New Insights into the Puzzling Pathogenesis of Calcific Aortic Stenosis

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1 New Insights into the Puzzling Pathogenesis of Calcific Aortic Stenosis Chen Li *, BSc., Songyi Xu *, MSc., Avrum I. Gotlieb, MDCM, FRCPC Departments of Pathology, Toronto General Research Institute, University Health Network, Ontario Canada M5G 1L5 Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada M5G 1L5 * Equal participation as first authors. Corresponding Author Avrum I. Gotlieb MDCM, FRCPC Faculty of Medicine Medical Sciences Building, 1 King s College Circle, Rm Toronto, Ontario M5S 1A8 Canada Telephone: (416) Fax: (416) avrum.gotlieb@utoronto.ca 1

2 1. The Human Aortic Heart Valve The valves of the human heart are fascinating structures that exist in a hostile environment. They are flow-regulating membranes inside a complex multichambered pump. The valve cusps of the aortic valve are attached at the base of the aortic outflow tract. The highly organized extracellular matrix (ECM) of valves serves to maintain physiologic structure and together with a heterogeneous population of constituent cellular elements regulate complex functions to give both flexibility and durability to the cusps. 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. A confluent monolayer of VECs lines the surface of the valve. VECs show regional characteristics as differences in gene expression have been identified when comparing VECs on the fibrosa to the ventricularis side of the aortic cusp. Underneath the endocardium VICs are located embedded in matrix which VICs secrete and which regulates cellular function as well. The matrix is distributed in the three histologically distinct layers of the valve the fibrosa on the sinus side, the spongiosa in the middle, and the ventricularis on the aortic outflow side. The fibrosa is rich in densely packed collagen fibers; the spongiosa is composed mainly of glycosaminoglycans (GAGs) and proteoglycans; and the ventricularis contains prominent elastin and collagen, GAGs, and proteoglycans. These provide the valve with a zone of stiffness, a zone which is compressible, and a zone which is elastic in nature, respectively. The fibrosa with its dense connective tissue provides strength when the valve is shut, the spongiosa with its 2

3 loose matrix of glycoproteins provides a cushion for the physical forces when the valve opens and shuts, and the ventricularis provides elasticity when the cusp changes shape during the cardiac cycle. This exquisite architecture utilizes a specific organization of cells and matrix which may then function for decades and decades without much tissue wear and free of clinical disease. All three layers are avascular, and the distinct layers are formed at least in part by the physical forces which are applied to the valve by hemodynamic sheer stress and mechanical forces. This was shown by a study of human valves. In the embryonic stage (up to 20 weeks) there are no layers present and then 2 layers appear. The trilaminar architectural pattern occurs after birth during the time that flow patterns are established postnatally. The cellular and molecular mechanisms that regulate the formation of the trilaminar compartmentalization are unknown. The mechanisms merit intense study since it is likely that disruption of the architecture promotes progression of valve dysfunction in some individuals which in turn promotes the progression toward clinical disease. 2. Clinical Features of CAS a) Clinical Presentation Calcific aortic stenosis (CAS) is the most common heart valve condition in adults in Western society and results in the second most common cardiovascular surgery performed. At present the pathogenesis of CAS is unknown; however, similar calcific aortic valve conditions occur in some cases of bicuspid aortic valves and in chronic rheumatic valve disease. The former usually appears 3

4 clinically in the 40s and 50s age range, about ten to twenty years before CAS while chronic rheumatic valve disease with prominent calcification is prevalent in developing and in under developed countries. CAS is a disease with high morbidity and mortality that is costly to the health care system and will become even more prominent in health economies as people live longer. It has precursor lesions that may remain asymptomatic for some time. During the precursor stage of the disease, aortic valve sclerosis occurs which results in cusp thickening without creating obstruction to the left ventricular outflow. With onset of clinical disease obstruction to left ventricular outflow occurs. Left ventricular hypertrophy, angina, cardiac arrhythmias, dizziness, syncope and congestive heart failure occur over time. CAS increases in prevalence with advancing age. 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. What leads to this progression from aortic sclerosis to CAS is unknown. The key to progression may be in a faulty valve repair system in some individuals. In these cases, there is excessive valve thickening due to sclerosis that promotes valve dysfunction. Valve cusp stiffening, alterations of hemodynamic shear stress and mechanical stress then lead to more sclerosis and thus a vicious positive loop occurs, leading to eventual clinical disease. However, risk factors have been identified and include hypertension, elevated low-density lipoprotein, male gender, smoking, and diabetes mellitus, which are similar to those of atherosclerosis. Statins, well-known therapeutic agents that effectively target atherosclerosis, have shown to be ineffective in a few 4

5 prospective clinical trials on CAS. It is still possible that treating CAS patients very early on will show a benefit in slowing down the progression of CAS. The mainstay of treatment for CAS is still surgical valve replacement, which results in a dramatic improvement of symptoms. The procedure is costly and carries a certain amount of risk. Cardiac fibrosis is inversely correlated with the amount of LV functional improvement after valve replacement surgery. 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. Transcatheter aortic valve implantation is a relatively new technique first done in humans in It is gaining acceptance in patients unsuitable for conventional open-heart surgery, generally older patients over 70 years of age with severe CAS. b) Histopathology The gross and microscopic features of CAS have been very well described in the cardiovascular literature. Detailed histopathological investigations by anatomic pathologists have characterized the microscopic features of CAS including changes in the number of VICs, accumulation of myxomatous and/or dense connective tissue matrix, inflammation with or without immune activity, neovascularization, necrosis, lipid deposition, calcification and bone formation. There are macrophages, foam cells, T lymphocytes present in CAS. Microthrombi may also be present on the surface of the cusps. 5

6 3. Pathogenesis of CAS The alterations of tissue architecture and content are due to active cellular processes that, taken together, suggest that a response to tissue injury regulated by VICs is occurring in the valve. The core processes that result in valve disease are part of the universal tissue repair processes present in human tissue and organs. Also aortic valve stenosis may be due to the reactivation of developmental programs that in the adult lead to valve calcification. These two concepts are not mutually exclusive and do overlap. VICs, the most prevalent cells in the valve, become activated in response to injury. In diseased valves they differentiate into myofibroblast type cells, expressing the marker alpha-smooth muscle actin (a-sma). These activated VICs 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 cusps with excessive remodeling, scarring, and calcification through unknown mechanisms. Thus 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 inflammation, neovascularization, oxidative stress, activation of the rennin-angiotensin system, matrix remodeling, and calcification and osteogenesis. 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. a) Cell Phenotypes 6

7 I. VICs The VICs have been increasingly studied by researchers investigating the pathogenesis of CAS. In 2007 we reviewed the literature on VICs and suggested that the VIC might indeed be the master cell that regulates valve structure and function. VICs are the most common cells in the valve and are distinct from other mesenchymal cell types in other organs. Thus to study VICs it is necessary to study cells from the valve and not other mesenchymal cell types, such as, fibroblasts, vascular or wound myofibroblasts. We presented a conceptual approach to the investigation of VICs by focusing on VIC phenotype-function relationships. Our review suggested that there are five identifiable phenotypes of VICs that can be defined by the current understanding of their cellular and molecular functions. These include embryonic progenitor endothelial/mesenchymal cells, quiescent VICs (qvics), activated VICs (avics), progenitor VICs (pvics), and osteoblastic VICs (obvics). Although these may exhibit plasticity and may convert from one form to another, compartmentalizing VIC function into distinct phenotypes is useful in bringing clarity to our understanding of VIC pathobiology and the pathogenesis of CAS. The embryonic progenitor endothelial/mesenchymal cells undergo endothelial-tomesenchymal transformation (EMT) that initiates the process of valve formation in the embryo. At the endocardial cushion of the embryo, progenitor endothelial/mesenchymal cells undergo the process of EMT, which is characterized by endocardial cells penetrating into the connective tissue matrix beneath to undergo transformation. Transforming growth factor (TGF)-, bone 7

8 morphogenic protein (BMP), Notch and vascular endothelial growth factor (VEGF) have important regulatory effects on EMT. Decrease in VEGF results in insufficient EC proliferation to promote EMT while increased VEGF maintains the EC phenotype. The qvics are the cells that are at rest in the adult valve and maintain normal valve physiology. The avics are the cells that regulate the pathobiological responses of the valve in disease and injury. The pvics consist of heterogeneous population of progenitor cells that may be important in repair. These include resident and/or constitutive stem cells, hematopoetic stem cells including endothelial progenitor cells, mesenchymal stem cells derived from the bone marrow and present in the circulation. The obvics regulate chondrogenesis and osteogenesis and they may be derived from resident VICs and from bone marrow progenitor cells. II. VECs VECs are endocardial cells with properties similar to those of thromboresistant endothelial cells (EC). They line the surface of the valve forming a confluent monolayer with adherens junctions holding the cells together. The cells are adherent to the subendocardial surface by actin containing stress fibers. The fibrosa and the ventricularis surfaces show differences in gene expression, which may promote disease conditions more readily on the fibrosa side. The fibrosa side shows underexpression of genes that regulate inhibition of calcification, including osteoprotegerin, C-type natriuretic peptide, and 8

9 parathyroid hormone. In addition, there is upregulation of members of the BMP family, which promotes osteogenesis. b) Proliferation The adult non diseased heart valve does not exhibit proliferations of VICs; however, cell proliferation is important in the response to tissue injury seen in CAS. There are significant alterations in cell number over time and space in heart valves. Cell proliferation is brisk during the early stages of valvulogenesis, but is significantly downregulated during subsequent valve remodeling and during physiological function in adults. With respect to space, the local mechanical microenvironment provides signals that modulate the level of cell proliferation in focal areas of the valve cusp. Overall, the regulation of cell number during development, physiological function, and disease is a finely orchestrated process involving the interplay of growth factors, transcription factors, mechanical cues, cell-cell interactions, and ECM. Increase in VIC number is a common histopathological feature of CAS. Increased cell proliferation marked by prominent phospho-histone H3 (PHH3) immunostaining in valves of CAS affected patients has also been reported. However, the mechanisms underlying the pathological proliferative responses are largely unknown. A growing body of literature suggests that the pathogenesis of CAS represents the recapitulation of some of the well characterized signalling pathways activated during valvulogenesis. During endocardial cushion formation, VEGF and nuclear 9

10 factor of activated T-cells cytoplasmic 1 (NFATc1) maintain EC proliferative potential and phenotype. During the proliferative expansion stage of valvulogenesis, there is a marked increase in the number of VECs and progenitor mesenchymal cells, the latter being facilitated by factors such as fibroblast growth factor 4 (FGF4), BMP regulated transcription factors Sox9 and Msx1/2, basic-helix-loop-helix (bhlh) transcription factor Twist1, and Wnt signalling. Once ECM stratification is initiated, the proliferation index is drastically reduced and VICs gradually become quiescent, as seen in normal adult valves. The negative regulation of mesenchymal proliferation is partly mediated by the downregulation of aforementioned pro-proliferative factors as well as expression of epithelial growth factor (EGF), which antagonizes the effects of BMP. The critical importance of these developmental genes is illustrated when in their absence in animal models, severe endocardial cushion defects occur. On the contrary, when these pro-proliferative genes become re-activated in adult valves, it is likely that disease would inevitably follow. Most notably, Sox9, Msx2, and Twist1 are shown to be expressed in areas of increased VIC proliferation in diseased aortic valves, suggesting that an embryonic trancriptional program is reinitiated in VICs when they become activated in disease. Cell number regulation is also closely associated with the spatial, mechanical cues in the microenvironment that VICs reside in. The first line of evidence comes from studies of VICs isolated from different regions of the mitral valve showing heterogeneity in mechanical loads and substrate makeup. Surprisingly these cells show diverse proliferative capacities, presumably in response to the 10

11 diverse physical stresses that they experience. It has been observed that when connective tissues are subjected to increased mechanical loading, fibroblasts increase their proliferative activity accordingly. To support this notion, there is evidence in porcine aortic valve cusp samples that shows enhanced proliferation as a result of cyclic stretch alone. Adding to the complexity of the regulatory network, VECs have been shown to play an indispensible role in mediating VIC proliferative response. The increased VIC proliferation in an organ culture model with endothelial denudation is reversed when VECs are seeded to the culture. The same growth stabilizing effect is observed in a VIC/VEC co-culture system, suggesting that VICs proliferate when not in communication with their respective endothelial cells. These two experiments have profound implications on CAS pathogenesis, highlighting the importance of an intact endothelium, which is often damaged in disease. It is well accepted that VICs become activated and undergo cell proliferation in the initial stages of CAS. Using a well characterized in vitro wound model, we reported an upregulation of TGF-β and augmentation of TGF-β signalling in VICs at the wound edge 24 hrs post wounding. This is consistent with the findings of TGF-β in human aortic valve lesions and its elevation in the plasma of aortic stenosis patients. We observed that TGF-β leads to increased VIC proliferation at the in vitro wound edge, leading to enhanced wound repair. Intriguingly, the same molecule, when added to a low density intact monolayer, inhibits VIC proliferation as demonstrated by the significant reduction in bromodeoxyuridine 11

12 (BrdU) incorporation. This growth inhibition is via a well studied mechanism in epithelial cells, namely retinoblastoma protein (prb) hypo-phosphorylation on Ser 807/811 residues resulting from decreased expression of cyclin D1/CDK4 and increased expression of an inhibitor of the complex, p27. Of interest, in both scenarios, Smad2/3 pathway is activated. One way to explain this apparent paradox is by postulating that VICs sense external stimuli and modulate the level of activation or suppression of TGF-β/Smad and TGF-β/non-Smad pathways. It is likely that the former inhibits proliferation and the latter promotes proliferation through ERK and PI3K pathways. The net result of the active modulation is either tissue homeostasis or wound repair, further illustrating the role of VICs as key players in the response to tissue injury paradigm. c) Inflammation Normal aortic valves do not contain inflammatory cells while diseased valves exhibit macrophages, T-lymphocytes and mast cells. It is assumed that these cells enter via the inflammatory cell adhesion pathway due to upregulation of cell adhesion molecules, such as ICAM, VCAM and selectins, on the valvular endocardial cells and/or due to injury and denudation of the surface endocardium. The upregulation of cell adhesion molecules is most prominent on the fibrosa side of the valve. The diseased valves show the presence of numerous proinflammatory cytokines such as TNF2, HLA-DR, BMP-2/4/7 and IL- 1 /2/6. Interleukin-1 receptor antagonist-mediated effects which reduce inflammation are dysfunctional when comparing stenotic CAS valves to normal 12

13 valves. TGF, which is considered an anti inflammatory mediator, is also ubiquitous in diseased valves, especially bound to matrix. It is likely that TGF regulates repair by attempting to reduce inflammation and promote fibrosis. This growth factor regulates VIC differentiation to cells which secrete matrix, matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and thus regulates scar formation. These VICs exhibit a myofibroblastic phenotype, expressing SMA. Foam cells are also present in the valve leaflets of diseased valves and accumulate excessive lipid. Lipid accumulation alters macrophage gene expression. The foam cells express scavenger receptors which are able to take up modified lipoproteins including oxidized LDL. Macrophages tend to die in these tissues and create cellular debris and release toxic oxidized lipids and numerous proinflammatory mediators. It is not yet known if CAS is an inflammatory disease or whether inflammation is an associated process occurring in injured and repairing tissues. Inflammatory mediators may be useful therapeutic targets to prevent and/or reduce the extent of calcification and osteogenesis, which can be triggered by inflammatory mediators. The osteogenic phenotypes of VICs can be induced by toll-like receptors 2 and 4. This phenotype is characterized by increase in alkaline phosphatase activity and upregulations of BMP-2 and Runx2. d) Neovascularization 13

14 The valve is normally avascular, thought to be due, at least in part, to the presence of chondromodulin-i, an antiangiogenic factor which is abundantly expressed in normal valves but markedly down-regulated in diseased valves. Its absence or down-regulation results in enhanced expression of the angiogenic factor VEGF as well as proteases that promote tissue remodeling including MMP-1, MMP-2, and MMP-13, thereby promoting angiogenesis. In advanced lesions, histopathology of the valves shows numerous new blood vessels associated with the expression of angiogenic factors such as VEGF and endothelial nitric oxide synthase. The origin of the cells that form these new vessels is poorly understood. It is hypothesized that some of the endothelial cells that form these neovessels may originate from several sources including surface VECs, circulating endothelial progenitor cells, and/or resident VICs. In early lesions, new-forming vascular sprouts are present associated with VICs expressing a-sma, VEGF receptor-2 and Tie-2. This suggests the presence of a transitional phenotype from mesenchymal- to endothelial-type cells, forming new capillaries with interendothelial junctions and partial basement membrane-like structure. Angiogenesis may indeed be an important independent process in valve remodeling. However, most investigators consider it to be part of the inflammatory process. In addition, calcification and bone formation may be regulated by angiogenic growth factors found in the diseased valve. e) Matrix remodeling 14

15 ECM is actively involved in the pathogenesis of CAS through the modulation of biochemical and biomechanical signals. In addition to providing structural support, ECM also influences a number of cellular processes by binding to surface receptors and transmembrane integrins, by regulating the availability of growth factors and cytokines which are normally bound to the matrix, by transducing hemodynamic forces, and by modifying its composition in response to cell-generated tractional forces. During embryogenesis, the valve ECM undergoes an extensive remodeling process that converts unassembled precursors of collagens, proteoglycans, and elastin into the highly organized laminar structure that confers strength and pliability. This process requires the controlled downregulation of early ECM components such as collagen 2 and remodeling enzymes as well as the upregulation of mature ECM proteins such as fibrillar collagens and elastin by many transcription factors that are active early in development, one of them being Twist1. It was shown in a mouse model that persistent Twist1 expression after embryogenesis leads to the formation of an unstructured and primitive matrix reminiscent of the immature ECM of endocardial cushion, which is enriched in collagen 2 and high in MMP-2 and MMP-13 activities. In addition, Twist1 expression co-localizes with regions of increased ECM disorganization in calcified human aortic valves, thereby providing strong evidence that favours the re-activation of developmental programs in CAS. Based on the findings in the literature, one may speculate that ECM is also implicated in disease progression through a positive feedback loop (Figure 1). 15

16 Under physiological conditions, VICs are protected from physical stress by the matrix that they continuously synthesize and remodel. However upon damage to the endothelium and injury to the VECs, the protective matrix is compromised. This exposes the VICs to supraphysiological mechanical loads, which stimulate them to develop corresponding properties to oppose the increased stress. For example, cells are known to increase their biosynthetic activity and tune their internal stiffness by cytoskeletal rearrangement. This may result in a stiffer ECM that now alters the conformation of the TGF-β latent complex to release more TGF-β ligands and in turn promotes myofibroblast differentiation. At the same time, ECM stiffening changes the biomechanical properties of the valve cusp and directly influences the local hemodynamics. The altered hemodynamic forces may result in the expression of disease-prone paracrine factors by the overlying VECs to increase fibrosis and further activate VICs. Furthermore, altered hemodynamics and consequently increased cyclic tension potentiate the effect of TGF-β signalling to increase the contractile and biosynthetic activity of activated VICs by directly acting on α-sma promoter. In an attempt to restore equilibrium, the expression of MMPs and TIMPs become markedly upregulated, as seen in patients with aortic stenosis. This self perpetuating cycle leads to progression of CAS with ECM disarray, cusp thickening and loss of elasticity. Interestingly, the processes discussed herein are all normal physiologic responses to injury that become excessive due to lack of inhibition for unknown reasons, ultimately leading to disease. It is important to recognize that ECM during injury can itself 16

17 initiate a cascade of cellular and molecular events that contribute to CAS progression. f) Calcification/Osteogenesis CAS is characterized by calcification and osteogenesis which result in the serious aortic stenosis that produce the clinical features of CAS. The pathogenesis of this calcification/bone formation has been under intense study. The pathogenesis of calcification and osteogenesis is likely due to two processes. Dystrophic calcification, a passive degenerative process characterized by the deposition of hydroxyapatite crystals associated with necrotic cells, is likely to be important. However, accumulating evidence is pointing towards osteogenic calcification, an active process mediated by genes involved in bone and cartilage development, as the predominant form of calcification in CAS. Initially it was thought that the origins of the obvics which regulate this process are not fully known; however, it is likely that precursor/progenitor cells are resident in the valve and become activated to promote calcification and bone formation. As well bone marrow derived progenitor cells are also likely to populate the injured/diseased valve. Some studies have demonstrated the presence of osteoblast-specific transcripts and osteogenic markers in calcified aortic valves. Examples include osteocalcin, Cbfa1 (Runx2), osteopointin, bone sialoprotein, BMP2/4 and collagen 10. Thus the thickened valve cusp allows for osteogenic transformation to occur and the obvic then regulates calcification and bone formation. 17

18 g) Physical Forces Valves are exposed to hemodynamic shear stress and to several mechanical forces during each cardiac cycle. The VECs and VICs have the capacity to sense and respond to tensile, compressive, and shearing forces in their local mechanical environment and alter their molecular and morphological profile, suggesting that physical forces play an important role in valve development and pathogenesis. To address its involvement in valvulogenesis, it was shown in developing zebrafish heart that lowered shear stress leads to abnormal valve phenotype, thereby demonstrating that hemodynamic force is the driving force for valve development. The relationship between physical forces and disease is best illustrated by the observation that sclerotic lesions occur preferentially on the aortic side of the leaflet, where the surface is exposed to low shear stress and disturbed hemodynamic oscillatory flow. Moreover, the aortic/fibrosa side of the aortic cusp is important to consider since it is where calcification usually is initiated. The occurrence of calcified lesions correlates with areas of high bending stresses, such as in the attachments of the cusps. On a molecular level, physical forces play an important role in regulating and modifying the cellular function of VICs and VECs. It has been shown that cells that experience greater stretch show more collagen synthesis, as evident by increased incorporation of [ 3 H]- proline. Similarly, as a result of transvalvular pressure, VICs isolated from the aortic valve are stiffer than those from the pulmonary valve. When the aortic pressure level is imposed on pulmonary VICs, as in the Ross operation, their 18

19 synthesis of ECM components increases significantly. VECs have been compared to vascular ECs and VECs on the fibrosa side have been compared to those on the ventricularis. The outcome of these studies show that valvular VECs have different phenotype than vascular ECs and that VECs on the fibrosa side express different genes than do VECs on the ventricularis side. These sidespecific differences can help to explain the initiation of disease on the aortic side. Whether this is due to hemodynamic mechano-transduction of VEC function is not known; however, the fibrosa surface is exposed to oscillatory shear stress while the ventricularis experiences a higher magnitude of shear stress which is rapid and pulsatile relative to the fibrosa surface. h) A unifying hypothesis A unifying hypothesis on the pathogenesis of CAS needs to consider and explain inflammatory processes in the valve tissue and VIC functions including activation, proliferation, migration, cytokine, growth factor and chemokine secretion, as well as collagen, glycosaminoglycans, proteoglycans, MMP and TIMP secretion. The hypothesis must consider lipid deposition, calcification and osteogenesis. It must also explain the regulatory influences of hemodynamic and mechanical forces on cusp structure and VEC and VIC function. How the trilaminar architecture is disrupted and how this promotes valve dysfunction at the gross and molecular level needs to be considered as well. We propose a theory to help guide future investigation into the pathogenesis of CAS. The focus 19

20 is on VEC and VIC activation, VIC proliferation and regulation of matrix formation, and osteogenic transformation (Figure 2). Puzzling questions that need to be answered to understand the pathogenesis of CAS a) What turns off endocardial-mesenchymal transformations and the genes that regulate valve development? b) What are the processes involved in creating the trilayered architecture of the valve cusp? c) What cellular processes are engaged in the maintenance of valve structure and renewal? d) How do physical forces regulate VEC/VIC function? e) What causes valve injury to result in valve cusp thickening? f) What initiates inflammation in the valve? g) What roles do lipoproteins and lipids play in valve injury and repair? h) How do cell-matrix interactions regulate repair? i) What initiates osteogenic transformation in the thickened fibrotic valve? j) What are the best models to use to study CAS? 20

21 References (A) Valve Development Hinton RB, Yutzey KE: Heart Valve Structure and Function in Development and Disease. Annu. Rev. Physiol :29-46 Combs MD, Yutzey KE: Heart Valve Development: Regulatory Networks in Development and Disease. Circulation Research 2009, 105: (B) VIC Structure/Function Mulholland DL, Gotlieb AL: Cell biology of valvular interstitial cells. Can J Cardiol 1996, 12: Liu AC, Gotlieb AL: Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells. Am J Pathol 2008, 173: Hajdu Z, Romeo SJ, Fleming PA, Markwald RR, Visconti RP, Drake CJ: Recruitment of bone marrow-derived valve interstitial cells is a normal homeostatic process. Journal of Molecular and Cellular Cardiology Gu X, Masters KS: Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation. Am J Physiol Heart Circ Physiol :H448-H458 Rodriguez KJ, Piechura LM, Masters KS: Regulation of Valvular Interstitial Cell Phenotype and Function by Hyaluronic Acid in 2-D and 3-D Culture Environments. Matrix Biol : Aikawa E, Whittaker P, Farber M, Mendelson K, Padera RF, Aikawa M, Schoen FJ: Human Semilunar Cardiac Valve Remodeling by Activated Cells from Fetus to Adult: Implications for Postnatal Adaptation, Pathology, and Tissue Engineering. Circulation : (C) Proliferation Chakraborty S, Wirrig EE, Hinton RB, Merrill WH, Spicer DB, Yutzey KE: Twist1 Promotes Heart Valve Cell Proliferation and Extracellular Matrix Gene Expression during Development in vivo and Is Expressed in Human Diseased Aortic Valves. Dev Biol : Blevins TL, Peterson SB, Lee EL, Bailey AM, Frederick JD, Huynh TN, Gupta V, Grande-Allen KJ: Mitral Valvular Interstitial Cells Demonstrate Regional, Adhesional, and Synthetic Heterogeneity. Cells Tissues Organs :

22 Li C, Gotlieb AI: Transforming Growth Factor-Beta Regulates the Growth of Valve Interstitial Cells in vitro. Am J Pathol : Ramhimi RA, Leof EB: TGF-beta Signaling: A Tale of Two Responses. J Cell Biochem : (D) Matrix Chen J-H, Simmons CA: Cell-Matrix Interactions in the Pathobiology of Calcific Aortic Valve Disease: Critical Roles for Matricellular, Matricrine, and Matrix Mechanics Cues. Circulation Research : Carthy JM, Boroomand S, McManus BM: Versican and CD44 in vitro valvular interstitial cell injury and repair. Cardiovascular Pathology 2011 [Epub ahead of print] Stephens EH, Shangkuan J, Kuo JJ, Carroll JL, Kearney DL, Carberry KE, Fraser Jr CD, Grande-Allen KJ: Extracellular matrix remodeling and cell phenotypic changes in dysplastic and hemodynamically altered semilunar human cardiac valves. Cardiovascular Pathology : e157-e167 [Epub ahead of print] (E) Calcification/Osteogenesis Yu Z, Seya K, Daitoku K, Motomura S, Fukuda I, Furukawa K-I: Tumor Necrosis Factor- Accelerates the Calcification of Human Aortic Valve Interstitial Cells Obtained from Patients with Calcific Aortic Valve Stenosis via the BMP2-DIx5 Pathway. The Jr Pharmacology and Environ Therapeu :16-23 New SEP, Aikawa E: Molecular Imaging Insights Into Early Inflammatory Stages of Arterial and Aortic Valve Calcification. Circulation Research 2011, 108: Rajamannan NM, Evans FJ, Aikawa E, Grand-Allen KJ, Demer LL, Heistad DD, Simmons CA, Masters KS, Mathieu P, O Brien KD, Schoen FJ, Towler DA, Yoganathan AP, Otto CM: Calcific Aortic Valve Disease: Not Simply a Degenerative Process. Circulation : Rajamannan NM: Calcific Aortic Valve Disease: Cellular Origins of Valve Calcification. Arterioscler Thromb Vasc Biol 2011, 31: Acharya A, Hans CP, Koenig SN, Nichols HA, Galindo CL, Garner HR, Merrill WH, Hinton RB, Garg V: Inhibitory Role of Notch1 in Calcific Aortic Valve Disease. PLOS One : Issue 11,

23 (F) Inflammation Lee JH, Meng X, Weyant MJ, Reece TB, Cleveland JC, Fullerton DA: Stenotic aortic valves have dysfunctional mechanisms of anti-inflammation: Implications for aortic stenosis. The Journal of Thoracic and Cardiovascular Surgery, , Mahler GJ, Butcher JT: Inflammatory Regulation of Valvular Remodeling: The Good (?), the bad, and the ugly. International Journal of Inflammation 2011 Article ID (G) Physical Forces Chan MW, Hinz B, McCulloch CA: Mechanical Induction of Gene Expression in Connective Tissue Cells. Methods Cell Biol : Butcher JT, Nerem RM: Vavular Endothelial Cells Regulate the Phenotype of Interstitial Cells in Co-culture: Effects of Steady Shear Stress. Tissue Eng : Butcher JT, Simmons CA, Warnock JN: Mechanobiology of the Aortic Heart Valve. J Heart Valve Dis : Cunningham KS, Gotlieb AI: The Role of Shear Stress in the Pathogenesis of Atherosclerosis. Lab Invest : 9-23 (H) CAS Pathogenesis Liu AC, Joag VR, Gotlieb Al: The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. Am J Pathol : Durbin AD, Gotlieb AL: Advances towards understanding heart valve response to injury. Cardiovasc Pathol :69-77 Schoen FJ: Mechanisms of Function and Disease of Natural and Replacement Heart Valves. Annu. Rev. Pathol. Mech. Dis : Markwald RR, Norris RA, Moreno-Rodriguez R, Levine RA: Developmental basis of adult cardiovascular diseases Valvular heart diseases. Ann. N.Y. Acad Sci : Li C, Xu S, Gotlieb AI: The response to Valve Injury: A paradigm to Understand the Pathogenesis of Heart Valve Disease. Cardiovasc Pathol :

24 Figure Legends Figure 1. ECM remodeling hypothesis of a positive feedback loop. Enhanced ECM synthesis and VIC contractility following valve injury result in a stiffer valve matrix, which promotes VICs to become activated and undergo myofibroblast differentiation. Myofibroblasts, through their intrinsic ability to increase ECM synthesis, lead to a positive feed back loop that continues to promote valve cusp thickening, creating a permissive substrate in which osteogenic transformation occurs, leading to progression of CAS. Figure 2. Proposed theory on the mechanism of the pathogenesis of CAS. Valve injury initiates VEC activation and inflammatory responses that lead to the release of factors involved in qvic activation. avics increase proliferation and ECM remodeling in response to a number of biomechanical and biochemical signals, leading to the disruption of valve architecture and subsequently cusp thickening/fibrosis. The upregulation of osteogenic pathways within the fibrotic valve contributes to calcification and bone formation via the activity of obvics. The result of this cascade of events is clinical valve disease. 24

25 Figures Figure 1. Valve Injury ECM Synthesis Cytoskeletal Rearrangement Contractile Forces ECM Stiffness Altered Mechanical and Hemodynamic Forces TGF-β VIC and VEC Paracrine Signalling Myofibroblast Differentiation 25

26 Figure 2. 26