DEMOCRITUS UNIVERSITY OF THRACE SCHOOL OF HEALTH SCIENCIES DEPARTMENT OF MOLECULAR BIOLOGY & GENETICS. Master s Programme of Studies

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1 DEMOCRITUS UNIVERSITY OF THRACE SCHOOL OF HEALTH SCIENCIES DEPARTMENT OF MOLECULAR BIOLOGY & GENETICS Master s Programme of Studies «Translational Research in Molecular Biology and Genetics» Title Neutrophil extracellular traps (NETs) and biomaterials: NETs are involved in the inflammatory response against coronary artery stents by Angelidou Evangelia-Iliana MASTER THESIS June

2 Neutrophil extracellular traps (NETs) and biomaterials: NETs are involved in the inflammatory response against coronary artery stents 2

3 Εσταριστίες. Καηά ηε δηάξθεηα ηεο πξνζπάζεηαο απηήο, ήηαλ πνιύ ζεκαληηθή ε πξνζθνξά ηνπ PhD βηνιόγνπ θ. Κσλζηαληίλνπ Κακπά, ν νπνίνο κε βνήζεζε ζεκαληηθά κε ηηο ηερληθέο ηνπ εξγαζηεξίνπ ζηα πξώηα κνπ βήκαηα. Ο θ. Κακπάο ζπλέβαιε ζεκαληηθά ζην ζρεδηαζκό ησλ πεηξακάησλ θαη ήηαλ πάληα πξόζπκνο λα ζπκβάιιεη ζηε δηεθπεξαίσζε ηπρώλ πξνβιεκάησλ. Δπίζεο, ζα ήζεια λα επραξηζηήζσ ηελ ηαηξό θα. Δηξήλε Απνζηνιίδνπ ηεο νπνίαο ε εκπεηξία ζην θιηληθό κέξνο ησλ κειεηώλ ήηαλ ηδηαίηεξα ρξήζηκε θαη επνηθνδνκεηηθή. Δπίζεο, ζα ήζεια λα αλαθεξζώ ζηνλ θ. Παλαγηώηε θέλδξν, επίθνπξν ηεο Α Παλεπηζηεκηαθήο Παζνινγηθήο Κιηληθήο ηνπ ΓΠΘ, ν νπνίνο ζπλέβαιε ζην θιηληθό κέξνο ησλ κειεηώλ θαη ήηαλ πάληα πξόζπκνο λα αθνύζεη ηνπο πξνβιεκαηηζκνύο κνπ. Ιδηαίηεξα ζεκαληηθή ήηαλ θαη ε βνήζεηα ηεο βηνιόγνπ θαη ηερληθνύ ηνπ εξγαζηεξίνπ θαο Βηθηώξηαο Σζηξνλίδνπ. Αθόκα, ζα ήζεια λα επραξηζηήζσ γηα ηελ βνήζεηα ηνπο θαη ηελ ακέξηζηε ζπκπαξάζηαζε ηνπ ηνλ κεηαπηπρηαθό θνηηεηή Θάλν Αξακπαηδηόγινπ θαη ηνπο ηειεηόθνηηνπο ηνπ ηκήκαηνο Μνξηαθήο Βηνινγίαο: Αιέμαλδξν Μήηζην, Γηώηα Μπνύηε, θαη Αζελά πγθνύλα. Καηά ηε δηάξθεηα ηεο κειέηεο θξίζεθε απαξαίηεηε ε ζπιινγή θιηληθώλ δεηγκάησλ. Με αθνξκή απηό ζα ήζεια λα επραξηζηήζσ ηνλ θαζεγεηή ηνπ ΓΠΘ, θ Γεκήηξην Μηθξνύιε, γηα ηελ παξνρή πλεπκνληθνύ ηζηνύ από ινβεθηνκή, απαξαίηεηνπ γηα ηελ απνκόλσζε αλζξώπηλσλ πλεπκνληθώλ ηλνβιαζηώλ. Χσξίο ηε ρξήζε ηνπ ζπλεζηηαθνύ κηθξνζθνπίνπ, ε παξαηήξεζε ησλ εμσθπηηάξησλ δηθηύσλ ρξσκαηίλεο ζα ήηαλ αδύλαηε. Γηα απηό ην ιόγν, επραξηζηώ ζεξκά ηελ αλαπιεξώηξηα θαζεγήηξηα Κπηηαξηθήο Βηνινγίαο ηνπ ΓΠΘ, θα Μαξία Κόθθα, γηα ηε βνήζεηα ζηε ρξήζε ηνπ ζπλεζηηαθνύ κηθξνζθνπίνπ. Γηα ηελ ηεξάζηηα ππνκνλή θαη εζηθή ηνπ ζπκπαξάζηαζε επραξηζηώ ζεξκά ηνλ ζύδπγό κνπ θαη θπζηθά ηνπο γνλείο καο. Μνπ παξείραλ όια ηα κέζα γηα λα κπνξέζσ λα αληαπεμέιζσ ζηηο αλάγθεο πνπ πξνέθππηαλ. Ιδηαίηεξα ζα ήζεια λα επραξηζηήζσ πνιύ ηνπο δύν κνπ γηνύο, πνπ κε ζηεξήζεθαλ ζε θάπνηα από ηα πξώηα βήκαηα ηεο δσήο ηνπο. Θα ήζεια ηέινο λα αλαθεξζώ ζηελ θαζνξηζηηθή ζπλεηζθνξά ηνπ θαζεγεηή Παζνινγίαο, θαη Γηεπζπληή ηνπ Δξγαζηεξίνπ Μνξηαθήο Αηκαηνινγίαο ηεο Α Παλεπηζηεκηαθήο Παζνινγηθήο Κιηληθήο θ. Κσλζηαληίλνπ Ρίηε, νη γλώζεηο ηνπ νπνίνπ ζηνλ ηνκέα ηεο «ζπλνκηιίαο» κεηαμύ θιεγκνλήο θαη εμσγελνύο ζπζηήκαηνο πήμεο, έλαο 3

4 ηνκέαο ζηνλ νπνίν έρεη εζηηάζεη ηηο έξεπλέο ηνπ ην Δξγαζηήξην Μνξηαθήο Αηκαηνινγίαο ηα ηειεπηαία ρξόληα κε πξσηνπόξεο δεκνζηεύζεηο, βνήζεζαλ θαηαιπηηθά ζηε δηεθπεξαίσζε ηεο κειέηεο. Οη ζπγθεθξηκέλεο κειέηεο δε ζα κπνξνύζαλ λα έξζνπλ ζε πέξαο δίρσο ηελ επίβιεςε θαη ζπκκεηνρή ηνπ. Σέινο ζα ήζεια λα εθθξάζσ ηηο επραξηζηίεο κνπ ζηελ θαζεγήηξηα ηνπ ηκήκαηνο Μνξηαθήο Βηνινγίαο θαη Γελεηηθήο ηνπ Γ.Π.Θ. θα. Μαξνπιάθνπ Ισάλλα, ε νπνία κε ηελ αλάζεζε απηήο ηεο κεηαπηπρηαθήο έξεπλαο κνπ έδσζε ζηελ πξαγκαηηθόηεηα ηελ επθαηξία λα πξνζεγγίζσ από θνληά ηνλ θόζκν ηεο βαζηθήο - κεηαθξαζηηθήο έξεπλαο θαη λα πινπνηήζσ έλα κεγάιν κνπ ζηόρν. Ήηαλ αξσγόο θαη ζεκαληηθή δάζθαινο γηα εκέλα, θαζώο κε βνήζεζε ζηελ αλάπηπμε ώξηκεο επηζηεκνληθήο ζθέςεο θαη ζην ζρεδηαζκό ησλ απαξαίηεησλ βεκάησλ γηα ηελ πξαγκάησζε ηεο επηζηεκνληθή κνπ ππόζεζεο. Σελ επραξηζηώ απεξηόξηζηα θαη ζα απνηειεί πάληα γηα εκέλα έλα παξάδεηγκα εζηθήο θαη επηζηεκνληθήο αθεξαηόηεηαο. 4

5 INDEX A. ABSTRACT...ζει.7 B. INTRODUCTION.9 1. CORONARY ARTERY DISEASE STENTS and RESTENOSIS TYPES OF STENTS, HISTOLOGY, EPIDEMIOLOGY, CLINICAL CHARACTERISTICS, PREDICTORS AND PATHOPHYSIOLOGY OF STENT RESTENOSIS MYOFIBROBLASTS (MFs) GENERAL INFORMATIONS ORIGIN/FUNCTIONS DIFFERENTIATION PATHWAYS ROLE OF MFs IN PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS ROLE OF MFs IN RESTENOSIS FROM WOUND HEALING TO FIBROSIS IL-17 IN WOUND HEALING/RESTENOSIS NETs NET MORPHOLOGY THE MECHANISM OF NET FORMATION NETs & COAGULATION IL-17 & NETs TISSUE FACTOR (TF) IN THROMBOSIS, INFLAMMATION AND FIBROSIS.38 5

6 9. AUTOPHAGY AND mtor...40 C. STUDY RATIONALE 45 D. MATERIALS AND METHODS..46 E. RESULTS..51 F. DISCUSSION 58 REFERENCES..61 6

7 A. ABSTRACT Background: Immediately after percutaneous coronary intervention (PCI) with stent placement, platelets, neutrophils and monocytes play a central role in promoting an inflammatory response in the stented segment of the vascular wall during a process that resembles to wound healing. It is known that neutrophil extracellular traps (NETs) released from neutrophils activated by specific stimuli have been implicated in inflammatory disorders. NETs are extracellular structures composed by chromatin filaments, histones and other cytoplasmic and granule proteins from the netotic neutrophils. NETs are also able to induce differentiation of fibroblasts (FBs) into myofibroblasts (MFs) thus implicated in fibrotic manifestations. It has been demonstrated that restenosis is an excessive wound healing process (fibrosis), where myofibroblasts and excess extracellular matrix (ECM) production play an important role. However, whether stents are able to induce NET release from neutrophils or being able to promote the initiation and propagation of fibrosis through differentiation of FBs to MFs is not known. The aim of this study is to investigate the in vitro effect of coronary artery stents on NET release and further, to FBs differentiation to MFs. Methods: Human lung fibroblasts were cultured in appropriate media. Two different types of stents, bare metal- and drug eluting- stent (BMS, DES respectively) were used to stimulate NET release from control neutrophils. Moreover, NETs were obtained by selective sampling from thrombotic arteries (ACS) of patients with acute myocardial infarction. BMS and DES were used in FBs cultures in order to study FBs differentiation. NETs released from stent-treated neutrophils and infarct-related arteries (IRA)-derived NETs were also added to FBs cultures to promote FBs differentiation. NET generation was assessed with immunofluorescence and MPO/DNA complex ELISA. FBs differentiation was assessed by alpha smooth muscle actin (a-sma) expression in confocal microscopy after appropriate staining. Results: Both stent types (BMS and DES) induced NET generation in control neutrophils. Further analysis revealed that stent-induced NETs were decorated with IL-17, while ACS NETs were decorated with tissue factor. None of stent types had 7

8 any direct effect on FBs differentiation assessed by a-sma expression. On the contrary, ex vivo ACS NETs, BMS- and DES-induced NETs promoted differentiation of FBs to MFs. However, the presence of DES in co-cultures of FBs and different types of NETs abolished FBs differentiation to MFs. Differentiation of FBs was also abolished after dismantling of NETs with DNase prior to FB stimulation from possible components that decorate those NETs. This suggests that the effects of stents in FBs were mediated by NETs. Conclusions: These data suggest that NETs are involved in the inflammation-induced fibrotic response of coronary stents. Interestingly our results indicate that DES stents seem to prevent FBs to MFs differentiation, this phenomenon could be attributed to autophagy induction in FBs. Thus, anti-netosis treatment may represent a potential therapeutic target in human fibrotic diseases such as in stent restenosis. 8

9 B. INTRODUCTION 1. CORONARY ARTERY DISEASE Epidemiology Coronary artery disease (CAD), is the most common type of cardiovascular diseases and despite advances in medicine remains the leading cause of mortality worldwide.[1-5] It affects the coronary arteries by producing narrowing of the vessels that supply the heart (figure 1). Figure 1. Atheroeclerotic plaque producing vessel norowing. CAD occurs when the inner and medial part of the vessel wall undergo thickening because of lipid accumulation and infiltration by inflammatory cells during the process of atherosclerosis (figure 2). Figure 2. Vascular layers: intima, media and adventitia. Elastic laminae represent layer borders Deposits of calcium phosphates, macrophages, neutrophils and smooth muscle cells are also found in atherosclerotic lesions. Acute coronary syndrome refers to the event 9

10 where a rupture of an atherosclerotic plaque occurs with subsequent exposure of the necrotic core to the circulation and thrombosis leading to vessel occlusion (figure 3). Figure 3.Vascular thrombosis after plaque rupture. Definitive diagnosis of CAD requires invasive methods such as coronary angiography. In most cases mechanical revascularization with angioplasty is mandatory to resolve symptoms of angina and/or vessel patency.[6] This initially involves coronary angiography to assess the presence, location, severity and extend of coronary stenosis (figure 4). Figure 4. Coronary angiography demonstrating a stenosis ~90% (Cardiology department of DUTH) 10

11 Percutaneous revascularization is performed by inserting a special catheter in the coronary arteries from the groin of the arm. Through this guiding catheter, the interventional cardiologist opens the vessel first by inflating a dedicated balloon following by stent implantation. However, although stents represented a breakthrough in the treatment of CAD, their use is associated with two severe complications named stent thrombosis and restenosis (figure 5). Figure 5. Coronary stent thrombosis occurs more often with DES (left) while restenosis occurs more often with BMS (right) 2. STENTS and RESTENOSIS The treatment of severe, symptom-producing atherosclerotic stenosis can follow different revascularization strategies, including stent implantation or Bypass surgery.[7, 8] Angioplasty is a safe and effective procedure to unblock stenotic coronary arteries. Initially, angioplasty was performed only with balloon inflation but the results were disappointing because of re-occlusionswithin 3 months. Today technical advances have been made and improved patient outcome has been achieved with the placement of small metallic spring-like devices called stents at the site of the blockage (figure 6). 11

12 Figure 6. Coronary stent The implanted stent serves as a scaffold that keeps the artery open. The first stents used are formed from pure metal (bare metal stents, BMS)[9, 10] which are replaced in the majority of cases with the newer drug-eluting stents (DES-see below on page 11).[11] Restenosis is a >50% reduction in lumen diameter after percutaneous coronary intervention, and is the result of arterial damage with subsequent neointimal tissue proliferation (figure 7).[12-16] Figure 7. Stent restenosis in vascular transection. Black points represent cutted stent struts. Stent thrombosis is an acute thrombotic stent occlusion presenting as an acute myocardial infarction.[17] It is possible that restenotic and thrombotic processes may occasionally coexist in some patients.[18] 3. TYPES OF STENTS, HISTOLOGY, EPIDEMIOLOGY, CLINICAL CHARACTERISTICS, PREDICTORS AND PATHOPHYSIOLOGY OF STENT RESTENOSIS During 1987, percutaneus transluminal angioplasty (PTA) technique with ballon expansion had significantly evolved with the introduction of bare-metal stents (BMS), 12

13 with the aim of obtaining a long-term patency of the stenotic vascular lumen. This evolution offered significant advantages, since ballon angioplasty alone was associated with increased rates of revascularization failure. Although BMS had a good initial result reflected in the first clinical studies assessing their short-term results, the long-term follow-up revealed a relevant percentage of patients that demonstrated instent restenosis, mainly related to inflammatory reaction and to neointima formation inside and through the stent. An initial effort was the improvements in strut configuration, thickness, and materials which have enhanced deliverability and reduced vessel damage (figure 8). Figure 8. The evolution of stent strut thickness and shape over the years. An important change in stent technology was the development of drug-eluting stents (DES), in which anti-proliferative drugs (e.g. paclitaxel, sirolimus, and the more recent zotarolimus and everolimus)[19] have been linked to the stent and gradually released for the prevention of restenosis (figure 9). 13

14 Figure 9. Drug eluting stent (DES) Subsequent studies of DES have demonstrtated impressive results in terms of restenosis reduction. However, the use of DES was accompanied by another longterm relevant side effect with respect to BMS, consisting in a delayed reendothelization after angioplasty, which leads to thrombosis, thus requiring a more effective and prolonged anti-platelet therapy.[20-25] A short history of stent types may be described below: Ballon angioplasty alone (1977) BMS (1987) BMS technical improvement DES (2003) DES technical improvement fully bioabsorbable stents (BVS) (now) Restenosis Histology Over the first 2 weeks after vascular injury, the vascular smooth muscle cells proliferate 3 to 5 times, accounting for 90% of the ultimate intimal proliferation. DES-treated compared to BMS-treated arteries demonstrate reduced smooth muscle cell proliferation, but have more histologic evidence of incomplete reendothelialization, chronic inflammatory cell infiltration, fibrin deposition, and platelet activation (figure 10).[26-29] 14

15 Figure 10. Re-endothelization after 30 days with BMS or DES stents. With BMS re-endothelization is complete after 30 days (upper panel) while with DES uncovered struts are observed in the same period (lower panel) Aside from delayed arterial healing, emerging evidence suggests that compared to BMS, DES impair endothelial function in arterial segments distal to the stented site.[30-33] Despite differences in proliferation, early histopathologic studies revealed that neointimal components after DES implantation were similar to those in BMS lesions in that the neointima was mainly composed of proliferative smooth muscle cells with proteoglycans-rich extracellular matrix.[34-37] However, despite efficacy in reducing neointimal proliferation and restenosis, DES failure and restenosis still occur even with the newer fully bioabsorbable vascular scaffolds (figure 11)[38-41] and is difficult to treat.[43, 44] Figure 11. Bioabsorbable vascular scaffold 15

16 Understanding the molecular mechanisms underlying the physiological healing response and the pathological restenosis response will help to develop novel therapeutic approaches to control the pathological formation of the neointima from one side and promoting endothelial coverage from the other thus eliminating the Achilles heels of current stent technology both restenosis and stent thrombosis. 16

17 Epidemiology The introduction of coronary stents marked a major turning point in the practice of interventional cardiology by reducing restenosis >50%.[45] As a result, by 1998 nearly 70% of coronary interventions performed involved the implantation of a BMS.[46] Today, despite advances in stent and delivery system design, procedural technique, and pharmacologic therapy, the overall rate of restenosis within implanted BMS remains tangible approximately 20% overall with reported rates of greater than 40% in certain, high-risk patient subsets such as diabetes mellitus, STEMI and overlapping multiple stents.[47-53] DES has dramatically reduced the rates of restenosis compared to BMS for ~50%.[54, 55] However, in-stent restenosis after DES still exists, and its prevalence is not negligible because the population treated with DES is large.indeed recent epidemiological studies report that the percentage of target vessel revascularization after DES is 11.8%,[56, 57] and there is an urge need for effective treatments to reduce restenosis rates. Course and clinical characteristics of in-stent restenosis Clinical studies demonstrate that peak restenosis rates are observed at 3 months after BMS implantation and remain relatively stable beyond 6 months.[58] In contrast, the polymeric coating on DES allowing for sustained release of embedded antiinflammatory and antiproliferative agents, delays the completion of the vessel reparative process by months to years.[59] Although some cases of in stent restenosis are clinically silent, the majority of restenosis lead to recurrent symptoms or an acute event.[60-69] The presentation of DES restenosis is similar to that of BMS.The time frame to restenosis after DES may indeed be longer than that after BMS because antiproliferative drugs can delay the biologic response to injury. Criteria usually used for definition of in stent restenosis are presented in Table1. 17

18 Table 1. Criterial for Clinical Restenosis as Requirement for Ischemia-Driven Repeat Revascularization 1. Diameter stenosis <50% and one of the following: Positive history of recurrent angina pectoris, presumably related to target vessel Objective signs of ischemia at rest (ECG changes) or during exercise test (or equivalent), presumably related to target vessel Abnormal results of any invasive functional diagnostic test (e.g., coronary flow velocity reserve, FFR <0.80); IVUS minimum cross-sectional area <4 mm^2 (and <6.0 mm2 for left main stem) has been found to correlate with abnormal FFR and need for subsequent TLR (5 7) TLR with diameter stenosis >70% even in absence of the above ischemic signs or symptoms ECG: electrocardiogram; FFR: fractional flow reserve; IVUS: intravascular ultrasound; TLR: target lesion revascularization Predictors of Stent Restenosis The widespread application of coronary stents (BMS and DES) resulted in an enhanced understanding and awareness of various factors that may increase the risk of clinical and angiographic restenosis.[70-72] Factors predictive of stent restenosis fall into three major categories: patient predictors, vessel predictors, and procedural predictors. Minimizing such high-risk features carries the potential to significantly reduce the incidence of restenosis following stent placement. Of all patient-related predictors identified, diabetes mellitus has consistently been shown to be a high-risk 18

19 clinical predictor of in-stent restenosis (ISR), amplifying the risk of developing restenosis by 30 50% following BMS implantation (figure 12) Figure 12. Patient risk stratification for stent induced complications. MACE: Major Adverse Cardiovascular Events Other predictive factors for DES restenosis include complex lesions (B2/C types of atherosclerotic plaques), small vessels, the reference-vessel diameter, longer stents, and stent underexpansion (Table 2).[73-75] Table 2. Predictors of ISR after DES implantation 19

20 Restenosis pathophysiology When a stent is placed in a blood vessel produces a vessel wall injury and a healing response initiates. During this healing process new tissue grows inside the stent, covering the struts of the stent. Initially, this new tissue consists of healthy cells from the endothelium (neointima). This is a favorable effect because development of normal lining over the stent allows blood to flow smoothly over the stented area without clotting since it is known that metallic struts activate platelets and trigger blood coagulation (figure 13). Figure 13. Vascular transection. Normal stent coverage Later, a scar tissue may form underneath the new healthy lining. In about 25% of patients, the growth of scar tissue underneath the lining of the artery may be so thick that it can obstruct the blood flow and produce an important blockage (excess healing response or stent restenosis (figure 14). Figure 14. Vascular transaction. Stent restenosis from excessive neointimal formation In-stent restenosis is typically seen 3 to 6 months after the procedure but it may be also occur in later phases.[76] 20

21 Animal models and human postmortem studies have shown that the mechanism of intimal hyperplasia is similar to wound healing.[77] The temporal wound-healing process following stent placement can be divided into 4 phases: 1) Hemostasis 2) Inflammatory phase (hours to days) 3) Granulation or cellular proliferation phase (days to weeks), and 4) Extracellular matrix remodeling phase (months). The arterial wall s healing response to mechanical injury comprises some key cell types: platelets, inflammatory cells, vascular smooth muscle cells. The major milestones in the temporal sequence of restenosis are platelet aggregation, inflammatory cell infiltration, release of growth factors, medial VSMCs modulation and proliferation, proteoglycan deposition and extracellular matrix remodeling (figure 15). [78] Figure 15. Four stages of normal wound healing The specific expression of these phases in the coronary artery leads to intimal hyperplasia at 1 to 4 months. 21

22 Similar changes are observed in the media of the vessel wall and in the adventitia with increase matrix deposition. Vascular remodeling represents a spatial reorganization of elements of the vascular wall that can be compensative, leading to vessel lumen enlargement, or constrictive, leading to lumen narrowing. Cellular mediators of restenosis Vascular inflammation following percutaneous coronary intervention involves complex interactions between multiple vascular cell types and under normal circumstances, the cellular and molecular processes that control vascular injury responses move towards repair and vascular healing. In pathological conditions, disregulation of vascular repair results in persistent vascular inflammation, neointimal proliferation, and restenotic obstruction of the stent lumen. The initial consequences immediately after stent placement are deendothelialization, crush of the plaque (often with dissection into the tunica media and occasionally adventitia), and stretch of the entire artery. It has been shown that immediately following PCI, platelets, neutrophils and monocytes play a central role in the initial inflammatory response (figure 16). [79] Figure 16. The initial inflammatory response. At the site of vascular injury (i.e. stent implantation following PCI,) endothelial cells are denuded and the subendothelial matrix is exposed to flowing blood. Platelets (green) immediately adhere the surface of the injured vessel. A multistep cascade of 22

23 platelet and leukocyte adhesion molecules direct leukocyte adhesion to the adherent platelets. Leukocyte capture and rolling are mediated by interaction between platelet P-selectin and leukocyte PSGL-1. Arrest and firm adhesion are mediated by platelet glycoprotien Ibα and leukocyte Mac-1. Chemokines stimulate transmigration into the extraluminal tissue to begin repair process. Next is a granulation or cellular proliferation phase. Over the first 2 weeks after injury growth factors are subsequently released from platelets, leukocytes, and vascular smooth muscle cells, which stimulate migration of smooth muscle cells from the media into the neointima. The vascular smooth muscle cells multiply 3 to 5 times, accounting for 90% of the ultimate intimal proliferation production.[80,81] The normally quiescent smooth muscle cells within the medial layer of the vessel wall are activated to migrate and proliferate in response to increased stimulatory growth factors and cytokines (PDGF, interleukin-1, interleukin-6, and tumor necrosis factorα.[82,83] Over longer time periods, the artery enters a phase of remodeling involving extracellular matrix (ECM) protein degradation (possibly through matrix metalloproteinases, MMPs) and resynthesis. Accompanying this phase is a shift to fewer cellular elements and greater production of extracellular matrix, composed of various collagen subtypes and proteoglycans.[84, 85] Thus, the neointima is initially consists of cellular components (vascular smooth muscle cells) and at later phases of excessive matrix elements (i.e. collagen produced by myofibroblasts). Drug eluting stents are coated with antiproliferative agents such as sirolimus, paclitaxel, zotarolimus and everolimus which possess potent anti-mitotic actions that strongly inhibit smooth muscle proliferation and matrix production[86-88] and thus reduce neointimal formation and restenosis. However, the antiproliferative agents used to prevent smooth muscle cell proliferation also delay reendothelialization and promote a sustained inflammation in the stented segment.[89, 90] DES-treated compared to BMS-treated arteries have more histologic evidence of incomplete reendothelialization, chronic inflammatory cell infiltration, fibrin deposition, and platelet activation. There is emerging evidence suggesting that chronic inflammation and/or incompetent endothelial may be an important mechanism of the late phase in stent-restenosis or thrombosis. An integrated view of the molecular and cellular events of in-stent restenosis has been proposed by Welt and Rogers (Figure 17).[91] 23

24 Figure 17. Schematic of an integrated cascade of restenosis. A, Atherosclerotic vessel before intervention. B, Immediate result of stent placement with endothelial denudation and platelet/fibrinogen deposition. C and D, Leukocyte recruitment, infiltration and SMC proliferation and migration in the days after injury. E, Neointimal thickening in the weeks after injury, with continued SMC proliferation and monocyte recruitment. F, Long-term (weeks to months) change from a predominantly cellular to a less cellular and more ECM-rich plaque. 4. MYOFIBROBLASTS 4.1. GENERAL CHARACTERISTICS Myofibroblasts (MFs) are mesenchymal cells demonstrating characteristics of both fibroblasts (FB) and VSMCs. Due to this double facing nature they are named MFs. They have fusiform shape and dentritic offshoots (figure18). Figure 18. Fibroblast and myofibroblast 24

25 The three elements that characterize MFs are i) the stress fibers (actin microfilaments) ii) the gap junctions θαη adherens junctions and iii) the fibronexus. These elements contribute in the synchronized contraction of MFs and extracellular matrix remodeling [94, 95]. Immunohistochemically, these cells express alpha-actin of the smooth mucle cells (alpha smooth muscle actin, a-sma, figure 19), vimentin and desmin.[95, 97] Figure 19. Expression of a-sma from myofibroblasts. Confocal microscopy 4.2. ORIGIN and FUNCTIONS Myofibroblasts (MFs), also defined as mesenchyme-like interstitial cells, are specialized cells originating in pathophysiological conditions and contributing to tissue repair during wound healing. They are responsible for granulation tissue contraction and soft tissue retractions. Their main activities are the production and modification of the extracellular matrix (ECM), the secretion of angiogenic and proinflammatory factors, and the generation of tensile force. MFs can derive from resident cells or from circulating bone marrow-derived precursors (figure 20).[98-25

26 103] Figure 20. Myofibroblasts origin. Myofibroblasts can originate from different cell types. Interestingly, activated MFs and smooth muscle cells share a number of markers including a-sma, SM22, caldesmon, vinculin and h-calponin[104, 105] desmin[ ] and myosin.[ ] 4.3. MF DIFFERENTIATION PATHWAYS Differentiation of FBs to MFs normally occurs during the wound healing process, under concomitant mechanical and biochemical signals. Three major ultrastructural features discriminate MFs from quiescent fibroblasts in tissues: (i) bundles of contractile microfilaments; (ii) extensive cell-to-matrix attachment sites, and (iii) intercellular adherens and gap junctions.[114] In this context, a-sma acts as a mechanosensitive protein that is recruited to stress fibres under high tension. The external stimuli and agents that are able to trigger the differentiation of MFs can be quite heterogeneous. Tissue injury, causing a loss of interaction between cells and ECM, can stimulate the formation of MFs aimed at a rapid wound healing. The Transforming Growth Factor beta-1 (TGF-beta-1) Transforming Growth Factor beta-1 (TGF-beta-1) is a cytokine with cytostatic and pro-apoptotic effects on most target cells. TGF-beta1 plays an important role in tissue 26

27 fibrosis and extracellular matrix remodelling, as it induces FB to MF differentiation through the induction of a-sma expression by binding to its receptors.[ ] A number of molecules induce the differentiation of local FBs to MFs,[126, 127] such as thrombin[128], endothelin-1(et-1),[129,130] platelet-derived growth factor, (PDGF) connective tissue growth factor (CTGF/CCN2) and angiotensin II (Ang II).[131, 132] 4.4. ROLE OF MFs IN PHYSIOLGICAL AND PATHOLOGICAL CONDITIONS MFs are first described in early 70s during wound healing mechanisms and thereafter they are considerate as crucial cells in the wound healing process. These wound healing-associated cells are in fact activated FBs expressing a-sma protein. Activated FBs migrate in the wound area to synthesize and secrete collagen in for the subsequent extracellular matrix remodeling. After remodeling was accomplished, these cells disapear probaply as a result of apoptosis (figure 21). Figure 21. Normal wound healing process Other physiological roles include embryogenesis [133], milk secretion, haemopoiesis and endometrial regeneration, and regulation of blood flow.[134] In pathological conditions, MFs are involved in tumor growth and in fibrotic manifestations such as pulmonary fibrosis,[135] pulmonary hypertension, asthma[136] scleroderma, pancreatic fibrosis[137] hepatic cirrhosis[138] renal fibrosis in diabetes and in intimal hyperplasia in in-stent restenosis.[139] The difference between normal tissue repair and fibrosis is at least in part due to defective clearance of MFs from the injured area (defective apoptosis), thus leading to sustained and exaggerated repair mechanisms.[140] 27

28 4.5. ROLE OF MFs IN- STENT RESTENOSIS The presence and the role of MFs in vascular (re)stenosis has been assessed in experimental animal models of revascularization, as well as in samples retrieved from patients during re-operation procedures. The large majority of studies highlighted the contribution of MFs to the neointima induced by vascular injury. Early studies reporting the presence and the role of MFs in animal models of vascular stenosis or in patients occasionally described these cells as activated mesenchymal cells or smooth muscle-like cells.[ ] In vitro studies revealed their constrictive potential together with their synthetic activity.[ ] The mechanisms at the basis of MF migration through the injured vascular wall involve a chemotactic recognition of MF receptors (i.e. VEGF-r, AT1-r) by specific chemokines and the synthesis of MMPs, in particular of MMP-1, but not of MMP-2.[ ] MFs can also migrate from the adventitia.[ ] Other migration signals include G-CSF, a potent hematopoietic cytokine produced by endothelium and immune cells, and is expressed at sites of vascular injury,[161,162] MMP-9,[163] stromal cell-derived factor-1 (SDF- 1).[163,164] 5. FROM WOUND HEALING TO FIBROSIS The human body can sustain a variety of injuries, including penetrating trauma, burn trauma and blunt trauma. All of these insults set into motion an orderly sequence of events that are involved in the healing response, characterized by the movement of specialized cells into the wound site. Fibrosis resembles greatly wound healing mechanism with the specific distinction that ultimately does not restore the dynamic balance between production and degradation ofextracellular matrix.[ ] The myofibroblasts remain active, collagen production continues, resulting in the destruction of the structure. Thus, fibrosis could be described as a process of uncontrolled healing (figure 22). 28

29 Figure 22. Normal healing and uncontrolled healing (Fibrosis). Normal healing leads to tissue restoration and normalization of O 2 delivery despite initial decrease tissue perfusion (left). Fibrosis and scaring leads to tissue dysfunction and continuous O 2 deficiency (right). As discussed earlier, acute wounds normally heal in a very orderly and efficient manner characterized by four distinct, but overlapping phases (healing cascade, figure): hemostasis (driven by platelets) inflammation (driven by immune cells), proliferation (MFs, vascular smooth muscle cells, epithelial cells, and endothelial cells) and remodeling (extracellular matrix/collagen). If too much collagen is deposited in the wound site, normal anatomical structure is lost, function is compromised and fibrosis occurs. Conversely, if an insufficient amount of collagen is deposited, the wound is weak and may dehisce (ulceration), (figure 23). 29

30 Figure 23. The healing cascade 6. IL-17 IN WOUND HEALING/RESTENOSIS IL-17A plays a critical role in neutrophil recruitment, angiogenesis, inflammation, and autoimmune disease including pulmonary and cardiac fibrosis via IL-17RA and MAPK signaling.[170] IL-17A exhibits the potential to recruit neutrophils and induces the expression of proinflammatory cytokines and chemokines (figure 24).[171, 172] 30

31 Figure 24. The central role of IL-17 in response to injury. IL-17 is produced by neutrophils through neutrophil extracellular trap (NET) release (see below), mast cell extracellular trap release and other IL-17 producing cells. A role for IL-17 in myocardial ischemic injury, hypertrophy, and remodeling has been suggested since cytokine IL-17 is a potent inducer of MMP-1 expression in primary human cardiac fibroblasts and stimulates MMP-1 expression independently of IL- 1β, IL-6, and TNF-α. More importantly, IL-17 induces human cardiac fibroblasts migration in an MMP-1-dependent manner. Since MMPs degrade ECM and facilitate migration, IL-17 may be potentially important in myocardial injury, remodeling, and failure although a specific role has not been found yet. Furthermore, IL-17 exhibits a remarkable capacity to modulate the immune response by cooperating with other cytokines to promote inflammation.[173,174] 31

32 7. NEUTROPHIL EXTRACELLULAR TRAPS (NETs) Neutrophils are terminally differentiated white blood cells that have a short life in circulation. If called into action, neutrophils leave the blood vessels and move toward the site of infection, following a chemotactic gradient produced by microbial or endogenous signals. At the inflammatory site, neutrophils are activated to perform several tasks, including cytokine secretion, degranulation, and phagocytosis. This process is of paramount importance in immunology. Neutrophils have two distinctive morphological characteristics: the shape of their nucleus and their granules. The nucleus of neutrophils is split into three to five lobules, hence the alternative name of polymorphonuclear often given to these cells (figure 25). Figure 25.Polymorphonuclear neutrophil (Grunwald-Giemsa staining) The evolutionary advantages of having a lobulated nucleus are not clear. Granules are specialized vesicles that contain a specific load, including many toxic molecules. Depending on their contents, granules are canonically classified into four groups: primary or azurophilic, secondary or specific, and tertiary or gelatinase, as well as secretory vesicles. Eosinophils, basophils, and mast cells also have granules, and together with neutrophils they make up the granulocyte family. Neutrophils are efficient phagocytes and engulf microbes into phagosomes that rapidly fuse with the granules, creating an inhospitable environment. There, microbes are exposed to many enzymes, including lysozyme, which breaks the bacterial wall; proteases; and phospholipases. Also, very cationic peptides, like bactericidal permeability increasing protein (BPI), defensins, and cathelicidins, are discharged into the phagolysosome. Simultaneously, reactive oxygen species (ROS), like superoxide and hydrogen peroxide, are generated by the NADPH oxidase complex at the phagosomal 32

33 membrane and released into its lumen. The biological activity of many of these components under defined in vitro conditions has been demonstrated numerous times but the relative contribution of each of them to neutrophil function in vivo remains to be determined. Neutrophils can also kill pathogens extracellularly by releasing neutrophil extracellular traps.[175] The impact of NETs derives from the combined antimicrobial activities of granular components, histones, and some cytoplasmic proteins. Eosinophils and mast cells, which are granulocytes closely related to neutrophils, granulocyte homologues in lower vertebrates, and even plants release extracellular traps. Hence, in addition to describing the function of NETs, we will also comment on the significance of extracellular traps in evolution NET MORPHOLOGY The ultrastructure of NETs is unusual; NETs consist of smooth filaments with a diameter of 17 nm composed of stacked, and probably modified, nucleosomes. [176] This backbone is studded with globular domains with a diameter of 50 nm made of granular proteins. This morphology in high-resolution scanning electron microscopy easily differentiates NETs from other fibrous structures such as fibrin. Interestingly, unfixed, fully hydrated NETs have a cloud-like appearance and occupy a space that is fold bigger than the volume of the cells they originate from, reflecting what they may look like in vivo when space is available, for example in the lung alveolus THE MECHANISM OF NET FORMATION NETs are the results of a unique form of cell death that morphologically is characterized by the loss of intracellular membranes before the integrity of the plasma membrane is compromised coined the term NETosis for neutrophil cell death that leads to the formation of NETs. To release NETs, activated neutrophils undergo dramatic morphological changes (figure 26). 33

34 Figure 26. Mechanism of NET release Minutes after activation, they flatten and firmly attach to the substratum. During the next hour, the nucleus loses its lobules, the chromatin decondenses, and the inner and outer nuclear membranes progressively detach from each other. Concomitantly, the granules disintegrate. After 1 h, the nuclear envelope disaggregates into vesicles and the nucleoplasm and cytoplasm form a homogenous mass. Finally, the cells round up and seem to contract until the cell membrane ruptures and the interior of the cell is ejected into the extracellular space, forming NETs.[177] Notably, despite the intermixing of cellular compartments, during the last phase of NETosis various proteins are present in NETs. Most of them originate from granules, few are from the nucleus, and cytoplasmic NET components are rare. NETosis is morphologically quite different from apoptosis and other forms of cell death. Other investigators have proposed alternative processes to the one just described that will, because of space limitations, not be discussed further. For example, one interesting observation is that NETs can result from the release of nuclear fragments and then their chromatin without compromising the plasma membrane. Importantly, however, granulocytes are particularly poor in mitochondria, and mitochondrial DNA is 100,000 times less abundant in extracellular traps than nuclear DNA; hence, the significance of this finding awaits further investigation. Many physiological inducers of NETosis have been reported. Infections due to various pathogens such as different bacteria, fungi, and viruses induce NETs. Molecularly, the few events that have been shown to be required, sequentially, are the production of ROS, the migration of the protease neutrophil elastase (NE) and later myeloperoxidase (MPO) from granules to the nucleus, the hypercitrulination of histones, and eventually the rupture of the cell. It is relevant to mention that the study of neutrophils is limited by the short life of these cells and the lack of 34

35 established cell lines that faithfully reproduce granulocyte biology, which rules out many conventional molecular approaches. In this section, we review our current knowledge about the mechanism of NET formation. NET formation requires the production of ROS. The NADPH oxidase enzyme complex (also called phagocytic oxidase; PHOX) assembles at the cell and phagosomal membrane and reduces molecular oxygen into superoxide anions by transferring electrons from NADPH. Superoxide dismutates into hydrogen peroxide, which in turn acts as substrate for one of the most abundant enzymes in the neutrophil s granules: MPO. MPO reacts with hydrogen peroxide to generate hypohalous acids, such as hypochlorous acid (HOCl). ROS oxidize various types of molecules including nucleic acids, lipids, and proteins. The most potent inducer of PHOX activation is PMA, which directly stimulates PKC. During NETosis, the segregation between eu- and heterochromatin is lost, and the nucleoplasm appears homogenous. This depends on the activity of NE and MPO, which are stored in azurophilic granules. NE is released, by an unknown mechanism, from granules and enters the nucleus, where it degrades the linker histone H1 and processes core histones.[178] NE activity is essential for NET formation because NEdeficient mice do not make NETs, which contributes to their immune deficiency. MPO also migrates to the nucleus later than NE, where it enhances chromatin decondensation. In agreement with this requirement, patients without MPO activity cannot produce NETs, and hypochlorous acid, the product of MPO, is sufficient for NET release. In addition to partial degradation by NE, histones undergo further modifications to decondense the chromatin structure. Upon neutrophil activation, the enzyme peptidylarginine deiminase 4 (PAD4) catalyzes the conversion of arginine residues to citrulline in three of the four core histones. In NETs and decondensed nuclei, but not in the nucleus of unstimulated neutrophils, histones are citrullinated. The relevance of PAD4 was tested pharmacologically in cell lines, which make few NETs, if any, but not in neutrophils. In PAD4-null mice, hypercitrullination of H3 was not detectable, and the strain failed to produce NETs.[179] Interestingly, in a S. pyogenes infection model, PAD4-null mice developed larger lesions than their PAD4- expressing siblings, but NET formation remains to be quantified in this model. Eventually, NETs are removed during the resolution of inflammation. NETs are susceptible to DNase1[180], an enzyme produced by the pancreas. It is not known 35

36 what happens to the debris left by DNase1 but perhaps phagocytes, macrophages, and neutrophils newly recruited to the inflammatory site clean up the mess. Colectively, NETs are extracellular fibrous structures composed of decondensed chromatin and lined with granule constituents of neutrophils, such as MPO and NE. During the last decade the release of NETs (NETosis) has been described as a novel antimicrobial mechanism in many infectious diseases. In addition, NETosis is considered as a new cell death modality distinguished from apoptosis and necrosis. Apart from infections a pathogenic role for NETs has been proposed recently in several neutrophil-mediated disorders, including thrombosis. Although molecular pathways which underline the formation of NETs are not clearly defined, peptidylarginine deiminase (PAD-4)-mediated hypercitrullination of histones, inflammatory stimuli (various cytokines, ROS), granular enzymes (MPO, NE) and induction of autophagy have been associated with the triggering of NETosis in sterile inflammation. On the other hand, NETs degradation by DNase I and phagocytic removal of NETs by macrophages, represent regulatory anti-inflammatory mechanisms aiming to balance excessive NETosis and restrict tissue injury. Methods to quantify NETs NETs are rather fragile structures, and some effort is required to unambiguously detect and quantify them. NET quantification should rely on their unique composition: chromatin tightly linked to neutrophil proteins such as NE, MPO, or calgranulin. This definition excludes chromatin released by other forms of cell death. Published methods of NET quantification include microscopy and DNA detection either with membrane-impermeable DNA dyes or by staining the DNA in the supernatant after releasing the NETs with a mild nuclease treatment. Immunostaining is an obvious way to detect NETs[175] but is prone to biases introduced by the observer. Automatic microscopy[180] is an objective and quantitative method to measure NET formation. Changes in nuclear morphology (loss of lobules and expansion of the nucleus) and composition (migration of NE and MPO to the nucleus(mpo/dna complex ELISA)) are specific and quantitative markers of the progress of NETosis. Anti-chromatin antibodies stain the compact nuclei of unstimulated neutrophils weakly, but the signal increases as the chromatin relaxes. In tissue sections and in secretions, NETs have been identified using the same markers 36

37 mentioned here. Computer-assisted analysis of the overlap between chromatin and neutrophil markers can quantify NETs in tissue sections. Although more technically challenging, NETs can also be identified in vitro and in vivo by measuring their size and detecting their antigens by scanning or transmission electron microscopy (figure 27). Figure 27. NET Visualization. NETs identified as chromatin and DNA filamens decorated with neutrophil elastase 7.3. NETS AND COAGULATION. Coagulation is a way to reduce blood loss after injury, but it also represents a primitive innate immune response that limits microbial spreading. Coagulation is an example of how the amount of NET formation can determine a good or bad outcome. NETs participate in timely clot formation, but if present in excess they induce massive coagulation that can stop the blood supply of organs, causing severe ischemia. Arterial blood clots are often induced by damage to the endothelium. In contrast, venous thrombi mainly develop when the blood flow is reduced for several hours. In both situations, neutrophils accumulate and adhere tightly to the endothelium. There, neutrophils produce NETs that serve as a scaffold for the stimulation of thrombus formation. Both NE and cathepsin G, two serine proteases that are expressed in the NETs, degrade inhibitors of coagulation. In mice deficient in both enzymes, during arterial thrombosis, fibrin deposition and clot formation are reduced, as is the case when the mice are treated with an anti-net antibody. 37

38 Interestingly, in experimental E.coli systemic infection, the proportion of bacteria sequestered in the microvasculature of the liver was higher in animals with functional NETs than in animals treated with an anti-chromatin antibody that blocks NET function, underlining the fact that coagulation also reduces bacterial spread to other organs. Together, these data indicate that clotting is enhanced by NETs, promoting defense against pathogens. Although the vessel is not damaged at the onset of venous thrombogenesis, numerous neutrophils and macrophages are recruited and play a major role during formation of the thrombus. There, activated platelets stimulate neutrophils to form NETs,[181] which serve as a prothrombotic scaffold and bind and activate FXII.[182] Consequently, NETs can be detected in venous thrombi.[183] 7.4. IL-17 and NETs IL-17 has been detected on NETs which have been release in response to different fibrotic stimuli or to the generic stimulus, PMA. NETs bearing IL-17 promote differentiation and collagen production form FBs. On the contrary, administration of pure recombinant IL-17 had no effect on FB function. IL-17 inhibition abolished NET-induced FB differentiation and CCN2 production from FBs.[184] This may imply that the histone/dna complex regulates FB differentiation while IL-17 is associated with fibrotic manifestations of FBs. 8. TF IN THROMBOSIS, INFLAMMATION AND FIBROSIS TF is a 47kDa transmembrane glycoprotein that shares high homology in secondary and tertiary structure with interferon γ receptors and is a member of the human classii cytokine receptor family. Currently, TF is considered as the main in vivo initiator of coagulation. The presence of a multitude of binding sites in the gene s promoter region indicates multi-potency of expression in a large variety of cells and under a vast array of stimuli. Under normal conditions TF is not expressed in endothelial cells, but only in sub-endothelial tissue, thus creating a protecting envelope between blood and sites of expression. However, under specific inflammatory conditions TF is expressed in endothelial cells and myeloid leukocytes,[185] There is emerging evidence indicating the presence of circulating TF in blood (blood-borne TF).One of the potential source of blood-borne TF are the peripheral blood cells. Although 38

39 monocytes have been reported to constitutively express TF and while this cell population is considered the main source of TF-bearing microparticles (MPs), there are emerging evidence indicating the possible implication of other cell populations in the generation of blood-bornetf. Nevertheless, apart from the role of the extrinsic coagulation system (TF-thrombin axis, figure 28) in thrombosis, this system has been implicated in several non-thrombotic models such as angiogenesis, tumor growth and metastasis, inflammation, and fibrosis. Figure 28. TF - Thrombin Axis Molecular mechanisms and the PARs The serine proteases of this pathway, namely TF/VIIa, Xa, and thrombin, are able to signal through the protease activated receptor (PAR) receptor family to produce intracellular signals [186] via phosphoinositide3-kinase (PI3K), Src tyrosine kinase, extracellular signal-regulated kinase (ERK), and mitogen-activated protein kinase (MAPK) pathways. The activation of these pathways results in the secretion of cytokines and chemokines implicated in several biological functions. Increased prevalence of venous thrombotic events is a long standing observation in patients suffering from infectious and sterile inflammatory disorders. Venous thrombosis constitutes a major morbidity and mortality factor in inflammatory diseases, including sepsis, systemic lupus erythematosus (SLE), inflammatory bowel disease, or vasculitis. Additionally, recent clinical data derived from patients with rheumatoid arthritis and SLE support the critical role of inflammation in accelerated atherothrombosis. 39

40 Experimental evidence links the observed thrombogenicity with TF-dependent activation of extrinsic coagulation cascade. Increased TF expression by endothelial and blood cells exposed to inflammatory mediators is proposed as an essential part of the pathogenic mechanism for arterial and venous thromboembolism that characterizes inflammatory disorders. These observations indicate a potential triggering role of inflammation in thrombosis. However, the relationship between inflammation and thrombosis is bidirectional since thrombosis can reignite inflammation creating a persistent or recurent inflammatory environment. TF-thrombin axis enhances the inflammatory response in several clinical models such as arthritis, antiphospholipid syndrome (APS), ischemia/reperfusion injury, and sepsis. Signaling through PARs plays a critical role for this reciprocal process. TF: FVIIa complex has been implicated in the induction of inflammation in the aforementioned clinical models. In an endotoxemic animal model, both TF deficiency and combined inhibition of thrombin and deficiency in PAR2 reduced inflammation. Further studies in animal models of sepsis demonstrated that extrinsic coagulation cascade inhibition with a varying range of anticoagulants [natural anticoagulants, Tissue Factor Pathway Inhibitor (TFPI), Protein C, and Antithrombin III] attenuated the persisting inflammation. Moreover, it has been recently shown that thrombin is able to generate biologically active C5a from C5 in the absence of C3, indicating a significant role in the reignition of inflammation. However, the physiological contribution of this pathway has to be further investigated. This data establish the reciprocal and close relationship between the thrombosis and inflammation. 9. AUTOPHAGY AND mtor Autophagy The term autophagy, derived from the Greek meaning eating of self, was first coined by Christian de Duve over 40 years ago, and was largely based on the observed degradation of mitochondria and other intra-cellular structures within lysosomes of rat liver perfused with the pancreatic hormone, glucagon.[187] Autophagy is a conserved pathway that maintains cellular homeostasis in response to 40

41 cellular stress. The main function of autophagy is to clear unfolded or aggregated proteins and organelles. During autophagy, cytosolic constituents are enclosed in a double-membrane vesicle, called autophagosome. The fusion of the early autophagosome with lysosomes is the terminal event of this process. LC3, a mammalian homolog of yeast Atg8, is the best-characterized autophagic marker. LC3BI has a diffuse distribution in the cytoplasm. When autophagy is induced, LC3BI is lipidated to form LC3BII, which integrates in the autophagic vacuole membrane. This conversion (LC3B-I to LC3B-II) is indicative of autophagic activity, whereas immunoblotting and fluorescence microscopy are essential tools for this detection. Mammalian target of rapamycin (mtor) is a serine-threonine kinase involved in multiple cellular processes and regulates autophagy negatively. The signaling of phosphatidylinositol 3-kinases (PI3K) participates in the initiation of autophagy, while PI3K inhibition by 3-methyladenine (3-MA), wortmannin and LY seems to inhibit this pathway. In recent years the scientific world has rediscovered autophagy, with major contributions to our molecular understanding and appreciation of the physiological significance of this process coming from numerous laboratories. Although the importance of autophagy is well recognized in mammalian systems, many of the mechanistic breakthroughs in delineating how autophagy is regulated and executed at the molecular level have been made in yeast of cytosolic components at the lysosome. Macro-autophagy delivers cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle, referred to as an autophagosome that fuses with the lysosome to form an autolysosome. In micro-autophagy, by contrast, cytosolic components are directly taken up by the lysosome itself through invagination of the lysosomal membrane. Both macro-and micro-autophagy are able to engulf large structures through both selective and non-selective mechanisms. In chaperone-mediated autophagy (CMA), targeted proteins are translocated across the lysosomal membrane in a complex with chaperone proteins (such as Hsc-70) that are recognized by the lysosomal membrane receptor lysosomal-associated membrane protein 2A (LAMP-2A), resulting in their unfolding and degradation. Autophagy begins with an isolation membrane, also known as a phagophore that is likely derived from lipid bilayer contributed by the endoplasmic reticulum (ER) 41

42 and/or the trans-golgi and endosomes (figure 29) [188], although the exact origin of the phagophore in mammalian cells is controversial. This phagophore expands to engulf intra-cellular cargo, such as protein aggregates, organelles and ribosomes, thereby sequestering the cargo in a double-membraned autophagosome. The loaded autophagosome matures through fusion with the lysosome, promoting the degradation of autophagosomal contents by lysosomal acid proteases. Lysosomal permeases and transporters export amino acids and other by-products of degradation back out to the cytoplasm, where they can be re-used for building macromolecules and for metabolism. Thus, autophagy may be thought of as a cellular recycling factory that also promotes energy efficiency through ATP generation and mediates damage control by removing non-functional proteins and organelles. Figure 29. Autophagy as a recycling mechanism for cell homeostasis The autophagy pathway was recently proposed to be required for NETosis. [189,190] When neutrophils are stimulated with PMA, they develop large vacuoles that are reminiscent of autophagosomes. Evidence for the involvement of this process in NETosis comes exclusively from pharmacological studies with wortmannin, which inhibits PI3 Kinases and PI3K-like enzymes and has low specificity. 42

43 mtor Autophagy as a cellular protective mechanism has received extensive attention from many scholars, and mtor as a major negative regulator of autophagy has been paid increasing attention. Mammalian target-of-rapamycin (mtor) is a recently discovered evolutionarily conserved protein kinase that is also an important signal transduction molecule. It senses environmental amino acids, ATP, growth factors, and insulin levels, and has an important regulatory role in cell growth. mtor exists in two complexes, termed mtor complex-1 (mtorc1) and mtorc2. mtorc1 is mainly involved in protein synthesis and cellular energy metabolism processes and is inhibited by rapamycin (figure 30). Figure 30. Central role of mtor in multiple cell functions mtorc2 plays an important role in the regulation of the cytoskeleton and is insensitive to rapamycin. Under basal conditions, the glomerulus seems to be maintained by extremely low mtor, whereas abnormal expression of mtor is found in kidney lesions. [191] Rapamycin, also known as sirolimus, is a natural antibiotic. By binding to FK506-binding protein of 12 kda (FKBP12), it is an acute specific inhibitor of mtorc1. Rapamycin is essential not only for the identification of mtor, but also for elucidating mtor-dependent signaling events and their role in metabolism and disease. 43

44 mtor regulation of autophagy TOR, as a central regulator of cell growth, plays a key role at the interface of the pathways that coordinately regulate the balance between cell growth and autophagy in response to nutritional status, growth factor and stress signals. Autophagy, as a cellular process mobilizing intracellular nutrient resource, plays an important role in contributing to survival during these growth unfavorable conditions. Eukaryotic cells have developed a mechanism through which autophagy induction is tightly coupled to the regulation of cell growth. Among the numerous components involved in the regulation of autophagy and growth, TOR (target of rapamycin) is a key component that coordinately regulates the balance between growth and autophagy in response to cellular physiological conditions and environmental stress. The function of TOR in yeast and higher eukaryotes encompasses regulation of translation, metabolism, and transcription in response to nutrients and growth factors. The broad cellular functions of TOR have made the protein kinase to be an important subject for study of cancer, metabolism, longevity, and neurodegenerative diseases. mtorc2 (mtor rictor complex), originally known as rapamycin insensitive but likely also targeted by rapamycin, is involved in the regulation of phosphorylation and activation of Akt/PKB, protein kinse C, serum- and glucocorticoid-induced protein kinase 1. Because Akt positively regulates mtorc1, it would be reasonable to speculate that mtorc2 acts as a negative regulator of autophagy. Indeed, mtorc2 inhibition induced autophagy and atrophy in skeletal muscle cells under a fasting condition. However, the autophagy induction by mtorc2 inhibition is mediated mainly by FoxO3, a transcription factor downstream of Akt, which is involved in autophagy gene expression. [192] 44

45 C. STUDY RATIONALE Vascular injury after stent placement is a trigger for the local activation of vascular healing mechanisms. Exaggerate healing response, as that triggered by bare metal stents (BMS) is often lead to excessive tissue accumulation, re-stenosis of the stented segment and repeat procedure is required. On the other hand, defective healing induced by drug eluting stents (DES) lead to incomplete re-endothelization, uncovered stent struts and thrombosis even months after stent placement. This risk of late thrombosis is anticipated by long periods of dual antiplatelet drugs treatment, increasing the frequency of hemorrhagic complications. For this reason, the study and understanding of the mechanisms responsible for restenosis or defective healing after stent placement, would trigger future research for new therapeutic targets aiming both at reduce restenotic phenomena and promote complete and early re-endothelization. A lot of studies have explored the role of neutrophils and NETs in inflammation and thrombosis. Exploring the role of neutrophils and NETs in vascular healing and restenosis, as well as thrombosis may help to develop novel drugs and/or biomaterials (e.g stents) against thrombo-inflammation, not intervening with primary hemostasis thus avoiding bleeding complications. In the present study we attempt to investigate the role of neutrophils and NETs in vascular response to stents. More specifically, we studied the effects of stents on neutrophils and human fibroblasts obtained from healthy donors (controls). We performed experiments with activation (using coronary artery stents and NETs) and inhibition (using DNase), on human fibroblast cultures and neutrophils in order to assess whether stents are able to induce NET formation in control neutrophils and further, if stents and/or NETs can differentiate fibroblasts into myfibroblasts. The recognition of the active participation of NETs in stent-related vascular remodeling may trigger further research on biomaterial- related fibrosis and thrombosis. 45

46 D. MATERIALS and METHODS Blood cell isolation and culture Peripheral blood neutrophils were isolated from heparinized blood from four healthy donors. Primary human lung fibroblasts were isolated from lung lobectomy samples derived from four donors at the Academic Hospital of Alexandroupolis, Greece, as previously described.[184] The study protocol design was in accordance with the Declaration of Helsinki and the procedures were approved by the local ethics committee. Cell isolation was performed as follows: 3ml of blood were dissolved in 3 ml of 0.9% NaCl and stown in 3ml Ficoll-histopaque 1119 (for PMN isolation) and in 3ml Ficoll- Histopaque 1077 (for monocyte isolation). A centrifurcation was performed at 500g for 30 min in room temperature. Each layer was washed with PBS, followed by a centrifurcation for 10 min in 300g. Supernatant was discarded and cells were measured and assessed for viability. Fibroblasts extraction protocol We used biopsies of lung tissue (4-7 sections of 2-3mm) transferred them in 50 ml tubes. After adding HBSS (full) and 300 U/ml antibiotics/antimycotic solution we rinsed the samples with 25 ml HBSS (full) and 300 U/ml antibiotics/antimycotic solution for 3-5 min and mixed them gently. We placed the tubes horizontally for 3 min at room temperature, allowing the tissues to settle and discard supernatant. This process was repeated 3 times. After the second rinse we transfer samples to a fresh 50 ml tube. After the third rinse, a centrifugation was performed at 200 x g for 10 min and the supernatant was discarded. Thereafter, we rinsed the tissue with 20 ml HBSS (without Ca and Mg) and 300 U/ml antibiotics/antimycotic solution and 1mM DTT (57µl DTT at 20 ml), and mixed gently before we placed the tubes horizontally (15 min at room temperature) and mix gently every 5 min.we then rinsed the tissues with 20 ml HBSS (without Ca and Mg) and 300 U/ml antibiotics/antimycotic solution, and mixed gently. We placed the tube horizontally for 3 min at room temperature, allowed tissue to settle down and discard supernatant. We repeated the procedure 3 times. 46

47 After the second rinse we transfered the samples to a fresh tube, we centrifuged at 200 x g for 10 min and discard the supernatant. We incubate at 37 C with HBSS (without Ca and Mg) and 300 U/ml antibiotics/antimycotic solution and 1mM EDTA for 30 min. we then allowed tissue to settle down and discard the supernatant. We rinsed with 25 ml HBSS (without Ca and Mg) and 300 U/ml antibiotics/antimycotic solution, and mixed gently. We allowed tissue to settle and discarded the supernatant. After the third rinse we added 10 ml RPMI and mixed gently to allow tissue to settle down and discarded the supernatant. We transfered the tissue to flask 75 cm2 and add 12 ml RPMI. Finaly, we transfered the flasks to the incubator at 37 C in 5% CO2. All experiments were carried out on LFs from passages 2 3. Fibroblasts were characterized by immunofluorescence staining using mouse monoclonal antibodies to alpha-smooth muscle actin (α-sma), vimentin, and desmin (Invitrogen, Carlsbad, CA, USA). Activated fibroblasts (myofibroblasts) were characterized by α-sma stress fibres, as assessed by immunofluorescence staining. All experiments were carried out on LFs from passages 2 3. Fibroblasts were characterized by Immunofluorescence staining using mouse monoclonal antibodies to alpha-smooth muscle actin (α-sma), vimentin, and desmin (Invitrogen, Carlsbad, CA, USA). Activated fibroblasts (myofibroblasts) were characterized by α-sma stress fibres, as assessed by immunofluorescence stainin. Verification of differentiation of LFs was performed before each experiment. Stimulation and inhibition studies Neutrophil stimulaiton Neutrophils from healthy individuals were cultured in 5% CO2 at 37 C in RPMI in the presence of 2% healthy donor serum. Neutrophils were treated with BMS and DES in order to release NETs. Neutrophils were also incubated with phorbol 12- myristate 13-acetate (PMA) (40 ng/ml; Sigma-Aldrich, St Louis, MO, USA), a generic inducer of NET release. NET structure generation and isolation To generate NETs, ^6 neutrophils were seeded in six-well culture plates (Corning Incorporated, New York, NY, USA) in low-serum RPMI medium (Gibco 47

48 BRL) and exposed to each of the aforemen biomaterials for 4 h. MPO/DNA complex ELISA (see below) was used to quantify NET release, MPO/DNA complex was measured in NET structures isolated from ^6 neutrophils. NET release was depicted as % increase compared with controls. Fibroblasts stimulation Human fibroblasts were cultured in 5% CO2, 37 o C in full DMEM (10% FBS, Full DMEM, Gibco BRL, New York, NY, USA) and low DMEM (2% FBS, Low DMEM) serum 24 h before each stimulation. Fibroblasts were stimulated with (biomaterials)coronary artery stents (both BMS and DES), with NETs derived from stimulation of PMNs with stents, with NETs obtained from patients with myocardial infarction (ACS NETs) and PMA-derived NETs As control NETs (ctrl NETs), NET structures from untreated PMNs were used. In inhibition studies, isolated NET structures were incubated with DNase1 (DNase-Ι, 10 U/ml, Fermentas, 798 Cromwell Park, USA). All substances used in this study were endotoxin-free, as determined by a Limulus amebocyte assay (Sigma-Aldrich) Immunofluorescence To assess the LF differentiation/proliferation, cells were stained for α-sma (Invitrogen) and visualized in a confocal microscope. Sections were counterstained with DAPI and visualized using confocal microscope. FBs / MFs were incubated for 48 hours in order to proliferate and attatched to round coverslips, placed in a 24-well plate with 500µl medium DMEM full (containing 10% FCS) per well. The cells were fixed in 4% PFA (paraformaldehyde). 500µl 8% PFA was added so that the final concentration in the well was 4%. Cells were incubated for 1 h at 4oC. The coverslip was removed carefully using a bent needle and a pair of bent forceps and put upside down on a drop of PBS 1x. Coverslips needed to be washed 3 times for 5 min at RT. Membrane permeability was achieved by the use of triton detergent. Then the coverslip was incubated in the same manner in a drop of 0.5% Triton X-100 for 1 min at room temperature and washed 3 times in PBS 1x. The coverslip was laid then on a drop of blocking buffer (5% goat serum in PBS) and incubated for 30 min at room temperature. Mouse-antihuman antibody (a-sma (1/200), vim, des) was diluted in blocking buffer (PBS +5% goat serum). The coverslip was transfered in the humid 48

49 chamber directly from blocking buffer onto a drop of primary antibody and incubated for 1 h at RT. The secondary rabbit-antimouse, antibody (488-Alexa fluor) was then diluted in blocking buffer. The coverslip was then transfer into the humid chamber on a drop of secondary antibody and incubated for 1 h at RT. Counterstaining with DAPI was then performed with incubation with 300 nm DAPI for 2.5 min in the dark and wash for 30 sec with distilled water. A 5µl drop of Mowiol was set onto a glass slide and coverslip was mounted upside down. The slide sould remain in dark overnight in order to dry the mowiol. Similarly, NETs obtained after stimulation of control PMNs with stents were examinated for the presence of tissue factor and IL-17 using immunofluorescence. NETs were stained with monoclonal antibodies for TF (ΣF, American Diagnostica, Greenwich, CT, USA or IL 17 (R&D Systems). A rabbit anti-mouse antibody Alexa fluor 488 or a goat anti-rabbit Alexa fluor 647 antibody (Invitrogen, Carlsbad, CA, USA) were also used for secondary antibody. DAPI counterstaining (Sigma-Aldrich) was used for DNA staining. Sample preparation and NET visualization was performed by immunofluorescence microscopy. Cell preparations were visualized in a fluorescence microscope (Leica DM2000) or a confocal microscope (Spinning Disk Andor Revolution Confocal System, Ireland) in a PLAPON 606O/TIRFM-SP, NA 1.45 and UPLSAPO 100XO, NA 1.4 objectives (Olympus, Hamburg, Germany). The percentage of NET-releasing cells was determined by examining 200 cells in a double-blind experi mental procedure. Verification and quantification of NETs with ELISA (MPO/DNA complex) To quantify NET release, MPO/DNA complex ELISA (R&D Systems) was used in NET structures isolated from neutrophils. NET release was depicted as % increase compared with controls. Micro plates with high binding F-bottom of the kit were covered with 50κl anti-mpo (5κg/ml) antibodies diluted in ζε PBS 1x (1/500) and with parafilm and stored at 4 o C overnight.the 2 nd day the solution was removed (hit and wash 3 times with 200κl PBS 1x). In each curve, 20 κl from the sample and 80 κl incubation buffer containing Anti-DNA Mab (1:25 dilution), were transferred, for assessment. The plate, covered with parafilm, was incubated for 2 hrs, shaking, at room temperature. Next, the solution was removed and washed 3 times with incubation buffer. Substrate was added (ABTS solution (poroxidase substrate)) and 49

50 the plate was incubated, shaking, at RT for 20 min resulting in the production of green colour. The intensity of the green colour was proportional to the quantity of MPO/DNA. Thereafter, the reaction was terminated by adding acid ABTS (Stop Solution). Each sample was measured in 405 nm. Collagen measurement The soluble collagen types (I V) were determined using a Sircol Collagen Assay Kit (Biocolor, Belfast, U.K.). Collagen production was measured in culture supernatants after 48 h of stimulation, based on opti mization experiments. More specifically, supernatants from MFs cultures were collected and incubated with polyethylene glycol in a Tris-HCl buffer, ph 7.6, a condensation media due to very low collagen concentrations at baseline (<5κg/ml). Following overnight incubation a centrifurcation was performed (12,000 rpm for 10 min) and supernatant was discarded. Thereafter, in each sample 1 ml of Picro-Sirius Red, a dye that specifically binds to collagen, was added and centrifurgated (12,000rpm, 10 min). Precipitates were washed with Acid-Salt (acetic acid, sodium chloride and surfactants, 750κl/sample), for removal of excess dye and a new centrifurgation was performed (12,000rpm, 10 min). Samples were agitated in vortex with alkaline solution (0.5 Μ sodium hydroxide, 250κl/sample). Collagen was measured after 5 min in 555nm Statistical Analysis Data are presented as means ±SD. Statistical analyses were performed using paired T- test. All statistical analyses were performed with origin Pro-8. P-values<0.05 were considered statistically significant. 50

51 E. RESULTS BMS and DES stents have no direct effects on fibroblast differentiation in vitro Coronary artery implants (stents) are associated with various degrees of restenosis, which represent an excess wound healing process. Since fibroblasts-myofibroblast transition is a key stage in fibrosis and also involved in restenosis, we first investigated whether stents were able to induce fibroblast differentiation to myofibroblast in vitro. For this reason, bare metal stents (BMS) and drug-eluting stents (DES) were co-incubated with human fibroblasts (FBs) for 48 hours in order to study any direct effect of these biomaterials on FBs differentiation. As first step in our experiments we performed immunofloresence for a-sma prior to stent coincubation to assure FBs phenotype (figure 31). Figure 31. Fibroblast Characterization. Undifferentiated, resting fibroblasts were isolated and identified using antibodies against a - actin of smooth muscle cells (a - SMA). Left: Fibroblasts observed with Giemsa staining. Right: Undifferentiated fibroblasts are showing negative staining for a- SMA, using Immunofluorecence staining for DAPI (DNA, blue) and a-sma (green), observed in confocal microscopy. After 48 hours of co-incubation with both stent types we did not observe phenotypic changes consistent with fibroblast differentiation. This indicates that stents cannot induce FBs differentiation directly (figure 32). 51

52 Figure 32. Stents cannot induce fibroblast (FBs) differentiation directly. FBs coincubated both with BMS and DES remained negative for a-sma staining. Left: FBs co-incubated with BMS; Right: FBs co-incubated with DES. No FB differentiation is observed. Immunofluorecence staining for DAPI (DNA, blue) and a-sma (green), observed in confocal microscopy. Stents induce the release of NETs, decorated with IL-17, from neutrophils in vitro Based on previous knowledge that neutrophils are involved in coronary stent restenosis, [193] and based on our previous results showing that stents are not able to induce fibroblast to myofibroblast differentiation directly, we hypothesized that stents may excert some of their restenotic effects on fibroblasts via interaction with neutrophils by inducing NETs. We thus first investigated whether various stents (biomaterials) were able to induce NETs from control neutrophils in vitro. For this reason, BMS and DES were co-incubated with control neutrophils for 2 hours and the effect of these biomaterials on NET formation was investigated. Indeed, both stent types induce NET generation from control neutrophils. Given that NET bound IL-17 and TF are implicated in fibrosis[184] and thrombosis[199] respectively, we examined the presence of IL-17 and TF on stent derived NETs. Interestingly, both stent types-induced NETs were decorated with interleukin-17 (IL-17, figure 33). Similarly, PMA-derived generic NETs also expressed IL

53 Figure 33. Both stent types induce NET generation from control neutrophils. Upper panel: Untreated control neutrophils stained for IL-17 and neutrophil elastase (NE). No intracellular IL-17 is detected as cells are IL- 17 negatives. Lower panel: Control neutrophils treated with DES (left) and BMS (right) stents released NETs. Note the presense of NETs positive for IL-17 in both cases. NE was used as neutrophil marker. (DAPI: blue, IL-17: green, NE: red) On the contrary, both stent types-induced NETs did not express tissue factor (TF), as assessed after apropriate staining (figure 34). These results demonstrate that stents, containing metal or various coatings (such as polymer and drugs) are able to induce IL-17-bearing NETs from resting neutrophils in vitro. 53

54 Figure 34. Stent-induced NETs did not express tissue factor (TF) Upper panel: Untreated control neutrophils stained for TF and neutrophil elastase (NE). Cells are TF-negative indicating the absence of intracellular TF expression. Lower panel: Control neutrophils treated with BMS (left) and DES (right) stents release NETs though these NETs were not decorated with TF. NE was used as neutrophil marker. (DAPI: blue, TF: green, NE: red) Our next question was to study the differences in the NETotic potential between BMS and DES from control neutrophils. In order to address this question we performed an experiment in which neutrophils were incubated with BMS and DES and after that we measured NET generation using MPO/DNA ELISA. We observed that DES induced more NET generation comparing to BMS. Differences in stent coating (DES used in our experiments, were coated with m-tor inhibitors) may account for the observed differences in NETotic potential between these two types of stents (figure 35). 54

55 Figure 35. DES induces more NETs compared to BMS. Assessment of NETotic potential of DES compared to BMS using MPO/DNA ELISA. (Control: unstimulated neutrophils, BMS: BMS stimulated neutrophils, DES: DES stimulated neutrophils, PMA: generic NET inducer, p-value<0.05 was considered stastistically significant). Stents-induced NETs promote fibroblast differentiation in vitro We next investigated whether both stent type-induced NETs, expressing IL-17, were able to induce fibroblast differentiation in vitro. For this reason, stent-derived NETs were co-incubated with fibroblasts. The differentiation process from fibroblast to myofibroblast was assessed by a-sma positive staining. As shown in figure 36, both stent types-derived NETs induced fibroblast differentiation. Figure 36. Stent-derived NETs induce fibroblast differentiation. a-sma positive cells (myofibroblasts, MFs) after incubation of resting fibroblasts (FBs) with stent-derived NETs (DAPI: 55

56 blue, a-sma: green).left: fibroblasts with BMS derived NETs. Right: fibroblasts with DES derived NETs. The rate of stent restenosis is greater in the setting of acute myocardial infarction, a state that induces abundant NET release.[194] We thus investigated whether NETs derived from patients presenting with myocardial infarction were able to promote differentiation of fibroblast to myofibroblast in vitro. For this reason, myocardial infarction-derived NETs were obtained from the infarct-related arteries (IRA-NETs) of patients with acute myocardial infarction. These NETs are found to express functional TF.[194] Incubation of fibroblasts with IRA-NETs resulted in fibroblast differentiation to myofibroblasts, as assessed by a-sma positive staining. Similarly, co-incubation of IRA-NETs and BMS with fibroblasts resulted in fibroblast differentiation to myofibroblasts, assessed by a-sma positive staining. On the contrary, no fibroblast (FB) differentiation was observed after adding DES into FBs and IRA-derived NETs co-cultures (figure 37). Figure 37. Myocardial infarction-derived NETs induce fibroblast differentiation in vitro. Upper Panel: A-SMA positive cells (myofibroblasts, MFs) after incubation of resting fibroblasts (FB) with IRA-derived NETs bearing tissue factor (DAPI: blue, a-sma: green). Lower Left: A-SMA positive cells (myofibroblasts, MFs) after incubation of resting fibroblasts (FBs) with IRA-derived NETs 56