Valve interstitial cell activation and proliferation are associated with changes in β-catenin

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1 Valve interstitial cell activation and proliferation are associated with changes in β-catenin by Songyi Xu A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto Copyright by Songyi Xu, 2012

2 Valve interstitial cell activation and proliferation are associated with changes in β-catenin Abstract Songyi Xu Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2012 Heart valve interstitial cells (VICs) undergo activation and proliferation in repair and disease, but the mechanisms are not fully understood. We hypothesize that the establishment of N- cadherin/β-catenin cell-cell contacts may decrease VIC activation, and that Wnt3a/β-catenin signaling may increase VIC proliferation. VIC cultures of different densities are stained for α- SMA, cofilin, TGF-β, psmad2/3, N-cadherin and β-catenin, and probed for phospho-β-catenin by Western blot. Low density VIC cultures are treated with exogenous Wnt3a and measured for cell number, proliferation, apoptosis, α-sma, β-catenin, and β-catenin-mediated transcription. β- Catenin sirna knockdown is used to assess β-catenin specificity. Increased staining of α-sma, cofilin, TGF-β, psmad2/3, nuclear β-catenin, and increased phospho-β-catenin are associated with few cell-cell contacts. Wnt3a increased VIC cell number, proliferation, nuclear β-catenin and β-catenin-mediated transcription without affecting activation and apoptosis, and proliferation is abolished by β-catenin sirna. Thus, N-cadherin/β-catenin cell-cell contacts may inhibit VIC activation and Wnt3a/β-catenin signaling may increase VIC proliferation. ii

3 Acknowledgments I was the first author of the published paper that is Chapter 2 (Xu S, Liu AC, Kim H, Gotlieb AI. Cell density regulates in vitro activation of heart valve interstitial cells. Cardiovasc Pathol [Epub ahead of print]) [170]. Hyunjun Kim and Amber Chang Liu carried out the α-sma and TGF-β studies and generated figures 1, 2 and 4. I carried out the cell contacts studies (Ncadherin, β-catenin and α-sma) as well as the psmad2/3, cofilin and phospho-β-catenin studies. I wrote the paper, handled all the reviewers comments, did additional experiments that were requested, and was responsible for the resubmission. I would like to thank Dr. Avrum I. Gotlieb for his scientific as well as personal guidance. I also appreciate the advice, guidance and support of my committee members Dr. Michelle Bendeck and Dr. Craig Simmons. As well, I would like to thank Dr. Alan Rosenthal for all his help and for keeping the lab running smoothly. In addition, I would like to thank Dr. Myron Cybulsky s lab for teaching me the Western blot and densitometry techniques. Moreover, I would like to thank Dr. Andras Kapus and his lab for helping me with the Western blot cell fractionation protocol. Also, I would like to thank Dr. Craig Simmons and his lab for helping me with the sirna transfection protocol. Furthermore, I would like to thank Dr. Benjamin Alman and his lab for providing me with the TOPFLASH, FOPFLASH and β-galactosidase DNA plasmids and helping me with the transfection and reporter assay techniques. Finally, I would not be where I am today without the love and support of my family and friends. Thank you. iii

4 Table of Contents Valve interstitial cell activation and proliferation are associated with changes in β-catenin... i Abstract... ii Acknowledgments... iii Table of Contents... iv List of Figures... vii List of Abbreviations... ix Chapter 1: Background The normal heart valve Heart valve disease Valve interstitial cells Activation Proliferation Apoptosis Role of TGF-β Role of cell-cell contacts Role of Wnt/β-catenin Rationale, hypothesis and general objective...12 Chapter 2: Cell density regulates in vitro activation of heart valve interstitial cells Introduction Materials and Methods VIC cultures Cell density and VIC activation Immunofluorescent staining and confocal microscopy...17 iv

5 2.2.4 Western blot analysis Statistical analysis Results α-sma expression in VICs decreases as cell density increases TGF-β signaling in VICs decreases concurrently with a decrease in α-sma expression as cell density increases Absence or loss of cell-cell contacts increases α-sma staining intensity Phospho-β-catenin is reduced in confluent VIC cultures Discussion...21 Chapter 3: Wnt3a/β-catenin increases proliferation in heart valve interstitial cells Introduction Materials and Methods Cell culture Treatment with exogenous Wnt3a Immunofluorescent staining and confocal microscopy Cell count Western blot Small interfering RNA transfection Quantification of proliferation Quantification of apoptosis TOPFLASH/FOPFLASH reporter assay Statistical analysis Results Wnt3a treatment increases β-catenin staining intensity in VICs Wnt3a treatment increases VIC cell number...32 v

6 3.3.3 Wnt3a treatment increases whole cell, cytosolic and nuclear protein levels of β- catenin Wnt3a treatment increases VIC proliferation; an effect dependent on β-catenin Wnt3a treatment does not affect VIC apoptosis Wnt3a treatment increases β-catenin-tcf-mediated transcription Discussion...33 Chapter 4: Discussion/Future Directions...35 References...39 vi

7 List of Figures Figure 1 TGF-β signaling pathways...64 Figure 2 Wnt/β-catenin signaling 65 Figure 3 Figure 4 Figure 5 Figure 6 α-sma staining decreases as cell density increases regardless of time in culture Percentage of VICs with High Intensity α-sma staining is less in high density cultures compared to low density cultures...67 Cofilin staining decreases as cell density increases...68 TGF-β staining decreases concurrently with α-sma staining as cell density increases...69 Figure 7 Nuclear Smad2/3 staining decreases as cell density increases...70 Figure 8 N-cadherin and β-catenin staining increases at cell-cell contacts, decreases in cytoplasm, and nuclear β-catenin staining disappears as cell density increases 71 Figure 9 Phospho-β-catenin is decreased in confluent cultures 72 Figure 10 β-catenin staining increases with Wnt3a treatment 73 Figure 11 Wnt3a treatment does not affect α-sma staining 74 Figure 12 Cell number appears to increase with Wnt3a treatment 75 Figure 13 Total cell count increases after Wnt3a treatment 76 Figure 14 Wnt3a does not affect cell viability 77 Figure 15 Wnt3a increases whole cell β-catenin level 78 vii

8 Figure 16 Wnt3a increases nuclear β-catenin level 79 Figure 17 Figure 18 Figure 19 β-catenin level decreases after sirna knockdown 80 Wnt3a increases VIC proliferation, an effect abolished by β-catenin sirna Wnt3a does not affect VIC apoptosis 82 Figure 20 Wnt3a increases β-catenin-mediated transcription 83 viii

9 List of Abbreviations AngII APC α-sma β-trcp BMP CAS angiotensin II adenomatous polyposis coli α-smooth muscle actin β-transducin repeat-containing protein bone morphogenetic protein calcific aortic stenosis CK1 casein kinase 1 ECM EGTA EMT GAG extracellular matrix ethylene glycol-bis (2-aminoethylether)-N, N N N -tetraacetic acid endothelial-to-mesenchymal transformation glycosaminoglycan GSK3 glycogen synthase kinase 3 MMP PBS PMSF PVDF TCF/LEF TGF-β matrix metalloproteinase phosphate buffered saline phenylmethylsulfonyl fluoride polyvinylidene fluoride T-cell factor/lymphoid enhancer factor transforming growth factor-β ix

10 VEC VIC avic obvic pvic qvic valve endothelial cell valve interstitial cell activated VIC osteoblastic VIC progenitor VIC quiescent VIC x

11 Chapter 1: Background 1

12 2 1.1 The normal heart valve The human heart has four chambers, an atrium and a ventricle on the right and the left side. The heart also has four valves, the tricuspid valve between the right atrium and right ventricle, the pulmonary valve between the right ventricle and the pulmonary artery, the mitral valve between the left atrium and left ventricle, and the aortic valve between the left ventricle and the aorta. Deoxygenated blood returns from circulation in the human body and fills the right atrium during diastole. When the pressure of the right atrium exceeds that of the right ventricle, the tricuspid valve opens, allowing the blood to flow into the right ventricle. When the right ventricle contracts during systole, the pulmonary valve opens and allows the blood to be pumped into the lungs for oxygenation. Similarly, oxygenated blood from the lungs fills the left atrium during diastole. When the pressure of the left atrium exceeds that of the left ventricle, the mitral valve opens, allowing the blood to flow into the left ventricle. When the left ventricle contracts during systole, the aortic valve opens to allow the blood to be pumped into the body. Contraction of the ventricles during systole exerts a back pressure on the tricuspid and mitral valves, causing them to close tightly. Similarly, after ejection of blood, back pressure from the blood in the pulmonary artery and aorta causes the pulmonary and aortic valves to close rapidly and completely, preventing backflow. The valves of the human heart are flow-regulating membranes inside a complex multichambered pump. The connective tissue of valves serves to maintain proper structure [1,2] and a heterogeneous population of constituent cellular elements regulates complex functions to give flexibility and durability to the leaflets [3]. The four types of cells found in valves are surface valve endothelial cells (VECs), valve interstitial cells (VICs), and towards the base of the valve, cardiac muscle cells and smooth muscle cells [4,5]. A confluent monolayer of VECs lines the surface of the valve [6]. Underneath the endocardium are VICs embedded in matrix which they secrete and which form the three histologically distinct layers of the valve the fibrosa, the spongiosa, and the ventricularis [6]. The fibrosa is rich in densely packed collagen fibers; the spongiosa is composed mainly of glycosaminoglycans (GAGs) and proteoglycans; and the ventricularis contains collagen, elastin and GAGs [7,8]. These provide the valve with a zone of stiffness, a zone which is compressible, and a zone which is elastic in nature, respectively [3].

13 3 The fibrosa with its dense connective tissue provides strength when the valve is shut, the spongiosa with its loose matrix of glycoproteins provides a cushion for the physical forces and mechanical strains and valve movement, and the ventricularis provides for elasticity when the cusp changes shape during the cardiac cycle. All three layers are avascular [9]. Heart valves are remarkably adapted to allow unidirectional and non-obstructed passage of blood without regurgitation, trauma to blood elements, thromboembolism, or excessive stress concentrations in the cuspal/leaflet or supporting tissue [7]. Valves are biologically dynamic structures, capable of considerable functional remodeling and repair of injury [7]. 1.2 Heart valve disease Heart valve disease results from valve dysfunction due to stenosis and/or regurgitation leading to progressive cardiac disease [10]. Heart valve disease has several different etiologies including congenital, genetic, infectious, inflammatory, drug-induced, and calcific [8,11-14]. It manifests as several different clinical conditions including calcific aortic stenosis (CAS), bicuspid aortic valve stenosis, acute and chronic rheumatic valve disease, valve prolapse (e.g. mitral valve prolapse), and annular calcification [8,15,16]. In many cases, the only available treatment is surgical intervention with heart valve replacement. In populations with access to costly cardiovascular surgery, over 285,000 patients undergo replacement worldwide annually, but 60% suffer from valve-related complications by 10 years after surgery [17]. Bicuspid aortic valve stenosis is the most common congenital heart defect [18]. The bicuspid valve typically consists of 2 unequal-sized leaflets, with the larger leaflet having a central ridge that results from fusion of cusps [18]. Bicuspid aortic valve stenosis is generally found with other cardiovascular defects such as coarctation of the aorta, ventricular septal defects, and atrial septal defects, suggesting a global disorder of cardiac development as the basis of the disease [18]. Symptoms typically develop in adulthood, and they include aortic valve stenosis, aortic valve incompetence, aortic valve calcification, aortic root dilation, and aortic root dissection [18]. Rheumatic valve disease develops after repeated episodes of acute rheumatic fever and affects primarily the mitral and aortic valves [16]. It results from an abnormal autoimmune response to bacterial antigens that cross-react with host connective tissue antigens, which leads to connective

14 4 tissue degeneration as part of the inflammatory reaction [16]. The inflammation triggers repair processes of the valve, leading to fibrosis, calcification, and eventual valve dysfunction [16]. In mitral valve prolapse, there is abnormal displacement of the valve leaflets into the left atrium during systole resulting in regurgitation and decreased cardiac output [16]. This is caused by myxomatous degeneration of the valve tissue characterized by an excessive accumulation of GAGs, loss of VICs and collagen, and elastic fragmentation [13,19]. CAS is the most common heart valve condition in adults in Western society [20]. It is a condition with high morbidity and mortality that is very costly to the health care system [21-23]. It is a chronic disease that is slowly progressive and has precursor lesions that may remain asymptomatic for some time [24]. During the initial stages of the disease, aortic valve sclerosis occurs which results in cusp thickening without creating obstruction to the left ventricular outflow [24]. This gradually progresses to CAS, obstructing flow [24]. Severe symptomatic aortic stenosis is associated with a life expectancy of less than 5 years [25]. While 20-30% of individuals over the age of 65 and 48% of individuals over 85 are affected by sclerosis, only 2% of individuals over 65 and 4% of individuals over 85 end up with CAS [20,22,26]. Risk factors for CAS include hypertension, elevated low-density lipoprotein, male gender, smoking, and diabetes mellitus [20], which are similar to those of atherosclerosis [27]. Statins, well-known therapeutic agents that target atherosclerosis, are being tested in prospective clinical trials on CAS, but the results are so far disappointing [28-31]. CAS is no longer considered to occur due to passive degeneration secondary to aging. Research has shown that the pathogenesis involves active cell and tissue processes that occur in a tissue response to injury including VIC accumulation, inflammation, neovascularization, oxidative stress, matrix remodeling, and calcification and osteogenesis [24,32-36]. These processes develop as the valve cusps thicken due to fibrosis. Irregular calcified nodules develop initially at sites exposed to high mechanical force in the fibrosa layer of the valve [27]. Typically, calcified nodules consist of calcium-phosphate mineral deposits, and a subset of end-stage human aortic valve lesions has been shown to contain bone [37-40]. Much research has focused on the pathogenesis of calcific aortic stenosis in an attempt to gain greater understanding to improve current treatments. VICs appear to increase their expression of osteoblast markers and increase

15 5 their rate of calcified nodule formation in response to a number of atherogenic factors [39,41,42]. Oxidized lipids have been found in areas of developing calcification and are shown to stimulate calcified nodule formation in vitro [39,43]. In addition, angiotensin II (AngII) is present in aortic valve lesions and has numerous lesion-promoting effects such as stimulating inflammation and macrophage-cholesterol accumulation, increasing oxidant stress, and stimulating expression of lipoprotein-retaining proteoglycan, biglycan [44-46]. There is a number of potential sources of AngII in diseased valves, including low density lipoprotein-associated angiotensin converting enzyme, macrophage-associated angiotensin converting enzyme, and mast cell chymase [44,47]. The major pathogenic receptor for AngII, the AngII type I receptor, is only present in VICs in valve lesions [44,47], providing further evidence supporting the possibility that AngII may play an important role in the pathogenesis of CAS. Furthermore, aortic valve lesions contain macrophages and T-lymphocytes that produce matrix metalloproteinases (MMPs) and proinflammatory cytokines such as interleukin-1β, tumour necrosis factor-α, transforming growth factor-β1 (TGF-β1), and bone morphogenetic protein 2 (BMP2), that promote calcification [39,42,48-54]. BMP2, present in human aortic valve lesions, can stimulate calcified nodule formation by upregulating transcription factor Msx2 which activates Wnt signaling or by upregulating the osteoblast-specific transcription factor Runx2/Cbfa1, which has been detected in calcified valves [33,55-58]. As well, mutations in Notch1, a transcription factor that normally represses Runx2/Cbfa1, are implicated in valve calcification [59]. It is possible that specific signaling mechanisms may be involved in valve calcification. Even though each heart valve disease has distinct gross and microscopic features, there are several pathological features that are common to most heart valve diseases, including VIC proliferation, inflammation, changes in the valve matrix, fibrosis, and eventual calcification. Despite research efforts focused on understanding the pathogenesis of valve disease, the knowledge gained has not been sufficient to lead to better prevention and treatment. 1.3 Valve interstitial cells Heart valve disease is considered to be a response to tissue injury that becomes excessive and leads to disruption of the leaflets/cusps with excessive remodeling, scarring, and calcification instead of proper cusp/leaflet repair. The tissue and cell processes important in valve function,

16 6 dysfunction and tissue engineering are best studied and understood as a response to tissue injury [5,60]. This response may be regulated by VICs, the most prevalent cells in the valve. They originate by endothelial-to-mesenchymal transformation (EMT) in the endocardial cushion and are primarily regulated by BMPs, TGF-β, Notch, and Wnt signaling in development [61]. BMPs and TGF-β play important roles in the initiation of endocardial cushion formation and EMT as well as the growth of endocardial cushions [62-75]. Wnt is important in endocardial cushion formation and EMT, especially the regulation of proliferation in endocardial cushion growth [70,76-78]. Notch plays a key role in EMT, particularly in the migration of endothelial cells into the cardiac jelly to become mesenchymal cells that will eventually form the mature heart valve [79,80]. Evidence shows that these developmental pathways, normally downregulated in adulthood, can become activated again in disease [81]. In general, there are five phenotypes that best represent the VIC family of cells, each of which exhibits specific sets of cellular functions essential in normal valve physiology and in pathological processes [32]. They include embryonic progenitor endothelial/mesenchymal cells, quiescent VICs (qvics), activated VICs (avics), progenitor VICs (pvics), and osteoblastic VICs (obvics) [32]. The embryonic progenitor endothelial/mesenchymal cells undergo EMT that initiates the process of valve formation in the embryo [82]. The qvics are the cells that are at rest in the adult valve and maintain normal valve physiology [4,83-85]. The avics are the cells that regulate the pathobiological responses of valve in disease and injury [86-88]. The pvics are the least well-defined and consist of a heterogeneous population of progenitor cells that may be important in repair [89-93]. The obvics regulate chondrogenesis and osteogenesis [24]. These phenotypes may exhibit plasticity and convert from one form to another [32] Activation Of particular interest are qvics that maintain normal structure and function in healthy adult valves as well as avics which regulate numerous processes in diseased or injured valves [32,94]. qvics become avics under conditions of pathological injury or abnormal hemodynamic/mechanical stress in which activated VECs and macrophages/foam cells arise, and a number of chemokines and growth factors stimulate qvic activation [32]. Activation of VICs is associated with increased ECM secretion and degradation, proliferation, migration, and

17 7 cytokine production, which are all important features of the wound repair process [60]. avics take on the features of myofibroblasts showing increased contraction, prominent actin stress fibers, and other contractile proteins [86]. The marker for avic is α-smooth muscle actin (α- SMA), a cytoskeletal isoform of actin, which is normally not found in qvic [32]. The assembly of α-sma into stress fibers is important for cell migration, force generation, and wound contracture both in vitro and in vivo [95,96]. Diseased heart valves show upregulation of α-sma staining in VICs [24,97-100]. Also, increased α-sma expression and stress fiber formation are observed in VICs along an in vitro wound edge of a wound made linearly across a confluent monolayer, suggesting that wounding activates the qvics [101]. avics likely play a key role in the pathogenesis of human clinical disease when the response to tissue injury becomes excessive and leads to disruption of the leaflets/cusps with excessive remodeling, scarring, and calcification through unknown mechanisms [8]. Thus, understanding the regulation of VIC activation and the associated cellular responses is critical to understanding the pathobiology of heart valve diseases Proliferation Under normal conditions, there is very little or no proliferation in adult valves [102]. The level of proliferation is much higher in heart valve development during cardiac cushion formation, but it is down-regulated in adult [103]. Evidence suggests that proliferation is increased in diseased valves, such as in areas around calcified nodules [102]. As well, in studies where organ cultures of porcine mitral valves are wounded with a linear superficial wound on the valve surface, increased proliferation is observed in wound edge VICs [104]. Furthermore, wound edge VICs along an in vitro scratch wound made linearly at the center of a confluent monolayer also exhibit increased proliferation compared to areas away from the wound edge [101]. Proliferation of fibroblasts and myofibroblasts has been shown to be inhibited by low doses of nitric oxide [105]. These findings suggest that proliferation is likely to be a response to tissue injury that leads to disease instead of successful repair when dysregulated. Thus, understanding the regulation of VIC proliferation is important to understanding valve function and disease.

18 Apoptosis Increased apoptosis is observed in wound edge VICs along an in vitro wound made linearly in the center of a confluent monolayer, indicating that cell turnover may be an important aspect of valve wound repair [101]. As cell populations rapidly proliferate during tissue reconstruction and remodeling in wound repair, proliferation is balanced by apoptosis. In addition, after completion of remodeling, many avics need to be eliminated by apoptosis [106]. In dermal wound healing, apoptosis is increased at the wound after wounding to eliminate dead or dying cells and to terminate regeneration and remodeling after healing is complete [107]. Dysregulation of apoptosis in wound healing can lead to a number of pathological processes, most notably hypertrophic scarring and keloid formation, two forms of hyperproliferative healing characterized by hypervascularity and hypercellularity [108,109]. In the same manner, if VICs did not undergo apoptosis, excessive ECM deposition and fibrosis would likely occur leading to valve scarring and dysfunction [101]. Thus, the regulation of VIC apoptosis is an important aspect of valve injury and disease. 1.4 Role of TGF-β The TGF-β superfamily of proteins is a family of cytokines and peptide growth factors that regulate biological functions in many systems including heart valves [27, Figure 1]. TGF-β1 is the predominant isoform of TGF-β exerting biological effects in the adult human and pig heart valve [101]. Activated TGF-β1 binds strongly to receptor type II, a serine/threonine kinase that phosphorylates the glycine-rich domain on receptor type I, which is another serine/threonine kinase that can then phosphorylate other downstream signaling molecules [110]. The best characterized signaling pathway involves the phosphorylation of Smad2 and Smad3 on the C- terminal Ser-Ser-X-Ser motif by receptor type I [110]. Once phosphorylated, Smad2 and Smad3 dissociate from the receptor and form a complex with Smad4 [110]. Afterwards, the Smad molecules can translocate to the nucleus to regulate gene expression [110]. TGF-β is secreted by numerous cell types including VICs with potent autocrine effects, one of which is VIC activation [101, ]. α-sma mrna and protein were upregulated concurrently with increased TGF-β in wound edge VICs along an in vitro wound [101]. Also, in

19 9 the same study, TGF-β neutralization reduced α-sma protein, mrna, and stress fibers whereas exogenous addition of TGF-β increased α-sma protein, mrna, and stress fibers [101]. TGF-β has also been found to induce α-sma protein and stress fiber expression in porcine aortic VICs cultured at low-cell density on collagen substrates [114]. In fibroblasts, TGF-β stimulated α- SMA gene expression through the TGF-β control element at -42 to -61 from the transcriptional start site of the α-sma promoter [115,116]. VIC activation by TGF-β induces dramatic augmentation of stress fiber formation and alignment leading to enhanced contractility, an important feature of wound healing [32,112]. Diseased valves exhibit increased levels of TGF-β. TGF-β is present in mitral valve prolapse [97,117] and CAS [98,118,119]. In addition, increased TGF-β and phosphorylated Smad2/3 were observed in wound edge VICs along an in vitro wound, and increased TGF-β concentration was detected in VIC-conditioned media collected at 24 hours after wounding [101]. Findings both in vivo and in vitro indicate that TGF-β is an important regulator of VIC activation and a key cytokine in valve repair and disease processes. 1.5 Role of cell-cell contacts Cell-cell adhesion is involved in all aspects of tissue morphogenesis [120,121]. Adhesive forces must be both resilient to maintain tissue architecture and dynamic to establish new contacts during wound repair [122]. Although on histological examination of the normal valve, VICs appear to reside as single cells, ultrastructural studies show that VICs extend cytoplasmic processes which adhere to neighbouring cells forming adherens junctions [123]. These adherens junctions are also present in confluent monolayers of VICs in cell culture [123]. Research shows that N-cadherin and β-catenin are present at the adherens junctions of VICs [124,125]. N- cadherin, a classical cadherin molecule, is a transmembrane glycoprotein that promotes calcium dependent homophilic binding with adjacent N-cadherin molecules on neighbouring cells via its extracellular domain [126,127]. It links to the cytoskeleton via molecules including β-catenin that bind to its cytoplasmic domain [126,127]. When freed from cell-cell contacts, β-catenin can also act as a transcriptional co-activator and bind to members of the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors in the nucleus [128].

20 10 Evidence suggests that cell-cell contacts are active regulators of epithelial-to-mesenchymal transition in epithelial and tumour cells, a key feature of which is α-sma expression [ ]. The loss of E-cadherin, a classical cadherin molecule found at adherens junctions of epithelial and some tumour cells, has been shown to promote epithelial-to-mesenchymal transition, while forced E-cadherin expression can restore the epithelial phenotype in transformed tumour cells [ ]. Cell contact-dependent activation of α-sma expression is also observed in kidney epithelial cells [135,136]. Furthermore, cell density, or the extent of cell-cell contacts, has been shown to regulate the cellular localization and transcriptional activity of β-catenin [137,138], and β-catenin signaling has been implicated in the induction and progression of epithelial-tomesenchymal transition in tumour cells [ ]. The role of cell-cell contacts in regulating VIC function has never been investigated before. Previous research from our lab shows that wound edge VICs along a linear mechanical injury in a confluent monolayer become activated and express α-sma, while VICs located away from the wound edge or VICs in non-wounded confluent monolayers are quiescent [101]. In addition, we reported that single VICs cultured at low density are activated and express α-sma [32]. It is possible that, similar to epithelial-to-mesenchymal transition, the disruption or absence of cellcell contacts in VICs may allow β-catenin to translocate to the nucleus and activate VICs and initiate early repair processes, while the establishment of cell-cell junctions may prevent β- catenin nuclear translocation and induce VIC quiescence. It is important to study the effects of N-cadherin/β-catenin cell-cell contacts on VIC activation because adherens junctions between VICs are disrupted in injury and disease. Since cadherin/β-catenin cell-cell contacts are important regulators of epithelial-to-mesenchymal transition, a key feature being the expression of α-sma, they might also regulate the activation of VICs. 1.6 Role of Wnt/β-catenin Free cytoplasmic β-catenin is a well-known mediator of the Wnt signaling pathway (Figure 2). For comprehensive reviews of the Wnt/β-catenin pathway, please refer to Logan and Nusse 2004, Clevers 2006, MacDonald et al, 2009, and Rao and Kuhl As well, updated information on Wnt signalling can be found on the Wnt homepage at Normally, the level of free cytoplasmic β-

21 11 catenin is kept low by proteasomal degradation. Upon binding of Wnt to its receptors, cytoplasmic β-catenin is stabilized and can travel to the nucleus to bind to the TCF/LEF family of proteins and mediate transcription. More details on the Wnt/β-catenin signaling pathway are covered in Chapter 3 of this thesis. Besides the canonical Wnt/β-catenin pathway, Wnt can also activate non-canonical pathways that are β-catenin-independent. The Wnt/JNK pathway regulates the establishment of planar cell polarity and involves the activation of Rho GTPases including rac, cdc42 and rho [143]. Wnt is also able to increase intracellular Ca 2+ and activate Ca 2+ -dependent enzymes such as protein kinase C important for cardiac differentiation, calcineurin that regulates gene expression through nuclear factor of activated T cells transcription factor, and calcium/calmodulin-dependent kinase II that activates nemo-like kinase and regulates histone acetylation and methylation [ ].The Wnt/β-catenin pathway has been implicated in the development of many tissues and organs, including heart valves where it was shown to regulate proliferation and EMT [103,70,76]. Several members of the Wnt family of proteins are shown to be involved in heart valve development, including Wnt2, Wnt3a, Wnt4, Wnt5b, Wnt7b, and Wnt9b [103]. Dysregulation of the canonical Wnt/β-catenin pathway has been implicated in many diseases, the most well-known being colorectal cancer, which results from constitutively activated β-catenin signaling [162]. Due to APC deficiency or β-catenin mutations that prevent its degradation, β-catenin-dependent transcription is upregulated in colon carcinoma cells, promotes cyclin D1 expression, and induces cell proliferation [164]. The role of β-catenin in cell proliferation has been well established in many cell types and under numerous conditions. For instance, hyperplastic cutaneous wounds contain prolonged periods of elevated β-catenin and increased β-catenin-dependent transcription [165]. Moreover, β-catenin has been shown to induce proliferation of vascular smooth muscle cells via increased cyclin D1 and decreased p21 levels [166]. Furthermore, in proliferating human keratinocytes, there is increased cytoplasmic/nuclear fraction of β-catenin [137]. In heart valves, increased expression of Wnt receptor Lrp5, stabilized β-catenin, and Wnt3 have been associated with CAS [167,168]. Since Wnt3 is specifically upregulated in disease and Wnt3a is among the Wnt proteins important in endocardial cushion formation, EMT and proliferation in valve developement, it is possible that the Wnt3a/β-catenin pathway may be activated in heart valve disease to increase VIC

22 12 proliferation as a response to injury. However, the regulation of the Wnt/β-catenin pathway on VIC proliferation in adulthood has not been investigated. 1.7 Rationale, hypothesis and general objective Our lab has been studying an in vitro model of VIC repair utilizing paradigms which are derived from findings in in vivo injured and repairing heart valves. Two important features of valve repair are VIC activation and VIC proliferation, both of which are upregulated in disease conditions [32]. The observation that VICs are activated only in low density cultures or along an in vitro wound edge suggests that the absence or loss of cell-cell contacts may stimulate VIC activation, while the establishment of cell-cell contacts may induce VIC quiescence [101]. Cellcell contact disassembly is found to induce β-catenin nuclear translocation and epithelial-tomesenchymal transition in epithelial and tumour cells, a key feature of which is α-sma expression, while the establishment of contact is shown to reverse the transition [ ]. When freed from cadherin contacts, β-catenin can travel to the nucleus and act as a transcriptional regulator [169]. It is possible for cell-cell contacts in VICs, which contain N- cadherin and β-catenin, to regulate VIC activation. This is worth investigating because cell-cell contacts between VICs are disrupted in injury and disease and may result in β-catenin nuclear translocation and act as an initiating signal to activate VICs to initiate wound repair. Cytoplasmic β-catenin is normally kept low through proteosomal degradation. It can be stabilized by Wnt and mediates the downstream signaling and transcriptional regulation of the Wnt/β-catenin pathway. The Wnt/β-catenin pathway is involved in many disease processes where it results in excessive proliferation [137, ]. Increased expression of Wnt receptor Lrp5, Wnt3 and β-catenin in diseased valves suggests that Wnt/β-catenin signaling may increase VIC proliferation in valve diseases as a repair response to injury. I hypothesize that N-cadherin/β-catenin cell-cell contacts regulate VIC activation and Wnt/βcatenin signaling regulates VIC proliferation. My objective is to study VIC activation in cultures of different densities, and therefore different degrees of cell-cell contact formation, as well as to study VIC proliferation and its regulation by Wnt3a/β-catenin signaling in low density cultures. To study cell-cell contacts and VIC activation, in cultures of different densities, I will observe α-

23 13 SMA and cofilin as markers of VIC activation, TGF-β and psmad2/3 as activation of the TGF-β pathway that regulates VIC activation, and N-cadherin and β-catenin in VIC adherens junctions to study the correlation between cell density and VIC activation. To study Wnt3a, β-catenin and VIC proliferation, I will treat low density VIC cultures with exogenous Wnt3a and measure the effects on total cell count, proliferation, apoptosis, β-catenin protein, and β-catenin-mediated transcription. I will also transfect VICs with sirna targeted against β-catenin to ensure that the effects observed are β-catenin-specific. Understanding the regulation of VIC activation by N- cadherin/β-catenin cell-cell contacts and proliferation by Wnt3a/β-catenin signaling will provide insight into the function of VICs and the pathobiology of heart valve diseases.

24 14 Chapter 2: Cell density regulates in vitro activation of heart valve interstitial cells (This is published Xu S, Liu AC, Kim H, Gotlieb AI. Cell density regulates in vitro activation of heart valve interstitial cells. Cardiovasc Pathol [Epub ahead of print] with minor modifications.) [170]

25 Introduction The regulation of cardiac valve function in both health and disease remains a mystery. To address this, in the last few years, studies on the cell and molecular biology of valve function have been initiated using several different in vitro model systems. One of the early successes has been the characterization of valve interstitial cell (VIC) phenotypes that regulate normal physiology and disease conditions [32,171,172]. VICs are the most prominent cell type in the heart valve and are known to have important roles in wound repair and heart valve disease [60,173]. In diseased heart valves, VICs are activated, characterized by α-smooth muscle actin (α-sma) expression, which is absent in normal heart valves [94,97,101,174]. In vitro studies show that VICs become activated upon wounding of a confluent monolayer and become quiescent upon repair of the wound [101]. In the earliest stages of wound repair, activated VICs at the wound edge undergo proliferation and apoptosis, as well as form α-sma stress fibers, leading to wound closure and repair 101]. Activated VICs require transforming growth factor β (TGF-β) to maintain activation through promoting α-sma protein expression [101,112]. Treatment with TGF-β neutralizing antibody leads to reduced VIC activation, α-sma messenger RNA production, proliferation, apoptosis, α-sma stress fibers formation, and wound closure, decreasing VIC wound repair [101]. Addition of exogenous TGFβ increases proliferation, stress fiber formation, and wound closure, enhancing VIC wound repair [101]. Thus understanding the factors that regulate VIC activation is important in understanding how repair occurs and how the structure and function of the valve leaflet are restored. We hypothesize that several conditions including cell density, time in culture, and the establishment of cell-cell contacts containing N-cadherin and β-catenin [124] are involved in regulating VIC activation in vitro and that this activation is associated with the presence of TGF-β. 2.2 Materials and Methods VIC cultures Porcine heart valves were obtained from a local abattoir, and explants were prepared from the distal third of the anterior leaflet of porcine mitral valves as previously described [83]. The atrial and ventricular surfaces of the explants were scraped with a scalpel blade and rinsed with

26 16 phosphate buffered saline (PBS ph 7.4) to remove valve endothelial cells. The explants were cut into 4x5-mm pieces, placed in 9-cm tissue culture dishes (Falcon; BD Biosciences, San Jose, CA, USA), and grown in standard medium, medium 199 (M-199), supplemented with 10% fetal bovine serum (FBS), and 2% penicillin, streptomycin, and Fungizone (GIBCO BRL, Life Technologies Inc, Rockville, MD, USA) in a humidified 95% air and 5% carbon dioxide atmosphere in an incubator at 37 C. VICs that grew out of the explants were detached with TrypLE Express (GIBCO; Invitrogen Corporation, Carlsbad, CA, USA) and subcultured. VICs between passages 3 to 5 were used Cell density and VIC activation VICs were plated on 22x22-mm glass coverslips (Corning Incorporated, Corning, NY, USA) in 35-mm tissue culture dishes at 17,000 cells per coverslip. VICs were incubated for 1, 2, 4, 7, and 10 days in standard media and were fixed and stained for α-sma. To rule out that time in culture to reach confluency affects activation, cells were plated at a 10-fold higher density in order to reach confluency more quickly. The extent of activation marked by staining of VICs plated at 170,000 cells per coverslip was compared to those plated at the lower density of 17,000 cells per coverslip at the same time points of 1, 2, 4, 7, and 10 days. To complement and confirm the activation of VICs indicated by α-sma staining, VICs were plated at 17,000 cells per coverslip; cultured for 2, 5, 8 and 14 days post-plating; fixed; and stained for cofilin. Cofilin is an actin binding protein that depolymerizes actin, which is an important process in actin turnover [175]. Recently, cofilin has been shown to play a role in α- SMA stress fiber formation, to increase in expression in activated VICs with prominent α-sma stress fiber expression, and to colocalize with α-sma in diseased valves [114]. To study the association of TGF-β with cell density and VIC activation, VICs were plated at 17,000 cells per coverslip; cultured for 2, 4, 6, and 8 days post-plating; fixed; and double stained for α-sma and TGF-β. To study the activation of the TGF-β signaling pathway, VICs were plated at 17,000 cells per coverslip; cultured for 2, 4, 6, 8, 10, and 12 days post-plating; fixed; and stained for psmad2/3.

27 17 To study the association of cell-cell contacts with VIC activation, subconfluent (6-7 days postplating) and confluent monolayers (10-11 days post-plating) of VICs were fixed and stained for β-catenin, N-cadherin, and α-sma Immunofluorescent staining and confocal microscopy VICs were fixed with 4% paraformaldehyde for 15 min, rinsed in PBS, incubated with 0.1% or 0.2% Triton X-100 in PBS for 5 minutes, and washed in PBS 3 times at 5-min intervals. The coverslips were incubated with anti-α-sma (1:500; Sigma Chemical Co., Germany), anti-cofilin (1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-tgf-β (1:100; Santa Cruz Biotechnology Inc.), anti-psmad2/3 (1:50; Santa Cruz Biotechnology Inc.), anti- -catenin (1:50; Santa Cruz Biotechnology Inc.), and anti-n-cadherin (1:200; BD Transduction Laboratories, Lexington, KY, USA) primary antibodies at room temperature. The coverslips were washed 3 times with PBS at 5-min intervals. Secondary antibodies donkey anti-mouse Alexa 488 (Invitrogen Corporation), goat anti-mouse Alexa 488 (Invitrogen Corporation), donkey anti-goat Alexa 488 (Invitrogen Corporation), goat anti-mouse Alexa 568 (Invitrogen Corporation), and donkey anti-mouse Cy3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) were applied to washed coverslips and incubated for 20 minutes. Nuclei were stained with Hoescht (Invitrogen Corporation). The coverslips were then washed again three times with PBS at 5-min intervals followed by dipping in distilled water. Fluoro Guard reagent (Bio- Rad Laboratories Inc., Hercules, CA, USA) or Prolong Gold (Invitrogen Corporation) was used for mounting of coverslips to glass slides for microscopic observation. For negative control, mouse immunoglobulin G was used as the primary antibody. Coverslips were examined using the 60x objective of a scanning confocal laser imaging system (BioRad MRC 1024; BioRad, Toronto, Ontario, Canada) fitted with an argon-krypton mixed-gas laser with excitation wavelengths of 488, 568, and 647nm, connected to a Nikon Optiphoto microscope (Nikon Canada), or Olympus FluoView 1000 Laser Scanning Confocal Microscope. Serial optical sections were taken at 0.5-μm thickness to include the entire thickness of the cells. The staining of TGF-β, β-catenin, and N-cadherin was described as strong or weak. For β-catenin and N-cadherin, peripheral and cytoplasmic staining was described, as well as nuclear staining

28 18 for β-catenin. The extent of α-sma staining of VICs was characterized as being of High, Low, or Absent. Fifteen fields were examined for each coverslip by starting observation five fields away from the edges of the coverslip and observing every other field of view to avoid examining the same cell more than once. A total of cells were examined per coverslip depending on cell density. VICs were plated in triplicate for each time point, and each experiment was repeated three times Western blot analysis VICs in 20%, 56%, and 100% confluent cultures were scraped into Triton lysis buffer [30mmol/L HEPES at ph 7.4, 100 mmol/l NaCl, 1 mmol/l EGTA, 20 mmol/l NaF, 1% Triton X-100, 1 mmol/l PMSF, 20 μl/ml Protease Inhibitor Cocktail (Roche, Germany), and 1 mmol/l Na 3 VO 4 ]. Samples were dissolved in Laemmli buffer and boiled for 10 minutes. Equal amounts (15 μg) of protein were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes using iblot protein transfer system (Invitrogen Corporation). The membranes were blocked with 0.5% non-fat dry milk (Bio-Rad Laboratories) and Phosphoprotein Blocker (Millipore Corporation, Billerica, MA, USA), and then blotted with anti-phospho-β-catenin (Ser33/37/Thr41) antibody (Cell Signaling Technology Inc.), anti-αtubulin antibody (Sigma-Aldrich, Inc., St. Louis, MO, USA), horseradish peroxidase (HRP)- conjugated anti-rabbit-igg antibody (Jackson ImmunoResearch Laboratories, Inc.), and HRPconjugated anti-mouse-igg antibody (Jackson ImmunoResearch Laboratories, Inc.) using Snap I.D. protein detection system (Millipore Corporation). Immunoreactive bands were visualized using Luminata Classico and Forte Western HRP Substrate (Millipore Corporation) Statistical analysis For each set of experiments, the staining intensity of cells in each cell density group was compared with those of other cell density groups at each time point and in between the time points using two-way analysis of variance. A value of p<0.05 was considered significant. The Bonferroni method was used to reflect multiple comparisons. These statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).

29 Results α-sma expression in VICs decreases as cell density increases VICs plated at 17,000 cells per coverslip are single cells upon plating (Figure 3A), are subconfluent at day 4 (Figure 3C), and become confluent at day 10 (Figure 3E). α-sma staining is at High Intensity at day 1 post-plating (Figure 3A). It decreases at day 2 compared with day 1 (Figures 3B and 4A-B), and decreases furthermore at day 4 compared to day 2 (Figures 3C and 4B-C). At day 7, VICs show α-sma staining of Low Intensity (Figure 3D). VICs do not show α- SMA staining in the confluent monolayer at day 10 post-plating (Figure 3E). VICs plated at a higher density of 170,000 cells per coverslip show a comparable pattern of loss of activation with increasing cell density (Figure 3F-J). Upon plating, some VICs are single cells while some are in contact with other cells (Figure 3F). They become confluent at day 7 postplating (Figure 3I). α-sma staining is at High Intensity at day 1 post-plating (Figure 3F). It decreases at day 2 compared with day 1 (Figure 3G). At day 4, VICs show α-sma staining of Low Intensity (Figure 3H). VICs show minimal α-sma staining in the confluent monolayer at days 7 and 10 post-plating (Figure 3I-J). To rule out that time in culture to reach confluency affects activation, the α-sma staining intensity of cells plated at 17,000 cells/coverslip is compared with cells plated at 170,000 cells/coverslip at days 1, 2 and 4 post-plating, which are the time points before the cultures reach confluence. At days 1, 2, and 4 post-plating, VICs show more High Intensity staining at each time point in cells plated at 17,000 cells/coverslip compared with those plated at 170,000 cells/coverslip (Figure 4A-C). The level of cofilin is high in low density VIC cultures at day 2 post-plating, and its level decreases over time as the density of VICs increases, indicating a decrease in VIC activation with increased density (Figure 5A-D). However, the change in cofilin staining is not as striking as α-sma. Since α-sma expression is the most widely used and accepted marker of VIC activation, it is used for the remainder of the study.

30 TGF-β signaling in VICs decreases concurrently with a decrease in α- SMA expression as cell density increases VICs plated at 17,000 cells/coverslip show strong TGF-β staining at day 2 post-plating (Figure 6B), at which time α-sma staining is at High Intensity (Figure 6A). At days 4 and 6 postplating, as VIC cell density increases, TGF-β staining intensity decreases compared with day 2 (Figure 6D, F), concurrently with decreases in α-sma staining intensity compared with day 2 (Figure 6C, E). At day 8 post-plating, VICs in the confluent monolayer show weak TGF-β staining (Figure 6H), parallel to Low Intensity α-sma staining (Figure 6G). At low cell density, which is associated with VIC activation and increased TGF-β staining, strong nuclear psmad2/3 staining can be observed in VICs (Figure 7A). As cell density increases, the number of VICs with nuclear psmad2/3 staining decreases, and for the VICs that still have nuclear psmad2/3 staining, the intensity decreases (Figure 7B-E). Finally, at confluency, which is associated with VIC quiescence and decreased TGF-β staining, psmad2/3 staining is predominantly present in the cytoplasm and not in the nucleus (Figure 7F) Absence or loss of cell-cell contacts increases α-sma staining intensity In single VICs, along with High Intensity staining for α-sma, strong staining for β-catenin and N-cadherin can be observed in the cytoplasm, especially around lamellapodia (Figure 8A, E, I, M). β-catenin staining is also found in the nuclear or perinuclear region of some cells (Figure 8A, I). As VICs establish contacts with each other, strong staining of β-catenin and N-cadherin is found to colocalize at cell-cell contacts, while α-sma staining remains predominantly at High Intensity (Figure 8B, F, J, N). In larger VIC islands, more cell-cell contacts are established among VICs, and compared with single VICs, the cytoplasmic staining for β-catenin and N- cadherin is weaker (Figure 8C, G, K). Also, nuclear or perinuclear staining for β-catenin can no longer be observed, and α-sma staining is much decreased (Figure 8C, G, K, O). Finally, in confluent monolayers, each VIC is completely surrounded by strongly stained cell-cell contacts with β-catenin and N-cadherin, and there is very weak cytoplasmic staining of β-catenin and N- cadherin, and Low Intensity staining of α-sma (Figure 8D, H, L, P).

31 Phospho-β-catenin is reduced in confluent VIC cultures When whole-cell lysates from subconfluent (20% and 56% confluence) and confluent VIC cultures are blotted for phospho-β-catenin at serine 33, serine 37, and threonine 41, decreased level of phospho-β-catenin is detected in confluent cultures (Figure 9). 2.4 Discussion We have shown that activation of VICs in vitro is associated with cell density of the culture and the establishment of cell-cell contacts. We also showed that time in culture does not influence activation. Although on histological examination of the normal valve, VICs appear to reside as single cells, we have previously shown using ultrastructural studies that VICs extend cytoplasmic processes which adhere to neighbouring cells forming adhesion junctions [123]. We have also shown that these adhesion junctions are present in confluent monolayers of VICs in cell culture [123]. However, the role of these adhesion junctions in regulating VIC function is unknown. We have shown that VICs are activated and express α-sma in response to a mechanical injury made in a confluent monolayer culture of early passage VICs [101]. These confluent cultures of early passage VICs are normally not activated even if they are treated with TGF-β, a potent promoter of mesenchymal cell activation [101]. On the other hand, we reported that single VICs cultured at low density are activated and express α-sma [32]. Together, our data suggest that absence of cell-cell junctions is a signal for VIC expression of α-sma and other genes that characterize VIC activation, while the establishment of cell-cell junctions in confluent monolayer culture may be important in regulating the switch of the VIC phenotype from the activated to the quiescent state. The data in this study showing increased VIC activation at low density when little cell-cell contact is present and VIC quiescence at confluency with full establishment of cell-cell contacts support this hypothesis. β-catenin and N-cadherin are present at the adhering junctions of VICs [124]. However, besides being a component of intact adherens junctions bound to the cytoplasmic tail of cadherins, β- catenin is also a well-known mediator of the Wnt signaling pathway [ ]. Normally, the level of free cytoplasmic β-catenin is kept low by the Axin/CK1/GSK3/APC destruction

32 22 complex, which phosphorylates it for proteosomal degradation [ ]. However, upon activation of the Wnt pathway, free cytoplasmic β-catenin is stabilized and can travel to the nucleus to act as a transcriptional co-activator where it associates with other factors including the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of proteins to regulate processes such as cell fate determination and proliferation [ ]. The Wnt/β-catenin pathway has been implicated in the development of many tissues and organs, including heart valves [70,76,103]. Dysregulation of this pathway, for example by mutations in β-catenin that allow it to escape degradation, has been implicated in diseases, especially cancers [ ]. Interestingly, there seems to be a degree of crosstalk between the Wnt/β-catenin and TGF-β pathways [176]. It has been shown that in confluent renal tubular epithelial cells, free β-catenin resulting from contact disassembly is associated with α-sma promoter activation and protein expression. In addition, TGF-β augments β-catenin mediated transcription by reducing proteosomal degradation of cytosolic β-catenin [135]. In the heart valve in vitro, Wnt3a has been shown to potentiate the ability of TGF-β to increase α-sma expression and activate VICs [177]. Results in this chapter show that β-catenin distribution pattern and TGF-β expression correlate with activation of the myofibroblast phenotype in VICs. Consistent with this model, at low cell densities with no cellcell contacts where there is highest VIC activation, β-catenin is localized to the cytoplasm and nucleus, and TGF-β expression is increased. In addition, upon confluence, when β-catenin is mostly present in the fully established cell-cell junctions, low in the cytoplasm, absent in the nucleus, with minimal TGF-β expression, VICs are quiescent. Thus, it is likely that the integrity of cell-cell junctions may be important in regulating VIC activation. Western blot analysis of whole-cell lysates from sub-confluent and confluent VIC cultures suggests reduced phosphorylation of β-catenin in confluent cultures. A change in total β-catenin protein levels in VIC cultures of different densities was not observed (data not shown). Even though confluent cultures show decreased levels of the form of β-catenin phosphorylated by CK1 and GSK3β and targeted for proteosomal degradation, this does not necessarily indicate decreased β-catenin degradation in confluent cultures, especially since a change in total β-catenin is not detected. There are many other mechanisms regulating the function of β-catenin besides phosphorylation at Ser33, 37 and Thr41 [169]. Also, merely decreasing the proteosomal degradation of β-catenin does not automatically result in increased nuclear translocation [169]. It

33 23 has been shown that unphosphorylated β-catenin can bind to both cadherins and TCF transcription factors [169]. In conclusion, VIC activation decreases as cell density increases and more cell-cell contacts are established, and this is associated with a concurrent decrease in the level of TGF-β. The highdensity and confluent cultures are considered to be an experimental model of the quiescent nondiseased heart valve, while the low-density cultures in which there is no or very minimal cell-cell interaction is considered to be the model of cell injury in which there is a loss of cell-cell contacts. Thus in valve repair where surgical procedures are becoming much more common, the initial response of VICs will likely be activation at the site of injury where there is less cell-cell contact initially. The surgeon expects that the physiological mechanisms present in the valve will limit the activation once more cells are present and result in a good healing outcome. Our model is being used to study this switch from activated to non-activated VICs. In addition, in the culture dish, we see that increased cell density and confluency turn off cell activation; however, in diseased valves, the cells continue to be activated. Thus, our tissue culture model is useful to study the mechanisms of de-activation of VICs, which could then be translated to reduce disease burden in the valve.

34 Chapter 3: Wnt3a/β-catenin increases proliferation in heart valve interstitial cells 24

35 Introduction β-catenin plays two roles in VICs. On one hand, it acts as an adhesion protein bound to the cytoplasmic tail of cadherins to establish cell-cell contacts [125,126]. On the other hand, it acts as a downstream signalling molecule of the Wnt pathway (Figure 2). For comprehensive reviews of the Wnt/β-catenin pathway, please refer to Logan and Nusse 2004, Clevers 2006, MacDonald et al, 2009, and Rao and Kuhl As well, updated information on Wnt signalling can be found on the Wnt homepage at Here is a brief description of the Wnt/β-catenin signalling pathway. Normally, the level of free cytoplasmic β-catenin is kept low by the Axin complex, which includes the scaffolding protein Axin, the tumour suppressor adenomatous polyposis coli gene product (APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). CK1 and GSK3 sequentially phosphorylate the amino terminal region of β-catenin, resulting in β-catenin recognition by β-trcp (beta-transducin repeat-containing protein), an E3 ubiquitin ligase subunit, and subsequent β-catenin ubiquitination and proteosomal degradation. However, in the presence of Wnt, Wnt binds to Frizzled and Lrp5/6 and the β-catenin degradation complex is disassembled, a process which involves phosphorylation of Lrp5/6, recruitment of Dishevelled, and recruitment of Axin away from the degradation complex. This leads to the stabilization of β-catenin in the cytosol, allowing it to travel to the nucleus to act as a transcriptional co-activator. The main partner for β-catenin in gene regulation is the T cell factor/lymphoid enhancer factor (TCF/LEF) family of proteins. In the absence of β-catenin, TCF/LEF transcription factors bind to Wnt response elements and recruit co-repressors such as Groucho and histone deacetylases. However, when β-catenin is in the nucleus, it binds to TCF/LEF, displaces transcriptional repressors, and recruits transcriptional activators such as chromatin remodelling complex swi/snf. The Wnt/β-catenin pathway has been implicated in the development of many tissues and organs, including heart valves where it was shown to regulate proliferation and endothelial-mesenchymal transition [70,76,164]. In valve diseases, genes and mechanisms important in development but downregulated in adulthood become upregulated [69,81, ]. For instance, there is very little or no proliferation in adult valves, but proliferation is increased in diseased valves, such as in areas around calcified nodules [102]. Also, only mild staining of Wnt3, Lrp5, and minimal

36 26 expression of β-catenin are found in normal valves [34,167]. However, in diseased valves, especially calcified valves, there is increased expression of Wnt3, Lrp5 and β-catenin [34,167]. Wnt/β-catenin pathway is implicated in regulating proliferation in several pathologies, including intestinal epithelial cells and smooth muscle cells, and downstream targets include c-myc, cyclin D1, and p21 [164,166,181,182]. Using an in vitro wound model, we have shown previously that VICs along a wound edge become activated, represented by increased expression of α-smooth muscle actin (α-sma) stress fibres, and undergo increased proliferation [101]. Therefore, it is possible for the Wnt/β-catenin pathway, upregulated in disease, to regulate proliferation in VICs in an attempt to mediate wound repair. 3.2 Materials and Methods Cell culture Porcine hearts were obtained from a local abattoir, and explants were prepared from the distal third of the anterior leaflet of porcine mitral valves as previously described [83]. The atrial and ventricular surfaces of the explants were scraped with a scalpel blade and rinsed with phosphate buffered saline (PBS ph 7.4) to remove valve endothelial cells. The explants were cut into 4x5- mm pieces, placed in 9-cm tissue culture dishes (Falcon; BD Biosciences, San Jose, CA, USA), and grown in standard medium, medium 199 (M-199), supplemented with 10% fetal bovine serum (FBS), and 2% penicillin, streptomycin and Fungizone (GIBCO BRL, Life Technologies Inc, Rockville, MD, USA) in a humidified 95% air and 5% carbon dioxide atmosphere in an incubator at 37 C. VICs which grew out of the explants were detached with TrypLE Express (GIBCO; Invitrogen Corporation, Carlsbad, CA, USA) and subcultured. VICs between passages 3 to 5 were used Treatment with exogenous Wnt3a Recombinant mouse Wnt3a (R&D Systems, MN, USA) was reconstituted in sterile PBS containing 0.1% bovine serum albumin. Media containing vehicle or 150 ng/ml of recombinant mouse Wnt3a was added to cultures [103]. Wnt3a was replenished every 48 hours if treatment length exceeded 2 days.

37 Immunofluorescent staining and confocal microscopy VICs were plated at 37,000 cells per 18-mm glass coverslip in 12-well tissue culture plates, and coverslips were fixed and immunofluorescently stained for β-catenin and α-sma at days 1, 3, 6, and 9 post-treatment with vehicle or Wnt3a. VICs were fixed with 4% paraformaldehyde for 15 minutes, rinsed in PBS, incubated with 0.1% Triton X-100 in PBS for 5 minutes, and washed in PBS 3 times at 5-minute intervals. The coverslips were incubated with mouse anti-α-sma (1:500; (Sigma Chemical Co., Germany) and anti- -catenin (1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) primary antibodies at room temperature for 1 hour. The coverslips were washed 3 times with PBS at 5-minute intervals. Secondary antibodies donkey anti-goat Alexa 488 (1:200; Invitrogen Corporation) and goat anti-mouse Alexa 568 (1:200; Invitrogen Corporation) along with nuclear dye Hoescht (1:500; Lonza, MD, USA) were applied to washed coverslips and incubated at room temperature for 20 minutes. The coverslips were then washed again three times with PBS at 5-minute intervals followed by dipping in distilled water. Prolong Gold (Invitrogen Corporation) was used for mounting of coverslips on glass slides for microscopic observation. Coverslips were examined using the 60x objective of Olympus FluoView 1000 Laser Scanning Confocal Microscope. Serial optical sections were taken at 0.5- μm thickness to include the entire thickness of the cells Cell count VICs were plated in 35-mm dishes at 20,000 cells per dish and allowed to grow for 3 days to 4-6% confluence. The cells were then treated with 150 ng/ml of Wnt3a for 4 days and then counted using Countess Cell Counter (Invitrogen Corporation) Western blot VICs were plated in 100-mm dishes at 200,000 cells per dish and allowed to grow for 3 days to 4-6% confluence. The cells were then treated with 150 ng/ml of Wnt3a for 4 days and then lysed. VICs were scraped into Triton lysis buffer to prepare whole cell lysates [30 mmol/l HEPES at ph 7.4, 100 mmol/l NaCl, 1 mmol/l EGTA, 20 mmol/l NaF, 1% Triton X-100, 1 mmol/l PMSF, 20 μl/ml Protease Inhibitory Cocktail (Roche, Germany), and 1 mmol/l Na 3 VO 4 ]. Whole cell lysate was centrifuged at 4 C at 13,000 rpm for 5 minutes and the supernatant was saved as

38 28 cytosolic lysate. Nuclear lysate was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to manufacturer s instructions (Thermo Fisher Scientific, USA). Prepared lysates were dissolved in Laemmli buffer and boiled for 10 minutes. Equal amounts of proteins were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to PVDF membranes using iblot protein transfer system (Invitrogen Corporation). The membranes were blocked with 0.5% non-fat dry milk (Bio-Rad Laboratories Inc., Hercules, CA, USA), and then blotted with rabbit anti-β-catenin antibody (Santa Cruz Biotechnology Inc.), mouse anti-tubulin antibody (Sigma-Aldrich, Inc., St. Louis, MO, USA), and mouse anti-histone H1 antibody (Santa Cruz Biotechnology Inc.) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit-igg antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and HRPconjugated anti-mouse-igg antibody (Jackson ImmunoResearch Laboratories, Inc.) using Snap I.D. protein detection system (Millipore Corporation, Billerica, MA, USA). Immunoreactive bands were visualized using Luminata Classico and Crescendo Western HRP Substrate (Millipore Corporation). Densitometry was performed using ImageJ software (NIH, USA). Values were presented normalized against loading control Small interfering RNA transfection Small interfering (si)rnas targeting β-catenin were obtained from Sigma-Aldrich (SASI_Hs_01_ , SASI_Hs_01_ ); negative control sirnas were obtained from Ambion (Streetsville, ON) ( ). The annealed duplexes were prepared as recommended by manufacturers. The sense strands of the synthetic oligonucleotide duplexes targeting β-catenin were CUCAGAUGGUGUCUGCUAU (SASI_Hs_01_ ) and GUUAUGGUCCAUCAGCUUU (SASI_Hs_01_ ). VICs were plated at cells/cm 2 in 6-well 35-mm diameter plates 24 hours prior to transfection. To silence β-catenin expression, VICs were transfected with 40 nm of a mixture of both sirna duplexes targeting β-catenin mentioned above (20 nm each) using N-TER TM nanoparticle sirna transfection system (Sigma-Aldrich, Inc., N2913) according to the

39 29 manufacturer s protocol. Cells only buffer treatment and 40 nm of negative control sirna were used as controls. VICs were transfected in complete medium for 30 hours. To assess the transfection efficiency, VICs were transfected with 20nM and 40nM of fluorescent oligonucleotides, siglo Red (Thermo Fischer Scientific, USA), using N-TER TM nanoparticle sirna transfection system in complete medium for 24 and 48 hours. Immediately after transfection, cells were fixed in 4% paraformaldehyde, counterstained with Hoescht (Lonza), mounted on microscope slides with Prolong Gold (Lonza), and viewed under Olympus FluoView 1000 Laser Scanning Confocal Microscope. To assess the efficiency of β-catenin knockdown, VICs grown in 6-well plates were immediately lysed after transfection in Triton lysis buffer as described above. Cell lysates were centrifuged at 4 C at 13,000 rpm for 10 minutes and the supernatant was analyzed by Western blotting as described above Quantification of proliferation VICs were plated at cells/cm 2 on 18-mm round glass coverslips in 12-well plates 24 hours prior to transfection. VICs were transfected with cells only buffer, negative control sirna, or β-catenin targeted sirna as described above for 30 hours. Afterwards, VICs were treated with vehicle or Wnt3a for 24 hours. Bromodeoxyuridine (BrdU) labeling agent (Sigma-Aldrich, Inc.) was added in 1:500 dilution to VIC cultures for 2 hours at 22 hours post-treatment with vehicle or Wnt3a. At 24 hours posttreatment, the coverslips were washed 3 times in serum-free M-199 media, fixed in ethanol:acetic acid (95:5) at 4 C for 20 minutes, and washed in PBS 3 times at 5-minute intervals. Then the coverslips were incubated with 0.2% Triton X-100 in PBS for 5 minutes and washed in PBS 3 times at 5-minute intervals. Afterwards, the coverslips were incubated in 2N HCl at 37 C for 30 minutes, washed in 0.1M sodium borate buffer (ph=8.5) twice at 5-minute intervals, and washed in PBS 3 times at 5-minute intervals. The coverslips were incubated with mouse anti-brdu (1:500; Sigma-Aldrich, Inc.) primary antibody at 37 C for 2 hours. The coverslips were washed 3 times with PBS at 5-minute intervals. Secondary antibody donkey antimouse Alexa 488 (1:200; Invitrogen Corporation) along with nuclear dye propidium iodide

40 30 (1:500; Sigma-Aldrich, Inc.) were applied to washed coverslips and incubated for 20 minutes at room temperature. The coverslips were then washed again three times with PBS at 5-minute intervals followed by dipping in distilled water. Prolong Gold (Invitrogen Corporation) was used for mounting of coverslips onto glass slides for microscopic observation and counting. Random counting of both total and BrdU-positive VICs was performed using the 40x objective of a fluorescent microscope (Carl Zeiss Inc., West Germany) starting five fields away from the edge of the coverslip and was continued linearly along the center of the coverslip counting cells in every other field of view to avoid examining the same cell more than once. Proliferation was calculated as a percentage of BrdU-positive cells out of the total number of VICs Quantification of apoptosis VICs were plated at cells/cm 2 on 18-mm round glass coverslips in 12-well plates 24 hours prior to transfection. VICs were transfected with cells only buffer, negative control sirna, or β-catenin targeted sirna as described for 30 hours. Afterwards, VICs were treated with vehicle or Wnt3a for 24 hours. Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) was performed using the TACS TdT-fluorescein in situ apoptosis detection kit (Trevigen Inc., MD) to identify apoptotic VICs according to the manufacturer s instructions. VICs were counterstained with Hoescht (Lonza). Both positive control and negative control were performed according to the manufacturer s instructions. Positive control was performed by treating the cells with nuclease to create DNA breaks in every cell. Negative control was performed by omitting the enzyme required to incorporate nucleotides into DNA breaks. Random counting of both total and TUNEL-positive VICs was performed using the 40x objective of a fluorescent microscope (Carl Zeiss Inc.) starting five fields away from the edge of the coverslip and was continued linearly along the center of the coverslip counting cells in every other field of view to avoid examining the same cell more than once. Apoptosis was calculated as a percentage of TUNEL-positive cells out of the total number of VICs.

41 TOPFLASH/FOPFLASH reporter assay VICs were plated on 12-well tissue culture plates at 300,000 cells per well and simultaneously transfected with TCF-responsive TOPFLASH reporter construct, which contains 3 TCF binding sites, or the corresponding FOPFLASH construct, which contains 3 mutated TCF sites. A β- galactosidase expression vector served as a control for transfection efficiency. TOPFLASH and FOPFLASH reporter constructs as well as β-galactosidase expression vectors were kindly provided by Dr. Benjamin Alman s lab. Transfections were performed using Lipofectamine 2000 (Invitrogen Corporation) transfection reagent according to the manufacturer s instructions. VICs were transfected for 30 hours in M-199 supplemented with 10% FBS, and 2% penicillin, streptomycin and Fungizone. Afterwards, VICs were allowed to recover in fresh media for 18 hours and then treated with vehicle or Wnt3a for 24 hours. VICs were lysed, and cell lysates were assayed for luciferase activity and β-galactosidase activity on a luminometer using the Dual-Light Chemiluminescent Reporter Gene Assay System (Applied Biosystems, MA, USA) according to the manufacturer s instructions. TOPFLASH and FOPFLASH luciferase activities were normalized to β-galactosidase activity and the background β-galactosidase activity was substracted. Reporter activity was calculated as a ratio of TOPFLASH:FOPFLASH Statistical analysis Statistical analyses were performed using GraphPad Prism version 5 software (GraphPad Software Inc., San Diego, USA). A value of P<0.05 was considered significant. One-way analysis of variance (ANOVA) or repeated measures ANOVA followed by Tukey s Multiple Comparison Test was used for comparison of multiple groups. T-test was used for comparison of two groups. 3.3 Results Wnt3a treatment increases β-catenin staining intensity in VICs Upon treatment of low density cultures with 150 ng/ml of Wnt3a, compared with control and vehicle-treated cultures, slightly increased staining of β-catenin is observed overall at day 1, which becomes more visible by day 3 (Figure 10A-F). However, it is difficult to ascertain the specific location of the increased staining. This increase in β-catenin staining becomes more

42 32 pronounced at days 6 and 9 post-treatment (Figure 10G-L). There seems to be increased cytosolic and nuclear β-catenin staining that contributes to the overall increase in staining intensity (Figure 10G-L). At all time points post-treatment, control and vehicle-treated cultures demonstrate similar intensity and pattern of β-catenin staining (Figure 10A, B, D, E, G, H, J, K). No effect is observed on VIC activation, assessed by α-sma staining (Figure 11) Wnt3a treatment increases VIC cell number After Wnt3a treatment, an increase in cell number can be observed starting at day 3 posttreatment compared with control and vehicle-treated cultures (Figure 12). At 4 days posttreatment, VIC cell counts show a significant increase (p<0.05) in cell number in Wnt3a-treated cultures compared with control and vehicle-treated cultures (Figure 13). No difference is observed between control and vehicle-treated cultures (Figure 12,13). Based on trypan blue staining, no difference in cell viability is observed among the 3 conditions (Figure 14) Wnt3a treatment increases whole cell, cytosolic and nuclear protein levels of β-catenin After 4 days of treatment with Wnt3a, whole cell lysates, cytosolic lysates, and nuclear extracts of VIC cultures show increased protein levels of β-catenin compared with control and vehicletreated cultures (Figure 15,16). The absence of histone H1 protein in cytosolic lysates and the absence of α-tubulin in nuclear lysates indicate good separation of cytosolic and nuclear proteins (Figure 15A, 16A). Densitometry measurements show a statistically significant (p<0.05) increase in β-catenin protein levels in Wnt3a-treated cultures in both whole cell and nuclear lysates (Figure 15B, 16B). The increase in nuclear β-catenin is especially prominent after Wnt3a treatment (Figure 16B). No difference is observed between control and vehicle-treated cultures (Figure 15,16) Wnt3a treatment increases VIC proliferation; an effect dependent on β-catenin To ascertain whether the effects observed with Wnt3a treatment are specifically due to the actions of β-catenin, sirna is used to knockdown β-catenin in VICs. Transfection efficiency is approximately 100% (data not shown). Compared with cells only and negative control sirna

43 33 cultures, which show similar levels of β-catenin protein, β-catenin targeted sirna decreases the level of β-catenin protein significantly (p<0.05) by approximately 50-60% (Figure 17). BrdU staining is used to determine whether the increase in total cell number observed with Wnt3a treatment is due to increased proliferation. In vehicle-treated cultures, the percentage of BrdU-positive VICs is similar in cells only and negative control sirna cultures, and only a slight decrease is observed in β-catenin targeted sirna cultures (Figure 18). Wnt3a treatment produces a significant increase (p<0.05) in the percentage of BrdU-positive VICs in cells only and negative control sirna cultures compared with vehicle treatment (Figure 18). The increase is similar in both cells only and negative control sirna cultures (Figure 18). However, this increase is completely abolished in the presence of β-catenin targeted sirna (p<0.05, Figure 18) Wnt3a treatment does not affect VIC apoptosis To determine whether apoptosis affects the increase in cell number observed with Wnt3a treatment, TUNEL assay is used to measure the percentage of apoptotic nuclei. An apoptosis rate of less than 5% is observed in all cultures (Figure 19). No difference is observed between vehicle and Wnt3a treated cultures (Figure 19). The percentage of apoptotic VICs appears slightly higher in sirna-treated cultures compared to cells only cultures in both vehicle and Wnt3a cultures, but this is not statistically significant (Figure 19). Positive control shows an apoptosis rate of approximately 100% and negative control shows an apoptosis rate of approximately 0% (data not shown) Wnt3a treatment increases β-catenin-tcf-mediated transcription There is a significant increase (p<0.05) in TOPFLASH/FOPFLASH reporter activity of at least 2 fold in Wnt3a-treated cultures compared with vehicle control cultures, indicating an increase in β-catenin-tcf-mediated transcription (Figure 20). 3.4 Discussion Wnt3a increases VIC cell number without affecting cell viability. More specifically, Wnt3a increases the percentage of BrdU-positive VICs, indicating an increase in proliferation. The

44 34 minimal level of apoptosis and the lack of effect by Wnt3a on the percentage of TUNEL-positive VICs further confirm proliferation as the process responsible for increases in VIC cell number after Wnt3a treatment. Proliferation is specifically abolished by β-catenin targeted sirna, suggesting a β-catenin specific effect. Interestingly, β-catenin sirna only produces a mild decrease in the percentage of BrdU-positive VICs in vehicle-treated cultures. This observation suggests that β-catenin is not be required for VIC proliferation under normal conditions but specifically mediates the proliferative effects of Wnt3a, which is elevated in disease conditions [34,167]. Wnt3a does not seem to affect VIC activation, assessed by α-sma staining. This is not surprising because recent research shows that Wnt3a alone is not sufficient to induce VIC activation [177]. Instead, it potentiates the ability of TGF-β to activate VICs such that there is a synergistic increase in VIC activation when Wnt3a and TGF-β treatments are combined [177]. Such a phenomenon is also demonstrated in other cell types [135]. Both immunofluorescent data and Western blot results indicate that Wnt3a increases β-catenin levels in VICs. Cell lysate fractionation and subsequent Western blot analysis show that Wnt3a increases β-catenin protein levels in whole cell, cytosolic and nuclear lysates, suggesting that Wnt3a treatment may lead to stabilization of cytosolic β-catenin, thereby allowing it to translocate to the nucleus to mediate transcription. Even though the increase in β-catenin protein level in the cytosol is not statistically significant based on densitometry analysis, cytosolic stabilization is a temporary step leading towards eventual nuclear translocation, therefore the increase in β-catenin protein might be not as dramatic. Importantly, after Wnt3a treatment, the increase in β-catenin is especially prominent in the nucleus and TOPFLASH/FOPFLASH reporter activity is significantly increased in VICs, indicating an increase in β-catenin-mediated transcription. In conclusion, the results obtained so far suggest that Wnt3a increases VIC proliferation through an increase in β-catenin-mediated transcription.

45 Chapter 4: Discussion/Future Directions 35

46 36 The common molecule involved in both VIC activation and proliferation appears to be β-catenin. It is both associated with VIC activation and increased TGF-β signaling and shown to regulate VIC proliferation downstream of Wnt3a signaling. The proliferative effect of Wnt3a/β-catenin signaling is somewhat an unexpected finding, because the increased VIC activation and nuclear staining of β-catenin in low density VIC cultures suggests a role for β-catenin in VIC activation. However, this can be explained by recent evidence that suggests Wnt3a alone is not sufficient to induce α-sma transcription or VIC activation [177]. Instead, the increase in β-catenin after Wnt3a treatment potentiates the ability of TGF-β to induce greater α-sma transcription and VIC activation than TGF-β treatment alone [177]. TGF-β-induced α-sma transcription and VIC activation is shown to depend on the availability of β-catenin [177]. A similar phenomenon is observed in other cell types. Research in kidney epithelial cells shows that β-catenin plays a strong potentiating and synergistic role in TGF-β-induced activation of α-sma promoter activity and protein expression [135]. TGF-β is shown to prevent proteosomal degradation of β-catenin and induce its nuclear translocation and β-catenin-mediated transcription [135,183]. The in vitro evidence in VICs and kidney epithelial cells suggests a crosstalk between TGF-β and Wnt signaling pathways in the regulation of VIC activation. A similar crosstalk might regulate VIC proliferation as well, since Wnt3a/β-catenin is shown to increase VIC proliferation in this study and TGF-β has been previously shown to increase VIC proliferation along an in vitro wound edge [101]. The in vitro findings are supported by observations in diseased valves where both TGF-β/Smad and Wnt/β-catenin signaling components are upregulated, along with VIC activation and proliferation. Thus, a crosstalk between TGF-β and Wnt signaling pathways may be involved in regulating VIC responses to injury in disease conditions [177]. The crosstalk between the TGF-β/Smad and Wnt/β-catenin pathways is poorly understood. TGFβ1 has been shown to promote the association of Smad4 and 3 with β-catenin [184]. Smad3 has been reported to interact with β-catenin to form a protein complex [184,185], and the phosphorylation of Smad2/3 is shown to be required for TGF-β1-induced β-catenin nuclear translocation [177]. The formation of a Smad3/β-catenin complex has been shown to protect β- catenin from proteasome degradation as well as facilitate β-catenin nuclear translocation and transcriptional activity [185]. Within the nucleus, Smad binding element and TCF/LEF can physically interact on binding to Smad3 and β-catenin, possibly initiating a cooperative

47 37 regulation [ ]. Therefore, it is possible that both pathways are activated in valve injury and disease and cooperate to regulate important repair mechanisms, such as VIC activation and proliferation. Future experiments should explore the possible synergistic regulation by TGF-β and Wnt3a in VICs in models of wound repair. Our lab has already investigated the effects of TGF-β on VICs in the in vitro scratch wound model. To extend upon those findings and the results in this thesis, Wnt3a treatment can be administered alone and together with TGF-β to measure VIC activation, proliferation, apoptosis, and wound closure at the wound edge, in areas away from the wound edge, and in confluent non-wounded monolayers. In addition, downstream genes and molecules involved in Wnt3a/β-catenin signaling and VIC proliferation can be explored, such as cyclin D1 and p21 [166]. These proposed future directions will study interactions and synergies operating to regulate VIC function and to identify proteins that need to be targeted to prevent the excessive repair which leads to clinical dysfunction. In addition, it is important to investigate whether Wnt3a/β-catenin signaling may regulate genes important in calcification since it is upregulated in CAS [34]. TGF-β, also upregulated in CAS, has been shown to induce calcification of sheep aortic VICs in culture and to increase the expression of alkaline phosphatase, a marker of osteoblasts and differentiating bone [189]. However, investigations on Wnt3a/β-catenin in CAS are lacking. In embryonic chicken aortic VICs in vitro, Wnt3a treatment does not induce expression of Runx2, osteocalcin and alkaline phosphatase, which are all markers of osteoblasts and differentiating bone [103]. However, Wnt3a signaling is shown to stimulate osteoblastogenesis and decreased Wnt signaling is associated with low bone mass [190,191]. It is important to investigate whether Wnt3a/β-catenin induces expression of genes important in CAS development in adult valves. My experiments are conducted with porcine valves in an in vitro system. Porcine valves are very similar to humans and are the model of choice for human surgical and tissue engineering considerations. However, even though my experiments provide useful and important information, there are limitations. First of all, in these studies, the VICs secrete their own matrix. The VICs are not plated on specific matrixes nor is the thickness or the stiffness of the matrix studied. Recent research suggests that TGF-β-induced β-catenin nuclear translocation and VIC

48 38 activation require a certain amount of matrix stiffness and increase with increasing stiffness [177]. In valve disease, the leaflets undergo fibrosis and they thicken and harden, increasing the stiffness of the environment that VICs reside in, making them more susceptible to activating agents [24]. In my experiments, VICs are directly plated on the surface of glass dishes, which is very stiff and in some ways resemble the stiff environment in disease conditions. However, it is still different from disease conditions. The in vitro system does not include interactions with matrix molecules such as matrix metalloproteinases 9 and 12 that are shown to cleave N- cadherin and induce β-catenin nuclear translocalization, cyclin D1 expression, and vascular smooth muscle cell proliferation [192]. Also, the in vitro system does not include inflammatory cells or hemodynamic forces, both of which are likely to play a role in valve diseases [104]. Hence, it is a simplification of a complex in vivo state [104]. Therefore, it is important to study VIC regulation in in vivo models such as mouse models [193]. In conclusion, the results in this thesis suggest that the loss or absence of cell-cell contacts involving N-cadherin and β-catenin is associated with VIC activation and increased TGF-β signaling, and that the Wnt3a/β-catenin pathway regulates, at least in part, VIC proliferation. Since increased VIC activation and proliferation are likely to be important in wound repair occurring in valve disease, the results increase our understanding of their regulation and suggest that TGF-β and Wnt3a may be important molecules in valve disease that deserve further study and may be targeted by therapy to design better treatments for patients.

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71 61 Figure 1. TGF-β signaling pathways. TGF-β binds to Receptor II, which phosphorylates Receptor I, which then phosphorylates Smad2 and 3 that are able to bind to Smad4 to form a complex that travels to the nucleus to mediate transcription. One of the downstream targets of this canonical pathway is α-sma. TGF-β is also able to signal through PI3K as part of the noncanonical pathway. TGF-β/Smad signaling can be inhibited by Smad6 or 7. Downstream effects of TGF-β signaling include myofibroblast transformation, proliferation, and ECM production. (Adapted from Rahimi and Leof, 2007) [110].

72 62 Figure 2. Wnt/β-catenin signaling. When Wnt is absent, β-catenin level is kept low by the axin degradation complex, which consists of Axin, APC, GSK3β, and CK1. CK1 and GSK3β sequentially phosphorylate serine/threonine residues at the amino terminus of β-catenin, which allows β-catenin to be recognized by β-trcp, an E3 ubiquitin ligase subunit, that then targets β- catenin for proteosomal degradation. When Wnt is present, it binds to Frizzled and LRP5/6, which recruits Dishevelled and disrupts the degradation complex, preventing β-catenin degradation. This allows β-catenin to translocate to the nucleus, displace transcriptional repressors, and bind to TCF/LEF transcription factors to regulate transcription of Wnt responsive genes. (MacDonald et al, 2009) [162].

73 63 Figure 3. Immunofluorescent photomicrographs of valve interstitial cells plated at 17,000 cells/coverslip (A-E) and at 170,000 cells/coverslip (F-J) immunostained for -smooth muscle actin at days 1 (A, F), 2 (B, G), 4 (C, H), 7 (D, I) and 10 (E, J) post-plating. Note that α-smooth muscle actin staining decreases as cell density increases regardless of time in culture. Magnification: 600x. White bar indicates 20 m. VIC, valve interstitial cell.

74 64 Figure 4. Percentage of valve interstitial cells expressing High Intensity -smooth muscle actin staining in monolayer culture at both low density and high density incubated for 1 (A), 2 (B) and 4 days (C) observed under confocal microscopy. The experiment is done in triplicate. Error bars denote standard deviation of the mean. Note that the percentage of High Intensity valve interstitial cells for high density is significantly less than the one for low density at each time point (*). VIC, valve interstitial cell; α-sma, α-smooth muscle actin.

75 65 Figure 5. Immufluorescent images of valve interstitial cells plated at 17,000 cells/coverslip, immunostained for cofilin at days 2 (A), 5 (B), 8 (C), and 14 (D) post-plating. Note that cofilin staining decreases as cell density increases. Magnification: 600x. White bar indicates 20 μm.

76 66 α-sma TGF-β Figure 6. Immunofluorescent photomicrographs of valve interstitial cells plated at 17,000 cells/coverslip, immunostained for -smooth muscle actin (green) and transforming growth factor-β (red) at days 2 (A, B), 4 (C, D), 6 (E, F), and 8 (G, H) post-plating. Note that transforming growth factor-β staining decreases concurrently with α-smooth muscle actin staining as cell density increases. Magnification: 600x. White bar indicates 20 m. α-sma, α- smooth muscle actin; TGF-β, transforming growth factor-β.

77 67 Figure 7. Immufluorescent images of valve interstitial cells plated at 17,000 cells/coverslip, immunostained for psmad2/3 at days 2 (A), 4 (B), 6 (C), 8 (D), 10 (E), and 12 (F) post-plating. Note that nuclear psmad2/3 staining decreases as cell density increases. Magnification: 600x. White bar indicates 20 μm.

78 68 β-catenin N-cadherin N-cadherin and β-catenin α-sma Figure 8. Immunofluorescent images of single valve interstitial cells (A, E, I, M), small groups of valve interstitial cells with recently established cell-cell contacts (B, F, J, N), valve interstitial cell islands (C, G, K, O), and confluent monolayers (D, H, L, P), immunostained for β-catenin (green; A-D), N-cadherin (green; E-H), β-catenin and N-cadherin (green and red respectively; I- L), and α-smooth muscle actin (green; M-P). Cell nuclei are stained blue with Hoescht. Note that as cell density increases, β-catenin and N-cadherin staining increases at cell-cell contacts and decreases in cytoplasm, and nuclear β-catenin staining disappears. Magnification: 600x. White bar indicates 20 µm. α-sma, α-smooth muscle actin.

79 69 Figure 9. Whole-cell protein expression of phospho-β-catenin at Ser33, Ser37, and Thr41 is determined by Western blotting for valve interstitial cell cultures at 20%, 56%, and 100% confluence. α-tubulin is used as the loading control. Note that phospho-β-catenin (Ser33/Ser37/Thr41) protein level is decreased at 100% confluence.

80 70 Control Vehicle Wnt3a Day 1 Day 3 Day 6 Day 9 Figure 10. Immunofluorescent images of valve interstitial cells in control cultures (A, D, G, J), vehicle-treated cultures (B, E, H, K), and Wnt3a-treated cultures (C, F, I, L) at days 1 (A, B, C), 3 (D, E, F), 6 (G, H, I), and 9 (J, K, L) post-treatment immunostained for β-catenin (green). Cell nuclei are stained blue with Hoescht. Note the increase in β-catenin staining in Wnt3a-treated cultures starting at day 3 that becomes more prominent as the treatment time increases. Magnification: 600x.

81 71 Control Vehicle Wnt3a Day 1 Day 3 Day 6 Day 9 Figure 11. Immunofluorescent images of valve interstitial cells in control cultures (A, D, G, J), vehicle-treated cultures (B, E, H, K), and Wnt3a-treated cultures (C, F, I, L) at days 1 (A, B, C), 3 (D, E, F), 6 (G, H, I), and 9 (J, K, L) post-treatment immunostained for α-smooth muscle actin (red). Note the similar staining in all three conditions at each time point. Magnification: 600x.

82 72 Control Vehicle Wnt3a Day 1 Day 3 Day 6 Day 9 Figure 12. Phase contrast images of valve interstitial cells in control cultures (A, D, G, J), vehicle-treated cultures (B, E, H, K), and Wnt3a-treated cultures (C, F, I, L) at days 1 (A, B, C), 3 (D, E, F), 6 (G, H, I), and 9 (J, K, L) post-treatment. Note the increase in cell number in Wnt3a-treated cultures starting at day 3. Magnification: 100x.

83 73 * Figure 13. Total cell number of valve interstitial cells relative to control at 4 days post-treatment in control, vehicle, and Wnt3a treated cultures. Three trials, each with 3 replicates, are performed. Note the increase in cell number after Wnt3a treatment. Error bars denote standard error of the mean. * indicates a statistical significance of p<0.05.

84 74 Percentage of viable cells Control Vehicle Wnt3a Figure 14. Percentage of viable valve interstitial cells based on trypan blue staining at 4 days post-treatment in control, vehicle, and Wnt3a treated cultures. Three trials, each with 3 replicates, are performed. Note the similar viability in all three conditions. Error bars denote standard error of the mean.

85 75 Figure 15. Representative Western blot of protein levels of β-catenin, α-tubulin and histone H1 in whole cell (A, first 3 lanes) and cytosolic lysates (A, last 3 lanes) of valve interstitial cells at 4 days post-treatment in control, vehicle, and Wnt3a treated cultures. Densitometry values relative to control for β-catenin protein levels in whole cell (B) and cytosolic (C) lysates at 4 days posttreatment in control, vehicle, and Wnt3a treated cultures. Results are from 3 experiments. Note the increase in β-catenin protein level in whole cell and cytosolic lysates in Wnt3a-treated cultures. Error bars denote standard error of the mean. * indicates a statistical significance of p<0.05. C, control; V, vehicle; W, Wnt3a.

86 76 Figure 16. A. Representative Western blot of protein levels of β-catenin, histone H1 and α- tubulin in nuclear lysates of valve interstitial cells at 4 days post-treatment in control, vehicle, and Wnt3a treated cultures. B. Densitometry values relative to control for β-catenin protein levels in nuclear lysates at 4 days post-treatment in control, vehicle, and Wnt3a treated cultures. Results are from 3 experiments. Note the increase in β-catenin protein level in nuclear lysates in Wnt3a-treated cultures. Error bars denote standard error of the mean. * indicates a statistical significance of p<0.05. C, control; V, vehicle; W, Wnt3a.

87 77 Figure 17. A. Representative Western blot of protein levels of β-catenin and α-tubulin immediately after 30 hours of sirna transfection in cells only, negative control sirna, and β- catenin targeted sirna cultures. B. Densitometry values relative to cells only for β-catenin protein levels after 30 hours of sirna transfection in cells only, negative control sirna, and β- catenin targeted sirna cultures. Results are from 3 experiments. Note the decrease in β-catenin protein level after β-catenin targeted sirna knockdown. Error bars denote standard error of the mean. * indicates a statistical significance of p<0.05. C, cells only; N, negative control sirna; β, β-catenin targeted sirna.

88 78 Figure 18. Percentage of proliferating valve interstitial cells after 30 hours of sirna transfection followed by 24 hours of treatment with vehicle or Wnt3a in cells only, negative control sirna, and β-catenin targeted sirna cultures. Three trials, each with 2 replicates, are performed. Note Wnt3a treatment increases proliferation of valve interstitial cells in cells only and negative control sirna cultures, but this effect is abolished by β-catenin targeted sirna. Error bars denote standard error of the mean. * indicates statistical significance of p<0.05. BrdU, bromodeoxyuridine.

89 79 Percentage of TUNEL-positive cells Vehicle Cells Only Vehicle Negative sirna Vehicle -Catenin sirna Wnt3a Cells Only Wnt3a Negative sirna Wnt3a -Catenin sirna Figure 19. Percentage of apoptotic valve interstitial cells after 30 hours of sirna transfection followed by 24 hours of treatment with vehicle or Wnt3a in cells only, negative control sirna, and β-catenin targeted sirna cultures. Three trials, each with 2 replicates, are performed. Note there is minimal apoptosis regardless of treatment conditions. Error bars denote standard error of the mean.

90 80 Figure 20. TOPFLASH/FOPFLASH reporter activity relative to vehicle-treated cultures in valve interstitial cells after vehicle or Wnt3a treatment. Results are from 3 experiments. Note the increase in reporter activity, indicating β-catenin-tcf mediated transcription, after Wnt3a treatment. Error bars denote standard deviation. * indicates a statistical significance of p<0.05.