The effect of microRNAs on the development of pulmonary arterial hypertension

THE EFFECT OF MICRORNAS ON THE DEVELOPMENT OF PULMONARY ARTERIAL HYPERTENSION A research report on the relationship between endothelial-tomesenchymal transition and pulmonary arterial hypertension

Maaike Dekker & Pascal Gunsch Pre-University College March 2015 Department of Molecular Cell Biology Supervised by prof. dr. Marie-JosĂŠ Goumans Leiden University Medical Centre (LUMC)

Preface Since October 2013, we have been following the Pre-University College at Leiden University.

This programme was founded to provide a broad, interesting and challenging course for top students at high school. This course includes lectures on the most diverse subjects, as well as projects that require individual or small group research and presentation. One of these projects is Block IV, in which students think of a research question and a research plan. Then, under the supervision of a professional in the field of interest, they carry out the research necessary to answer their question.

Our research question was: “What is the effect of miR-23 and miR-155 on the endothelialmesenchymal transition in idiopathic and heritable pulmonary arterial hypertension patients?” We were assigned to one of the leading experts on this area: prof. dr. Marie-José Goumans. She works at the Department of Molecular Cell Biology at the Leiden University Medical Centre (LUMC).

Though this research report is written as a part of the Pre-University College programme, it also serves as our profielwerkstuk. This is some sort of ‘high school thesis’. To make this report clear for other students (and teachers) at our school, we also added a general part on pulmonary arterial hypertension and microRNA. We learned a lot from this experience and it was a great pleasure to do this research.

Maaike Dekker & Pascal Gunsch March 2015

Preface

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Table of contents Preface...............................................................................................................................................2 Table of contents ..............................................................................................................................3 Pulmonary arterial hypertension.....................................................................................................4 Causes and subtypes ....................................................................................................................4 Symptoms .....................................................................................................................................6 Treatment .....................................................................................................................................7 Consequences ...............................................................................................................................9 The Pathogenesis of PAH ...............................................................................................................10 Vascular remodelling ..................................................................................................................10 Endothelial-to-mesenchymal transition ...................................................................................11 The influence of microRNA ............................................................................................................14 Biogenesis of microRNA .............................................................................................................14 Function of microRNA ................................................................................................................16 The Research ...................................................................................................................................17 Endothelial-to-mesenchymal transition in endothelial cells .......................................................18 Introduction ................................................................................................................................18 Methods ......................................................................................................................................19 Results .........................................................................................................................................20 Discussion....................................................................................................................................23 MicroRNAs and the development of pulmonary arterial hypertension .....................................25 Introduction ................................................................................................................................25 Methods ......................................................................................................................................25 Results .........................................................................................................................................26 Discussion....................................................................................................................................28 Conclusion .......................................................................................................................................30 Acknowledgements ........................................................................................................................31 References.......................................................................................................................................32 Protocols..........................................................................................................................................36

Table of contents

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Pulmonary arterial hypertension Pulmonary hypertension is a disease caused by increased blood pressure in the blood vessels of the lungs. There are five main categories of lung diseases that are associated with an increased blood pressure in the lungs. Among these are well-known diseases such as chronic obstructive pulmonary disease (COPD), but there are some rare diseases as well. One example of a rare disease is pulmonary arterial hypertension. Patients suffering from this disease have an elevated systolic blood pressure in the pulmonary arteries.

Causes and subtypes Pulmonary arterial hypertension (PAH) is defined as a sustained pulmonary arterial pressure of >25 mmHg at rest, or of >30 mmHg with exercise.[1] In addition, significant left heart disease, lung disease and chronic thromboembolic disease must be absent.[2] The incidence is estimated at 52 per million per year.[3] The World Health Organisation (WHO) has set up a classification system for pulmonary hypertension. It placed PAH in WHO group I,[4] which consists of three categories: idiopathic PAH, heritable PAH and PAH associated with other risk factors.[5] 1. Idiopathic PAH Idiopathic PAH (IPAH) is a form of PAH with no demonstrable, or spontaneous, causes. The incidence rate of IPAH is estimated at 2–3 per million per year, and women are more affected as the ratio female:male is 2.3:1.[6] This increased prevalence in women is explained by a possible influence of oestrogen on the disease.[7] Interestingly, the progression of the disease is worse in man compared to women, it is more severe and there is a higher mortality.[8] The exact mechanisms underlying the initiation and progression of IPAH are still unknown.

2. Heritable PAH Heritable PAH (HPAH) is a form of PAH associated with a genetic defect. This is very often a mutation in the bone morphogenetic protein receptor II (BMPR2). Although the disease is inherited in an autosomal-dominant way, only 20% of the carriers develop clinical PAH.[9] This suggest that just having the mutation is not enough, but other factors are important in developing the clinical symptoms of PAH. Furthermore, this autosomal dominance suggests a haploinsufficiency of the BMPR2 gene.[10] Haploinsufficiency means that just one good allele is not enough to produce a functional protein or the right amount of the protein.

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Hereditary haemorrhagic telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, is an autosomal-dominant genetic disorder that is known to lead to PAH.[2] A mutation that is sometimes found in this disease is a mutation in the activin-like kinase 1 (ALK-1) gene. This mutation is also found in PAH patients.[11] The ALK-1 receptor is important in BMP signalling and thus comparable with BMPR2. The question remains whether the ALK-1 mutation causes the development of HHT, or whether HHT causes PAH. 3. PAH associated with other risk factors PAH associated with other risk factors is PAH associated with other diseases, for example HIV infection, collagen vascular diseases, congenital heart disease, (accidental) use of particular drugs and toxins, connective tissue diseases or schistosomiasis.[9] To date, it is becoming clear that merely having the BMPR2 mutation is not enough to develop PAH. Environmental factors have been associated with disease progression. This is also referred to as the second hit model. In this model, the BPMR2 mutation only makes it more likely to develop the disease. One important inducer seems to be appetite suppressants, more specifically, those which contain aminorex, dexfenfluramine, or fenfluramine.[2] Amonirex fumarate was linked to an increase and fall of PAH in the late 1960s in Austria, Switzerland and Germany. A quite recent widespread of fenfluramine and its derivatives in Europe shows an increase in PAH cases.

Other studies also suggested that rape seed oil and diet pills have a positive effect on the development of PAH.[7] These appetite suppressants may increase local levels of serotonin.

The activity of serotonin transporter is associated with pulmonary artery smooth muscle cell proliferation, one of the events resulting in PAH. The L-allelic variant of the gene, connected to increased expression of this transporter, is also found in 65% of patients with IPAH to 27% of the controls.[11] Certain stimulant drugs such as amphetamine, methamphetamine, and cocaine are thought to influence serotonin transport as well and so to also cause PAH.[2] On the other hand, medication with selective serotonin reuptake inhibitors increases local serotonin levels, but has not been linked to increase of PAH in adults, an increase in mortality in established PAH patients nor promotion of smooth muscle cell proliferation in vitro.[2] Prevalence Pulmonary arterial hypertension is a quite rare disease: a study done in Scotland in 2007 shows that there were 52 cases per million every year.[3] An earlier research in France suggests 15,0 cases per million and 2,4 cases per million per year.[12] See Figure 1.

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The prevalence of PAH in certain groups is higher: patients with HIV for example have a 0,5% prevalence.[13] As stated before, the prevalence of the disease in women is higher than in men. The disease is 2–4 times more prevalent in women than in men.[14]  Figure 1. Prevalence of PAH. Prevalence

of different subtypes of PAH. Data was analysed from three sources in France and Scotland: white is the French national registry, grey is the Scottish hospitalisation records and black is the Scottish Pulmonary Vascular Unit database. IPH: idiopathic pulmonary hypertension (PH); FPH: familial PH; CTD-PH: connective tissue disease-associated PH; CHD-PH: congenital heart disease-associated PH; PPHTN: porto-pulmonary hypertension; APH: PH associated with other diseases or things. Image from Peacock AJ, Murphy NF, McMurray JJV et al. An epidemiological study of pulmonary arterial hypertension. Eur Respir J 2007;30:104–109.

Symptoms Symptoms linked to PAH include dyspnoea on exertion, fatigue, dizziness, angina pectoris particularly during physical activity and peripheral oedema.[2,15] These symptoms are all due to failure of the right heart ventricle. These symptoms are all rather general phenomena and so they may be linked to another cause, making diagnosing PAH difficult. In time, the symptoms get worse and so the fatigue and dyspnoea may become constant. Observations also state that inflammation is part of the disease, since autoantibodies, pro-inflammatory cytokines and inflammatory infiltrates have been found in some cases of PAH.[11] Although looking at the symptoms may be one way the disease can be discovered, but since they are so general, it is hard to draw the right conclusion and diagnose the patients using these criteria. Therefore, persons who are known to potentially develop PAH can be screened to monitor the presence and possible progress of the disease. People with a predisposition to PAH and advised to be screened, are:     

People with a genetic mutation connected to PAH People with a first-degree relative with idiopathic PAH People with scleroderma People with congenital heart disease and systematic-to-pulmonary shunts People with portal hypertension

Of course, an incidental discovery is also still a way to find out about the disease.[1]

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Treatment Since no cure for the disease has been found yet, all the treatment is aimed at relieving the symptoms and slowing down the progress of the disease. There are currently 9 medical therapies which have already gained regulatory approval or are under regulatory review.

These therapies are targeted at the prostacyclin pathway, the nitric oxide pathway, and the endothelin pathway. By targeting two or all three of these pathways, the therapy becomes more effective.[16] In the 1980s, the median survival was 2.8 years after diagnosis. Progress has been made, but a real cure remains unknown. Patients are discouraged to not engage in any physical activity anymore, since this would stimulate deconditioning and worsening of the disease. The physical activity in which a patient engages must, however, be appropriate to the condition of the patient. Contraceptives are recommended to women with this condition, since pregnancy and labour puts more pressure on the cardiopulmonary system.[17] Symptomatic benefit Oxygen Supplemental oxygen therapy can be prescribed, to maintain an oxygen saturation level of over 90%. Since chronic hypoxemia can develop due to PAH, this oxygen therapy is needed.[17] Anticoagulants This is nowadays a preferred treatment, since it showed positive outcomes. These positive outcomes showed an increase in three-year survival rate.[16] However, uncertainties remain.

The treatment affects in situ thrombosis.[17] Diuretics The diuretic therapy reduces the right ventricular preload, what thus leads to a lower blood pressure in the arteries. There are however still uncertainties about which substance best to use and whether or not this therapy really works.[17] Calcium Channel Blockers This was most favourable until the 1980s. Until then, people tried to reduce the pulmonary arterial pressure with vasodilators. Most favourable and effective were high oral doses of Calcium Channel Blockers (CCBs), but only in part of the patients. This therapy is merely effective in patients with acute vasodilator challenge.[16,17]

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Advanced therapy Endothelin receptor antagonists Endothelin is a substance that inter alia stimulates vascular smooth muscle cell proliferation and exercises a direct vasoconstrictor effect. This substance has two receptors: the ETa and ETb receptor. When the A-version is stimulated, vasoconstriction and vascular smooth-muscle cell proliferation occur. But when the B-version is stimulated, pulmonary endothelin clearance is induced as well as the production of prostacyclin and nitric oxide by endothelial cells. This principle is used in the oral drug bosentan, which has a negative effect on both receptors. Bosentan had very good outcomes in both life quality and clinical worsening. This drug has very little side effects, apart from some in the liver. Monthly screening of the liver is therefore mandatory, but there are no cases of patients with liver dysfunction or so in the over 12000 patients who have already received the drug. Research is now conducted with selective blockers of the ETa-receptor, such as sitaxsentan and ambrisentan. These, however, may cause acute hepatitis.[17] Prostacyclin therapy Prostacyclin induces relaxation of vascular smooth muscle by stimulating the production of cyclic AMP (cAMP) and inhibits the growth of smooth-muscle cells. In addition, it is a powerful inhibitor of platelet aggregation. There are different ways this therapy can be executed: one can become an epoprostenol infusion. This has very good results in both life quality and survival rate, but this method is very expensive, complicated and very uncomfortable, since there are many side-effects. An alternative may be treprostinil or beraprost. Treprostinil works quite similarly as epoprostenol, but beraprost is different. This medicine can be taken orally and shows improvement in condition, but not in the overall cardiovascular haemodynamics. Another option would be inhaled iloprost. This has positive outcomes in studies, but it has short time of action so it would have to be inhaled 6 to 12 times a day. Another disadvantage is that the long-term effects of iloprost are still unknown.[17] Nitric oxide This is at the moment not a therapy yet, but it could become one. This substance is known as a vasodilator and since PAH is linked to a defect in the production of nitric oxide, it has potential. Short-term inhalation of nitric oxide has had positive outcomes, but long-term inhalation not.

Phosphodiesterase-5 inhibitors This therapy is an extension on the one described above. Intracellular cyclic guanosine monophosphate (cGMP) is a nitric oxide-dependent substance that also induces vasodilation.

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cGMP is broken down by phosphodiesterase-5, so if the break down is prevented, the vasodilation can increase. This is indeed shown by studies. The short-term data of sildenafil is promising, yet no long-term data is available.[17] Sildenafil is a medicine that blocks phosphodiesterase-5 and is normally used in men with erectile dysfunction.

Surgical intervention Balloon atrial septostomy This means to create a hole in the septum between the right and left atria of the heart, creating a better flow of the blood and hence a lower blood pressure. However, this is at cost of lower oxygen levels in the blood.

Transplantation Lung transplantation and/ or heart and lung transplantation is seen as a final solution. During other therapies, patients remain under control to see whether or not they need transplantation.

Consequences The disease leads to the obstruction of the pulmonary arteries, due to vasoconstriction, local thrombosis, and remodelling of the arteries.[18] This leads to high blood pressure and bad exchange of substances in the lungs. The disease always leads to an early death. As stated before, other conditions may occur such as chronic hypoxemia or in situ thrombosis. It is clear that a better understanding of the disease could lead to better treatment and better survival.

Despite the wide offer of therapies, there is still no cure for the disease. All our current therapies merely improve quality of life and increase life expectancy. To find this cure, a better understanding of the disease is necessary. Many investigation that are currently running try to improve our understanding of the disease, in order to find a better medicine in the near future.

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The Pathogenesis of PAH The pathogenesis of PAH is still largely unknown, though there are many new insights into the molecular mechanisms underlying the disease. The symptoms are caused by vascular remodelling, a process that resembles endothelial-to-mesenchymal transition and involves different pathways, including the TGF-β and BMP pathways. However, the exact role of environmental and genetic factors and their impact on these pathways remains unclear.

Vascular remodelling Though the different types of PAH differ in origin, they are all caused by vascular remodelling of the arteries in the lungs.[19] Normally, the arterial wall has three different layers: the intima, consisting of a tightly packed layer of endothelial cells (ECs); the media, consisting of neatly arranged smooth muscle cells (SMCs); and the adventitia, mainly consisting of fibroblasts and collagen fibres. In patients with PAH, various changes occur in the composition of the artery, due to excessive proliferation of the cells in the vascular wall, dysfunction of endothelial cells and resistance of smooth muscle cells to apoptosis.[20–22] There may also be endothelial injury, inflammation or fibroblast like cells in the intima.[23] All these changes result in the formation of a neointima that narrows the lumen substantially,[24] leading to chronic vasoconstriction (Figure 2). Because of this, the thick layer of cells makes gas exchange more difficult.

 Figure 2. Vascular remodelling

in PAH. On the left is the cross-section of a healthy artery. The three distinct layers are represented by different cell types. Due to excessive proliferation and other factors, the lumen narrows, causing chronic vasoconstriction (on the right). Adapted from Pugliese SC, Poth JM, Fini MA, Olschewski A, El Kasmi KC, Stenmark KR. Am J Physiol Lung Cell Mol Physiol 2015;308(3):L229–L252.

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Endothelial-to-mesenchymal transition The vascular remodelling that occurs in PAH is probably caused by a process known as endothelial-to-mesenchymal transition (EndoMT).[25] EndoMT is important in the development of the embryo, as it is involved in cardiovascular developments, such as heart formation.[26] In essence, during EndoMT, endothelial cells are induced to change to a mesenchymal or fibroblast-like phenotype.[27] The cells become motile and start to express typical mesenchymal markers, such as α smooth muscle actin (α-SMA) and type I collagen, rather than endothelial markers, such as the platelet endothelial cell adhesion molecule (PECAM).[28,29] Research has shown that EndoMT can be induced by a cytokine known as Transforming growth factor (TGF)-β.[27] TGF-β signals using the TGF-β pathway.

The TGF-β pathway TGF-β controls a great variety of cellular processes, such as cell proliferation, migration, differentiation and apoptosis.[30,31] It is for this reason that TGF-β is important during embryonic development as well as in adult life, and that it is deregulated in many diseases, including cancer and vascular diseases.

The TGF-β protein is part of a large family of proteins called the TGF-β superfamily. It includes, amongst others, TGF-β, activin and bone morphogenetic proteins (BMP). Each protein can bind to a specific receptor complex, containing type I and type II receptors. Type I receptors are also known as activin receptor-like kinases (ALKs). There are different types of type I and type II receptors and most proteins can bind to more than one receptor. In order for a signal to be transduced from the membrane to the nucleus, a protein first forms a complex with two type I receptors and two type II receptors. TGF-β binds to the TGF-β receptor type II (TGFβR2) and to ALK-1, ALK-2 or ALK-5. BMPs bind to the bone morphogenetic protein receptor type II (BMPR2) and to ALK-1, ALK-2, ALK-3 or ALK-6.[31–34] The type II receptor activates the type I receptor which in its turn activates a Smad protein by phosphorylation. Smads are the signal transducers. TGFβR2 and ALK-5 typically activate Smad2 or Smad3, while BMPR2, ALK-1, ALK-2, ALK-3, ALK-6 activate Smad1, Smad5 or Smad8.

These are referred to as receptor-mediated Smads (R-Smads). Two phosphorylated R-Smads will then bind to Smad4 (also known as the common or co-Smad).[31–34] The formed complex migrates to the nucleus and binds to other transcription factors and one of the co-activators p300 or CBP. This complex will attach to a specific part of the DNA and then transcribe specific target genes. With these genes, the Smads will exert their regulatory function. The BMP-activated Smads target genes like the inhibitor of differentiation 1 (ID-1) gene, while the TGF-β-activated Smads target genes like plasminogen activator inhibitor 1 (PAI-1) and connective tissue growth factor (CTGF).[35] The Pathogenesis of PAH

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Several mechanisms can inhibit the TGF-β or BMP signal transduction via Smads. One of these processes involves the inhibitory Smads (I-Smads), which are Smad6 and Smad7.[34] The I-Smads bind to the type I receptors and thus interfere with the phosphorylation of the R-Smads. As a result, no complexes are formed that can regulate DNA transcription. Another family of molecules involved with Smad inhibition are the Smad ubiquitination regulatory factors (Smurfs). Smurf1 interacts with Smad1 and Smad5, to interfere with the BMP signalling, while Smurf2 is less specific and interferes both with the BMP and TGF-β signalling.[32] See Figure 3.

 Figure 3. Overview of the TGF-β pathway. The pathway starts with TGF-β and BMP

molecules. These bind with type I and type II receptors to activate specific R-Smads, which couple with the co-Smad and enter the nucleus to interfere with DNA transcription. Image from http://www.cellsignal.com/ contents/science-pathway-researchstem-cell-markers/tgf-smad-signaling-pathway/pathways-tgfb.

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The BMP signalling is very important in the development of the vascular system and also plays a key role in the pathogenesis of most vascular diseases.[36] The role of these BMPs, however, is complex. Different BMPs can exert similar or very different effects. It has been demonstrated that BMP2 and BMP4 promote endothelial cell and SMC proliferation in vitro.[35] BMP9 binds to BMPR2 and ALK-1. Research has shown that BMP9 activates Smad1, Smad2 and Smad5. Via these Smads, it induces the expression of endothelial factors.[37] TGF-β and BMP signalling can also happen without the phosphorylation of Smads, but using the so called non-Smad pathways. These include mitogen-activated protein kinase (MAPK) pathways, phosphatidylinositol-3-kinase (PI3K) and Akt pathways and Rho-like GTPase pathways.[32] The effects of the non-Smad pathways can vary by cell type, but may include proliferation, apoptosis and regulation of Smad-pathways. These non-Smad pathways can be activated by the receptors that are also involved in Smad signalling. An example of non-Smad signalling involves c-Jun N-terminal kinase (JNK) and p38 MAPK. These work when receptors activate TGF-β activated kinase 1 (TAK1), which activates a MAPK pathway. However, it may also activate Smads and thereby interfere with the Smad pathways.[38,39] Deregulation of signalling in PAH In patients with PAH, EndoMT seems to be stimulated, which leads to the typical vascular remodelling. Mutations or changes in different parts of the TGF-β pathway may cause this. As described before, a large group of HPAH patients have a BMPR2 mutation, leading to haploinsufficiency. Though less common in patients, an ALK-1 mutation has also been associated with the development PAH.[36,40] Normally, BMP2 and BMP4 signal through the BMPR2 to stimulate the expression of endothelial factors and supress the proliferation of SMCs. In patients with a BMPR2 mutation, significantly lower levels of BMPR2 are expressed. This means reduced activation and signalling of Smads. Furthermore, in vitro studies have shown that a loss of Smad signalling is compensated by an increase in non-Smad signalling, notably involving the p38 MAPK pathway. These pathways cause cell proliferation.[19,38,39] A loss of BMPR2 therefore causes the endothelial cells to become SMCs. Of course, in reality the processes involved in deregulated BMPR2 signalling are not that simple. The exact mechanisms underlying this process are still not fully understood, and much research has to be done to clarify this process.

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The influence of microRNA Research has shown that microRNAs are associated with the development of PAH. MicroRNAs (miRNA) are small, non-coding molecules of RNA that are about 20–25 nucleotides long. They interfere with mRNA by binding to the 3' untranslated region (3'-UTR).

MiRNAs regulate the expression of specific genes after transcription by either cleaving and degrading the mRNA or inhibiting the initiation of translation, also known as translational repression. An upregulated or downregulated expression of certain types of these molecules could be one of the contributing factors to the development of the disease. Researchers have identified several miRNAs that are important in the development of PAH, including miR-23 and miR-155.

Biogenesis of microRNA The formation of miRNA is a complex process involving multiple steps. There are different ways for a gene to become miRNA, however, there is one way that prevails. First, if the miRNA gene is intergenic, the gene is simply transcribed by RNA polymerase II. The result is a RNA molecule called the primary miRNA (pri-miRNA). One pri-miRNA molecule can give rise to several different miRNAs. It is also common that the miRNA genes are on the introns of other genes. They are removed from the coding mRNA by the spliceosome. In that case they are called intronic.

The pri-miRNA molecule has one or more structures that look like hairpin loops, caused by parts of complementary RNA that form a double strand of RNA. The loops of these structures are recognised by the nuclear protein DGCR8, which activates the enzyme Drosha. Drosha is an RNase enzyme that processes the pri-miRNA by cutting the RNA near the base of the hairpins to form precursor miRNA (pre-miRNA).

The pre-miRNA is transported out of the nucleus into the cytoplasm by Exportin-5 and RanGTP. In the cytoplasm, the endonuclease Dicer cleaves the pre-miRNA hairpin into an imperfect double-stranded miRNA:miRNA* duplex. This duplex contains the mature miRNA and its imperfect complementary strand (miRNA*). The duplex is bound to the argonaute protein Ago2, in which the mature miRNA is formed. This strand is also indicated with the suffix 5p. Most of the times, the complementary strand (denoted by the suffix 3p) degrades, though they may also have a regulatory function.

The mature miRNA is then incorporated into the RNA-induced silencing complex (RISC). This complex binds the miRNA to the complementary RNA sequence the 3'-UTR of the mRNA. If The influence of microRNA

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the binding is complete, the mRNA is cleaved and degraded. However, if the binding is incomplete, the initiation of translation is inhibited, resulting in gene silencing by translational repression. (Figure 4). Because of the imperfect binding, the miRNA may be complementary to several different types of mRNA and thus bind to and regulate the expression of different types of genes. The process of gene regulation by miRNA is known as RNA interference (RNAi).[41–43]

 Figure 4. MiRNA biogenesis. RNA

polymerase II transcribes a gene (1). The miRNA gene is either intergenic and coding for one type of miRNA (2a) or multiple types of miRNA (2b), or intronic and found on the introns of other mRNA strands (2c). The pri-miRNA is processed by Drosha into pre-miRNA, which is then transported out of the nucleus by Exportin-5 and Ran-GTP (3). Dicer (4) further processes the miRNA to form the miRNA:miRNA* duplex (5). Next, this duplex will bind to the RISC complex (6). The newly formed complex will recognise and bind to the 3'-UTR of the target mRNA (7) and regulate gene expression by translational repression (8a) or cleavage of the mRNA (8b). Adapted from Araldi E, Schipani E. MicroRNA-140 and the silencing of osteoarthritis. Genes & Dev 2010;24:1075–1080.

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Function of microRNA Many different miRNAs have been associated with the vascular remodelling witnessed in PAH patients. These include upregulation of miR-138, miR-367, miR-27b, miR-302b, miR-145 and miR-450a and downregulation of miR-204.[43] There also is a change in the expression of miR-24 and miR-155, which we will explain in more detail. miR-24 An important target of miR-24 is Tribbles-like protein 3 (Trb3).[44] Trb3 is an important modulator of BMP signalling. Trb3 binds to BMPR2 and promote the degradation of Smurf1.

A decreased expression of Smurf1 causes an increased expression of Smad1 and in lesser extent the other R-Smads. miR-24 decreases Trb3 causes a decreased expression of Smads and therefore a lack of TGF-β and BMP signalling.[44,45] The reduction of TGF-β and BMP signalling promotes proliferation in SMCs.[46] miR-155 The main targets of miR-155 are Smad2 and Smad5.[47,48] Overexpression of miR-155 therefore decreases Smad2 and Smad5 concentrations. This inhibits TGF-β and BMP signalling. It has also been demonstrated that miR-155 affects non-Smad signalling by increasing phosphatidylinositol (3,4,5)-triphosphate (PIP3) levels.[49] PIP3 activates the MAPK and Akt pathways. Furthermore, Ras homolog gene family A (RhoA) has been proposed as a target of miR-155.[50,51] RhoA induces its own signalling pathway and causes cell junction formation and stability. If RhoA is decreased, the cell junctions are partially lost and a SMC-like phenotype occurs.

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The Research Our research question was: “What is the effect of miR-24 and miR-155 on the endothelial-tomesenchymal transition in idiopathic and heritable pulmonary arterial hypertension patients?”.

To investigate this, we first wanted to culture cells from healthy controls and patients with IPAH and HPAH caused by a BMPR2 mutation. Then, we wanted to stimulate these cells with TGF-β, miR-24 and miR155, to look at the effect of these signal molecules on the EndoMT. Our hypothesis is that TGF-β, miR-24 and miR-155 all stimulate EndoMT. TGF-β because it is a natural inducer of EndoMT, and the miRNAs because they target different factors that result in changed signalling. The result of these changes in signalling also cause EndoMT. In essence, this means that α-SMA expression would increase, while PECAM expression would decrease. Furthermore, changes in the phenotype of the cells should be visible. The nice and ‘packed together’ endothelial cells will become more loose and spindle like.

However, soon after we had started doing our research, we stumbled upon some major problems. First of all, the patient material required to carry out the research, failed to grow in culture. Therefore, we could only continue the experiments with the control group.

Secondly, we were even more unfortunate when we noticed that these cells didn’t respond to the different stimulations. In the end, the research we did was aimed at finding out why the cells didn’t react.

Luckily, someone else was carrying out a similar experiment and we could get their data. This way we could still answer our original research question. However, since we carried out a series of experiments to find out why our cultures didn’t react, we also had to answer a second research question: “Why didn’t the cell cultures react to the stimulation?” We answered these questions in two different experiments, which can be found in this report.

The first experiment, “Endothelial-to-mesenchymal transition in endothelial cells”, is about the original cells that didn’t react. The second experiment, “microRNAs and the development of pulmonary arterial hypertension”, is about our original research question.

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Endothelial-to-mesenchymal transition in endothelial cells Introduction Functioning as a control group, endothelial cells were stimulated with TGF-β, BMP2, BMP9, LDN193189 and SB431542, or not stimulated at all. As a result, some of the cells were supposed to show endothelial-to-mesenchymal transition (EndoMT), while for other cells, this process should be inhibited.

All of the above mentioned molecules play a role in the TGF-β signal transduction pathway.

This pathway is known to initiate EndoMT, as it regulates other factors too, such as cell proliferation, differentiation and apoptosis. The pathway starts with a signalling molecule binding to a receptor complex. Next, this complex of receptors will initiate a cascade of reactions involving many different molecules. These molecules will change the expression of specific genes regulated by the signalling molecule.

TGF-β is one of the principal proteins involved in the TGF-β pathway. It binds to a complex of TGFβR2 and either ALK-1, ALK-2 or ALK-5. BMP2 is another protein belonging to the TGF-β superfamily. It binds to the receptors BMPR2 and ALK-2, ALK-3 or ALK-6. BMP9 binds to BMPR2 and ALK1. See Figure 5. Both BMPs induce the expression of endothelial factors and supress the expression of smooth muscle like factors. LDN193189 is an artificial inhibitor of BMP signalling, as it targets ALK-1, ALK-2, ALK3 and ALK-6. SB431542 is an artificial inhibitor of signalling with TGF-β. It is known to inhibit ALK-5.  Figure 5. Signal transduction during EndoMT. TGF-β

binds to a complex TGFβR2 and a TGFβR1 (ALK-1, ALK-2 or ALK-5). BMPs bind to a complex of the BMPR2 and ALK-2, ALK-3 or ALK-6. LDN193189 and SB431542 target specific ALKs to inhibit signalling. The binding of TGF-β or BMPs change the structure of the receptor complex and as a result, phosphorylate R-Smads. Numerous reactions follow that cause changes in transcription.

Image from Wagner M, Siddiqui MA. Signal transduction in early heart development (II): ventricular chamber specification, trabeculation, and heart valve formation.

Exp Biol Med (Maywood) 2007;232(7):866-80.

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The different signal molecules in the TGF-β signalling pathway target different genes. The BMP signals target genes like the inhibitor of differentiation 1 (ID-1) gene, while TGF-β targets genes like plasminogen activator inhibitor 1 (PAI-1). One of the genes that is expressed in the cells after EndoMT, is α smooth muscle actin (α-SMA). Another phenotypical difference that occurs during EndoMT is that cells lose the expression of PECAM, which is specific for endothelial cells, and gain actin filaments.

In this research, we will look at the effects of the ligands TGF-β, BMP2, BMP9, and the kinase inhibitors LDN193189 and SB431542 on the expression of PECAM and actin filaments using focal microscopy. We will also analyse the gene expression of α-SMA, ID-1 and PAI-1.

Methods Stimulation After seeding the cells on gelatine coated wells, they were stimulated with TGF-β by adding 1 ng/mL to the culture medium for 3 days, as a positive control, and with dilution buffer as a negative control. Other cells were stimulated with BMP2, BMP9, LDN193189 and SB431542 to look at their effect on the expression of markers specific for endothelial cells and cells that underwent EndoMT. Focal microscopy Endothelial cells were cultured on glass coverslips coated with gelatine. After 3 days of culture, the cells were fixed with 4% paraformaldehyde and incubated with and anti-rabbitPECAM antibody followed by a secondary anti-mouse antibody coupled to FITC. Actin filaments were stained by incubating the cells with phalloidin coupled to TRITC. The nucleus was stained using DAPI to allow the identification of the cells within the well. Pictures of the cells were taken using fluorescent focal microscopy. mRNA expression The expression of α-SMA, ID-1 and PAI-1 mRNA was determined using qPCR. The expression levels of the mRNA was compared to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the household gene. This way, it was possible to determine the relative normalised expression of these genes.

Statistical analysis The data in the histograms represent the mean and the standard error of the mean. The data was analysed with a two-tailed student’s t-test for unequal variances. Differences were considered significant with a p-value of p < 0.05.

Endothelial-to-mesenchymal transition in endothelial cells

19

Results Endothelial cells were stimulated with TGF-β, BMP2, BMP9, LDN193189 and SB431542. After 3 days of culture, PECAM and actin filaments were stained green and blue respectively. The nuclei were stained to identify the individual cells. The cells were photographed, see Figure 6.

A Negative control

B TGF-β

C BMP2

D BMP9

E LDN193189

F SB431542

 Figure 6. Stained

cells. Nuclei were stained blue with DAPI, PECAM-antibodies green with antibodies coupled to FITC and actin filaments red with phalloidin coupled to TRITC. Representative images are shown (n = 2).

Endothelial-to-mesenchymal transition in endothelial cells

20

In all the cell cultures, cell nuclei were successfully stained blue with DAPI. It is clearly visible that the cultures stimulated with TGF-β and BMP2 express relatively more PECAM on their cell membrane than the other cell cultures. Furthermore, cells stimulated with BMP9 hardly showed any antibody binding to PECAM antibodies. The other conditions showed only sporadic expression of PECAM. The culture stimulated with TGF-β expressed only low amounts of filamentous actin. Filamentous actin seems relatively high in the cells stimulated with BMP9, LDN193189, SB431542, and the negative controls.

Using qPCR, the expression of α-SMA, ID-1 and PAI-1 mRNA was investigated. The results can be seen in Figures 7, 8 and 9 respectively. TGF-β and SB431542 seem to stimulate the expression of α-SMA, though these results are not significant. There was no expression of α-SMA measured when the cells were stimulated with BMP9 or LDN193189. The expression of ID-1 is significantly stimulated by BMP9 and significantly inhibited by LDN193189 stimulation. BMP2 also had a positive effect on the expression of ID-1 (p = 0.054), though this was not considered significant. For PAI-1, the expression was significantly stimulated by BMP9. Adding BMP2, LDN193189 or SB431542 to the cell culture significantly decreased the expression of PAI-1. TGF-β had no significant effect.

α-SMA expression 3,5 3,0 2,5 2,0 1,5 1,0 0,5 0,0 control

TGF-β

BMP2

BMP9

LDN

SB

 Figure 7. Relative normalised expression of α-SMA mRNA. mRNA corresponding with the α-SMA gene was

measured using qPCR. LDN: LDN193189; SB: SB431542.

Endothelial-to-mesenchymal transition in endothelial cells

21

ID-1 expression 4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5 0,0 control

TGF-β

BMP2

BMP9

LDN

SB

 Figure 8. Relative normalised expression of ID-1 mRNA. mRNA corresponding with the ID-1 gene

was measured

using qPCR. LDN: LDN193189; SB: SB431542.

PAI-1 3,0

2,5

2,0

1,5

1,0

0,5

0,0 control

TGF-β

BMP2

BMP9

LDN

 Figure 9. Relative normalised expression of PAI-1 mRNA. mRNA corresponding with the

SB

PAI-1 gene was

measured using qPCR. LDN: LDN193189; SB: SB431542.

Endothelial-to-mesenchymal transition in endothelial cells

22

Discussion An important initiatory of PAH pathology is the endothelial-to-mesenchymal transition (EndoMT). Different factors can induce or inhibit this transition. In this experiment, we analysed the effect of TGF-β, BMP2, BMP9, LDN193189 and SB431542 on this process. The EndoMT can be shown in two different ways: by phenotypical changes of the cells, from a cobble to a spindle morphology, or by changes in gene expression. In this experiment we looked at both ways. Phenotypical change was assessed with staining and microscopy, gene expression with qPCR. To look at the phenotypical changes, the nucleus, PECAM and actin filaments were stained.

The brighter and more omnipresent a specific colour is, the more PECAM proteins on the cell membrane or actin filaments there are. The nucleus was stained to identify the different cells. Since an endothelial cell differs in shape from a mesenchymal cell, the shape can also be used to determine whether or not the transition has taken place. The results in Figure 6 show that cells stimulated with TGF-β and BMP2 express relatively more PECAM, since they are more green, while BMP9 only reacted slightly. TGF-β stimulated cells showed low amounts of actin filaments, and for the others, it was relatively high. It should, however, be noted that the actin filaments are in two different forms: cobble and spindle. Spindle actin is better visible than PECAM, but cobble actin may be covered by PECAM and difficult to see.

For the focal microscopy assay, we employed a technique involving small glass plates. These small glass plates had to be washed between every treatment. During rinsing, some glass plates broke or fell in the fluid. It was also possible that the thin layer of cells was washed off.

When the glass plates fell, we couldn’t always determine again on which side the cells were, so some of the glass plates were placed upside down in the new plate. Hence, some cells were not able to bind to the added stains. This explains the odd observations in the photographs of the cells and their unreliability.

Furthermore, these results are based on analysis of microscopy images, whereas true methods of quantification would be more accurate to base these results on. Since we analysed two samples per group, this study could be made more reliable by increasing the number of samples.

From previous studies[35,51] we hypothesised that there is a change in ID-1 and PAI-1. ID-1 is thought to increase when adding BMP2 and BMP9. PAI-1 is thought to increase when adding TGF-β. After stimulation with LDN193189, no BMP mediated effects are expected, and after stimulation with SB431542, no TGF-β mediated effects are expected. In the control cells, no increase nor decrease is anticipated in both of the genes. Furthermore, an increase

Endothelial-to-mesenchymal transition in endothelial cells

23

in α-SMA expression is expected when EndoMT should have occurred. This is the case for TGF-β, BMP2 and BMP9.

To look at the gene expression of α-SMA, ID-1 and PAI-1, qPCR was used. Since αSMA, ID-1 and PAI-1 are regulated by the TGF-β pathway (see Figure 10), we expected changes in the expression of these genes. The results are shown in figures 7, 8 and 9.  Figure 10. TGF-β and BMP9 gene activation. TGF-β

activates PAI-1, while BMPs activate ID-1. Image from Cunha SI, Pietras K. ALK1 as an emerging target for antiangiogenic therapy of cancer. Blood 2011;117(26):6999–7006.

These results are quite interesting. There was no significant change in α-SMA expression.

There were, however, elevated levels of α-SMA in the TGF-β and SB431542 samples. Two other samples, BMP9 and LDN193189, actually did not show any expression. This explains why morphologically there was no EndoMT observed in these cell cultures. The lack of expression in BMP9 and LDN193189 stimulated cells probably have to do with the technique used. The level of expression was so low that no data could be acquired. Another explanation is that the cells were stimulated too shortly, or that the used concentration was too low.

Expression of the ID-1 gene was significantly inhibited by LDN193189, while significantly induced by BMP9 and also induced by BMP2, though not significant. This is consistent with the hypothesis and confirms that these endothelial cells react to BMP stimulation. The samples stimulated by the other ligands showed no significant changes in expression of ID-1, as was expected from the literature.

BMP2, LDN193189 and SB431542 significantly inhibited PAI-1 expression. Interestingly, PAI-1 expression was significantly increased after stimulation with BMP9. TGF-β had no significant effect. The defect in signalling is probably caused by a defect in a part of the TGF-β signalling, be it the receptor, co-receptor or the following R-Smads.

The results of this experiment are not consistent with the hypothesis that TGF-β induces EndoMT. Therefore, the hypothesis must be rejected. However, since n = 1 does not count in biomedical since, this experiment has to be repeated at least two more times before definitive conclusions can be made, maybe also using another endothelial cell line to make sure this one does not have genetic or epigenetic defects.

Endothelial-to-mesenchymal transition in endothelial cells

24

MicroRNAs and the development of pulmonary arterial hypertension Introduction Pulmonary arterial hypertension (PAH) is a rare disease affecting the lung vasculature. It can be subdivided in different categories. Two of the most common categories are idiopathic PAH (IPAH) and hereditary PAH (HPAH). Most of the times, HPAH is caused by a mutation in the BMPR2 gene.

The BMPR2 receptor plays an important role in the TGF-β pathway. For instance, this pathway regulates cell proliferation, differentiation and apoptosis. The pathway starts with a signalling molecule binding to a receptor complex. Next, this complex will initiate a cascade of reactions involving many different molecules. These molecules will change the expression of specific genes. In PAH, the pathway seems to be deregulated. As a result, vascular remodelling and vasodilation occur.

The process resembles endothelial-to-mesenchymal transition (EndoMT). The process can be induced by stimulation with TGF-β. During EndoMT, endothelial cells lose their typical endothelial properties and become more like fibroblasts. This means that they lose the expression of PECAM-1 and their tightly packed phenotype, and that they start to express αSMA and become more spindle-like.

Apart from the mutation in the BMPR2 gene, a secondary genetic factor has been identified.

Certain microRNAs (miRNAs), more specifically miR-24 and miR-155,[45,52] are known to interfere with various molecules needed for signalling in the TGF-β pathway. As a result, EndoMT may or may not occur.

In this research, we will look at the effect of miR-24 and miR-155 on the EndoMT in cells of patients with IPAH and HPAH caused by a mutation in the BMPR2 gene.

Methods Participants For this investigation, endothelial cells from three different donors were used. The first donor was healthy and serves as the control. The second donor suffered from HPAH caused by a mutation in the BMPR2 gene. The third donor suffered from IPAH.

MicroRNAs and the development of pulmonary arterial hypertension

25

Stimulation The samples were stimulated with TGF-β as a positive control and with nothing as a negative control. Furthermore, they were transfected with miR-24 or miR-155 to assess the effect of these microRNAs on the endothelial-mesenchymal transition in the cells.

Luciferase assay A plasmid vector containing the BMPR2 gene and the luciferase gene downstream of the BMPR2 gene was inserted in cells of the HeLa cell line. The cells were treated with miR-24, miR-155 or a combination. An empty vector, only containing the luciferase gene, was inserted to determine the background level of expression. mRNA expression The expression of PECAM-1 and α-SMA mRNA was determined using qPCR. The expression levels of the mRNA were compared to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the household gene.

Statistical analysis The data in the histograms represent the mean and the standard error of the mean. The data was analysed with a two-tailed student’s t-test for unequal variances. Differences were considered significant with a p-value of p < 0.05.

Results A luciferase assay was performed to be sure that miR-24 and miR-155 act on the 3'-UTR of the BMPR2 gene. See Figure 11. Stimulation with miR-24, miR-155 and those two combined showed significant repression of transcription of the BMPR2 gene. miRNAs on 3'-UTR of BMPR2 700000

luciferase activity

600000 500000 400000 300000 200000 100000 0 control

empty

miR-24

miR-155 miR-24 and -155

 Figure 11. Luciferase activity in

endothelial cells. Using plasmid vectors, the luciferase gene was placed downstream of the 3'-UTR of the BMPR2 gene. When the microRNAs interfered with the BMPR2 mRNA, they would also interfere with the mRNA corresponding with the luciferase enzyme. The empty vector only contained the luciferase gene and gives an indication of the background transcription. Data acquired from MJTH Goumans.

MicroRNAs and the development of pulmonary arterial hypertension

26

PECAM-1 expression 9 8

PECAM-1 mRNA

7 6 5 4 3 2 1 0 control HPAH

IPAH control HPAH

IPAH control HPAH

control

TGF-β

miR-24

IPAH control HPAH

IPAH

miR-155

 Figure 12. Relative normalised expression of PECAM-1 mRNA. The mRNA corresponding with the PECAM-1 gene

was measured in endothelial cells after treatment with microRNA. TGF-β served as a positive control. HPAH: patient with HPAH caused by a BMPR2 mutation; IPAH: patient with IPAH. Data acquired from MJTH Goumans.

α-SMA expression 7 6

α-SMA mRNA

5 4 3 2 1 0 control HPAH

IPAH control HPAH

IPAH control HPAH

control

TGF-β

miR-24

IPAH control HPAH

IPAH

miR-155

 Figure 13. Relative normalised expression of α-SMA mRNA. The mRNA corresponding with the

α-SMA gene was measured in endothelial cells after treatment with microRNA. TGF-β served as a positive control. HPAH: patient with HPAH caused by a BMPR2 mutation; IPAH: patient with IPAH. Data acquired from MJTH Goumans.

MicroRNAs and the development of pulmonary arterial hypertension

27

Endothelial cells were isolated from a healthy control, a patient with HPAH caused by a BMPR2 mutation, and a patient with IPAH. The cells were stimulated with TGF-β, miR-24 or miR-155. Using qPCR, the expression of PECAM-1 and α-SMA mRNA was measured. The results are shown in Figures 12 and 13.

The expression of PECAM-1 did not change significantly after stimulation with TGF-β, miR-24 or miR-155. Though it seems that miR-155 inhibits the expression of PECAM-1, p = 0,201.

There was, however, significantly more expression of PECAM-1 in the IPAH cells that were not stimulated. The stimulation with TGF-β, miR-24 and miR-155 all significantly increased the expression of α-SMA. The IPAH cells reacted more to miR-24 and miR-155, while the HPAH cells reacted more to stimulation with TGF-β.

Discussion Research has shown that certain miRNAs regulate the expression of genes that are important in the EndoMT. This process plays a major role in the development of PAH. Two of these miRNAs are miR-24 and miR-155. In this experiment, we looked at the effect of miR-24 and miR-155 on the development of EndoMT in patients with HPAH caused by a BMPR2 mutation and IPAH patients.

The cells of the healthy controls, HPAH patient and IPAH patient were successfully cultured.

The data from the luciferase assay are shown in Figure 11. They show a significant decrease in the BMPR2 mRNA when adding the microRNAs. Both miR-24 and miR-155 show a decrease in the mRNA, but the decrease in the cells stimulated with miR-155 is the biggest. The control cells showed a bigger expression than the cells stimulated with the empty vector. This means that the background expression is less than the BMPR2 expression. The concentration of the mRNA after stimulation with the miRNAs was decreased, meaning that it had a significant effect and was not just based on the background transcription.

The results of the qPCR are shown in Figures 12 and 13. Figure 12 shows there was a slight decrease in PECAM-1 mRNA after stimulation with the miRNAs. This would mean that the cells partially lose their endothelial properties, which is a characteristic of the EndoMT. Interestingly, the concentration of PECAM-1 mRNA was significantly increased in unstimulated cells of the patients with IPAH and HPAH. This could just be an anomaly because we use cells isolated from patients. More samples from different patients (at least two more) should be included to make the obtained results more reliable.

Figure 13 shows the concentration of α-SMA mRNA. TGF-β, miR-24 and miR-155 all significantly increased the α-SMA mRNA concentration. There was no significant difference in

MicroRNAs and the development of pulmonary arterial hypertension

28

α-SMA expression in the unstimulated samples. It is interesting to notice that TGF-β had more effect on the HPAH cells, while the miRNAs had more effect on IPAH cells.

A possible explanation for this could be that the cells with the BMPR2 mutation are already used to the haploinsufficiency. These cells have adapted to cope with the loss of BMPR2 and are therefore less affected. The cells without the BMPR2-mutation in PAH patients are not used to this haploinsufficiency, and therefore directly react to it. Analysing the TGF-β receptor profile might provide an answer for this question.

In brief, the results show that TGF-β, miR-24 and miR-155 stimulate the EndoMT in endothelial cells with PAH. IPAH cells reacted more to the miRNAs than HPAH cells.

The results confirm our hypothesis. TGF-β, miR-24 and miR-155 increased EndoMT in the endothelial cells of PAH patients. However, these results show two stimulating microRNAs: This makes miR-24 and miR-155 possible targets for future therapies. The counterparts or inhibitors of these miRNAs could be working as medicine. Future researches may indicate if this is indeed possible.

MicroRNAs and the development of pulmonary arterial hypertension

29

Conclusion Pulmonary arterial hypertension is a rare disease, affecting the pulmonary arteries. A high blood pressure occurs in the arteries, and so vasoconstriction occurs. A big part of this vasoconstriction is caused by endothelial cells becoming more mesenchymal or fibroblastlike. This change in phenotype is also known as endothelial-to-mesenchymal transition.

This transition can be induced by TGF-β. MicroRNAs are known to be able to interfere with this transition as well. In this research, we looked at the influence of miR-24 and miR-155.

Our hypothesis was that these would also cause EndoMT. Although there were some odd results, most of the results matched the hypothesis.

The research could have been improved in many different ways: the control experiment was conducted in duple. For more accuracy, more cells could have been used. Another thing is the use of glass plates: since they easily fell and broke, more samples could have been used to make it more likely that the experiment succeeded.

The outcomes may be used to develop a better target for medicines. At present, therapy for PAH patients does not fully cure the disease, and is aimed at improving the living conditions and survival term of the patients.

Better understanding of the underlying mechanisms could lead to better medication.

Medicines targeting these two miRNAs may contribute to this, after future research has been done. Further research could also be done to the failed experiment, to find the cause of why the cells didnâ&#x20AC;&#x2122;t react.

Conclusion

30

Acknowledgements We would like to thank our supervisor Marie-JosĂŠ Goumans for her indispensable guidance and critical support through our research. Before, during and after our experiments she helped us understand and explore the subjects in great detail. We would also like to show our appreciation for JosĂŠ Maring, who guided us during the experiments and taught us the basic skills needed for work in a lab. Furthermore, we want to express our gratitude to Annabeth Simonsz, who was a big support for us. She helped us out when needed and helped organise the internship. Not to forget our supervisors at school: thank you for the understanding and assistance concerning deadlines and working pressure. At last, we would like to thank those who contributed to our investigation without our knowledge.

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Protocols The following pages contain the protocols we used for the experiment called ‘Endothelial-tomesenchymal transition in endothelial cells’.

Endothelial cell culture  Remove culture medium  Wash the cells 2x with EDTA or PBS  Remove EDTA or PBS  Add Trypsine (0.25% from Gibco, final concentration we use for culturing is The frozen portions (2 ml) of Trypsin are diluted 5x with EDTA) (T25: 0.5 ml, T75: 1 ml)  Put the cells back in the incubator and wait for a couple of minutes.  Check under the microscope if cells are dissociating (when cells still adhere “tap” against the flask.  When all cells are dissociated add 2.5 ml culture medium (T25) or 4 ml medium (T75) to inactivate the trypsine  Spin 10min 1000 RPM (RT)  Remove the supernatant  Add new culture medium and transfer the cells to the new culture flasks coated with 0.1% gelatine Experiment  Day 1: Split cells and seed in 6-wells plate or 24 wells plate for staining  Day 2: stimulate cells with TGF-β (1 ng/mL) and/or other substances  Day 4: Check cells for change in morphology and if effect, stop experiment - Fix cells for staining - Lyse cells for PCR (RNA analysis)

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Staining Lysis  Take pictures of the cells (6 well plates).  Wash cells 1x with ice-cold PBS  Add 200 μl 1x Lysis buffer + inhibitors (on ICE)  Incubate for 30 min (on ICE)  Scrape cells from plate and pipet them in an eppendorf tube (on ICE)  Spin for 5 min at highest speed (4 C)  Transfer 180 μl of supernatant to a new eppendorf tube (on ICE) Cell staining  Rinse cells 1x PBS  Fix cells with 4% paraformaldehyde for 15 min at room temperature (300 μl/well  Wash cells 2x PBS 5 min on shaker  Permeabilise cells with 0.5% triton in PBS, 10 min at RT (300 μl/well)  Wash cells 3x with PBS  Block with 0.1% Triton, 0.5% BSA in PBS (blocking solution) 1h at RT  Incubate slides with first antibodies: 1:500 anti PECAM + 1:300 anti α-SMA in blocking solution at 4°C overnight in humidified chamber.  Place the slides on a clean 24 well plate in order to rinse them with TBST after the incubation with the first antibody  Wash 3x with TBST for 5 min on shaker  Incubate with secondary antibodies 1:500 (Anti rabbit IgG- Alexa 488 (PECAM) and Anti mouse IgG- Alexa 594 (α-SMA)) for 45 min at RT in the dark!!!, in blocking solution  Wash 3x with TBST for 5 min on shaker  Wash 1x PBS  Mount the slides on microscope slides  Leave drying overnight in the dark at RT  Take pictures using the microscope

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RNA preparation Extraction  Add 1 ml TriPure per 50–100 mg tissue  Homogenise sample in tissue homogeniser  Incubate 5 min at 15 to 25 °C to dissociate nucleoprotein complexes  Add chloroform (0.2 ml per 1 ml TriPure)  Shake vigorously 15 s  Incubate 2–15 min at 15 to 25 °C  Centrifuge 12,000 x g, 15 min at 4 °C  The result is a tube with 3 phases: - aqueous (containing RNA) colourless - interphase (containing DNA) white - organic (containing protein) red Isolation of RNA  Transfer aqueous phase to new tube  Precipitate with isopropanol (0.5 ml per 1 ml TriPure)  Mix by inversion  Incubate 5–10 min at 15 to 25 °C  Centrifuge 12,000 x g, 10 min at 4 °C  Discard supernatant  Wash pellet 1 x with 75% EtOH (1 ml EtOH per 1 ml TriPure)  Centrifuge 7500 x g, 5 min at 4 °C  Discard supernatant  Air dry pellet 30 min  Resuspend in RNase-free water  Incubate 10–15 min at 55–60 °C to resuspend  Store RNA at –15 to –25°C

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cDNA synthesis Digestion of Genomic DNA  Prepare the following mixture: Component Final concentration Total RNA 1 μg 10x Incubation Buffer 1.1 μl DNase I recombinant, RNase-free 0.025 μl RNase-free water up to 11 μl  Incubate at 25 to 37 °C for 15–20 min  Stop the reaction by adding 2 μl of 0.2 M EDTA (pH 8.0) to a final concentration of 8 mM and heating to 75 °C for 10 min. The concentration of EDTA has to be taken into account for all subsequent applications cDNA synthesis  Prepare the following reaction mixture in a tube on ice: Component Final concentration Total RNA 10 ng – 5 μg + or poly(A) RNA 1 ng – 0.5 μg or specific RNA 0.01 pg – 0.5 μg Oligo(dT)18 primer (0.5μg/μl) 1 μl or sequence-specific primer 15–20 pmol DEPC-treated water to 12 μl  Mix gently and spin down for 3–5 sec in a microcentrifuge  Incubate the mixture at 70 °C for 5 min, chill on ice and collect drops by brief centrifugation  Place the tube on ice and add the following components in the indicated order: Component Final concentration 5x reaction buffer 4 μl Ribonuclease Inhibitor (20 u/μl) 1 μl 10 mM dNTP mix 2 μl  Mix gently and collect drops by brief centrifugation  Add RevertAid™ H Minus M-MuLV Component Final concentration Reverse Transcriptase (200u/μl) 1μl (final volume 20μl)  Incubate the mixture at 42 °C for 60 min  Stop the reaction by heating at 70 °C for 10 min. Chill on ice.  The first strand cDNA synthesized can be used directly for amplification by PCR. Protocols

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qPCR  Synthesize cDNA from 1 μg RNA (if possible), treated with DNase  Try to keep amount of RNA constant for all samples, especially if less RNA is available  Dilute the cDNA 20x for use in qPCR - This can be done for the entire batch, or of half the amount of cDNA. Keep in mind that for multiple qPCRs it best to do these from 1 batch of diluted cDNA.  Control PCR (optional) - Perform a control PCR on the cDNA samples and some/all RNA samples to confirm the synthesis of cDNA and the absence of DNA contamination in the RNA samples  Prepare the following mixture (mastermix) (for 1 sample): - 4 μl SYBRGreen - 0.5 μl Forward Primer (10 μM) - 0.5 μl Reverse Primer (10 μM) - 1 μl mQ  Make a pipetting scheme - Samples are always measured in triplicates. Besides your samples, take along a mQ (no template control – NTC) along for every primer pair.  Add 2 μl cDNA (20x diluted) to the wells according to scheme  Add the mastermix to the wells according to scheme  Spin down 2 min at 1000 RPM in plate centrifuge (S2-)  Go to the qPCR machine. Use the following protocol: - 95 °C for 3 min First denaturation - 95 °C for 10 sec Denaturation - 60 °C for 30 sec Annealing and amplification - Repeat step 2+3 39 times - Melt curve: 65 °C to 95 °C; increase 0.5 °C /5 sec, constant read

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