ivabradin-Therapie ein regelmäßiges Monitoring auf Vorhofflimmern (VHF) durchzuführen. Bei neu auftretendem VHF unter Ivabradin sollten Nutzen und Risiken sorgfältig abgewogen werden. • Der Einsatz von Verapamil oder Diltiazem ist in Kombination mit Ivabradin kontraindiziert. • Die Ivabradin-Anfangsdosis sollte 2 × 5 mg/d und die Ziel- bzw. Erhaltungsdosis 2 × 7,5 mg/d nicht übersteigen. • Vor dem Beginn oder Hochdosieren von Ivabradin sollte eine sorgfältige Bestimmung der Herzfrequenz erfolgen (mehrfache Ruheherzfrequenzmessungen oder Ruhe-EKG oder 24-h-EKG). • Fällt die Ruheherzfrequenz unter Ivabradin auf <50/min oder tritt eine symptomatische Bradykardie auf, muss die Dosis reduziert werden (die niedrigste Dosis beträgt 2 × 2,5 mg/d). Diese Empfehlungen basieren auf einem Review der finalen Daten der SIGNIfY-Studie, in der Ivabradin bei 19.000 Patienten mit stabiler KHK, aber ohne Zeichen einer Herzinsuffizienz, evaluiert wurde. Hinsichtlich des primären kombinierten Endpunkts, kardiovaskulärer Tod oder nicht tödlicher Myokardinfarkt, war SIGNIfY neutral. In dieser Studie erhielten die Patienten entgegen dem üblichen IvabradinDosierungsschema von zu Beginn 2 × 5 mg/d mit sukzessiver Steigerung auf die Zieldosis von 2 × 7,5 mg/d bereits eine Anfangsdosis von 2 × 7,5 mg/d und viele Patienten erhielten später 2 × 10 mg/d. Die Firma Servier hat alle Fachkreise über die neuen Empfehlungen informiert. Nach dem jetzt von der EMA ab-

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geschlossenen Review-Verfahren wird in Kürze eine entsprechende Anpassung der Procoralan®-Fachinformation erfolgen. F. S.

Sorin Group expandiert weiter Die Sorin Group, ein global agierendes Medizintechnikunternehmen mit Hauptsitz in Mailand, ist weltweiter Marktführer in der Herstellung von Produkten zur Behandlung von HerzKreislauf-Erkrankungen. Der Fokus liegt dabei auf 2 Therapiefeldern: • der Herzchirurgie (kardiopulmonale Bypass-Lösungen für Operationen am offenen Herzen sowie für den Herzklappenersatz bzw. die -reparatur) sowie • dem Herzrhythmus-Management (z.B. Schrittmacher, Defibrillatoren) Rund ein Drittel der weltweit implantierten künstlichen Herzklappen werden von der Sorin Group hergestellt und bei 50 % aller Herzoperationen weltweit kommen Geräte oder Systeme des Unternehmens zum Einsatz. Mit einem Weltmarktanteil von 70 % ist Sorin führend in der Herstellung von Herz-Lungen-Maschinen. Alle Herz-Lungen-Maschinen der Sorin Group werden in Deutschland am Standort Münchnen produziert. Die Anzahl der gefertigten Systeme wuchs in den letzten 4 Jahren um rund 26 %, sodass das im Jahr 2000 bezogene Werk im vergangenen Jahr auf insgesamt 11.000 m² erweitert wurde. Neben den Herz-Lungen-Maschinen inklusive Zubehör werden in München auch Autotransfusionssysteme und Hypothermiegeräte entwickelt und gefertigt. Hier sind derzeit rund 250 Mitarbeiter für das Unternehmen tätig. Die Sorin Group verpflichtet sich zur Einhaltung höchster Qualitätsstandards – beginnend bei Forschung & Entwick-

Abbildung 1: S5, die neueste Generation der Herz-Lungen-Maschinen von Sorin (© Sorin Group)

Abbildung 2: Mit dem neuen Autotransfusionssystem XTRA® lässt sich zum einen aus gesammeltem Wundblut ein Konzentrat gewaschener Erythrozyten gewinnen, zum anderen bietet es aber auch die Möglichkeit, Thrombozyten aus Vollblut zu separieren (© Sorin Group)

lung über den Herstellungsprozess bis hin zum fertigen Produkt – sowie höchster rechtlicher und operativer Standards. Das Compliance-Programm der Sorin Group gilt als Vorzeigemodell der Industrie. S. M.

ABSTRACTS

Abstracts* of the 29 Annual Meeting of the German Atherosclerosis Society March 26–28, 2015 Tagungsstätte der Universität Gießen Rauischholzhausen th

The effect of 15-deoxy-∆12,14prostaglandin J2 on proteolytic activity and migration of human macrophages M. Abhari, M. Schubert, S. Becher, S. Lorkowski Institute of Nutrition, Friedrich Schiller University Jena, Germany Background: Macrophages play an important role in arterial remodeling during atherogenesis. Particularly, classically activated M1 macrophages in the shoulder region of atherosclerotic plaques produce large amounts of proteinases, such as matrix metalloproteinases (MMPs), leading to the destruction of the extracellular matrix (ECM) of the fibrous caps, thus causing plaque rupture. 15-DeoxyΔ12,14-prostaglandin J2 (15dPGJ2) is a multifunctional anti-inflammatory lipid metabolite which is abundantly produced by macrophages. We have shown that 15dPGJ2 downregulates the expression of MMP8 and MMP14 by macrophages, but the underlying signalling pathways and functional consequences are not understood. Aim: We aim to unravel the signalling pathways involved in balancing the proteolytic activity of macrophages by 15dPGJ2. Methods and results: Our results revealed that 15dPGJ2 decreases doseand time-dependently the expression of MMP8 and MMP14 at the mRNA and protein level in human THP-1 macrophages as well as in human primary macrophages. Whereas regulation of * The abstracts are listed alphabetically according to the name of the first author Perfusion 01/2015

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MMP14 by 15dPGJ2 takes place independently of PPARγ, expression of MMP8 is regulated in part via PPARγ. PPARγ-independent signalling pathways were investigated by treatment of mature macrophages with various inhibitors of key signalling proteins, such as PI3K, JAK, JNK and MAPKs, in combination with 15dPGJ2. Effects of the inhibitors were examined at the mRNA and protein level of MMP8 and MMP14 using RT-qPCR and Western blotting, respectively. While investigating PPARγ-independent signalling pathways, none of the inhibitors tested so far was able to block the downreg ulation of MMP8 by 15dPGJ2. In contrast, preliminary results on MMP14, which still need further confirmation, provide evidence that the mTOR pathway is involved in the downregulation of MMP14 by 15dPGJ2. First results using in vitro scratch-wound assays show that 15dPGJ2 blocks migration of macrophages, possibly by downregulating MMP14. Conclusions: 15dPGJ2 regulates macrophage behavior with respect to their proteolytic activity and their migratory behavior. Our studies may help for identifying new approaches in preventing myocardial infarction by preventing atherosclerotic plaque rupture.

Inhibition of IL-17A attenuates and stabilizes advanced murine atherosclerosis and is involved in human atherosclerosis M. Akhavanpoor1, D. Okuyucu1, S. Wangler1, A. Dietz1, L. Zhao1, K. Stellos2, K.M. Little3, F. Lasitschka4, A. Doesch1, M. Hakimi5, T.J. Dengler6,

T. Giese7, E. Blessing1, C.A. Gleissner1, H.A. Katus1, C. Erbel1 1 Department of Cardiology, University of Heidelberg, Germany 2 Institute of Cardiovascular Regeneration, University of Frankfurt am Main, Germany 3 Illumina, Inc., San Diego, CA, USA 4 Institute of Pathology, University of Heidelberg, Germany 5 Departments of Vascular and Endovascular Surgery, University of Heidelberg, Germany 6 Department of Cardiology, SLK-Hospital Heilbronn, Bad Friedrichshall, Germany 7 Institute of Immunology, University of Heidelberg, Germany Objective: Atherosclerosis is believed to be a chronic inflammatory disease regulated by a complex network of cytokines and chemokines. Interleukin17A plays an important role in host defense and is involved in the pathology of different autoimmune and inflammatory diseases. Recent studies demonstrated an association of IL-17A to atherosclerosis. Interestingly, the precise role of IL-17A in murine and human atherosclerosis still remains controversial. In the current study we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. Methods: 26-weeks old apolipoprotein E-deficient (ApoE–/–) mice were fed a standard chow diet and treated either with anti-murine IL-17A mAb (n=15) or control IgG (n=10) for 16 weeks. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied by in vitro and ex vivo experiments. Results: Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (p=0.001) by reducing inflammatory burden and cellular infiltration (p=0.01) and improved lesion stability (p=0.01). In vitro experiments showed that IL-17A plays a role in monocyte adhesion and sensitization of antigenpresenting cells toward pathogenderived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. © Verlag PERFUSION GmbH

ABSTRACTS

Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a pro-inflammatory milieu and up-regulation of molecules expressed by the IL-17A-induced macrophage subtype. Conclusions: We here show for the first time that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabili zation in advanced lesions in ApoE–/– mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte/macrophage lineage. In addition, translational experiments suggest a relevance of IL-17A in human system.

Multiplexed microarray protein analysis in small volume samples N. Al-Fakhri1, M. Bomert2, G. Köllisch3, P.I. Pfefferle4 1 Center of Transfusion Medicine and Hemotherapy, Philipps University Marburg, Germany 2 Department of Anaesthesiology, Philipps University Marburg, Germany 3 Institute of Immunology, Philipps University Marburg, Germany 4 Biobank, Philipps University Marburg, Germany Aims: Multiplexed analyte measurement offers many advantages over the conventional ELISA format when applied in large scale studies or clinical trials. In the present study we set out to define the reliability and consistency of a suspension multiplexed protein array, the cytometric bead array (CBA), in a large-scale, longitudinal cytokine study. Methods: The cytokines IL-5, IL-10, TNF-α and IFN-γ were measured in samples from a childhood immunology study. Analytical performance of CBA was determined in sample supernatants and CBA was compared to conventional ELISA. Results: Within-run and total imprecision were between 5.2–10.8 % and 5.6–13.2 %, respectively, at three different concentrations for all cytokines. Slopes of dilution linearity were between 1.01 and 1.31 for the four cyPerfusion 01/2015

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tokines. The recovery rate at two different concentrations of the cytokines was between 97 % and 113 %. Lower limits of detection and quantification as well as functional sensitivity were determined. Comparison of the multiplex array and solid phase method showed good correlation with r between 0.82 and 0.93. The sample volume required for the multiplex format was 25 % of the ELISA sample volume. Conclusions: CBA analytical evaluation and comparison to an ELISA format demonstrated high reproducibility, sensitivity and good applicability for multiplex protein analysis in small volume samples.

Inflammation, but not recruitment, of adipose tissue macrophages requires signalling through Mac-1 (CD11b/CD18) in diet-induced obesity (DIO) N. Anto Michel1, D. Wolf1, N. Bukosza1, D. Engel2, M. Poggi2, C. Weber3, C. Bode1, K. Peter4, E. Lutgens2, 5, A. Zirlik1 1 Atherogenesis Research Group, University Heart Center, University of Freiburg, Germany 2 Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands 3 Institute for Cardiovascular Prevention, Ludwig Maximilians University, Munich, Germany 4 Atherothrombosis and Vascular Biology, Baker IDI Heart and Diabetes Institute, Melbourne, Australia 5 Department of Medical Biochemistry, Subdivision of Experimental Vascular Biology, Academic Medical Center, University of Amsterdam, The Netherlands Objective: Cell accumulation is a prerequisite for adipose tissue inflammation. The leukocyte integrin Mac-1 (CD11b/CD18, αMβ2) is a classic adhesion receptor critically regulating inflammatory cell recruitment. Here, we tested the hypothesis that genetic deficiency and therapeutic modulation of Mac-1 regulates adipose tissue inflammation in a mouse model of dietinduced obesity (DIO).

Methods and results: C57Bl6/J mice genetically deficient (Mac-1–/–) or competent for Mac-1 (WT) consumed a high fat diet for 20 weeks. Surprisingly, Mac-1–/– mice presented with increased diet-induced weight gain elevated glucose levels, decreased peripheral insulin sensitivity, dyslipidemia, and signs of aggravated hepatic steatosis. Unexpectedly, accumulation of adipose tissue macrophages (ATMs), the main cell fraction expressing Mac-1 in adipose tissue, was unaffected. Expression of pro-inflammatory genes, such as of IL-6 and MCP-1, was reduced in Mac-1–/– mice. Conversely, treatment of ATMs with an agonistic anti-Mac-1 antibody, M1/70, induced pro-inflammatory genes in cell culture. In vivo, treatment with M1/70 induced a hyper-inflammatory phenotype, increased cell accumulation, loss of anti-inflammatory T-regulatory cells, and increased expression of IL-6, demonstrating that Mac-1 engagement is required for activation, but not for accumulation of ATMs. Finally, specific inhibition of Mac-1’s adhesive interaction to its biased agonist CD40L by the novel peptide inhibitor cM7 reduced T cell activation, but hardly affected myeloid cell accumulation. Conclusions: We present the surprising finding that adhesive properties of the leukocyte integrin Mac-1 are not required for macrophage accumulation in adipose tissue. Instead, agonism of Mac-1 potently modulates inflammation. These findings question the net effect of integrin blockade in cardiometabolic disease.

Soluble klotho and mortality in patients with acute coronary syndrome: The Ludwigshafen Risk and Cardiovascular Health Study V.M. Brandenburg1, M.E. Kleber2, M.G. Vervloet3, T. Larson4, A. Tomaschitz5, S. Pilz6, 7, T. Stojakovic8, G. Delgado2, T.B. Grammer2, W. März2, 8, 9, H. Scharnagl8 1 Department of Cardiology, University Hospital of the RWTH Aachen, Germany © Verlag PERFUSION GmbH

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Medical Clinic V, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany 3 Department of Nephrology, VU University Medical Center, Amsterdam, The Netherlands 4 Division of Renal Medicine, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden, Department of Nephrology, Karolinska University Hospital, Stockholm, Sweden 5 Specialist Clinic for Rehabilitation, PV Bad Aussee, Austria 6 Department of Cardiology, Medical University of Graz, Austria 7 Department of Epidemiology and Biostatistics and EMGO Institute for Health and Care Research, VU University Medical Center, Amsterdam, The Netherlands 8 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria 9 Synlab Center of Laboratory Diagnostics, Heidelberg, Germany 2

Background: Klotho is the co-receptor of fibroblast growth factor 23 (FGF23) particularly in the kidney and hence participates in important regulatory pathways in phosphate and vitamin D metabolism. Moreover, excess FGF23 levels have consistently been shown to be independently associated with dismal cardiovascular outcome (especially heart failure) in various patient cohorts. Noteworthy, klotho is not expressed in the myocardium, so it is unclear to what extent klotho participates in mediating FGF23-related cardiac event rate. Moreover, it is unknown if soluble klotho (s-klotho) levels as reflector of the functionality of the klotho-FGF23-axis influence cardiovascular outcome independently. Methods: We therefore sought to investigate 1) the associations of s-klotho with traditional cardiovascular risk factors and particularly with FGF23 levels and 2) the impact of s-klotho upon long-term mortality in a large cohort of patients referred for coronary angiography without severe kidney disease. We examined whether s-klotho levels at baseline were associated with cardiovascular and total mortality in 2948 patients from the Ludwigshafen Perfusion 01/2015

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Risk and Cardiovascular Health Study (LURIC). We particularly investigated if s-klotho levels interact with FGF23 levels regarding outcome prediction. Results: Mean age of participants was 63±10 years; median s-klotho levels were higher in women compared to men (419 [412–426] versus 435 [424–446] pg/mL, p=0.025] but independent from age. Diabetes (in n=1136 patients) was associated with higher s-klotho levels: 440 (430–449) versus 414 (406–421) pg/mL in non-diabetics (p<0.001). After stratification of patients according to quartiles of GFR S-klotho levels revealed a significant decrease in parallel to declining kidney function and a linear increase among quartiles of FGF23 levels. During a median follow-up of 9.9 years, 884 deaths (30 %) occurred, 545 (18 %) of which were cardiovascular. Age- and sex-adjusted hazard ratios (HRs) in the fourth quartile compared to the first quartile of s-klotho were 1.03 (95% CI 0.85–1.23; p=0.797) for all-cause mortality and 0.93 (95% CI 0.73–1.18; p=0.532) for cardiovascular mortality. Excess mortality as seen with high levels of baseline FGF23 was not modified by adjustment to baseline s-klotho levels. Conclusions: In patients undergoing coronary angiography baseline s-klotho levels decline with decreasing GFR but do not predict the risk for all-cause and cardiovascular mortality over longterm follow-up. S-klotho levels do not modify the association between high FGF23 levels and increased event rate. S-klotho levels appear to be of limited importance in terms of future cardiovascular risk prediction in patients undergoing coronary angiography with no or minor chronic kidney disease.

Regulation of monocyte/macrophage polarization by extracellular RNA during atherosclerosis development H.A. Cabrera-Fuentes1, 2, K.T. Preissner1, 2 Institute of Biochemistry, Medical School, Justus Liebig University, Gießen, Germany 2 Department of Microbiology, Kazan Federal University, Kazan, Russian Federation

Aims: Monocytes/macrophages respond to external stimuli with rapid changes in the expression of numerous inflammation-related genes to undergo polarization towards the M1 (proinflammatory) or M2 (anti-inflammatory) phenotype. We have previously shown that, independently of Toll-like receptor activation, extracellular RNA (eRNA) could exert pro-thrombotic and pro-inflammatory properties in the cardiovascular system and provoke cytokine mobilization. Results: Here, mouse bone marrowderived-macrophages (BMDM) differentiated with mouse macrophagecolony-stimulating factor (M-CSF) were found to be skewed towards the M1 phenotype when exposed to eRNA. This resulted in up-regulated expression of inflammatory markers such as TNF-α and Il-6, together with Il-12 and iNOS, whereas anti-inflammatory genes such as chitinase-like proteins (Ym1/2) and macrophage mannose receptor-2 (Cd206) were significantly down-regulated. Human peripheral blood monocytes were treated with eRNA and analysed by micro-array analysis of the whole human genome, revealing an up-regulation of 79 genes by at least four-fold; 27 of which are related to signal transduction and 15 are associated with inflammatory response. Conclusion: In accordance with the proposed actions of eRNA as a pro-inflammatory “alarm signal”, these data shed light on the role of eRNA in the context of chronic inflammatory diseases such as atherosclerosis.

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Modulation of atherogenesis by adaptive immunity in BALB/c mice F. Cheng1, 2, L. Twardowski2, K. Winter1, A. Canisus3, E. Pross1, K.J. Lackner3, K. Reifenberg4, M. Torzewski2 1 Dr. Margarete Fischer Institute of Clinical Pharmacology, Stuttgart, Germany 2 Department of Laboratory Medicine, Robert Bosch Hospital, Stuttgart, Germany 3 Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center, Johannes Gutenberg University, Mainz, Germany 4 Animal Laboratory Services, German Cancer Research Center, Heidelberg, Germany Aim: In our study, immunogenic genesis of atherosclerosis was studied in immunodeficient transgenic mice of the Th2-prone BALB/c background, which represent a suitable model for the generation of humanized mice. Methods and results: BALB/c-Ldlr–/– (C-Ldlr–/–) mice were used as controls. We generated two immunodeficient strains, referred to as C-Ldlr–/–Rag1–/– and C-Ldlr–/–Rag1–/–Il2rg–/–, which show severe combined B- and T-cell immunodeficiency. The second strain additionally has a complete loss of NK cells because of γ-chain inactivation. All mice were fed an atherogenic Western type diet (WTD) for 12 or 24 weeks, respectively. Serum cholesterol of both immunodeficient mice was significantly increased compared to controls. The atherosclerotic lesion development and composition were quantitatively analysed. Compared to controls (25,285/24,975 µm2, p<0.01), double (55,916/53,488 µm2) and triple (44,522/34,736 µm2) mutants developed significantly more atherosclerosis after 24 weeks on WTD as indicated by plaque area of aortic sinus (median/interquartile range). Triple mutants show significant increase of lesional macrophages (Mø 53.56/22.32 %, p<0.01) and a decrease of smooth muscle cells (SMCs 8.54/5.3 %, p<0.01) compared to controls (Mø 23.57/18.38 %, SMCs 16.52/4.7 %) and double mutants (Mø 22.12/14.54 %, SMCs 17.41/9.6 %). Conclusions: In summary, a comPerfusion 01/2015

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bined B and T cell immunodeficiency in a Th2-prone BALB/c background significantly accelerates atherosclerotic lesion progression. Furthermore, combined immune and γ-chain defects have the potential to change atherosclerotic cellularity to a more unstable phenotype.

Inhibition of miR-92a improves re-endothelialization and prevents restenosis following vascular injury J.-M. Daniel1, J. Dutzmann1, W. Bielenberg1, A. Bonauer2, R.A. Boon2, A. Fischer2, E. van Rooij3, J. Bauersachs1, S. Dimmeler2, D.G. Sedding1 1 Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany 2 Institute for Cardiovascular Regeneration, Centre of Molecular Medicine, Goethe University, Frankfurt, Germany 3 miRagen Therapeutics Inc., Boulder, CO, USA Objective: MicroRNA (miR)-92a is an important regulator for endothelial proliferation and angiogenesis after ischemia, but the function of miR-92a in re-endothelialization after endovascular injury is widely unknown. We tested the effects of highly specific LNA-based inhibitors of miR-92a on endothelial recovery and neointimal lesion formation in a wire-induced injury model. Methods and results: MiR-92a was significantly up-regulated in neointimal lesions after wire-induced vascular injury in C57/Bl6 mice as detected by qPCR. In vitro, transfection of vascular cells with pre-miR-92a inhibited proliferation and migration of endothelial cells (ECs) but did not exert significant effects on smooth muscle cells (SMCs). In vivo, inhibition of miR-92a expression with highly effective, specific LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area at 10 days following injury and attenuated neointimal lesion development at 21 days after injury. Moreover, the enhanced endothelial recovery in LNA-92a treated mice was

associated with a reduced inflammatory response and attenuated proliferation of SMCs within the neointimal and medial layer at 14 days after injury. Conclusions: Our data indicate that inhibition of miR-92a attenuates neo intimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury.

Effects of docosahexaenic acid on disease activity and cardiovascular risk factors in patients with rheumatoid arthritis. A double-blind, placebo-controlled, randomized cross-over study: microalgae oil vs. sunflower oil C. Dawczynski1, 5, M. Dittrich1, T. Neumann2, K. Knoll2, A. Welzel2, M. Kiehntopf3, P. Oelzner2, S. Völker4, A. Köberle4, O. Werz4, S. Lorkowski5, G. Jahreis1 1 Department of Nutritional Physiology, Institute of Nutrition, Friedrich Schiller University of Jena, Germany 2 Department of Internal Medicine III, Jena University Hospital, Germany 3 Department of Clinical Medicine and Laboratory Diagnostics, Jena University Hospital, Germany 4 Department of Pharmaceutical/Medicinal Chemistry, Friedrich Schiller University of Jena, Germany 5 Department of Biochemistry and Physiology, Institute of Nutrition, Friedrich Schiller University of Jena, Germany Background: The potential of fish oil as supplier for eicosapentaenoic acid (C20:5n3, EPA) and docosahexaenoic acid (DHA, C22:6n3) regarding reduction of cardiovascular risk factors and support of therapy of chronic inflammatory diseases, respectively, are well described in the literature. Fatty cold water fishes are the main source of these n-3 long chain poly unsaturated fatty acids (LC-PUFA). So far, less is known about the physiological effects of the single compounds EPA or DHA. Aim and methods: The present placebo-controlled double-blind human © Verlag PERFUSION GmbH

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intervention study in cross-over design investigated the effect of a long-term consumption of foods enriched with microalgae oil as source of DHA for patients with rheumatoid arthritis (n=35; age 60±11 years; BMI: 26.3±4.4). The daily product portfolio consisted of 30 g milk powder, 60 sausages and 8 g tomato spread. The patients consumed the foods (DHA-fortified foods, including 8 g algae oil = 2.1 g DHA) and the placebo products enriched with 8 g sun-flower oil) daily over 10 weeks (10 week wash-out between both periods). Results: The daily consumption of the DHA-enriched products caused a significant increase of n-3 index and in particular DHA concentrations and a significant decrease of the ratios arachidonic acid (C20:4n6, AA)/EPA and AA/DHA in plasma and erythrocyte lipids. The proportion of LC-PUFA in the membranes influence inflammatory processes because the LC-PUFA with more than 20 C-atoms are precursor for metabolites such as prostaglandins, leukotrienes, resolvines, protectins, etc. The observed shift of n-3/n-6 LC-PUFA level as response to the DHA-intervention indicates their antiinflammatory potential. The observed changes in concentrations of LTB4, 5-HETE, as well as adhesion molecules (sPECAM-1, sICAM-3) and cytokines (IFN-γ, IL-12p70, Il-1β, IL-8, IL-13) in response to the DHA intervention substantiate the anti-inflammatory potential of the intervention. Due to consumption of the products fortified with DHA the disease activity assessed by Disease Activity Score DAS 28, 68, the US-7-Score (ultrasound of 7 joints), as well as different life quality questionnaires was significantly improved. The support of therapy of this chronic inflammatory disease was not shown in response to consumption of the placebo products. Moreover, the daily consumption of the DHA-rich algal oil resulted in a significant increase of HDL as well as a significant reduction of LDL/HDL ratio and TAG. Conclusion: Concluding, the daily Perfusion 01/2015

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consumption of DHA-rich alga oil can support therapy of patients with rheumatoid arthritis, and additionally reduce cardiovascular risk factors. Algae oil as valuable source of n-3 LC PUFA which is suitable to cover n-3 LC-PUFA demand in order to counteract overfishing.

Saturated fatty acids are not off the hook: How dietary saturated fat influences cardiovascular risk C. Dawczynski1, 6, M. Kleber2, W. März2, 3, 4, G. Jahreis5, 6, S. Lorkowski1, 6 1 Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 th V Department of Medicine (Nephrology, Hypertensiology, Endocrinology, Diabetology, Rheumatology), Medical Faculty Mannheim, University of Heidelberg, Germany 3 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria 4 Synlab Academy, Synlab Services GmbH, Mannheim, Germany 5 Institute of Nutrition, Friedrich Schiller University Jena, Germany 6 Competence Cluster for Nutrition and Cardiovascular Health (nutriCARD), Friedrich Schiller University Jena, Germany The recent meta-analysis of Chowdhury et al. (2014) postulates among other findings the lack of association between circulating blood levels and intake of total saturated fatty acids (SFA) with coronary outcomes. On closer examination, it is evident that two of the eight included studies focused on the proportion of pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) or their sum in serum lipid esters (approx. 0.5–1.0 % of the fatty acids [FA] in total phospholipids) and their impact on cardiovascular disease (CVD) risk. The odd-chain FA are markers for milk or ruminant fat intake. Both studies indicate negative associations between milk-fat intake and first-ever myocardial infarction. Neither of the two studies described the association between total circulat-

ing blood SFA on coronary outcomes. In contrast to the cardio-protective effects of dairy consumption, an elevated intake of C16:0 and C18:0 and/or their increased de novo synthesis may raise CVD risk. Thus, it is of particular importance to differentiate the effects of individual circulating SFA to cardiovascular outcomes. Excluding the studies that evaluated the association of milk fat FA and cardiovascular outcomes, revealed a positive association of total SFA blood levels and coronary outcome (RR 1.21, 95% CI 1.04–1.40). Therefore, results obtained from studies of C15:0 and C17:0 cannot be mixed with results from studies on other SFA because of the opposite physiological effects of regular consumption of foods rich in C16:0 and C18:0 compared to high intake of dairy products. In our opinion, it is vital to analyse the impact of individual SFA on CVD incidence in order to draw the right conclusions.

The gut hormone Glucagon like peptide 1 (GLP-1) is increased during acute myocardial infarction and improves left ventricular function S. Diebold1, F. Kahles1, M. Schwarz1, M. Berger1, J. Möllmann1, R. Stöhr1, C. Lebherz1, A.C. Foldenauer2, H.A. Katus3, E. Giannitsis3, N. Marx1, M. Lehrke1 1 Department of Internal Medicine I, University Hospital of the RWTH Aachen, Germany 2 Institute of Medical Statistics, University Hospital of the RWTH Aachen, Germany 3 Department of Internal Medicine III, University Hospital Heidelberg, Germany Purpose: GLP-1 is an incretin hormone which is released in response to nutritional stimuli from the gut causing glucose-dependent insulin secretion from the pancreas while also holding cardioprotective properties. Interestingly, GLP-1 secretion was recently found to be induced by inflammatory stimuli including endotoxin in mice while increased concentrations of circulating GLP-1 have been reported in patients with coronary artery disease. © Verlag PERFUSION GmbH

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Methods and results: To follow up on this observation we have assessed GLP1 plasma levels in 1419 patients with coronary artery disease. Higher GLP1 levels were found in 1181 patients presenting acute myocardial infarction (39.47±22.19 pM) in comparison to 238 patients holding stable coronary artery disease (17.52±14.91 pM) (p<0.001 under further consideration of sex and age). To investigate whether GLP-1 would similarly be increased by myocardial infarction under experimental conditions we performed LAD ligation in C57/Bl6 mice. Indeed, GLP-1 was increased in a time dependent manner from baseline (11.29 pM) to a maximum of 29.73 pM after 6 hours (n=3; p<0.05). To evaluate the functional relevance of endogenous GLP-1 induction during myocardial infarction we pretreated mice with the GLP-1 receptor antagonist exendin-9 (100 nM/kg mouse). This led to an impairment of left ventricular function in exendin-9 treated (6469 dp/dt) versus control animals (7818 dp/dt under dobutamine stress as assessed by millar catheter; p<0.05) 6 hours post LAD ligation. Conclusion: GLP-1 levels are increased in the context of acute myocardial infarction in mice and men and lead to improved left ventricular function. This provides a new cross talk between the gut and the heart in context of acute coronary syndrome.

The effects of Dl-homocysteine or Dl-homocysteine thiolactone on cardiac acetylcholinesterase activity and plasma antioxidant enzymes in rats D. Djurić1, M. Čolović2, J. Jakovljević1, O. Stanojlović1, M. Djuric1, V. Jakovljević3, D. Krstić4 1 Institute of Medical Physiology “Richard Burian”, Faculty of Medicine, University of Belgrade, Serbia 2 Vinča Institute of Nuclear Sciences, University of Belgrade, Serbia 3 Department of Physiology, Faculty of Medical Sciences, University of Kragujevac, Serbia Perfusion 01/2015

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Institute of Medicаl Chemistry, Faculty of Medicine, University of Belgrade, Serbia

Objective: It is known that DL-homocysteine (DL-Hcy) and DL-homocysteine thiolactone hydrochloride (DLHcy TLHC) induce adverse effects on cardiovascular system. Considering the possible role of enzyme acetylcholinesterase (AChE) and oxidative stress in these effects, the aim of this study was to assess the effects of these compounds on rat cardiac tissue AChE activity, antioxidant enzymes activity (catalase/CAT, glutathione peroxidase/ GPx, superoxide dismutase/SOD) and index of lipid peroxidation (MDA) in plasma. Methods: Male Wistar rats (weighing 180–250 g) were divided into three groups (5–7 rats per group); control group (1 ml 0.9 % NaCl, i.p.), DL-Hcy group (8 mmol/kg b.m., i.p.) or DLHcy TLHC group (8 mmol/kg b.m., i.p.). An hour after the administration rats were euthanized by decapitation, the whole blood was collected for plasma parameters analysis, and the heart was excised. The buffered and homogenized parts of the cardiac tissue were used for measurement of AChE activity by the Ellman method. Plasma MDA level and antioxidant enzymes activity were determined by standard spectrophotometrical method. Results: The acute administration of DL-Hcy and DL-Hcy TLHC induced significant inhibition of AChE activity in rat cardiac tissue homogenate (0.023±0.002 or 0.021±0.002 ΔA/min/ mg of tissue, respectively) vs. control (0.039±0.003 ΔA/min/mg of tissue) (AChE activity inhibition of 41 % vs. 46 %, respectively). Following acute i.p. administration of DL-Hcy or DLHcy TLHC, the plasma concentration of antioxidant enzymes was considerably increased (CAT: 26.49±4.22 or 51.51±6.79 U/ml of plasma, respectively, vs. 16.40±2.11 U/ml in controls, GPx: 1.76±0.28 or 3.26±0.31 U/ml of plasma, respectively, vs. 1.09±0.14 U/ ml in controls, SOD: 30.41±0.71 or 21.35±1.98 U/ml of plasma, respec-

tively, vs. 25.31±0.96 U/ml in controls). At the same time plasma MDA level was decreased (6.09±0.85 or 8.86±1.84 nmol/ml of plasma, respectively, vs. control (15.37±1.41 nmol/ml of plasma). Conclusion: Our findings probably indicate that DL-Hcy or DL-Hcy TLHC have pro-oxidant effects taking into consideration significantly increased activities of antioxidant enzymes (CAT, GPx, SOD) in plasma following intraperitoneal administration of these substances in rat. However, decrease of plasma MDA level during the same period leads to the conclusion that there was an inverse response to the raise of antioxidant enzymes. Both substances inhibit activity of AChE in rat cardiac tissue homogenate however DL-Hcy TLHC induced stronger effects than DL-Hcy.

Large-scale reverse siRNA transfection: A novel tool to detect regulators of cholesterol metabolism in human macrophages G. Domschke1, F. Linden1, A. Hafner1, C. Erbel1, H. Erfle2, H. Runz3, H.A. Katus1, C. Gleissner1 1 Department of Cardiology, University of Heidelberg, Germany 2 BioQuant, ViroQuant-Cell Networks RNAi Screening Facility, University of Heidelberg, Germany 3 Institute of Human Genetics, University of Heidelberg, Germany Background: Macrophages are known key regulators in atherosclerotic plaque formation. In the vascular intima, macrophages develop toward foam cells through intake of low-density lipoproteins and their derivatives, accumulate in the plaque and support inflammatory processes by producing chemokines and cytokines. To identify novel therapeutic targets it is essential to understand which genes regulate this process. Furthermore, potential differences between healthy individuals and coronary artery disease (CAD) patients are of great interest in order to develop © Verlag PERFUSION GmbH

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specific therapies. We therefore sought to develop an siRNA-based high throughput platform that allows identifying genes involved in macrophage foam cell formation in cells isolated from individual patients. Methods: We chose a set of 89 genes previously described as being up-regulated during macrophage foam cell formation. For each gene, three predesigned siRNAs were used for transfection and plated onto 384 well plates. Transfection mixture and scrambled siRNA were used as negative controls. Human monocytes were isolated from human whole blood by negative bead isolation and differentiated toward macrophages using monocyte colonystimulating factor. The freshly isolated cells where seeded onto the 384-well plates where they were reversely transfected by siRNA. After three days, DiI-labeled LDL was added and LDL uptake was measured by wide-field fluorescence microscopy and subsequent automatic image analysis. In the primary screening the whole gene set was investigated in six male patients with severe CAD and six healthy agematched control individuals. In a subsequent confirmatory run, ten CAD patients and ten control individuals were compared. Results: We found that human macrophages can be successfully exposed to reverse siRNA transfection and that knock-down-related alterations in LDL uptake can be efficiently quantified by fluorescence microscopy. The primary screening revealed that down-regulation of several genes, including ALDH1A2, APOC1, CMTM6, FABP4, EPHX1, WBP5 and ZYX, significantly altered LDL uptake. Four of these genes (APOC1, CMTM6, FABP4 and WBP5) could be confirmed to decrease LDL uptake in macrophages, both from healthy individuals and CAD patients with a more pronounced down-regulation in cells derived from CAD patients as compared to healthy controls. Conclusions: We present a novel siRNA-based high-throughput screening tool that may help to assess the efPerfusion 01/2015

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fects of single genes on macrophage foam cell formation at the single cell level. Using this novel method, we can identify novel regulatory genes of cholesterol metabolism in human macrophages. Our method may contribute to the identification of novel therapeutic targets in atherosclerosis.

Gaining insights into the redox regulatory and anti-apoptotic activity of APEX1 in endothelial cells N. Dyballa-Rukes1, J. Haendeler1, 2, C. Goy1, A. Eckers1, J. Altschmied1 1 IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany 2 Central Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty, University of Düsseldorf, Germany Aims and methods: Apurinic/Apyrimidinic endonuclease/Redox factor-1 (APEX1) is a multifunctional protein that has both DNA repair and redox regulatory activity. Its redox activity allows it to reduce specific cysteine residues in the DNA binding domain of several transcription factors, including NF-κB. As a number of these transcription factors are regulators of cancer progression, APEX1 has emerged as promising target in cancer research. However, not much is known about APEX1 functions in the cardiovascular system. Since APEX1 can mediate the reduction of transcription factors independent of its intrinsic redox activity it probably recruits other reducing molecules, like thioredoxin-1 (TXN). TXN is one of the most important anti-oxidative proteins in the endothelium and significantly counteracts oxidative stress and apoptosis in endothelial cells (EC). We therefore investigated in primary human EC if APEX1 exhibits antiapoptotic capabilities by itself, if transcription factors are redox-controlled by APEX1 under oxidative stress in EC, and which role the interaction of APEX1 with TXN plays in the redox homeostasis of EC.

Results: Overexpression of human APEX1 in EC inhibited H2O2-induced apoptosis. To characterize the antiapoptotic properties of APEX1 we generated mutants that lack the C-terminal DNA repair domain (APEX1ΔC 1-127) or the N-terminal nuclear localization signal (NLS) including parts of the redox domain (APEX1ΔN 21-318). We could demonstrate that full-length APEX1 (APEX1wt) and APEX1ΔC 1-127 showed a predominant nuclear localization and prevented H2O2-induced apoptosis, whereas the mutant APEX1ΔN 21-318 was mainly localized in the cytosol and exhibited no longer anti-apoptotic properties. Thus, the anti-apoptotic activity of APEX1 in EC critically depends on its nuclear localization and redox-activity. Since it is known that the p65 subunit of NF-kB is activated by pro-apoptotic stimuli in EC, we analysed the impact of APEX1 on p65 in DNA binding assays. Under oxidative stress, overexpression of APEX1wt and APEX1ΔC 1-127 reduced DNA binding of p65, whereas the NLS-deficient mutant seems to enhance DNA binding capacity. Conclusion: These data lead to the assumption that APEX1 acts antiapoptotic at least partially due to suppression of NF-kB dependent, pro-apoptotic gene expression programs. In future studies we will analyse the impact of APEX1 on other transcription factors in concert with TXN.

The aryl hydrocarbon receptor reduces vessel functionality and shortens healthy aging A. Eckers1, S. Jakob1, C. Heiss3, C. Goy1, V. Brinkmann1, N. Ale-Agha1, J. Altschmied1, N. Ventura1, 2, J. Haendeler1, 2 1 IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany 2 Central Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty, University of Düsseldorf, Germany 3 Department of Cardiology, Pneumology and Angiology, University Hospital Düsseldorf, Germany © Verlag PERFUSION GmbH

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Aims and methods: Development of age-associated diseases like atherosclerosis depends not only on genetic disposition but also on environmental influences. The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed transcription factor known to activate detoxifying enzymes. It has been shown that the known ligands of the AhR dioxin and benzo[a]pyrene (BaP) promote atherosclerosis. In addition, recent studies demonstrated that recovery of the blood flow after hindlimb ischemia was significantly enhanced in AhR-deficient mice demonstrating increased angiogenesis in the absence of AhR. Thus, AhR seems to be linked to vessel functionality and age-related diseases. To investigate the role of the AhR regarding aging and age-related vascular processes, we analysed health-span in AhR-deficient Caenorhabditis elegans (C. elegans), vessel stiffness in AhR-deficient mice and human subjects as well as functional parameters in primary human endothelial cells (EC) and we investigated the expression of the AhR in blood from young and old healthy human subjects. Results: To determine if the AhR is relevant for aging, we analysed motility and life span in an AhR mutant C. elegans strain. Vascular stiffness of AhR-knockout mice and their wildtype (wt) littermates as well as of young and old human subjects was determined by measuring pulse wave velocity (PWV). Phosphorylation of the endothelial nitric oxide synthase (eNOS) in mice aortas and endothelial cells was analysed by western blot. EC were analysed for migration, apoptosis and proliferation in the presence of the AhR agonist BaP and the AhR antagonist MNF (3’-Methoxy-4’-nitroflavon). Expression of the AhR in blood of human subjects was determined with realtime PCR using TaqMan. The AhR-deficient strain of C. elegans showed an enhanced motility and an extended life span compared to wt worms. Old (17–20 months) wt mice showed increased PWV, which is inPerfusion 01/2015

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dicative of impaired vessel function and enhanced stiffness. In AhR-deficient mice, we observed reduced PWV in both old and young (3–6 months) mice, suggesting reduced vessel function already at young age. Analysis of eNOS phosphorylation showed increased eNOS activity in thoracic aortas of AhR-deficient mice. In line with this finding, the AhR activator BaP increased eNOS phosphorylation at threonine 495 in EC demonstrating reduced eNOS activity. Moreover, BaP reduced migration of EC, which was reversed by the addition of the antagonist MNF without any changes in proliferation or apoptosis. Thus, loss of active eNOS by AhR activation especially inhibits the migration of EC. Finally, we demonstrated not only a positive correlation between age and PWV in human subjects but also between AhR expression and PWV. Conclusion: Our data demonstrate that loss of AhR extends life span as well as health span in C. elegans. Knock out of AhR in mice leads to improvement of vessel functionality by decreasing vessel stiffness. Finally, the PWV in humans correlates not only with age but also with the expression of the AhR in blood cells. Thus, AhR expression may be useful as a new predictor of healthy aging from nematodes to humans.

S-Adenosylhomocysteine: A novel non-traditional cardiovascular risk factor in chronic kidney disease? I.E. Emrich1, A.M. Zawada1, K. S. Rogacev1, S. Seiler1, R. Obeid2, J. Geisel2, D. Fliser1, G.H. Heine1 1 Department of Internal Medicine IV – Nephrology and Hypertension, Saarland University Medical Center, Homburg, Germany 2 Clinical Chemistry and Laboratory Medicine/ Central Laboratory; Saarland University Medical Center, Homburg, Germany Background: Although homocysteine has been discussed as a cardiovascular risk factor in patients with chronic kidney disease (CKD), interventional studies in which homocysteine was

lowered via B vitamin supplementation failed to demonstrate a survival benefit. Recently, it has been suggested that the homocysteine metabolite S-adenosylhomocysteine (SAH), which is a potent inhibitor of methylation reactions and thus a central epigenetic regulator, may be the real culprit in cardiovascular disease and thus explain the hitherto paradoxical findings. Against this background we now aimed to analyse (1) to which degree SAH accumulates in CKD and (2) whether SAH, compared to homocysteine, is associated more strongly with prevalent cardiovascular disease. Methods: Plasma homocysteine and SAH concentrations were assessed among 297 CARE FOR HOME participants suffering from CKD (K/ DOQI stages G1–G5). SAH was analysed using a Waters 2795 alliance HT, coupled to a Quattro micro API tandem mass spectrometer. Homocysteine was assessed by using a fluorescence polarization immunoassay on the Abbott AxSym system. Kidney function was determined as estimated glomerular filtration rate (eGFR), using the MDRD equation. Results: Among the 297 CKD patients, mean eGFR was 44±19 ml/ min/1.73 m², and mean plasma SAH and homocysteine were 50.1±30.1 µmol/l and 18.4±7.2 nmol/l, respectively. 32 % of patients had prevalent cardiovascular disease at baseline. eGFR correlated more strongly with plasma SAH (r=0.497) than with plasma homocysteine (r=0.424). Patients with prevalent cardiovascular disease had higher plasma SAH than patients without prevalent cardiovascular disease (p=0.007). Nevertheless, plasma SAH did not independently predict prevalent cardiovascular disease in logistic regression analysis. Discussion: In CKD, plasma SAH accumulates to a higher degree than plasma homocysteine. Follow-up of study participants will reveal whether SAH independently predicts future cardiovascular events. Moreover, further studies are needed to examine if © Verlag PERFUSION GmbH

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an increase of plasma SAH represents a novel non-traditional cardiovascular risk factor and to identify strategies to lower SAH, after B vitamins failed to reduce SAH levels.

Galectin-3 binding protein plasma levels are independently associated with cardiovascular mortality C.A. Gleissner1, 2, C. Erbel1, 2, F. Linden1, G. Domschke1, M. Akhavanpoor1, A.O. Doesch1, M. Kleber5, H.A. Katus1, 2, W. Maerz3, 4, 5 1 Department of Cardiology, Angiology and Pneumonology, Heidelberg University Hospital, Germany 2 DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg, Germany 3 Medical Clinic V (Nephrology, Hypertensiology, Rheumatology, Endocrinology, Diabetology), Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany 4 Medical University of Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria 5 Synlab Academy, Synlab Services GmbH, Mannheim, Germany Aims: Galectin-3 binding protein (Gal-3BP) plasma levels have been associated with subclinical carotid atherosclerosis. We sought to test whether Gal-3BP levels are associated with coronary artery disease (CAD) and cardiovascular outcome. Methods and results: Gal-3BP plasma levels were measured by ELISA in 2,922 patients from the Ludwigs hafen Risk and Cardiovascular Health (LURIC) study (age 62.7±10.6 years, 62.7 % male, 2,268 with angiographically confirmed CAD). During the follow-up period (8.8±3.0 years), 866 individuals died, 654 of cardiovascular causes. Gal-3BP levels in CAD patients were 6.7±3.2 versus 6.2±3.2 mg/ ml in controls (p<0.001). Overall, Cox proportional hazards regression of Gal3BP quintiles showed an increasing risk for all-cause and cardiovascular mortality after adjustment for gender, age, and cardiovascular risk factors (HRQ5 for all-cause mortality 1.292 Perfusion 01/2015

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[1.033–1.618, p=0.025]; HRQ5 for cardiovascular mortality 2.626 [1.088– 6.336, p=0.032]). After further adjustment for glomerular filtration rate, troponin-T, NT pro-BNP, and C-reactive protein levels, hazard ratios for cardiovascular mortality remained significantly elevated in patients with stable CAD (HR per unit increase of Gal-3BP 1.392 [1.061–1.825, p=0.017]), but not in unstable CAD. Gal-3BP was significantly associated with parameters of lipid metabolism (triglycerides, HDL, apolipoproteins B, A-I, and A-II) and inflammation (high-sensitivity CRP, fibrinogen, LPS-binding protein, and IL-6). Conclusions: We show that (1) Gal3BP plasma levels independently correlate with the diagnosis and the severity of coronary atherosclerosis, (2) elevated Gal-3BP levels are independently associated with both all-cause and cardiovascular mortality, and (3) Gal-3BP levels are associated with markers of metabolic and inflammatory distress. These findings establish a novel role for Gal-3BP in CAD.

Redox homeostasis in the aged endothelium: to be or not to be? C. Goy1, S. Jakob1, N. Dyballa-Rukes1, A. Eckers1, J. Altschmied1, J. Haendeler1,.2 1 IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany 2 Central Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty, University of Düsseldorf, Germany Aims and methods: Reactive oxygen species (ROS) are viewed as a double-edged sword. They act on one hand as signalling molecules, but on the other hand ROS can damage macromolecules, including DNA, lipids and proteins within cells. Therefore, each cell contains oxidative and anti-oxidative systems to maintain the redox homeostasis. During the process of aging of an organism as well as in senescent cells the ROS levels are increased. Whether these ROS levels are causally involved

in senescence induction or only innocent bystanders is still unclear in the vascular aging field. In the endothelium pro-oxidative and pro-inflammatory stimuli, which increase intracellular ROS levels, are known to damage the endothelium, lead to endothelial dysfunction and increase the numbers of senescent endothelial cells. Moreover, in humans, endothelial function declines with age. The objective of this study was to investigate whether the redox homeostasis of primary endothelial cells is disturbed in stress-induced senescence and if such a disturbance is causal for senescence induction. Therefore, thioredoxin-1 (Trx-1), as one important anti-oxidative protein and NADPH oxidase 4, whose sole purpose is to produce ROS and which shows the highest expression in endothelial cells, were investigated in stress-induced premature senescence. For that purpose endothelial cells were treated for two weeks with 50 µM H2O2. Results: This treatment induces senescence-associated β-Galactosidase and nuclear p21 as typical features of senescence. This was accompanied by an increase in total as well as mitochondrial ROS, Indeed, an imbalanced redox homeostasis is detected by elevated NOX4 and decreased Trx1 levels. Senescence induction can be abrogated by lentiviral reexpression of Trx-1. Moreover, the lysosomal protease cathepsin D is „over-activated“ in the senescence model used, which explains the reduced Trx-1 protein levels. Inhibition of “over-active” Cathepsin D by the specific, cell-permeable inhibitor pepstatin A abolishes increase in nuclear p21 protein, ROS formation and degradation of Trx-1 protein, thus leading to blockade of stress-induced premature senescence by stabilizing the cellular redox homeostasis. Moreover, in vivo aortic Trx-1 levels are decreased and cathepsin D activity is increased in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium when compared to their wildtype littermates. Conclusions: In conclusion, our data © Verlag PERFUSION GmbH

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demonstrate that loss of Trx-1 and upregulation of NOX4 importantly contribute to the imbalance in the redox-status of senescent endothelial cells ex vivo and in vivo. In future studies we will analyse if the disturbed redox homeostasis in NOX4 transgenic mice also affects their lifespan.

Cyclodextrin promotes murine atherosclerosis regression by dissolution of cholesterol crystals and LXR-mediated macrophage reprogramming A. Grebe1*, S. Zimmer2*, N. Bode2, D. De Nardo1, L.I. Labzin1, A. Kerksiek3, N. Niyonzima4, T. Espevik4, C. Hempel5, M.T. Heneka6, J.-Å. Gustafsson7, T. Ulas8, J.L. Schultze8, S.D. Wright9, G. Nickenig1, D. Lütjohann3, E. Latz1, 4, 10, 11 1 Institute of Innate Immunity, University Hospital Bonn, Germany 2 Department of Internal Medicine II, University Hospital Bonn, Germany 3 Institute of Clinical Chemistry und Clinical Pharmacology, University Hospital Bonn, Germany 4 Centre of Molecular Inflammation Research at the Norwegian University of Science and Technology, Trondheim, Norway 5 Addi and Cassi Fund, Reno, NV, USA 6 Department of Neurology, University Hospital Bonn, Germany 7 Center for Nuclear Receptors and Cell Signaling, University of Houston, TX, USA 8 Genomics and Immunoregulation, Life and Medical Sciences Institute, University of Bonn, Germany 9 CSL Behring, King of Prussia, PA, USA 10 Department of Infectious Diseases and Immunology, UMass Medical School, Worcester, MA, USA 11 German Center of Neurodegenerative Diseases (DZNE), Bonn, Germany authors contributed equally * Both Aims: Inflammation is the driving force in the pathogenesis of atherosclerosis. Cholesterol crystals (CCs) are a hallmark of atherosclerotic lesions and represent a potent inflammatory stimulus. 2-Hydroxypropyl-β-cyclodextrin (CD), a clinically used oligosaccharide, Perfusion 01/2015

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enhances cholesterol solubility and mediates cholesterol depletion from cell membranes. Thus, we studied the potential of CD treatment in mouse models of atherosclerosis. Further, we aimed to decipher the mechanisms of CD function in murine atherosclerosis by studying its capability to mediate CC dissolution and cholesterol efflux. Methods: ApoE–/– mice fed a high-fat diet for 8 weeks received subcutaneous CD injections twice a week. To study atherosclerosis regression ApoE–/– mice were fed a high-fat diet for 8 weeks to induce advanced atherosclerosis prior to CD treatment for 4 weeks. Atherosclerotic plaque size and CC-load were assessed by confocal-laser-reflection microscopy. Isotope labeled CC and GC-MS-SIM analyses were used to assess crystalderived cholesterol metabolism and localization. Reverse cholesterol transport from macrophages loaded with isotope-labeled CC was analysed in C57BL/6 mice. CD-induced transcriptional changes in CC-loaded macrophages were assessed by genome-wide mRNA profiling. Results: In an atherosclerosis mouse model CD treatment reduced atherosclerosis development and promoted atherosclerosis regression as determined by a reduction in atherosclerotic plaque size and CC plaque load. In vitro, CD solubilized CCs and promoted cholesterol ester and oxysterol production in macrophages resulting in liver X receptor (LXR)-mediated transcriptional reprogramming. CD increased cholesterol efflux from macrophages and enhanced reverse cholesterol transport in vivo. Conclusions: CD is an effective therapy for the prevention and treatment of murine atherosclerosis. Further, our data suggests that the beneficial effects of CD on murine atherosclerosis are mediated by its ability to dissolve CCs and induce the metabolism and efflux of crystal-derived cholesterol from macrophages in vitro and in vivo. Thus, CD may be used in clinical trials to pharmacologically reduce atherosclerosis burden.

The Toll-like receptor 2/6 ligand MALP-2: A novel option for vascular regeneration K. Grote1, H. Schuett2, J. Schuett1, R. Oberoi1, M. Luchtefeld1, B. Schieffer2 1 Philipps University Marburg, Department of Internal Medicine – Cardiology, Marburg, Germany 2 University Hospital Gießen and Marburg GmbH, Location Marburg, Department of Internal Medicine – Cardiology, Marburg, Germany Aims: Toll-like receptors (TLR) are initially identified as important sentinels of the innate immune system, recognizing invading pathogens in order to initiate the body´s immune defense. Growing evidence exist that cardiovascular expressed TLRs play crucial roles in pathophysiological processes in the myocardium and the vasculature. We here investigated a novel opportunity for a TLR2/6 dependent pathway to promote vascular regeneration. Methods: The TLR ligand MALP-2 (naturally occurring in Mycoplasma spec.) was used to specifically stimulate a TLR2/6-dependent pathway. Gene expression was studied by qRTPCR, Western blot, ELISA and protein array. Angiogenesis was investigated by migration assays (transwell), proliferation (BrdU-incorporation) and in matrigel assay in vitro and in vivo in mice. Superoxide was detected by dihydroethidium and NAPDH oxidase activity by electron spin resonance spectroscopy. Angiogenic properties of mesenchymal stem cells (MSCs) were investigated in a sheep model of tissue engineering. Reendothelialization and neointima formation was assessed after vascular injury in mice. Results: We observed that MALP2 exhibits a considerable angiogenic potential in vitro and in vivo. We further provide evidence that the induction of angiogenesis is mediated by the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF), mainly released by endothelial cells and monocytes in a TLR2/6-dependent © Verlag PERFUSION GmbH

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manner. Interestingly, we found that the release of GM-CSF from endothelial cells is controlled by a Nox2-containing NAPDH oxidase. In addition, we present evidence that TLR2/6-dependent stimulation of MSCs with MALP2 enhanced angiogenesis in vitro in a paracrine manner. Subsequent transplantation of these MSCs significantly enhanced capillary density in engrafted tissue transplants. Furthermore, we observed that TLR2/6-dependent stimulation by MALP-2 promotes reendothelialization and inhibits neointima formation after experimental vascular injury via enhanced proliferation and migration of endothelial cells. Conclusions: Taken together, we identified the activation of a TLR2/6dependent pathway by MALP-2 as a potent stimulus for the local release of growth factors such as GM-CSF and as a novel therapeutic option to promote angiogenesis and endothelial regeneration after vascular injury.

Selective inhibition of BET bromodomains as therapeutic option to prevent negative vascular remodeling M. Haertlé, S. Weisheit, J. Dutzmann, J.-M. Daniel, K. Donde, J. Bauersachs, D. Sedding Department of Cardiology and Angiology, Hannover Medical School, Germany Background: The response of vascular cells to acute injury determines the extent of negative vascular remodeling and neointima formation. Epigenetic regulation of evolutionary conserved protein-interaction modules that recognize acetylated lysines called bromodomains, is critically involved in the transcriptional control of functionregulating gene sets. To date it is not known, if epigenetic modulation contributes to altered signalling responses in activated vascular cells. Therefore, we sought to determine the impact of bromodomain-dependent signalling on proliferation, migration and apoptosis of primary human coronary artery Perfusion 01/2015

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smooth muscle cells (SMC) and endothelial cells (EC). Methods and results: The effect of selective bromodomain-inhibition on cellular function was observed in primary SMC and EC in vitro. Selective inhibition of bromodomains and extraterminal (BET) bromodomains by (+)-JQ1 and I-BET151 dose-dependently prevented the growth medium-induced proliferation of SMC (100±12.73 % vs. 16.81±3.71 %; p<0.0001 [1 µM (+)-JQ1] and vs. 3.49±0.58 %; p<0.0001 [1 µM IBET151]) as well as EC (100±15.43 % vs. 46.04±8,12 %; p<0.0001 [1 µM (+)JQ1] and vs. 29.63±4.44 %; p<0.0001 [1 µM I-BET151]). Subsequent flow cytometry analysis of propidium jodidestained cells revealed that bromodomain-inhibition resulted in a cell cycle arrest in the G0/G1, whereas apoptosis was not induced. (+)-JQ1 and I-BET151 also attenuated migration of SMC and induced a mature, differentiated SMC phenotype. Conclusion: In conclusion, BET bromodomain-containing proteins are suggested to be critically involved in cell cycle regulation and proliferation of SMC and EC and therefore vascular remodelling. But the complex regulation still remains elusive. Moreover, selective inhibition of BET bromodomains by the specific compounds (+)JQ1 and I-BET151 might represent a novel therapeutic approach to prevent negative vascular remodeling.

accumulation of leukocytes and lipids in the vessel wall. B cells have been shown to influence these processes in different ways. Natural IgM producing B1a cells protect from atherosclerosis, while B2 cells aggravate plaque formation. The role of a recently described GM-CSF-producing B cell population, the innate response activator (IRA) B cell, in atherosclerosis is unknown. Methods and results: We show by flow cytometry, RT-PCR and immunohistology that GM-CSF-producing IRA B cells progressively arise in mice and humans with atherosclerosis and preferentially accumulate in secondary lymphoid organs via Myd88-dependent signalling. Mixed bone marrow chimeras lacking B cell-derived GM-CSF develop smaller plaque lesions with fewer macrophages and T effector cells. Mechanistically, IRA B cell derived GM-CSF promotes the generation of IL-12-producing classical dendritic cells that mount an adaptive TH1 cell response. Proatherogenic IFN-γ-producing TH1 cells accumulate in the aorta and stimulate isotype switching of atherosclerosis-related immunoglobulins against oxidized lipoproteins. Conclusion: IRA B cells represent a previously unknown regulatory node in atherosclerosis-related immunity that direct DC to prime an antigen-specific TH1 response thereby aggravating atherosclerosis.

GM-CSF producing IRA B cells shift the adaptive immune response towards a TH1 milieu and aggravate atherosclerosis

Selective inhibition of the histone lysine methyltransferase G9a preserves differentiation and inhibits calcification in vascular smooth muscle cells

I. Hilgendorf1, I. Theurl1, L.M.S. Gerhardt1, C.S. Robbins1, A. Zirlik2, J.L. Witztum3, P. Libby4, M. Nahrendorf1, R. Weissleder1, F.K. Swirski1 1 Massachusetts General Hospital, Boston, MA, USA 2 University Heart Center Freiburg, Germany 3 Department of Medicine, UCSD, La Jolla, CA, USA 4 Brigham and Women’s Hospital, Boston, MA, USA

F. Kahles1, J. Marx1, A. Makowska1, M. Lehrke1, N. Marx1, H.M. Findeisen1, 2 1 Department of Internal Medicine I – Cardiology, University Hospital Aachen, Germany 2 Department of Cardiology and Angiology, University Hospital Münster, Germany

Purpose: Atherosclerosis is a chronic inflammatory disease driven by the

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cells (SMCs) at sites of vascular injury is regulated by epigenetic mechanisms. Epigenetic histone methylation has been recognized as a dynamic mark controlling many biological processes in health and disease. Here we have identified a histone methyltransferase inhibitor as a selective modulator of SMC differentiation. Methods and results: We studied the effects of several epigenetic modifiers on smooth muscle cell proliferation, inflammation and TNF-α-mediated dedifferentiation. UNC0638, a specific inhibitor of the G9a histone methyltransferase controlling histone h3 lysine 9 dimethylation was found to selectively modulate SMC differentiation. UNC0638 had no effect on TNF-αinduced MCP-1 expression or PDGFinduced SMC proliferation as detected by cell counting and BrdU incorporation. However, UNC0638 treatment significantly attenuated TNF-α-induced downregulation of the SMC marker gene SM22α, suggesting that UNC0638 reduces SMC dedifferentiation. This effect was detectable up to 72 hours after the initial treatment and associated with a strong and equally sustained reduction of the respressive histone mark (h3k9me2) at the SM22α promoter. Based on these data we analysed the impact of UNC0638 treatment on CaPO(4)-induced SMCcalcification, an in vitro model of SMC transdifferentiation. Consistent with the previously observed preservation of the SMC-differentiation status, UNC0638 treatment significantly attenuated SMC calcification as detected by von Kossa-staining. This was associated with a reduced expression of SMC calcification markers including ALPL, Runx2, BMP-2 and osteocalcin while anti-calcification markers including PPAR-γ and MGN were upregulated. Conclusion: In summary, our data suggest pharmacologic modulation of histone methylation as a promising approach to target SMC phenotypes and differentiation status in vascular diseases like in-stent restenosis or atheroPerfusion 01/2015

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sclerosis and warrant further research to dissect histone methylation dependent mechanisms in SMCs and to investigate in vivo applications of small molecule inhibitors.

The incretin hormone GIP is modulated by inflammatory stimuli and downregulated in critically ill ICU patients: central relevance of interleukin 1β F. Kahles1, C. Meyer1, S. Diebold1, R. Stöhr1, J. Möllmann1, C. Lebherz1, H.M. Findeisen1, 2, A. Koch3, F. Tacke3, N. Marx1, M. Lehrke1 1 Department of Internal Medicine I – Cardiology, University Hospital Aachen, Germany 2 Department of Cardiology and Angiology, University Hospital Münster, Germany 3 Department of Internal Medicine III – Gastroenterology, University Hospital Aachen, Germany Purpose: The incretin hormone GLP-1 was recently found to be increased in response to inflammatory stimuli leading to insulin secretion and prevention of hyperglycemia in context of critical illness. We here study the relevance of the other main incretin hormone glucose-dependent insulinotropic peptide (GIP) as a regulator of glucose metabolism under inflammatory conditions. GIP is known to be released in response to food intake from endocrine intestinal cells leading to glucose-dependent insulin secretion. Methods and results: Low dose lipopolysaccharide (LPS) injection (100 μg/kg) – used as an inflammatory stimulus – time-dependently increased GIP secretion in C57BL/6J mice. Interestingly, this was only apparent at a low LPS dose (4.1 fold increase with 10 µg/ kg; p<0.05) and lost with median LPS dosage (1.1 fold increase with 1 mg/kg; p=0.72) while high LPS concentrations led to a trend towards decreased GIP levels (0.6 fold decrease with 2 mg/kg; p=0.2). To elucidate the relevant mechanisms we injected mice with inflammatory cytokines known to be released in response to endotoxin. GIP levels

significantly increased in response to IL-1β (1.9 fold; p<0.01) and showed a trend for IL-6 (1.6 fold; p=0.16) but not for TNF-α (all 4 μg/kg) administration. Using IL-1-receptor- and IL-6 knockout mice we found LPS-mediated GIP secretion to be selectively dependent on IL-1 but not on IL-6 signalling. To evaluate the functional relevance of inflammatory GIP secretion we pretreated mice with the GIP-receptor antagonist (Pro3)GIP (25 nmol/kg). This however did not affect LPS-induced insulin secretion or blood glucose lowering. Nevertheless, (Pro3)GIP markedly blunted LPS-induced TNF-α (p<0.01) and IL-6 secretion (p<0.05) suggesting that GIP may have proinflammatory effects in states of acute inflammation. We next asked whether a similar inflammatory GIP regulation is present in humans. We thereby found circulating GIP levels to decline in response to an inflammatory stimulus (cardiac surgery with extracorporal circulatory support; n=18; baseline 44.4±5.7 pg/ ml to 29.3±4.9 pg/ml after 24 hours; p<0.05). Furthermore, GIP serum levels were significantly decreased (5.8 fold) in critically ill ICU patients (n=68) in comparison to healthy controls (p<0.001). Conclusion: GIP provides a novel link between the immune system and the gut. Although GIP seems to hold minor relevance for the regulation of glucose metabolism under inflammatory conditions it acts as an inflammatory immune modulator. This requires further characterization.

Optimization of the transfection of human macrophages A.-A. Keller1, M.B. Maeß1, K. Rennert2, A. Mosig2, S. Lorkowski1 1 Institute of Nutrition, Friedrich Schiller University Jena, Germany 2 Center for Sepsis Control and Care (CSCC), University Hospital Jena, Germany Background: Common protocols for cell detachment apply enzymes or © Verlag PERFUSION GmbH

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enzyme-mixes like accutase I. These approaches achieve cell detachment by degradation of extracellular matrix (ECM) proteins cleaving the linkage of cells to the ECM and to other cells. This may be harmful to some cells or may have detrimental side-effects on analyses, as the enzymes may also degrade cellular surface proteins, which affects the functionality of cells or removes specific markers to be targeted in further analyses. In order to avoid enzymatic cell detachment the Nunc™ UpCell™ product line has been developed. These cell culture materials are coated with a thermo-sensitive polymer, which changes its properties with temperature. At 37°C the polymer is slightly hydrophobic allowing cell attachment and growth. Below 32°C the polymer becomes hydrophilic generating an aqueous film between cells and polymer surface. Consequently, detachment with intact ECM proteins and cell surface proteins occurs. Aim: Confirmation of the comparability of Nunc™ UpCell™ plates with an established accutase I based enzymatic detachment method. Methods and results: We have subsequently optimized differentiation conditions to achieve premature adherent human THP-1 macrophages. Thus, we have established an electroporation protocol for transfection of these cells using Lonza Nucleofector technology. Here, we tested the suitability of the UpCell plates for detaching premature THP-1 macrophages for nucleofection, as detachment of the cells is vital for this transfection protocol. We present data verifying proper cell morphology and vitality and high transfection efficiency for macrophages cultured on UpCell plates determined by flow cytometric analysis. Appropriate macrophage behavior was confirmed by measuring markers of macrophage differentiation and polarization by RTqPCR. Both differentiation and culture conditions were optimized for THP-1 cells in order to improve their response to polarizing stimuli. Conclusion: Our data indicate that Perfusion 01/2015

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the UpCell plates yield results at least as good as the enzymatic approach. In conclusion Nunc UpCell materials are a viable alternative to enzymatic detachment. Furthermore, optimized differentiation and culture conditions improve the response of transfected THP-1 macrophages to polarizing stimuli.

Identification of 4 pc plasmalogens as lipidome signature in patients with coronary artery disease and acute coronary syndromes R. Klingenberg1*, I. Sutter2 ,3*, A. Othman2, 4, L. Rohrer2, U. Landmesser1, 3, C.M. Matter1, 3, T.F. Lüscher1, 3, A. von Eckardstein2, 3, 4, T. Hornemann2, 3 1 Department of Cardiology, University Heart Center, University Hospital Zurich, Switzerland 2 Institute of Clinical Chemistry, University and University Hospital of Zurich, Switzerland 3 Competence Center for Integrated Human Physiology, University of Zurich, Switzerland 4 Competence Center for System Physiology and Metabolic Diseases, ETH Zurich and University of Zurich, Switzerland authors contributed equally * Both Background: Coronary artery disease (CAD) is a chronic disease resulting from the formation of atherosclerotic lesions within the arterial wall. Glycerophospholipids and sphingolipids play an important role in lipoprotein metabolism, plaque formation and therefore in the development of atherosclerosis. Objectives and methods: Here, we show the association of plasma levels of glycerophospholipids and sphingolipids as well as sphingosine1-phosphate (S1P) species and sphingoid bases of sphingolipids with CAD and acute coronary syndrome (ACS). In this study, we included comparison of statin-treated and untreated CAD patients. Furthermore, we described the distribution of phosphatidylcholines (PCs) and sphingomyelins (SMs) among plasma lipoproteins. Plasma lipids were measured using three different liquid chromatography-mass spectrometry methods.

Results: The glycerophospholipid and sphingolipid profile was markedly altered in plasma of patients with CAD and ACS, respectively. Specifically, 45 out of 65 quantified glycerophospholipid and sphingolipid species, 16:1S1P as well as four sphingoid bases were lower in plasma of CAD patients as compared to healthy controls. Similarly, plasma levels of 42 glycerophospholipids and sphingolipids, 16:1-S1P, 18:1-S1P as well as six sphingoid bases were lower in ACS patients, relative to healthy subjects. Statin therapy did not have any significant effect on the plasma concentrations of glycerophospholipids and sphingolipids. The oddchain PCs, PC33:1, PC33:2, PC33:3 and PC35:3, which are PC plasmalogens, were the most significantly and consistently altered species in plasma of both patients with CAD and ACS, respectively. The PC and SM species were abundant exclusively in lipoprotein particles, whereas LPCs were mainly present in the lipoprotein-free fraction. Conclusion: Marked alterations in the plasma concentrations of glycero phospholipids and sphingolipids were found in patients with CAD and ACS, among which four PC plasmalogens figured most prominently. Since the vast majority of glycerophospholipids and sphingolipids are present in lipoproteins, our observations are likely to be related to changes in composition, number and/or oxidative state of lipoproteins.

The α-tocopherol long-chainmetabolite α-13’-COOH regulates the expression of ABCA1 in primary fibroblasts S. Kluge1, 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 © Verlag PERFUSION GmbH

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Background: α-Tocopherol (α-TOH) is metabolized in the liver by side-chain truncation initiated by CYP3A4-dependent ω-hydroxylation which results in the formation of long-chain-metabolites (α-LCM) hydroxychromanol (α-13‘-OH). Subsequent α‑oxidation in peroxisomes forms the α-LCM carboxychromanol (α-13‘-COOH) which also occurs in human plasma. Vitamin E is known to modulate cholesterol homeostasis but almost nothing is known on the effects of α-13’-COOH on cholesterol homeostasis. Aim: In our study we want to unravel the regulatory effects of α-13’-COOH on the expression of ABCA1. Methods and results: We found that in primary fibroblasts α-13’-COOH has antagonistic effects on the expression of ABCA1 mRNA and protein. We were therefore interested in the pathways by which α-13’-COOH modulates the expression of this cholesterol exporter. We investigated the signalling pathways mediating these effects at the mRNA level using real-time RT-PCR and at the protein level using Western blotting. Initially, we focused on blocking experiments of the MAPK signalling cascade which is known to regulate ABCA1 expression. We found that blocking of ERK 1/2 results in an upregulation of the ABCA1 protein expression while ERK-phosphorylation is increased in α-13’-COOH treated cells. We obtained further evidence for an involvement of arachidonic acid in the regulation of ABCA1 by α-13’COOH. We are currently analyzing the missing parts of the proposed ERK-dependent signalling involved in ABCA1 regulation via α-13’-COOH. Conclusion: We here provide first evidence for a contribution of ERK signalling to the α-13’-COOH dependent regulation of ABCA1. Further experiments are required to elucidate the full regulatory pathway.

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The sodium-hydrogen exchanger NHE1 in endothelial cells regulates angiogenesis N. Kryeziu1, 2, J.-C. Hennings2, S. Lindenmüller1, C. Hübner2, R. Heller1 1 Institute for Molecular Cell Biology, Centre for Molecular Biomedicine (CMB), University Hospital Jena, Germany 2 Functional Genetics, Institute of Human Genetics, University Hospital Jena, Germany Introduction: New blood vessel formation from pre-existing ones, angiogenesis, occurs continuously throughout the life of an organism and requires active involvement of endothelial cells. The tissue in need for a better blood supply is ischemic and a number of growth factors are released, which among other effects trigger ion fluxes through the endothelial cell membrane. Sodium-hydrogen exchanger NHE1 regulates intracellular pH (pHi), affects enzymatic activities and cytoskeleton dynamics, and could thus be important in endothelial cell proliferation, migration and survival, modulating the complex process of angiogenesis. The aim of this study was to characterize the role of NHE1 in angiogenesis in vitro and in vivo in Nhe1 knockout mice. Methods: Experiments were performed in primary human umbilical vein endothelial cells (HUVEC) or in mice lung endothelial cells (MLEC). HUVEC were treated with specific siRNA to downregulate NHE1. Transfection efficiency was verified by Western blot and immunofluorescence on the protein level, and by pHi measurement for functional analysis. Basal pHi and pHi recovery after acidification with NH4Cl in bicarbonate-free HEPESbuffered media was measured using BCECF, a pH-sensitive fluorescent dye. To characterize angiogenic capabilities of HUVEC in vitro, proliferation (cell counting), survival (flow cytometry: subG1 cell cycle analysis and Annexin V-PI), migration (trans well migration assay), and sprouting from endothelial spheroids were analysed. Total Nhe1knockout mice in comparison to lit-

termate wildtype mice were used to study angiogenesis in vivo employing the Matrigel plug assay. In this assay, matrigel, a mixture of basement membrane proteins, with or without VEGF was injected subcutaneously to allow plug formation and vascularisation. The plugs were excised 7 days later and analysed immunohistochemically to quantify new blood vessel formation. Results: NHE1 was crucial for pHi control in endothelial cells under bicarbonate-free conditions since total absence of NHE1 in MLEC prepared from Nhe1 knockout mice resulted in lower basal pHi and abolished recovery after intracellular acidification. NHE1 was successfully knocked down in HUVEC as shown at the protein (Western blot and immunofluorescence) and the activity level (pHi measurement). Downregulation of NHE1 led to cell cycle arrest in G0/G1 after 24 hours of siRNA treatment and reduced cell numbers after 48 hours (minus 35 % versus control cells); survival was not altered. In addition, sphingosine-1-phosphatetriggered migration of cells with NHE1 knockdown was inhibited by 32 % compared to control cells and VEGFinduced sprouting from NHE1-depleted spheroids was reduced by 44 %. The data obtained in vitro were confirmed by studies of in-vivo angiogenesis. Nhe1-knockout mice showed significantly less angiogenesis in response to VEGF than their wildtype littermates (minus 21 %). Conclusion: The results from in-vitro and in vivo-assays demonstrate that NHE1 plays a supportive role in angiogenesis and may serve as a target to interfere with angiogenic processes.

AMPK controls systemic inflammatory response: effects on endothelial permeability and liver function S. Lindenmüller1, 2, K. Spengler1 , C. von Loeffelholz2, 3, L. Bloch2, 4, A. Thuy2, 3, M. Gräler2, 3, O. Huber2, 4, R. Bauer1, 2, B. Viollet5, R. Heller1, 2 1 Institute of Molecular Cell Biology, University Hospital Jena, Germany © Verlag PERFUSION GmbH

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Integrated Research and Treatment Center, Center for Sepsis Control and Care, University Hospital Jena, Germany 3 Department of Anesthesiology and Intensive Care Medicine, University Hospital Jena, Germany 4 Department of Biochemistry II, University Hospital Jena, Germany 5 Institut Cochin, Université Paris Descartes, CNRS, France 2

Aim: Sepsis and systemic inflammatory response syndrome (SIRS) are characterized by a generalized dysregulated inflammatory response. One hallmark is microvascular dysfunction which is linked to endothelial dysfunction and which results in decreased organ perfusion and subsequent development of organ failure. We hypothesized that the energy-sensing enzyme AMP-activated kinase (AMPK), an important regulator of cell metabolism and homeostasis, can limit inflammatory responses of the endothelium under the condition of systemic inflammation and sepsis and protect against the development of vascular dysfunction. Methods: Experiments were performed in wildtype mice and in mice, in which the catalytic subunit AMPKa1 was knocked out. SIRS was induced by intraperitoneal injection of LPS. 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-increasing compounds is evaluated. To investigate differences in wildtype and knockout animals blood was taken to analyse plasma cytokine levels and livers were prepared to obtain protein lysates and tissue sections. Immunofluorescence studies were performed to evaluate liver endothelial cells using antibodies against CD31, an endothelial marker, and claudin 5, an endothelial-specific tight junction protein. Furthermore, a survival study after LPS injection was performed. Results: Mice, in which the AMPKa1subunit is knocked out, showed a highPerfusion 01/2015

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er LPS-induced mortality than wildtype mice. In addition, they developed a more pronounced systemic inflammation as shown by considerably higher plasma levels of IFN-γ, IL-1β, IL-10, TNF-α, IL-6 and MCP-1. Induction of SIRS led to an increased vascular permeability in organs, especially in liver. When compared to wildtype mice, a higher vascular leakage was observed in AMPKa1-knockout animals. Exvivo investigation of liver sections by immunofluorescence (CD31 and claudin 5 stainings) showed a damage of the liver sinusoidal endothelium and a disrupture of tight junction complexes. The breakdown of claudin 5 was also seen in Western blot analyses. The LPS-induced effects on endothelial morphology were markedly pronounced in AMPKa1-knockout animals when compared to wildtype mice. Conclusion: AMPK protects endothelial cells and vascular barriers from inflammatory alterations by stabilizing endothelial tight junctions and may thus contribute to the maintenance of organ function. AMPK may represent a pharmacological target to prevent or treat endothelial dysfunction under conditions of systemic inflammation or sepsis.

In patients with severe aortic stenosis increased plant sterol deposition in vascular tissue characterizes patients with concomitant coronary artery disease A. Luister1, H.F. Schött3, D. Lütjohann3, C. Husche3, H.-J. Schäfers4, M. Böhm1, J. Plat5, U. Laufs1, O. Weingärtner1, 2 1 Department of Internal Medicine III, Saarland University Medical Center, Homburg, Germany 2 Department of Cardiology, University Hospital Oldenburg, European Medical School Oldenburg-Groningen, Oldenburg, Germany 3 Institute of Clinical Chemistry und Clinical Pharmacology, University Hospital Bonn, Germany 4 Department of Thoracic and Vascular Surgery, Saarland University Medical Center, Homburg, Germany

Department of Human Biology, Maastricht University, The Netherlands

Purpose: The aim of the study was to evaluate the relationship of phytosterols, oxyphytosterols and other markers of cholesterol metabolism in relation to concomitant coronary artery disease in patients with severe aortic stenosis scheduled for elective aortic valve replacement. Methods: Besides markers for cholesterol metabolism (plant sterols and cholestanol as markers for cholesterol absorption and lathosterol as an indicator of cholesterol synthesis) oxyphytosterols were determined in plasma and aortic valve cusps of 104 consecutive patients with severe aortic stenosis (statin treatment: 68 patients; no statin treatment: 36 patients). Coronary angiography prior to aortic valve replacement determined the extent of coronary artery disease. Results: Patients treated with statins were characterized by lower cholesterol, lower cholestanol and lower lathosterol plasma levels. Statin treatment did not affect sterol concentrations in cardiovascular tissue. Absolute values for the cholesterol absorption markers (sitosterol and campesterol) were significantly higher in vascular tissue of patients with documented coronary artery disease compared with those without concomitant CAD. Campesterol oxides were significantly higher in tissue of aortic valve cusps and oxidized sitosterol-to-cholesterol ratios were significantly higher in plasma of patients with coronary artery disease. Interestingly, neither the cholesterol absorption marker cholestanol nor the ratio of cholestanol-to-cholesterol was associated with CAD. Conclusions: Patients with concomitant coronary artery disease are characterized by increased concentrations of plant sterols and their respective oxides in vascular tissue. The fact that cholestanol was not associated with CAD further strengthens the hypothesis that plant sterols are athero genic. © Verlag PERFUSION GmbH

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Evaluation of the second worldwide sterol and oxysterol harmonization trial D. Lütjohann1, H.-F. Schött1, A. Lövgren-Sandblom2, U. Diczfalusy2, W.J. Geilenkeuser3, I. Björkhem2 and the ENOR oxysterol evaluation group 1 Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Germany 2 Department of Laboratory Medicine, Division of Clinical Chemistry, Karolinska University Hospital, Karolinska Institute, Huddinge, Sweden 3 Reference Institute for Bioanalytics, Bonn, Germany Aims: Serum concentrations of lathosterol and the plant sterols campesterol and sitosterol as well as the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Cholesterol metabolites such as 7α-, 7β-, 24S-, 25-, and 27-hydroxycholesterol and 7-ketocholesterol are monooxygenated cholesterol metabolites (oxysterols) and bile acid precursors. Furthermore, oxysterols are used as prognostic and diagnostic tools for the description of dyslipidemic and neurodegenerative states in mammalian subjects as well as reactive oxygen species (ROS) markers. There are conflicting results on basic serum or plasma levels of these parameters from specialized laboratories around the world. Methods: Twenty-two laboratories specialized in either gas-liquid or high performance-liquid chromatography (GLC or HPLC), agreed to participate in a second worldwide survey under the expertise of the reference institute for bioanalytics (RfB) located at Bonn, Germany. A set of two plasma samples (A and B) was sent to each participant for measurement of oxysterol concentrations and a further set of two different lyophilized pool sera (C and D) was sent to each participant for measurement of cholesterol and non-cholesterol sterols (NCS). In contrast to the first international survey test, each participant received standard solutions with defined concentrations of cholesterol, NCS and oxysterols. Perfusion 01/2015

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Results: The results were sent back from 21 participants and evaluated by RfB. The different sterols and oxysterols were quantified either by GC-flame-ionization detection, GLCor HPLC-mass selective detection. Each laboratory used its own internal standard. The data were given in the individual 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) in its used unit. Unfortunately, again cholesterol appeared to be autoxidized as proofed by highly varying concentrations of 7αand 7β-hydroxycholesterol as well as by increased 7-ketocholesterol. Those oxysterols, which are exclusively enzymatically produced (24S-, 25- and 27-hydroxycholesterol), were in the range as known from the literature. The lowest coefficient of variation is presented for cholesterol (C: 28.9 % and D: 33.0 %). For non-cholesterol sterols there are coefficients of variation between 43.5 % and 79.6 %. For 7-oxygenated cholesterols, coefficients of variation vary between 47.2 % and 129 % and for 24S-, 25- and 27-hydroxycholesterol between 56.6 % and 111 %. Conclusions: In comparison with the first world-wide survey trial we found a slight improvement in the variation of the data. However, we need further careful considerations and proposals to harmonize the methods for GC- and LC-analytics of cholesterol, non-cholesterol sterols and oxysterols. This includes the work-up procedure, safety parameters, use of standards and internal standards, chromatographic separation and detection mode and finally the unit to present each parameter.

The role of proteasomal protein degradation in endothelial senescence O. Meçe1, N. Kryeziu1, T. Grune2, A. Simm3, R. Heller1 1 Institute for Molecular Cell Biology, Centre for Molecular Biomedicine (CMB), University Hospital Jena, Germany 2 German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany 3 Department of Cardiothoracic Surgery, University Hospital Halle, Halle (Saale), Germany Aim: Endothelial cells (EC) underlie senescence in vivo, which is believed to contribute to endothelial dysfunction and vascular diseases. Senescence may be triggered by sustained cell replication, known as replicative senescence or stress-induced pathways, which may especially take place in certain inflammatory or oxidative microenvironments. One way through which senescence may be elicited or aggravated is the induction of a functional decline in protein degradation systems leading to the accumulation of protein aggregates. The current project aims to characterize protein degradation pathways in endothelial cells undergoing replicative senescence and to investigate the effect of proteasomal inhibition on endothelial senescence. Methods: The study was performed in human umbilical vein endothelial cells, which were cultured for up to 22–25 passages. Several parameters evaluating oxidative stress (protein carbonylation in Western blots) or senescence (senescence-associatedβ-galactosidase (SA-β-gal) staining, lipofuscin autofluorescence, cell size, granulation and growth) were assessed. The expression of the proteasomal subunits was analysed by Western blotting using specific antibodies. Proteolytic capacities were determined by means of fluorigenic substrates specific for peptidyl-glutamyl-like (β1 subunit), trypsin-like (β2 subunit) and chymotrypsin-like (β5 subunit) activities of the 20S proteasome. To investigate long term effects of proteasome inhibition on endothelial senescence, © Verlag PERFUSION GmbH

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the compounds MG132 or bortezomib were added to growth medium on a daily basis for 4 or 2 consecutive days, respectively. Subsequently, cells were allowed to recover and senescence markers were analysed after 6 inhibitor-free days. Results: Our results show that endothelial cells acquire a senescent phenotype after 22–25 passages in culture as verified by growth stop, SA-β-gal, increase in size and granulation as well as lipofuscin accumulation. Studies on the expression of proteasomal subunits revealed a decline of the 20S core components including β5 and β2 subunits in senescent cells versus early passage cells. Concordantly, proteasomal activities, in particular chymotrypsin-like activity, declined progressively with time of endothelial cells in culture. Transient inhibition of the proteasome by MG132 or bortezomib led to development of premature senescence as demonstrated by the appearance of senescence markers. In parallel, a persistent accumulation of carbonylated proteins was observed suggesting that proteasome inhibition leads to oxidative stress, which may be causally related to the development of senescence. Conclusion: Collectively, our data show that endothelial cells acquire a senescent phenotype, which is characterised by a decline of the proteasomal protein-degradation system. Proteasome inhibition alone is able to trigger senescence in vitro, possibly via inducing oxidative stress, suggesting a causal relationship between catabolic insufficiency and senescence. Further studies addressing the molecular links between proteolytic decline and senescence will yield a better understanding of catabolic insufficiency in age-associated pathologies of the cardiovascular system.

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The PDE-4 inhibitor roflumilast increases hepatic mitochondrial biogenesis and respiratory capacity leading to reduced body weight J. Möllmann1, F. Kahles1, C. Bäck2, R. Stöhr1, K. Hess1, H.M. Findeisen3, S. Krüger4, C. Lebherz1, F. Tacke2, N. Marx1, M. Lehrke1 1 Department of Internal Medicine I, University Hospital Aachen, Germany 2 Department of Internal Medicine III, University Hospital Aachen, Germany 3 Department of Cardiology and Angiology, University Hospital Münster, Germany 4 Department of Pneumology, FlorenceNightingale-Hospital Düsseldorf, Germany Introduction: Roflumilast is a selective PDE-4 inhibitor, which was recently found to reduce body weight and improve glucose metabolism in mice and men with yet unknown mechanism. Methods and results: To further elucidate the relevant mechanism of body weight reduction and improved glucose metabolism under PDE-4 inhibition we treated C57/BL6 mice (n=15/group) a high fat diet with or without roflumilast (21 mg/kg) for 11 weeks. Roflumilast significantly reduced body weight (32 g [control] vs. 28 g [roflumilast]; p<0.001) with a trend to increased food intake. This was associated with reduced hepatic steatosis leading to lower hepatic cholesterol (p<0.01) and triglyceride (p<0.05) content with decreased macrophage infiltration (p<0.05) and lower plasma levels of AST and ALT (both p<0.05). Mechanistically, roflumilast significantly increased PKA activity and CREB phosphorylation leading to augmented expression of the mitochondrial biogenesis factor PGC1alpha. This was paralleled by a significant increase in the expression of mitochondrial DNA, respiratory chain and mitochondrial fission and fusion gens (p<0.05). Furthermore, roflumilast increased mitochondrial respiratory capacity in hepatocytes under in vitro conditions from 100 % (control) to 127 % (p<0.05) in a PKA-dependent manner (mean of two

experiments each performed in n=5/ condition). Conclusion: The PDE-4 inhibitor roflumilast increases mitochondrial biogenesis and respiratory capacity and leads to weight loss and reduced steatohepatitis. This holds promise for a new treatment option for metabolic diseases.

Deficiency of the sialyltransferase St3Gal4 reduces Ccl5-mediated myeloid cell 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 German Centre for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Germany 6 AMC, Amsterdam, The Netherlands 7 Cardiovascular Research Institute Maastricht, The Netherlands * Both authors contributed equally Aims: Sialylation by α2-3 sialyltransferases 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 receptors. As the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of the sialyltransferase 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-deficient myeloid cells showed a reduced binding of Ccl5 and impaired Ccl5© Verlag PERFUSION GmbH

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triggered integrin activation. Correspondingly, Ccl5-induced arrest on TNF-α-stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4–/– mice. In contrast, St3Gal4-deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance 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. Conclusions: In summary, these findings identify St3Gal4 as a promising target to reduce inflammatory leukocyte recruitment and arrest.

Deficiency of endothelial Cxcr4 reduces reendothelialization and enhances neointimal hyperplasia after vascular injury in atherosclerosis-prone mice H. Noels1*, B. Zhou1*, P.V. Tilstam1, W. Theelen1, X. Li1, L. Pawig1, C. Schmitz2, S. Akhtar1, S. Simsekyilmaz1, E. Shagdarsuren1, A. Schober2, R.H. Adams3, J. Bernhagen4, 5, E,A. Liehn1, 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 Münster, 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 Perfusion 01/2015

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German Centre for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Germany authors contributed equally * Both

Aims: The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neo intimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation. Methods and results: β-Galactosidase staining using Bmx-CreERT2 reporter mice and double immunofluorescence revealed an efficient and endothelialspecific deletion of Cxcr4 in BmxCreERT2+ compared to Bmx-CreERT2Cxcr4-floxed ApoE–/– mice (referred to as Cxcr4EC-WTApoE–/– and Cxcr4EC-KO ApoE–/–, respectively). Endothelial Cxcr4 deficiency significantly increased wire injury-induced neointima formation in carotid arteries from Cxcr4EC-KOApoE–/– mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4EC-KOApoE–/– carotids compared to Cxcr4EC-KOApoE–/– controls. Furthermore, stimulation of human aortic endothelial cells with Cxcl12 significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the Cxcr4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1+Flk1+Cd31+ and of Lin-Sca1+ progenitors in Cxcr4EC-KOApoE–/– mice following vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, VEGF, S1P or Flt3-ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4EC-KO ApoE–/– mice.

Conclusion: Endothelial Cxcr4 is crucial for efficient reendothelialization following vascular injury through endothelial wound-healing and proliferation, and through the mobilization of Sca1+Flk1+Cd31+ cells, often referred to as circulating endothelial progenitor cells.

Modulation of angiogenesis through platelet-mediated mechanisms H. Nording, J. Patzelt, M. Olbrich, F. Emschermann, R. Schleicher, S. Gekeler, M. Gawaz, H.F. Langer Cardioimmunology Rresearch Group, Department for Internal Medicine III, Faculty of Medicine, University Hospital Tübingen Background: Endothelial cells play a key role in angiogenesis. For some years, it has been known that platelets also have the capacity to modulate angiogenesis. Methods and results: In a model of growth factor-induced angiogenesis we were able to show in vivo that platelets exert an antiangiogenic effect. Furthermore, we were able to show in vitro that the complement receptor C5aR expressed on platelets inhibits endothelial migration and tube formation. C5aRdeficient mice are known to display a phenotype of enhanced angiogenesis. We were able to demonstrate that the angiogenesis-inhibiting effect of the C5a receptor in C5a receptor-deficient mice was partially reversible by administration of wild type platelets. This effect was inhibited by pharmacological platelet depletion or inhibition of the C5a receptor. In vivo, systemic platelet depletion in C5a receptor knockout mice lowers their level of angiogenesis to the level of wild type mice. The supernatant of platelets stimulated with C5a inhibits endothelial migration and tube formation in vitro. Conclusion: In conclusion, upon stimulation with complement components platelets seem to exert an antiangiogenic effect.

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Lipocalin 2 (LCN2) mediates proatherosclerotic processes and is elevated in patients with coronary artery disease R. Oberoi, E.P. Bogalle, K. Grote, B. Schieffer, J. Schütt, M. Luchtefeld Department of Internal Medicine – Cardiology, University Hospital Gießen and Marburg, Marburg, Germany Objective: Lipocalin 2 (LCN2) is known to be associated with multiple acute and chronic inflammatory diseases but the underlying molecular and cellular mechanisms remain unclear. Here, we investigated not only whether LCN2 – upon its release from macrophages – contributes to pro-atherosclerotic processes but also whether LCN2 serum levels are associated with the severity of coronary artery disease progression in humans. Results: In an autocrine-paracrine loop, tumour necrosis factor-α (TNF-α) promotes the release of LCN2 from murine bone-marrow derived macrophages (BMDM) and vice versa. Moreover, LCN2 stimulation of BMDM led to an up-regulation of M1 macrophage marker mRNA, e.g. inducible NO synthase (iNOS), while M2 macrophage markers were not altered. In addition, enhanced migration of J774A.1 cells towards LCN2 was observed. Furthermore, LCN2 increased the expression of the scavenger receptor Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) as well as the scavenger receptor class A-1 (SRA-1) and induced the conversion of macrophages into foam cells. In atherosclerotic lesions of low density lipoprotein receptor-deficient (ldlr−/−) mice fed a high cholesterol diet, LCN2 was co-localized with macrophages in the shoulder region of the atherosclerotic plaque. In addition, LCN2 serum levels were significantly increased in plasma samples of these mice. Finally, LCN2 serum levels correlated with the severity of CAD in patients as determined by coronary angiography. Conclusion: Here we demonstrated Perfusion 01/2015

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that LCN2 plays a pivotal role in early atherosclerotic processes by promoting polarization, migration of monocytic cells and development of macrophages towards foam cells. Moreover, LCN2 may be used as a prognostic marker to determine the status of CAD progression.

Targeting tumour necrosis factoralpha: Interplay between myeloid and vascular cells in atherosclerosis R. Oberoi, J. Schütt, M. Luchtefeld, K. Grote, B. Schieffer Department of Internal Medicine – Cardiology, University Hospital Gießen and Marburg, Marburg, Germany Introduction: Atherosclerosis is a lipid related chronic inflammatory disease. One of the key mediators of inflammatory response is tumour necrosis factor-alpha (TNF-α). Endothelial cell (EC) activation is one of the initial steps in plaque formation. Activated endothelium captures leukocytes on its surface, leading to their transmigration into sub-endothelial space. Epidemiologic data suggest that TNF-α blockade, and inflammatory suppression in general, might have beneficial effects on vascular outcomes in patients with inflammatory arthritis, however detailed studies are still lacking. This study aims to elucidate macrophage derived repertoire of cytokines and its effect on endothelial activation under pro-atherosclerotic conditions. Methods and results: In this study, phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein (ox-LDL, 10 µg/mL and 25 µg/mL) for 48 hours. There was significant release of TNF-α (p<0.001, n=3) in a concentration-dependent manner as analysed by enzyme linked immunosorbent assay. This TNF-α rich conditioned medium activated human umbilical vein endothelial cells (HUVEC) to express adhesion molecules. There was enhanced mRNA expression

of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin (qRTPCR, p<0.01, n=3). Addition of TNF-α inhibitor (Adalimumab, 1 µg/mL) to the conditioned media, reduced mRNA expression of VCAM-1, ICAM-1 and E Selectin (qRT-PCR, p<0.05, n=4) as compared to IgG control. Conclusion: Our preliminary results show that pro-inflammatory factors (viz. TNF-α) derived from macrophages under atherosclerotic environment lead to endothelial activation and addition of TNF-α inhibitor down-regulated this activation status.

Expression of anaphylatoxin receptors on platelets in patients with coronary heart disease J. Patzelt1, 2*, K. Mueller2*, S. Breuning2, A. Karathanos2, R. Schleicher1, P. Seizer2, M. Gawaz2, H.F. Langer1,.2+, T. Geisler2+ 1 Section for Cardioimmunology, Eberhard Karls University Tübingen, Germany 2 University Hospital, Department of Cardiovascular Medicine, Eberhard Karls University Tübingen, Germany * Both authors contributed equally + These authors share senior authorship Aims: Inhibition of components of the complement system or of its receptors has been postulated as a concept for primary and secondary prevention in atherosclerosis and was applied in clinical trials. Although the anaphylatoxin-receptors C3aR and C5aR are commonly associated with inflammatory cells, in vitro studies suggested their expression also on platelets. Methods: Expression levels of C3aR and C5aR were measured by flow cytometry in a collective of 302 patients with documented coronary artery disease (CAD) including patients with stable CAD (n=152), unstable angina (n=54), acute myocardial infarction (AMI; Non-ST elevation myocardial infarction n=70; ST elevation MI, n=26) or healthy controls (n=21). Results: Patients with stable CAD, un© Verlag PERFUSION GmbH

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stable angina or AMI had significantly higher expression of C5aR on platelets in comparison to healthy controls (MFI 14.68 [5.2], 14.56 [5.18] and 13.34 [4.52] versus 10.68 [3.1]; p<0.001). In contrast, the expression of C3aR on platelets was significantly enhanced in patients with stable and unstable CAD but not in patients with AMI compared to controls. While there was a strong correlation between the soluble ligands of these receptors C3a and C5a, we observed only a weak correlation with their receptors on platelets. Similarly, agonist induced aggregation (MEA, ADP, and TRAP) showed only a weak correlation with the expression level of anaphylatoxin receptors on platelets. Of note, the expression of both anaphylatoxin receptors on platelets strongly correlated with platelet activation as assessed with the surface activation marker P-selectin (r=0.47, p>0.001 for C3aR; r=0.76 for C5aR, p<0.001). Likewise, we observed a positive correlation of C3aR with other molecules associated with platelet activation such as SDF-1. Conclusions: In summary, we observed a positive correlation between the expression of anaphylatoxin-receptors C3aR and C5aR with platelet activation in patients with CAD. Further investigations are needed to study the clinical and mechanistic relevance of these findings.

The chemokine receptor CXCR4 and atherosclerosis L. Pawig1, H. Noels1, Y. Döring2, C. Weber2 1 Institute for Molecular Cardiovascular Research (IMCAR), RWTH Aachen University, Germany 2 Institute for Cardiovascular Prevention, Ludwig Maximilians University Munich, Germany Objective: Cardiovascular diseases with atherosclerosis as their underlying pathology are the leading cause of morbidity and death in industrialised countries. Atherosclerosis is a chronic inflammatory disease that causes a narrowing of arteries, which can ultiPerfusion 01/2015

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mately lead to total vessel occlusion, myocardial infarction or stroke. The accumulation of lipids as low-density lipoprotein (LDL) in the subendothelial space and their uptake by macrophages is a major event in the development of atherosclerosis. A protein expressed on most cells present in the atherosclerotic plaque including macrophages and neutrophils is the chemokine receptor CXCR4. Preliminary in vitro experiments showed an increased uptake of fluorescently-labelled LDL by THP1derived macrophages following stimulation with stromal-cell derived factor 1 (SDF-1, also known as Cxcl12), an important ligand of CXCR4. Therefore, we aimed here to examine whether CXCR4 influences the lipid accumulation which leads to plaque development. Methods and results: The atherosclerotic phenotype in CXCR4-deficient mice was investigated using the LysM-Cre+ CXCR4flox/flox ApoE–/– mouse model, which carries a knockout of CXCR4 on monocytes, macrophages and neutrophils. Flow cytometry and differential blood count showed that neutrophil levels in peripheral blood of LysM-Cre+ CXCR4flox/flox ApoE–/– mice were elevated, indicating a potential pro-inflammatory state. Atherosclerotic plaque development was induced by 12 weeks of high-fat diet and plaques were characterized by Oil-Red-O and immunofluorescent stainings. No difference in plaque size in the aortic root and total aorta could be observed between wildtype mice and LysM-Cre+ CXCR4flox/flox ApoE–/– mice. Additionally, the deposition of lipids in the plaques and their uptake by plaque macrophages, as measured by a combination of a Nile Red and macrophage staining of atherosclerotic lesions, was not altered. Conclusion: In conclusion, our results indicate that CXCR4 does not play a major role in lipid uptake by macrophages in atherosclerotic lesions. Furthermore, CXCR4-deficiency in macrophages and neutrophils does not affect lesion size, despite an in-

creased neutrophil mobilization in the blood. The latter observation supports previous reports which revealed an increased mobilization of neutrophils from the bone marrow upon blockage of CXCR4. However, disruption of the Cxcl12/CXCR4 axis has previously been shown to aggravate atherosclerotic plaque formation by elevated neutrophil infiltration into the plaque and neutrophil hyperactivation. The absence of altered atherosclerosis in our mouse model suggests that these pro-atherosclerotic effects of CXCR4blocked neutrophils could be counteracted by anti-atherogenic effects of CXCR4-deficiency in monocytes and macrophages. Further functional studies of the role of CXCR4 in macrophages and monocytes are required to examine this hypothesis.

A prospective, randomized, placebocontrolled, double-blind trial evaluating the influence of a balanced diet containing L-arginine and B vitamins on endothelial function in subjects with mild to moderate blood pressure elevation H. Robenek1, D. Menzel2 1 University Hospital Münster, Germany 2 Stuttgart Background: Mediated by nitric oxide (NO), intact endothelial cells control vascular homeostasis by regulating vascular tone and preventing smooth muscle cell proliferation, monocyte adhesion to the endothelium and platelet aggregation, thereby protecting blood vessels from the formation of atherosclerosis. Hence, the endothelial function is considered a reliable indicator of vascular health. The vascular endothelium constitutes a primary sensitive target for the damaging effects of atherogenic risk factors such as high cholesterol, high blood pressure (BP) or smoking which can all disrupt the homeostasis and lead to endothelial dysfunction (ED) and the initiation of atherosclerosis. ED can be considered © Verlag PERFUSION GmbH

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the earliest stage of cardiovascular disease. The assessment of ED can detect vulnerable patients at risk for future cardiovascular events. ED is characterized by reduced bioavailability of NO and the impairment of endothelium-dependent vasodilation. NO is formed from the semi-essential amino acid L-arginine by the endothelial nitric oxide synthase (eNOS) enzyme if sufficient B vitamins B6, folic acid and B12 are provided. The latter being co-factors in the NO synthesis, e.g. by increasing the availability of tetrahydrobiopterin (BH4), are synergistic with L-arginine. Objective: The aim of the trial was to investigate the influence of a balanced diet consisting of a unique synergistic combination of L-arginine with B vitamins B6, folic acid and B12 on the change of endothelial function induced by a standardized fat meal (FM), assessed by non-invasive peripheral arterial tonometry (EndoPAT TM, Itamar Medical Ltd., Israel) in volunteers with slightly to moderately elevated BP not requiring antihypertensive drug treatment. Methods: The trial was performed as a confirmatory, prospective, randomized, placebo-controlled, double-blind, single-center study with parallel design and an open follow-up phase. 81 eligible subjects aged 40 to 65 years (37 % female; all non-smokers) were randomly assigned to receive either the dietetic food product TAP (Telcor® Arginin plus; 2 capsules b.i.d.; total daily amounts: 2.4 g L-arginine, 3 mg vitamin B6, 0.4 mg folic acid, 2 µg vitamin B12; active group, n=40) or a matching placebo (microcrystalline cellulose; placebo group, n=41) for 3 months followed by an open follow-up phase of an additional 3 months with the active product being administered in both groups. The primary study endpoint was the postprandial (FM-induced) change of endothelial function assessed by means of the validated reactive hyperemia peripheral arterial tonometry index (RHI) at 3 months compared with study start (RHI post-pre, Perfusion 01/2015

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visit 3 to visit 1; ΔΔRHI). Secondary efficacy parameters included BP and homocysteine levels. Safety parameters were assessed throughout the entire study period. Results: 80 subjects (age 53.8±5.8 years, BMI 25.3±2.7 kg/m²) completed the study protocol (one drop-out in the placebo group). The compliance exceeded 95 % in both study phases. The primary efficacy analysis revealed superiority of TAP over placebo by a statistically significant (p=0.0349) difference between the two groups in reducing the deterioration of endothelial function caused by a fat meal. While in the active group ΔRHI increased to 0.371±0.740, almost no change could be detected in the placebo group (0.031±0.602), which demonstrates a significant protection of the endothelium in the active group. Over the course of the study period, the BP and homocysteine levels were reduced by TAP. There was no difference between the groups in the frequency of adverse events. There were no serious adverse events or clinically relevant laboratory changes in either group. In both groups, most of the participants rated the tolerability as good, reaching 94 % after 6 months. The findings confirmed the excellent safety profile of TAP. Conclusion: This trial confirmed the effective and safe use of the dietary L-arginine/B vitamin combination product TAP. The primary efficacy analysis demonstrated a statistically significant superiority of the L-arginine/B vitamin combination over placebo in improving and restoring impaired endothelial function after fat load. The result was achieved by a daily supplementation of 2.4 g of L-arginine in combination with B vitamins which confirms the practical relevance of the synergism between these nutrients. In conclusion, the nutritional combination of L-arginine and B vitamins can successfully be used to reduce cardiovascular risk factors, to improve vascular health and for the dietary treatment of early stages of atherosclerosis.

Interactions of human arterial smooth muscle cells with perivascular and subcutaneous fat cells U. Schmidt1, B. Schreiner1, A. Königsrainer4, H.-E. Schaller5, H.-U. Häring1, 2 ,3, D.I. Siegel-Axel1, 2, 3 1 Department of Internal Medicine, Division of Endocrinology, Diabetology, Angiology, Nephrology and Clinical Chemistry, University of Tübingen, Germany 2 Institute of Diabetes Research and Metabolic Diseases (IDM), University of Tübingen, Germany 3 German Center for Diabetes Research (DZD), Neuherberg, Germany 4 Department of General Visceral and Transplantation Surgery, University of Tübingen, Germany 5 Department of Plastic, Hand and Reconstructive Surgery, BG-Trauma-Center, University of Tübingen, Germany Introduction: Visceral, subcutaneous and perivascular adipose tissue (PVAT) possess not only supportive function, but represent endocrine and paracrine organs that modulate vascular function and also play an important role in the pathophysiology of cardiovascular diseases. (Pre)-adipocytes not only act in an autocrine and endocrine manner but produce adipokines, cytokines and vasoactive substances that exert paracrine effects on endothelial cells and smooth muscle cells (SMC) of the arterial vessel wall. In the present in-vitro study, we examined mRNA expression of arterial SMC, perivascular and subcutaneous fat cells in mono- and cocultures to characterize the cross-talk of these cell types via the blood stream of the whole organism, or via the vasa vasorum pervading the vessel wall in detail. Methodology: Human perivascular (PVPA) and subcutaneous (SPA) preadipocytes, as well as human arterial smooth muscle cells were freshly isolated from blood vessels and adipose tissue deriving from different patients staying at the BG Trauma Center or the Clinic of General Surgery of the UKT. PVPA were characterized and tested for purity by FACS analysis and SMC by immunostaining against α-smooth muscle actin. Preadipocytes were fur© Verlag PERFUSION GmbH

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ther differentiated in a special medium for 2 weeks to transform into mature adipocytes. Complete differentiation was proven by oil red staining. The fat cells were co-cultured either alone, or in transwell systems with SMC for 24, 48, 72 and 96 hours. Then, mRNA of both cell types was extracted and analysed by real-time PCR. To quantify the secretion of various angiogenic and proinflammatory proteins in the supernatants, specific ELISA were used. Results: Both PVPA, as well SPA expressed at 24, 48, 72 and 96 hours in both mono- and co-cultures proinflammatory and growth factors, such as IL-6, IL-8, IL-1b, MCP-1, TGF-b and TSP-1, but also angiogenic factors such as bFGF, VEGF and HGF. SMC showed a high basal mRNA expression of IL-6, but remained unchanged by co-cultivation with fat cells, whereas in fat cells themselves, the pro-inflammatory factors IL-6, IL-8, IL-1b and MCP-1 were strongly upregulated by co-culture with SMC. The angiogenic factors VEGF and bFGF were upregulated. In SMC, an increased expression of IL-8 and IL-1b was observed. Similar results were also found at the protein level. Conclusions: Both perivascular, as well as subcutaneous fat tissue, which increases in patients with obesity and diabetes and which is altered in function and in its secretory behaviour, affects the function of vascular wall cells, such as SMC and endothelial cells. Since PVAT is located adjacent to the adventitia of the vessel wall without any barrier, there is a close contact with the vasa vasorum, which promote neovascularization following vascular injury and inflammation. Thus, the transport of vasoactive substances such as VEGF, bFGF, etc., but also of cytokines or chemokines (interleukins, MCP-1), which were increased by co-cultivation in our investigations, is facilitated in the direction of the media and the endothelium. The involvement of the adventitia (in addition to the media and the endothelium) in atherosclerosis has been demonstrated Perfusion 01/2015

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many times. Current studies are focusing more and more on interactions of adipose tissue with the vessel wall and their importance in atherosclerosis.

Formation of different lipid-laden human macrophages in response to terminally oxidized LDL (oxLDL) and enzymatically modified LDL (eLDL): a multi-omics approach G. Schmitz, E. Orsó Institute for Laboratory Medicine and Transfusion Medicine, University Hospital Regensburg, Germany Aim: Formation of macrophage foam cells is a critical step in atherogenesis. Monocytes, as precursors for macrophages, rapidly shift their transcriptomic and lipidomic profiles from activation of SREBP-2 target genes required for cholesterol synthesis to activation of SREBP-1c target genes required for fatty acid (FA) biosynthesis, upon macrophage colony-stimulating factor (M-CSF)-dependent in vitro differentiation of primary human monocytes to macrophages. This transcriptional regulation is accompanied with dramatically increased fatty acid synthesis for enhanced phospholipid synthesis, parallel with the switch of the major lipid class from cholesterol in monocytes to phosphatidylcholine in macrophages. Based on these results we supposed that different modified lipoproteins (e.g. oxLDL and eLDL) may also induce diverse cellular responses in monocyte-derived macrophages. Methods: Dissection of the in vitro oxLDL- and eLDL-induced cellular responses was performed by transcriptomic (microarray) and lipidomic (mass spectrometry) profiling of cells as well as electron microscopy. Results: Oxidized LDL preferentially up-regulates scavenger receptors required for its internalization, induces preferential lipid storage in the acidic compartment with formation of lamellar bodies, resembling drug-induced

endolysosomal phospholipidosis, parallel with increased cellular content of the endolysosomal signature lipid bis(monoacylglycero)phosphate (BMP), pro-apoptotic signalling and appearance of ceramide-enriched surface membrane microdomains. By contrast, challenge of macrophages by eLDL leads to expanded cholesterol- and sphingomyelin-enriched surface membrane microdomains, up-regulation of diverse pattern recognition receptors required for phagocytosis of eLDL, parallel with extensive lipid droplet formation, increased endoplasmic reticulum (ER)-stress and membrane contact site formation for interorganelle trafficking and signalling, and enhanced cellular content of the mitochondrial lipid cardiolipin (CL) and the major droplet lipid cholesteryl ester (CE). Conclusion: Taken together, foam cell formation occurs exclusively in response to eLDL as characteristic CEdroplet storage. By contrast, in response to oxLDL only few lipid droplets are formed, while the endolysosomal compartment is filled with undigested lipids parallel with the basification of luminal pH and accumulation of BMP, specific for phospholipidosis.

Influence of HDL isolation conditions on HDL size and functional read outs L. Schmölz1, T. Bernscherer2, S. Lorkowski1, A. Ritsch2 1 Institute of Nutrition, University of Jena, Germany 2 Department of Internal Medicine, Innsbruck Medical University, Austria Aims: In the clinical routine different methods have been established to isolate HDL from plasma and serum samples. The gold-standard is the isolation via subsequent density gradient ultra centrifugation, which is time-consuming and needs a high amount of plasma. These two facts make this method not practicable for large-scale studies based on limited sample volumes from patients. Methods: The method used instead is © Verlag PERFUSION GmbH

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based on a precipitation mixture containing polyethylene glycol (Quantolip). The ApoB-containing lipoproteins are pelleted and the supernatant is used as HDL-rich fraction. This method is fast and needs only small sample volumes. Therefore removing ApoBcontaining lipoproteins using Quantolip is the standard method to prepare serum samples to be used in measurements of serum HDL efflux capacity in large-scale studies. Results: In a systematic screening of these methods by FPLC analysis, we found a marked decrease in HDL size using the Quantolip conditions, while the size of HDL isolated by ultracentrifugation was in good accordance with that of native HDL. We were able to confirm this observation using Lipogel electrophoresis and ultrafiltration, respectively. As Quantolip-derived HDL is routinely used in large-scale cholesterol efflux studies, we searched for an alternative method that does not influence HDL size. We were indeed able to find a method for precipitation of ApoB-containing lipoproteins based on sodium phosphotungstate and magnesium chloride which was able to fulfill all our criteria. We also controlled the usability of this method for measurement of serum HDL efflux capacity in serum samples. Conclusions: We feel that the protocol for preparation of HDL-enriched serum samples described in this work will help to improve results of serum HDL efflux capacity measurements in future clinical studies.

Shedding light on the regulation of MMP 14 protein shedding in human macrophages M. Schubert, S. Lorkowski Institute of Nutrition, Friedrich Schiller University Jena, Germany Background: Matrix metalloproteinases (MMPs) are a group of zincdependent enzymes that degrade inter alia several components of the extraPerfusion 01/2015

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cellular matrix. Hence, they play a pivotal role for the stability of atherosclerotic plaques, a critical factor for the outcome of vascular disease. Active MMPs are therefore unfavorable for the stability of the atherosclerotic plaque. Proteolytic activity of membrane-bound MMPs is regulated by autocatalytic processing, homodimerization, internalization, binding of endogenous inhibitors and shedding of their extracellular parts. MMP14 is a membrane-bound protease of 60 kDa in its active state that is known to be shed in several ways resulting in different protein fragments. Autocatalytic processing leads to a 44 kDa form of the protein. Despite this, there are two more shedding events known, leading to soluble fragments of 50 kDa and 30 kDa respectively. Aim: The aim of our studies is to better understand shedding and function of MMP14 in macrophages. Methods and results: For our investigations we used macrophages obtained from the monocytic cell line THP-1. THP-1 macrophages were generated from monocytes using phorbol 12-myristate 13-acetate. Mature THP-1 and primary macrophages expose MMP14 as the known zymogen (63 kDa) and the active form (60 kDa). Also different fragments of shed MMP14 of 50 kDa and lower molecular mass can be detected using Western blotting. In addition to these known isoforms of MMP14, we found a new membrane-bound MMP14 fragment of approx. 50 kDa which has not been described so far, neither in THP1 macrophages nor in other cell types. Characterization and identification of this MMP14 form may contribute to better understanding the regulation of this protease in macrophages. We first hypothesized that this new MMP14 isoform occurs due to alternative splicing. To identify possible MMP14 splice variants in THP-1 macrophages, we used an RT-PCR approach using a set of primer pairs, specially designed for that purpose. First results do not confirm that MMP14 is alternatively

spliced in THP-1 macrophages. This leads to the conclusion that the modification occurs at the protein level. To get first insights into a proposed unknown shedding mechanism, we performed Western blots with antibodies directed against different domains of the protein, for example, two antibodies binding at different sites of the catalytic domain. To identify the remaining membrane-bound fragments, we used an antibody directed against the cytoplasmic domain. The shed soluble fragments of MMP14 were analysed by Western blot analyses of the cell culture supernatants. Further characterization of MMP14 protein isoform formation in THP-1 macrophages was performed with the help of inhibitors. For example, a furin inhibitor was used to identify active and inactive MMP14 isoforms. Conclusions: We identified a new MMP14 protein isoform in human THP-1 macrophages and primary human macrophages. The functional relevance and the underlying regulatory shedding mechanisms are not known. Insights in the regulation of MMP14 shedding may help to better understand the regulation of MMP14 function.

OxLDL induced Jab1/COPS5 protein expression in human macrophages with impact on inflammatory signalling and foam cell formation A. Schwarz, G. Bonaterra, L. Mey, R. Kinscherf Institute for Anatomy and Cell Biology, Philipps University of Marburg, Germany Aim: Atherosclerosis is an inflammatory disease involving recruitment of macrophages (MΦ), which are the most abundant cell type in atherosclerotic plaques. Oxidized low-density lipoprotein (oxLDL) mediates the transformation of MΦ to cholesterolrich foam cells and the release of proinflammatory cytokines, events which are constituents of vulnerable plaques. Jab1/COPS5 (c-Jun activation domain binding protein-1) is expressed in all © Verlag PERFUSION GmbH

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stages of human plaques, involved in the activation of TLR-mediated activation of p38 MAPK and controls NF-κkB activation. Thus, we were interested to investigate the role of Jab1/ COPS5 during foam cell formation and release of pro-inflammatory cytokines after ox-LDL treatment of human MΦ. Methods and results: Differentiated U937 MΦ were incubated with oxLDL (50 µg/ml) for 4 hours and 24 hours, to induce foam cell formation confirmed by Oil Red O staining. Using Western blotting we show after 4 hours and 24 hours oxLDL stimulation a significant 1.54-fold and 1.42-fold increase of Jab1/COPS5 protein level in comparison with the control. Additionally, the Iκ-Bα protein level was 2-fold increased (p<0.005) after 4 hours, but 7-fold decreased (p<0.005) after 24 hours in conjunction with a significant (p<0.005) nuclear translocation of NF-κB after 24 hours oxLDL-treatment as shown by Western blot and immunofluorescence staining. Moreover, we investigated p38 activation by phosphorylation (Thr180, Try182) and found a significant (p=0.01) increase after 4 hours oxLDL-treatment in parallel with an up-regulation of TNF-α (4.5-fold; p=0.008) and IL6 (52-fold; p=0.017) mRNA expression, quantified by real time PCR. Finally, we demonstrated the interaction of JAB1/COPS5 with p38 after 24 hours oxLDL incubation by co-immunoprecipitation and immunofluorescence staining, where the phosphorylation of p38 (Thr180, Try182) was not detectable. Conclusion: Our results suggest an oxLDL-mediated regulation of Jab1/ COPS5 in human MΦ, which influences the p38 MAPK pathway and probably defers the Iκ-Bα degradation with consequences for foam cell formation.

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Angiogenesis is dependent on endothelial autophagy – the role of AMPK K. Spengler1, N. Kryeziu1, S. Lindenmüller1, O. Mece1, A. Weiland1, L.-O. Klotz2, R. Heller1 1 Institute for Molecular Cell Biology, Centre for Molecular Biomedicine (CMB), University Hospital Jena, Germany 2 Institute of Nutrition, Friedrich Schiller University of Jena Germany Aim: AMP-activated protein kinase (AMPK), an important energy-sensing enzyme, is known to regulate cell metabolism and homeostasis and may protect endothelial cells from cellular stress. One protective pathway triggered by AMPK is autophagy, which plays a role in the degradation of longlived or damaged proteins and organelles and contributes to stress adaptation and cell survival in an adverse environment. AMPK has been shown to be activated by angiogenic growth factors and to play an essential role in regulating angiogenesis but the underlying mechanisms are still unknown. The aim of this study was to understand whether AMPK-driven autophagy contributes to the control of angiogenesis. Methods: Experiments were performed in human umbilical vein endothelial cells. Cells were stimulated with the specific AMPK activator A769662 or with vascular endothelial growth factor (VEGF). Angiogenesis was evaluated by quantifying sprouting from endothelial spheroids. To monitor autophagy, phosphorylation of ULK1, which represents an initial event, and conjugation of LC3B, which characterizes autophagic flux when measured in the presence of the lysosomal inhibitor bafilomycin A1, were analysed in Western blots. Inhibition of autophagy was achieved by siRNA-mediated downregulation of the autophagy proteins ULK1 and Beclin1. Cell growth and apoptosis (subG1 fraction) were characterized by cell cycle analysis via flow cytometry. Oxidative stress was analysed by alterations of intracellular GSH levels and protein carbonylation.

VEGF-induced signalling events were detected in Western blots using specific antibodies against the phosphorylated kinases Akt and Erk1/2. Results: Angiogenesis stimulated by VEGF was strictly dependent on autophagy since VEGF-induced sprouting was inhibited in cells, in which ULK1 and Beclin1 were downregulated. Under these conditions proliferation and cell survival were also decreased. In parallel, cells exhibited oxidative stress as shown by reduction of intracellular GSH and protein carbonylation suggesting that autophagy exerts a stress-protecting role. In contrast, VEGF-induced signalling events (Akt and Erk1/2 phosphorylation) remained unchanged. To investigate a possible role of AMPK, which is activated by VEGF, in autophagy induction, a specific activator of AMPK, A769662, was employed. A769662 induced both angiogenesis and autophagy as shown by phosphorylation of ULK1, an autophagy initiator, and LC3B conjugation, a marker for autophagosome formation. Similar to VEGF-triggered sprouting A769662-induced angiogenesis was dependent on efficient autophagy. Conclusion: Our data underline the importance of autophagy for functional angiogenesis. In addition, they show that AMPK activation stimulates autophagy, which is required for AMPKinduced angiogenesis. Since angiogenic growth factors lead to activation of AMPK, the subsequent stimulation of autophagy by AMPK may protect cells from stress and ensure the functionality of the endothelium, which is prerequisite for angiogenic processes.

The E3 ligase ITCH modulates lipid deposition and plaque development in ApoE–/– mice R. Stoehr1, A. Marino1, R. Menghini1, G. Melino2, M. Federici1 1 Department of Systems Medicine, University of Rome Tor Vergata, Italy 2 Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Italy © Verlag PERFUSION GmbH

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Aim: To assess the role of ITCH, an E3 Ubiquitin Ligase involved in T-cell differentiation in the development of atherosclerosis. We have previously shown that mice lacking ITCH show reduced weight gain and a tendency to M2 (anti-inflammatory) polarization, thus protecting them from the effects of a high fat diet. Methods: We crossbred ITCH−/− with the hypercholesterolemic mouse model ApoE to generate double knockout mice (ApoE−/− ITCH−/−) and fed the animals a western diet (WD). Histological analysis of the aortic roots was used to compare atherosclerotic burden. RNA expression analyses were used to investigate cholesterol and triglyceride metabolism. FACS analysis characterized the circulating inflammatory cells. To examine the effects of ITCH−/− in the immune compartment alone we performed Bone Marrow Transplant (BMT) experiments. Results: After 12 weeks of WD ApoE−/− ITCH−/− mice were lighter and showed improved fasting glycaemia. FACS analysis revealed an increase in circulating M2 macrophages (CD115+ CD11b+ Gr1–). Histological analysis of the aortic roots of ApoE−/− ITCH−/− showed reduced plaque and Oil-red-O staining compared to ApoE−/− animals (90402±9534 μm2 vs. 372382±35540 and 88921±28288 vs. 277590±23305 μm2, n=8, p<0.0001 and p<0.001, respectively). Liver histology showed ApoE−/− ITCH−/− to be protected from fatty infiltration with a concomitant reduction in the triglyceride content (62.71±3.356 vs. 159.4±4.059 mg/gram of tissue, p<0.0001, n=4). Metabolic phenotyping through the use of metabolic cages indicated that ApoE−/− ITCH−/− have a higher oxygen consumption (2192±24.38 vs. 2011±20.38, p<0.0001, n=4) coupled to a higher CO2 production (1922±23.76 vs. 1848±20.22, p<0.0187, n=4) than ApoE−/− mice, suggesting an increased basal metabolic rate. Furthermore, the respiratory exchange ratio was reduced in ApoE−/− Perfusion 01/2015

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ITCH−/− mice (0.8658±0.003673 vs. 0.9095±0.003741, p<0.0001, n=4), suggesting a higher reliance on fatty acids as the main energy source. RTPCR gene expression analysis of the liver showed an increased expression of genes involved in lipid metabolism such as PGC1b, STAT6, FABP4 and CPT1a. Furthermore we found an increase in genes regulating mitochondrial biogenesis and respiration including Nrf1 and Tfam. In addition to the effects on metabolism ITCH downregulation has been shown to promote a Th2 bias. To dissect the effects of ITCH−/− on the immune system from those on the liver we performed bone marrow transplantation (BMT) experiments. FACS analysis of the KO recipients showed an increase in T-regulatory cells with a concomitant increase in circulating M2 macrophages. Aortic root analysis after 8 weeks of western diet revealed a much less pronounced decrease in lesion size than in the whole body knockout (145348±2717 vs. 189593±20831 μm2, p=0.1, n=6). Conclusion Taken together our data suggests that downregulation of ITCH may be beneficial in the setting of atherosclerosis by not only polarizing the immune system towards a less inflammatory phenotype but also affects lipid metabolism by increasing mitochondrial oxidation.

On the pathogenesis of valvular aortic sclerosis: presence of enzymatically modified LDL in early lesions L. Twardowski1, F. Cheng1, 3, J. Michaelsen2, S. Winter3, U. Hofmann3, E. Schaeffeler3, S. Müller3, M. Sonnenberg3, K. Steuer3, G. Ott4, M. Schwab3, 5, U.F.W. Franke2, M. Torzewski1, 3 1 Department of Laboratory Medicine, Robert Bosch Hospital, Stuttgart, Germany 2 Department of Cardiovascular Surgery, Robert Bosch Hospital, Stuttgart, Germany 3 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany

Department of Pathology, Robert Bosch Hospital, Stuttgart, Germany 5 Department of Clinical Pharmacology, University Hospital Tuebingen, Germany 4

Background: We have demonstrated previously enzymatically degraded LDL (eLDL) as an essential causative component for the initiation of atherosclerosis. Herein, we investigated early human valvular cusp lesions for the presence of eLDL and effectors of the innate immune system to discover possible pathways leading to aortic valve sclerosis. Methods and results: Immunohistochemical staining demonstrated extensive extracellular deposits of eLDL. Complementary analysis of lipid composition revealed higher concentrations of the decisive components of eLDL, i.e. unesterified cholesterol and linoleic acid, compared to internal control tissues. Furthermore, the complement component C3d and terminal complement complexes co-localized with eLDL, which is compatible with the proposal that subendothelially deposited eLDL is enzymatically transformed to a complement activator at early stages in valvular cusp lesion development. Gene expression profiles of proteases and complement components corroborated by immunohistochemistry demonstrated an upregulation of the protease cathepsin D (a possible candidate for LDL degradation to eLDL) and the complement inhibitor CD55. Surprisingly, there was no substantial CRP expression in early valvular cusp lesions as investigated by microarray analysis, RT-PCR analysis and immunohistochemistry. Finally, we demonstrated cellular uptake of eLDL by valvular interstitial cells (VICs)/myofibroblasts. Conclusions: The present study is a startup of a hypothesis on the pathogenesis of aortic valve sclerosis declaring extracellular lipoprotein modification, subsequent complement activation and cellular uptake by VICs/myofibroblasts as integral players. © Verlag PERFUSION GmbH

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Platelet microvesicles in vascular inflammation T. Vajen1, E.M. Vasina1, J.W.M. Heemskerk1, L.J. Schurgers1, C. Weber2, T.M. Hackeng1, R.R. Koenen1 1 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands 2 Institute for Cardiovascular Prevention (IPEK), Ludwig Maximilians University Munich, Germany Background: Microvesicles are gathering increased attention not only as biomarkers but also as mediators of cell communication and as integral effectors of disease. Platelets present a major source of microvesicles and release these microvesicles either spontaneously or upon activation. Plateletderived microvesicles (PMV) retain many features of their parent cells and have been shown to exert modulatory effects on vascular and immune cells. We hypothesize that PMV play a role in the interaction with vascular smooth muscle cells with respect to vascular remodeling. Methods: PMV were isolated from stored platelet rich plasma. Platelets were removed by centrifugation at 2570 × g for 5 minutes. The plateletfree plasma was centrifuged at 20,000 × g for 60 minutes. The pellet containing PMV was resuspended in Hepes buffer. Impurities of residual platelets, PMV aggregates and other contaminants were removed from the resuspended pellet by filtration (0.8 µm pore size) and pelleted again at 20,000 × g for 40 minutes. PMV were quantified and characterized by flow cytometry using antibodies against Annexin A5/ phosphatidylserine and CD41a/GPIIb. Size calibrated microbeads were used to quantify the absolute amount of PMV/ml. Human smooth muscle cells (SMC) from coronary arteries were cultured at 37°C in DMEM medium, supplemented with 20 % foetal bovine serum and 1 % penicillin/streptomycin. Cell migration experiments were performed using a chemotaxis µ-slide. Proliferation of SMC was measured by Perfusion 01/2015

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the BrdU-cell proliferation kit. Platelet receptors implicated in PMV-SMC interaction were identified by blocking antibodies. Results: In presence of PMV a gradient was generated which resulted in significant migration of the SMC in the direction of the gradient. Both, the total displacement distance and the velocity of movement of SMC were significantly increased compared to the control condition without PMV. The PMV binding to SMC was specifically abrogated with the integrin αIIbβ3 inhibitor (integrilin) pointing to an integrindependent mechanism of interaction. A proliferative effect on SMC could be measured 48 hours after incubation with PMV. The proliferative effect of PMV relied on interactions via integrin αMβ2, CD40 as well as on P-selectin. Conclusion: Isolated microvesicles have shown to exert an immunomodulatory activity on various cell types. The present data indicate a role of PMV in SMC chemotaxis and proliferation, which might contribute to vascular atherogenesis, in particular vascular remodeling. Further, PMV may induce a switch of SMC phenotypes.

Development of a specific extra cellular CyPA inhibitor S.N.I. von Ungern-Sternberg1, G. Fischer2, E. Kremmer3, M. Gawaz1, A.E. May1, P. Seizer1 1 Medical Clinic III, Cardiology and Circulatory Disorders, Eberhard Karls University Tübingen, Germany 2 Max Planck Research Unit for Enzymology of Protein Folding, Halle/Saale, Germany 3 Helmholz Center Munic, Institute for Molecular Immunology, Munic, Germany Introduction: Cyclophilin A (CyPA) is an abundantly expressed intracellular protein, where it exerts a variety of functions due to its peptidyl-prolyl cis-trans isomerase (PPIase) activity. Recent data indicate that extracellular CyPA significantly contributes to cardiovascular inflammation, myocardial ischemia and reperfusion injury, and

myocardial remodelling processes. Thus, CyPA appears to represent a novel target to treat vascular and myocardial inflammation. Here we investigate the new specific extracellular CyPA inhibitor 8H7-mAb for its effect on platelets and monocytes during CyPA stimulation in vitro. Methods and results: Mice and rats were immunized with peptides containing the EMMPRIN-binding side sequence and anti-CyPA antibodies were generated. Then, the anti-CyPA antibodies were screened in a chemotaxis assay with monocytes. According to this setting the hybridoma supernatant clone 8H7 revealed the best inhibition of monocytic migration to CyPA. Western blot analysis showed that 8H7 binds preferentially to CyPA compared to CyPB. For further in vitro characterization freshly isolated human platelets were stimulated with recombinant CyPA and analysed by flow cytometry for the expression of p-selectin. The CyPA-dependent activation of platelets was abrogated by the CyPA inhibitors 8H7-mAb (p<0.05). In addition perfusion of CyPA-stimulated whole blood showed an enhanced adhesion of platelets on fibrillar type I collagen which was reduced by the CyPA inhibitor 8H7. As expected, migration of human monocytes towards CyPA was nearly abolished by purified 8H7-mAb. Moreover, in zymography the CyPA-induced MMP-9 activity of foam cells was reduced by 8H7 to base line level. Notably, the inhibitory effect of 8H7 was similar to established cyclosporin-A-derived CyP-inhibitors like NIM811 or MM284. Conclusion: Our data indicate that the novel antibody 8H7-mAb represents an effective novel pharmacological tool for a specific inhibition of extracellular CyPA.

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Role of protein tyrosine phosphat ases in the development of antiangiogenic therapy resistance in renal cell carcinoma A. Weiland, F. Weber, W. Müller, F.D. Böhmer, R. Heller Institute for Molecular Cell Biology, Centre for Molecular Biomedicine, University Hospital Jena, Germany Aims: Targeted antiangiogenic drug therapies interfering with the signalling pathway of vascular endothelial growth factor (VEGF) have shown beneficial but limited effects in patients with renal cell carcinoma. Limitations are often due to the development of an acquired drug resistance, whose mechanisms are incompletely understood. Protein tyrosine phophatases (PTPs) are key regulators of protein tyrosine phosphorylation and are thus critically involved in the regulation of growth factor-induced angiogenesis. This study is aimed at investigating whether downregulation or inhibition of PTPs via tumour-associated factors may contribute to reduced sensitivity of endothelial cells against tyrosine kinase inhibitors targeting VEGF receptor 2 (VEGFR2). In particular, the receptor-like subtype DEP-1 (density-enhanced phosphatase) as well as the cytosolic subtype PTP1B, which are both known to regulate VEGFR2, are examined Methods: Experiments were performed with primary human endothelial cells from umbilical cord veins. Downregulation of PTPs was achieved by PTP1B-or DEP-1-targeting siRNAs and verified by immunoblotting. To monitor the effect of VEGF stimulation alone or in combination with the VEGFR2-targeting inhibitor sunitinib, Western blot analyses using antibodies against phosphorylated (Tyr1175) or non-phosphorylated VEGFR2 were performed. Furthermore, cell proliferation was determined by means of the Cell Proliferation ELISA, BrdU (Roche) and angiogenesis was analysed in a sprouting assay using endothelial Perfusion 01/2015

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cell spheroids. To test the influence of tumour cell-conditioned medium on the expression of PTPs cell culture medium from the renal cancer cell line 786-O was collected for 24 hours and incubated with endothelial cells. Results: Treatment of endothelial cells with specific siRNA led to a downregulation of PTP1B or DEP-1 by 94 % or 92 %, respectively. A decreased expression of these phosphatases resulted in an increased VEGF-induced phosphorylation of VEGFR2 confirming their role in the regulation of receptor activation. Sunitinib pretreatment (10–100 nM, 30 min) repressed VEGFR2 phosphorylation in response to VEGF in a dose-dependent manner. The inhibitory effect of sunitinib was decreased in cells with a combined downregulation of PTP1B and DEP-1 compared to control cells. In parallel, the reduction of VEGF-induced cell proliferation by sunitinib was lower in PTP-depleted cells. Importantly, conditioned medium from the renal cancer cells led to a reduced expression of DEP-1 and PTP1B in endothelial cells suggesting that tumour cells release factors that amplify VEGF signalling via repression of PTPs. In parallel, the phosphorylation of VEGFR2 was significantly increased in endothelial cells treated with tumour cell conditioned medium. This was accompanied by increased sprouting from endothelial cell spheroids. Conclusions: Our data reveal that PTP1B and DEP-1 are negative regulators of VEGFR2 signalling in endothelial cells. We also show that a combined downregulation of PTP1B and DEP-1 reduces sensitivity towards sunitinib and that conditioned medium of tumour cells affects the expression of PTP1B and DEP-1. From these data we suggest that downregulation of PTPs, which may be induced by tumourassociated factors, might contribute to antiangiogenic therapy resistance.

Vascular effects of intraperitoneal applicated sterols, phytosterols and oxyphytosterols in ApoE–/–mice O. Weingärtner1, D. Lütjohann2, H.-F. Schött2, T. Speer3, J. Plat4, U. Laufs1 1 Department of Internal Medicine III, Saarland University Medical Center, Homburg, Germany 2 Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Germany 3 Department of Internal Medicine IV, Saarland University Medical Center, Homburg, Germany 4 Department of Human Biology, Maastricht University, The Netherlands Purpose: Plant sterol esters (PSE) are used as food supplements to reduce serum cholesterol levels. Both plant sterols and cholesterol are prone to oxidation and convert to oxysterols and oxyphytosterols. The effects of oxysterols and oxyphytosterols on reactive oxygen species (ROS), endothelial function and atherogenesis in ApoE–/– mice are not known. Methods: Male ApoE–/– mice were subjected to intraperitoneal application of cholesterol, sitosterol, 7-beta-hydroxycholesterol, 7-beta-hydroxysitosterol or cyclodextrin solution as control over a time period of 4 weeks. All animals had access to ad libidum water and cholesterol free normal chow. After 4 weeks we determined sterol, oxysterol, phytosterol and oxyphytosterol levels in serum, ROS activity assessed by spin trap in aortic tissue, endothelial function of aortic rings and atherosclerosis in the aortic sinus (n=10 per group). Results: Compared to control i.p. application of cholesterol showed no difference in regard to plasma cholesterol levels (379±111 mg/dl vs. 381±41 mg/ dl), but resulted in a significant decrease in lanosterol levels (156±54 vs. 118±6 µg/dl). Likewise, the i.p. application of 7-beta-hydroxy-cholesterol (0.013±0.094 mg/dl vs. 0.196±0.094 mg/dl) and 7-beta-hydroxysitosterol (346±167 vs. 4899±1111 ng/ ml) increased respective plasma levels compared to controls. However, this © Verlag PERFUSION GmbH

ABSTRACTS

effect was not seen for the phytosterol sitosterol (40±27 vs. 16±6 ng/ml). In regard to oxidative stress in aortic tissue we found a significant increase in 7-beta-hydoxy-sitosterol treated mice compared to control (increase compared to control by 157.7±48.9 %), but no effect by cholesterol (91.9±67.5 %), sitosterol (106.4±12.5 %) and 7-beta-hydroxycholesterol (109.0±52.1 %). Compared to controls, neither cholesterol nor sitosterol, or 7-beta- hydroxyl-cholesterol and 7-beta-hydroxy-sitosterol affected endothelial function. Atherosclerotic lesions were evaluated by oil-red-Ostaining in the aortic sinus. Compared to controls there was no difference in cholesterol treated mice (17.2±8.5 vs. 14.5±9.8 %), sitosterol treated mice (17.0±9.2 %), 7-beta-hydroxy-cholesterol (7.9±4.5 %) and 7-beta-hydroxysitosterol treated mice (10.1±6.4 %). Conclusions: Increased oxyphytosterol plasma levels increase ROS activity in aortic tissue, but do not affect endothelial function and atherosclerosis in ApoE–/–mice.

Effects of high fat feeding and diabetes on regression of athero sclerosis induced by low-density lipoprotein receptor gene therapy in LDL receptor-deficient mice F. Willecke1, C. Yuan2, L. Grauer2, K. Oka3, L. Chan3, Y. Hu1, S. Barnhart4, K.E. Bornfeldt4, I.J. Goldberg1, E.A. Fisher2 1 Division of Endocrinology, Diabetes and Metabolism, New York University Langone Medical Center, New York, USA 2 Cardiology, New York University Langone Medical Center, New York, USA 3 Department of Medicine and Molecular and Cellular Biology, and the Diabetes Research Center, Baylor College of Medicine, Houston, TX, USA 4 Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, Diabetes and Obesity Center of Excellence, University of Washington, Seattle, WA, USA Objective: We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or Perfusion 01/2015

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insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr–/–) mice. Methods and results: Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAdLDLR). After injection of the HDAdLDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. Conclusion: In conclusion, HDAdmediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr–/– mice in different metabolic states.

Deficiency of the co-stimulatory molecule CD27 impairs regulatory T cell survival and exacerbates atherosclerosis H. Winkels1, 2, E. Smeets2, S. Meiler2, L. Beckers2, A. Dandl1, C. Spitz1, C. Bürger1, C. Weber1, E. Lutgens1, 2, N. Gerdes1, 2 1 Institute for Cardiovascular Prevention, Ludwig Maximilians University Munich, Germany 2 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands

Objective: Atherosclerosis, an inflammatory disease of large arteries, is – through its clinical manifestations stroke and myocardial infarction – the leading cause of morbidity and mortality in the industrialized world. Adaptive immunity and co-stimulatory signals play a pivotal role during all stages of the disease. Recently, regulatory T cells “Treg” were attributed an anti-inflammatory and anti-atherogenic role. The interaction of CD70, a member of the tumour necrosis factor super family “TNFSF” with its receptor CD27 modulates Treg development but also affects T cell proliferation, differentiation, and activation at the sites of antigen priming and at effector function. We hypothesized an increased atherosclerotic burden and an exacerbation of disease upon CD27 deficiency. Methods and results: Cd27–/– mice were crossed with ApoE–/– mice. Cd27–/– ApoE–/– and littermate controls (Cd27+/+ ApoE–/–) were sacrificed at the age of 18 and 28 weeks. Cryosections of the aortic sinus were prepared and analysed for atherosclerotic lesion size, histology and cellular composition. 18 week-old Cd27–/– ApoE–/– mice have a trend towards bigger atherosclerotic lesions displaying a 2.5-fold higher macrophage content. Flow cytometry revealed a significant decrease in the abundance of splenic (26 %) and aortic (27 %) Tregs and increased apoptosis of Treg in the thymus (60 %) of Cd27–/– ApoE–/– mice. In contrast, 28 week-old Cd27–/– ApoE–/– mice did not differ in splenic Treg content and had similar atherosclerotic plaque size and phenotype compared to their littermate controls. Furthermore, bone marrow transplantation of Cd27–/– ApoE–/– and littermate controls into ApoE–/– recipient mice revealed a 2.3-fold increase in atherosclerotic plaque size and a pronounced pro-inflammatory plaque phenotype accompanied by reduced frequency of aortic (54.1 %) and splenic (17.7 %) Tregs. Cd27–/– ApoE–/– Tregs showed the same suppressive and migratory capacity as those isolated from controls. © Verlag PERFUSION GmbH

ABSTRACTS

Conclusion: Taken together, our data reveal that deficiency for CD27 impairs thymic Treg development thereby exacerbating early atherogenesis and increasing the macrophage content of atherosclerotic lesions. However, later stages of atherosclerosis were not affected by a CD27 deficiency.

Prevalence of M4 macrophages within human coronary athero sclerotic plaques is associated with features of plaque instability A. Wolf1, C. Erbel1, 2, F. Lasitschka3, F. Linden1, G. Domschke1, M. Akhavanpoor1, A.O. Doesch1, 2, H.A. Katus1, 2, C.A. Gleissner1, 2 1 Department of Cardiology, University of Heidelberg, Germany 2 DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg, Germany 3 Institute of Pathology, University of Heidelberg, Germany Background: The platelet chemokine CXCL4 induces monocyte differentiation resulting in a macrophage phenotype called M4, which co-expresses CD68, MMP7, and S100A8. It is unknown whether M4 macrophages are associated with plaque destabilization. Methods: Atherosclerotic arteries were obtained from hearts with severe coronary artery disease (CAD, n=32) and controls (dilated cardiomyopathy with mild CAD, n=19) explanted during allograft transplantation. Coronary arteries were stained with H&E, immuno-fluorescence was performed for CD68, MMP7, and S100A8. Results: Both CD68+ macrophages representing the entire macrophage population and MMP7+S100A8+CD68+ M4 macrophages could be reproducibly identified within all arterial lay-

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ers. The average proportion of M4 macrophages of all macrophages was 31.7±16.2 %. The highest number of M4 macrophages was found in the adventitia, followed by the intima. CD68+ and M4 macrophage numbers were significantly higher in patients with severe CAD. Presence of M4, but not of all CD68+ macrophages within the intima and the media was significantly associated with plaque instability as determined by Stary class. Multivariate analysis of the determinants of plaque instability showed a highly significant contribution of cardiovascular risk factors (p=0.008), there were trends for age (p=0.060) and prevalence of M4 macrophages (p=0.098). Conclusions: We demonstrate for the first time that M4 macrophages can be reproducibly found in coronary artery plaques. The prevalence of M4 macrophages is associated with plaque instability, most likely representing a surrogate marker of inflammatory activity. These findings make M4 macrophages an interesting therapeutic target to stabilize atherosclerotic plaques.

Mechanisms of PVAT dysfunction in diet-induced obese mice N. Xia, S. Horke, A. Habermeier, E.I. Closs, G. Reifenberg, U. Förstermann, 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 function. Under conditions of diet-induced obesity, however, PVAT becomes dysfunctional. The present study was conducted to study the mechanisms

of PVAT dysfunction in diet-induced obese mice. Methods: Obesity was induced in male C57BL/6J mice with high-fat diet (HFD, 60 % energy from fat). Results: In aortic PVAT from control mice, immunohistochemical staining of endothelial nitric oxide synthase (eNOS) was observed in adipocytes. PVAT NO production was detected by 4,5-diaminofluorescein diacetate (DAF2-DA) fluorescence with a Zeiss Laser Scanning Microscope. In the PVAT from mice fed a HFD for 20 weeks, PVAT NO production was reduced. Vasodilator response of PVATcontaining aorta to acetylcholine was inhibited by diet-induced obesity. 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, likely due to an inhibition of Akt. In addition, the production of reactive oxygen species in PVAT was reduced by the eNOS inhibitor LNAME, indicating eNOS uncoupling. Indeed, an induction of arginases and a deficiency of L-arginine were found in PVAT of obese mice. Finally, eNOS dysfunction was also associated with an enhanced eNOS acetylation and reduced activity of the NAD+-dependent deacetylase sirtuin 1 (SIRT1). Ex vivo incubation of PVAT-containing aorta with NAD+ improved the acetylcholine-induced vasodilation. Conclusions: eNOS uncoupling, reduced eNOS phosphorylation and enhanced eNOS acetylation are involved in PVAT dysfunction in diet-induced obese mice.

Mitteilungen

Neues Warnsignal für Schlaganfall entdeckt Jedes Jahr erleiden in Deutschland ca. 260.000 Menschen einen Schlaganfall. Typische Symptome sind Sehstörungen, vorübergehende Taubheitsgefühle, Schwindel oder starke Kopfschmerzen. Nun haben Forscher neue Signale entdeckt, die auf ein erhöhtes Schlaganfallrisiko hinweisen können. Der Blick auf die Gefäße lohnt sich: Niederländische Wissenschaftler fanden heraus, dass – unter Berücksichtigung der Faktoren BMI, Bluthochdruck und Diabetes – das Risiko für einen Schlaganfall bei Menschen mit Gedächtnisstörungen um 20 % erhöht ist [1]. Hierfür analysierten sie die Daten von 9153 Probanden. Eine mangelnde Durchblutung der relevanten Hirnareale kann häufig zunächst zu Gedächtnisstörungen führen, bevor die typischen Symptome eines Schlaganfalls auftreten. Zusätzlich konnte eine Verbindung mit dem Bildungsgrad festgestellt werden. Je höher das Bildungsniveau ist, desto höher ist auch das Schlaganfallrisiko. Ein möglicher Grund dafür ist, dass Personen mit höherem Bildungsniveau kognitive Leistungsstörungen vermutlich besser ausgleichen können, sodass Veränderungen der Gefäße dadurch erst später bemerkt werden. Zuvor hatte eine Studie sogar gezeigt, dass das Schlaganfallrisiko bei Personen mit kognitiven Leistungsstörungen um 40 % erhöht ist [2]. Auch hier konnten die Forscher keine Begründung liefern und vermuten ebenfalls, dass es bei den Probanden durch Gefäßschädigungen schon vorher zu kleinen unbemerkten „Mini-Infarkten“ im Gehirn mit der Folge eines echten Hirnschlags kam. Bei Gedächtnisproblemen sollte daher immer auf die Gefäße geschaut werden, um das Schlaganfallrisiko zu verringern. E. W. Quellen 1 Sajjad A et al. Subjective memory complaints and the risk of stroke. Stroke 2015;46:170-175 2 Lee M et al. Cognitive impairment and risk of future stroke: a systematic review and meta-analysis. CMAJ 2014;186:E536E546 Perfusion 01/2015

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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

Schriftleitung: Univ.-Prof. Dr. Dr. Edzard Ernst, Emeritus Professor of Complementary Medicine, University of Exeter, Peninsula Medical School, Salmon Pool Lane, Exeter EX2 4SG, UK E-Mail: Edzard.Ernst@pms.ac.uk Tel: +44 (0) 1392 726029 Fax: +44 (0) 1392 421009 Die Zeitschrift erscheint 6-mal im Jahr; Jahresabonnement 27,–; Einzelheft 5,50, inklusive MwSt., zuzüglich Versandspesen. Der Abonnementpreis ist im voraus zahlbar. Stornierungen sind bis 6 Wochen vor Ablauf eines Kalenderjahres möglich. Abonnementbestellungen direkt beim Verlag.

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