The aortic valve was long considered a passive structure that opens and closes in response to changes in transvalvular pressure. Recent evidence suggests that the aortic valve performs highly sophisticated functions as a result of its unique microscopic structure. These functions allow it to adapt to its hemodynamic and mechanical environment. Understanding the cellular and molecular mechanisms involved in normal valve physiology is essential to elucidate the mechanisms behind valve disease. We here review the structure and developmental biology of aortic valves; we examine the role of its cellular parts in regulating its function and describe potential pathophysiological and clinical implications.
Journal of Advanced Research (2010) 1, 5–12 University of Cairo Journal of Advanced Research REVIEW ARTICLE Cellular regulation of the structure and function of aortic valves Ismail El-Hamamsy, Adrian H Chester, Magdi H Yacoub * Harefield Heart Science Center, National Heart and Lung Institute, Imperial College London, United Kingdom KEYWORDS Aortic valve; Endothelium; Mechanotransduction; Aortic valve calcification; Interstitial cells Abstract The aortic valve was long considered a passive structure that opens and closes in response to changes in transvalvular pressure Recent evidence suggests that the aortic valve performs highly sophisticated functions as a result of its unique microscopic structure These functions allow it to adapt to its hemodynamic and mechanical environment Understanding the cellular and molecular mechanisms involved in normal valve physiology is essential to elucidate the mechanisms behind valve disease We here review the structure and developmental biology of aortic valves; we examine the role of its cellular parts in regulating its function and describe potential pathophysiological and clinical implications ª 2009 University of Cairo All rights reserved Introduction The aortic valve lies at the junction between the left ventricle and the aorta It opens and closes >100.000 times daily For a long time, the aortic valve was believed to be a passive structure that opens and closes in response to changes in transvalvular pressures Although this is partly true, recent evidence clearly dem* Corresponding author Address: Harefield Heart Science Center, Harefield Hospital, Hill End Road, Harefield, Middlesex, United Kingdom Fax: +44 1895 828 900 E-mail address: m.yacoub@imperial.ac.uk (M.H Yacoub) 2090-1232 ª 2009 University of Cairo All rights reserved Peer review under responsibility of University of Cairo 2090-1232 ª 2009 University of Cairo All rights reserved Peer review under responsibility of University of Cairo Production and hosting by Elsevier doi:10.1016/j.jare.2010.02.007 onstrates that the aortic valve has a tightly regulated and highly conserved architecture which allows it to perform sophisticated functions, in turn affecting systolic blood flow, ventricular workload and coronary blood flow, among other things In addition, the cellular components of the valve play an important role in maintaining normal valve architecture and composition Dysregulation in one of the elements can lead to dysfunction and dysmorphic changes of the valve In this review, we will discuss the unique structure of the aortic valve We will then focus on the role of aortic valve endothelial cells in maintaining valve structure and function and their possible role in calcified aortic valve disease, with an emphasis on clinical implications Aortic valve structure Macroscopic structure The aortic valve mechanism is composed of four inter-related component parts which form a functional unit: the aortic annulus, the aortic cusps, the sinuses of Valsalva and the sinotubular junction The normal aortic valve is composed of three semi-lunar cusps that are attached to a crown-shaped annulus at their base Adequate and coordinated opening of the aortic valve is essential to ensure unobstructed laminar blood flow from the left ventricle and decrease ventricular workload in systole [1–4] Similarly, proper closure of the cusps in diastole preserves the shape of the root and contributes to the creation of vortices in the sinuses of Valsalva, an important determinant of adequate coronary blood flow in diastole and in systole [5,6] The aortic valve lies in a unique hemodynamic and mechanical environment exposing the cusps to a wide range of stresses during the cardiac cycle, which range from pressure, to tension and bending forces Although aortic valve cusps are extremely thin structures, yet both sides of the valve are exposed to different stresses, in particular shear stress which is a major stimulus for valve endothelial cells (VECs) The ventricular side of the cusps is exposed to high-shear stress due to a systolic, high velocity, laminar blood flow, whereas the aortic side of the cusps is exposed to lowshear stress secondary to diastolic, low velocity, disturbed blood flow VECs have the ability to sense changes in shear stress and to translate these mechanical stimuli into biological responses (mechanotransduction) [7] Previous studies focusing on the vascular endothelium show that different patterns of flow can greatly influence the response of underlying tissues through activation of specific mechanotransduction pathways in the endothelium, which can lead to structural changes at the level of the vessel wall Cellular structure In addition to the VECs which line both sides of the cusp, the body of the aortic valve is composed of valve interstitial cells (VICs) lying within the extracellular matrix (ECM) (Fig 1) VICs are composed of different cell types, namely fibroblasts, smooth muscle cells and myofibroblasts [8] Smooth muscle cells can exhibit both secretory and contractile properties Along with the fibroblasts, these secretory properties are responsible for generation, maintenance and repair of the ECM which is mainly composed of elastin, collagen and proteoglycans [9] The three-dimensional microscopic architecture of I El-Hamamsy et al the aortic valve cusps is highly preserved between species and consists of three layers distinguishable by their chemical composition and mechanical properties [10] (Fig 1) On the aortic side lies the fibrosa, a layer rich in collagen fibers which provides most of the tensile strength to the valve On the ventricular side is the ventricularis which is rich in elastin fibers, thus providing elasticity to the aortic valve Between these two layers is the spongiosa, which represents about 60–70% of the thickness of the cusp and is primarily composed of proteoglycans Proteoglycans are highly hydrated, thus acting as ‘‘shock absorbers’’ during the different parts of the cardiac cycle In addition to this basic structure of aortic valves, a population of resident stem cells lying within the cusps has recently been recognized [11] They appear to originate from the mobilization of hematopoietic-derived stem cells towards cardiac valves [12] Their role is not yet fully understood but they are thought to contribute to valve repair, cell regeneration, as well as participating in valve calcification in some disease states as will be discussed later Although aortic valves are avascular structures which extract their nutrients by extraction from surrounding blood, they are richly innervated by a highly preserved network of afferent and efferent nerves which contribute to valve structure and function [13–15] Cell lineage and developmental biology Aortic valve originate from endocardial cushions in the developing embryo Endocardial cushions result from the migration of endothelial cells into the cardiac jelly, followed by a process of endothelial-to-mesenchymal transformation (EMT) which initiates what will eventually become a tri-layered aortic valve structure Migration, differentiation and delamination of these cells is a tightly controlled series of processes which depend on the activity of specific signaling molecules such as NOTCH1, transforming growth factor b (TGF-b) and the wnt/b-catenin pathway [16–19], as well hemodynamic cues [20] Importantly, the embryonic outflow tract which would ultimately yield the aortic and pulmonary valves as well as the ascending aorta and pulmonary artery are composed of cells derived from the neural crest [21] NOTCH1 is also involved in regulating the migration and differentiation of these cells in the embryo [17] Understanding the developmental biology of the aortic valve and root has important implications for understanding normal and diseased valve physiology because most of the signals that are operational during morphogenesis continue to influence growth and adaptation in postnatal life [22] For instance, EMT continues into adult life as demonstrated by the differentiation of mature VECs into mesenchymal cells expressing smooth muscle a-actin, a process which could contribute to valve repair and interstitial cell regeneration [23] In addition, defining cell lineages helps explain differences in cell behaviour in response to common stimuli [24] Aortic valves lie in a unique hemodynamic environment Figure Histological section showing the triple layer architecture of a normal aortic valve Endothelial cells form a monolayer on each side of the cusp Interstitial cells, a mix of fibroblasts, smooth muscle cells (SMCs) and myofibroblasts fill the body of the cusp They possess contractile and secretory properties and are responsible for the synthesis and repair of the surrounding extracellular matrix Blood flowing through the vasculature generates shear stress on the luminal side of the vessel, which is entirely lined by endothelium Shear stress represents the frictional force per unit area The magnitude of shear stress can be estimated in most of the vasculature by Poiseuille’s law stating that wall shear stress is proportional to blood flow viscosity and volu- Cellular regulation of the structure and function of aortic valves metric flow rate and inversely proportional to the third power of the internal radius [25] While actual wall shear stress is very difficult to estimate, mean shear stress along an artery is estimated at 20 dynes/cm2 Unlike blood vessels, the aortic valve is not a cylindrical structure, therefore estimation of shear stress levels on either side of the valve requires more complex modelling Nevertheless, the pattern of flow on the aortic and the ventricular sides of the valve has been recognized for a long time Leonardo da Vinci was the first to accurately describe the laminar flow on the ventricular surface of the valve and the disturbed vortex flow in the sinuses of Valsalva to which ECs on the aortic side are exposed Estimates of actual valve shear stresses have varied significantly in the literature [26–30] Some have suggested that mean shear stress along the ventricular surface of the aortic valve is around 20 dynes/cm2 [30,31], while others estimate actual peak shear stress on the ventricular surface at 80 dynes/cm2 whereas it oscillates between +10 and À8 dynes/cm2 on the aortic surface [32] Flow along the ventricular surface is a high-shear laminar flow whereas it is a low-shear disturbed flow on the aortic side continuity The most striking difference between valvular and vascular endothelial cells is cell alignment with regards to flow orientation Whereas the vascular endothelium throughout the body aligns with the long axis of the cell parallel to flow [34] (except in areas of turbulent flow) [35], VECs are aligned perpendicular to the direction of flow [31,36] (Fig 2) This was first described by Deck [36] by electron microscopic analysis of explanted aortic valves and further validated by in vitro studies [31] Cultured porcine aortic VECs were compared to aortic (vascular) endothelial cells from the same animal in response to unidirectional non-pulsatile laminar flow at 20 dynes/cm2 Whereas vascular cells were aligned parallel to flow after 24 h, instead, VECs aligned perpendicular to flow even without the presence of an aligned substrate [37] These adaptations were dependent on cytoskeletal reorientation, a process involving different mechanotransduction pathways in each type of endothelium Laminar flow induced activation of Rho-kinase, phosphatidylinositol-3-kinase and Endothelial cell heterogeneity The endothelium is a monolayer of cells lining blood and lymphatic vessels which sits at the interface between all body organs and blood They possess anti-thrombogenic, antiadhesive, anti-proliferative and vasodilatory properties which mainly result from the synthesis and release of nitric oxide (NO), its major biosynthetic product and prostacyclin (PGI2) Nevertheless, although all endothelial cells throughout the body share the same basic properties, studies have demonstrated a considerable amount of heterogeneity between the endothelium from different regions of the body both at the structural and functional levels [33] Structurally, although most endothelial cells have a typical cobblestone appearance, they can vary significantly in thickness, ranging from 0.1 lm in capillaries and veins to lm in the aorta In addition, the number, distribution and properties of tight and adherens junctions between them varies significantly from one vascular bed to another reflecting endothelial adaptation to its hemodynamic and metabolic environment Junctions are tighter in large vessels exposed to high-shear stresses and flow rates than small arterioles, capillaries or venules Finally, the endothelium can be continuous (large arteries and veins) or discontinuous (liver sinusoidals), fenestrated or non-fenestrated to allow filtration and transendothelial transport Similarly, endothelial cells exhibit marked functional heterogeneity on several levels: permeability, mechanotransduction pathways in response to mechanical stimuli and angiogenesis among other functions Aortic valve endothelial cells exhibit unique properties Mechanotransduction and alignment For a long time, aortic VECs were thought to play a minor role in valve physiology because valves were considered passive structures that opened and closed in response to changes in transvalvular pressure More recently, several studies focusing on the structural and functional properties of aortic VECs suggest that valvular endothelium possesses unique properties which distinguish it from other endothelial beds, particularly the endothelium lining the aorta with which it lies in direct Figure (a) Scanning electron microscopy of aortic valve endothelial cells forming a monolayer on the surface of the aortic cusps (1000·) The cells are tightly joined and aligned thus constituting the link between the blood milieu and the interstitial space (b) Scanning electron microscopy of aortic valve endothelial cells on the aortic side of the cusp at higher magnification (5000·) Microcilia on the surface of the cells are visible and likely act as sensors of changes in hemodynamic shear stress, in turn activating different intracellular mechanotransduction pathways 8 I El-Hamamsy et al calpain pathways in vascular endothelial cells, whereas VECs did not require activation of the latter for cytoskeletal reorganization [31] Gene expression profile VECs also appear to have a higher proliferation rate than vascular endothelial cells [38] Furthermore, both sets of cells possess different transcription profiles In a study examining the transcription profiles of 847 genes, there was common expression of 55 activated genes whereas a further 48 genes were differentially activated Among those genes with a higher activation in the valvular ECs were transcription factors associated with higher proliferation rate such as jun D and protein kinase C [38] Notably, both vascular endothelium and VECs expressed markers linked with calcification such as osteonectin, bone morphogenic protein-7 and -9 (BMP-7 and BMP-9) Differences in gene expression profile between vascular and valvular endothelium were further validated by another study showing different gene expression profiles in response to shear stress stimulation of cultured porcine aortic endothelial cells or VECs [37] In that study, Butcher et al showed preferential expression of genes associated with chondrogenesis by VECs whereas vascular endothelial cells expressed more genes associated with osteogenesis Shear stress reduced the expression of osteogenic genes [37] Aortic valve endothelial cells are different on both sides of the valve Aortic valve calcification is a major clinical problem in elderly patients and those with bicuspid aortic valve disease With increased life expectancy, improved diagnostic techniques and better global access to health care, the incidence of aortic valve calcification is expected to triple within the next 40 years [39], making it one of the major sources of cardiovascular disease In recent years, it has become increasingly clear that aortic valve calcification is an active cell-driven process that shares many similarities with atherosclerosis Among those, early endothelial dysfunction has been shown to act as an initial occurrence in the cascade of events leading to valve calcification [40] Histologically, aortic valve calcification occurs exclusively on the aortic side of the valve suggesting that perhaps VECs on the aortic side are less resistant to calcification than those on the ventricular side (Fig 3) Simmons et al developed an innovative technique to separately analyze gene expression of VECs from either side of the valve [41] This modified Hautchen technique for en face isolation of VECs allowed reliable extraction of high quality mRNA for analysis of gene expression profiles between both aortic and ventricular VECs The authors reported the differential expression of 584 genes in situ between both sides of the valve [42] VECs derived from the aortic side expressed fewer genes associated with inhibition of calcification such as oteoprotegerin, parathyroid hormone and chordin, a protein that inhibits the osteoinductive effects of BMPs [42] In addition, aortic-sided VECs showed increased expression of transcripts linked with bone formation including BMP4 However, this was balanced by a higher expression of antioxidative and anti-inflammatory genes on the aortic side Notably, there were higher levels of endothelial nitric oxide synthase (eNOS) on the aortic side of the valve Because the mRNA obtained in this study originated directly from freshly Figure Histological section of a calcified human aortic valve from a patient with calcified aortic valve stenosis Tripp–McKay silver impregnation staining shows the calcium nodule on the aortic side of the cusp, below the endothelial layer explanted aortic valves, it represented a good picture of in situ gene expression in normal valves However, the gene array used was a human array and only covered 12,000 genes Currently, porcine gene arrays that cover >24,000 genes are commercially available and could provide additional clues into the functional side-specificity of aortic VECs This will be greatly enhanced following the full sequencing of the pig genome which is expected towards the end of 2009 We have recently succeeded in separately isolating aortic VECs from either side of the valve and culturing them in vitro We are currently studying their intrinsic properties in vitro in an effort to determine their responses to various mechanical and pharmacological stimuli as well as modifications in gene expression profiles Differences in flow patterns between both sides of the valve as described earlier are sensed by a thin layer of glycoproteins on the luminal surface of endothelial cells called the glycocalyx, which communicates with the cytoskeleton and can activate several signaling pathways in response to flow [25,43–45] This process of translating mechanical stimuli into biological signals is commonly termed mechanotransduction Studies on vascular endothelial cells have demonstrated that differences in shear stress translate into activation of different signaling pathways, illustrated by the presence of atherosclerotic plaques in areas of low wall shear stress in the vasculature such as the carotid artery bifurcation One of the major activated signaling pathways is the nuclear factor-kB (NFkB), a highly conserved transcription factor which translocates to the nucleus when activated, triggering the production of proinflammatory molecules [46,47] To date, the glycocalyx has not been identified or characterized on the surface of aortic valves Identification of the specific cell-surface molecules which contribute to mechanotransduction in VECs could open new avenues into understanding disease processes in conditions of abnormal shear stresses on the valves The different mechanotransduction pathways involved on either side of the valve in response to different patterns of flow have yet to be described (Fig 4) Recently, a flow apparatus consisting of a cone and plate was developed to allow in vitro reproduction of aortic and ventricular flow patterns Cellular regulation of the structure and function of aortic valves Figure Illustration of some of the mechanostransduction pathways activated inside VECs in response to shear stress Cells sense shear stress through mechanosensors on their surface which trigger activation of intracellular mediators such as nuclear factor-kB (NFkB) or Rho, which lead to synthesis of various signaling molecules which are either directly released by the cells or translocate to the nucleus and induce modifications in gene expression The pathways activated result in different effects including modified chemotaxis, cell adhesion and expression of inflammatory markers (adapted from [46,62]) on aortic valves [32] Preliminary studies using that system show that unlike vascular endothelium, VECs on both the aortic and ventricular side were not activated by exposure to bidirectional oscillatory flow (reproducing flow on the aortic side), as illustrated by an absence of vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin and BMP4 [48] However, exposure of aortic-sided endothelium to high-shear laminar flow (ventricular flow) resulted in endothelial activation characterized by expression of these inflammatory markers [48] This underscores the notion that both sets of VECs activate different mechanotransduction pathways in response to similar mechanical stimuli Endothelial cells play an important role in normal aortic valve function In addition to their traditional role in preserving an anti-adhesive and anti-proliferative surface, VECs are actively involved in the regulation of aortic valve function As discussed previously, mechanical strain on the surface of the cusps as well as coronary blood flow are both affected by the shape and stiffness of the aortic valve cusps [5,6,49,50] Aortic valve calcification has been shown to occur in areas of high mechanical strains on the valve, namely the commissures, the base of the cusps and the free edges [51] Changes in the stiffness of the aortic valve cusps could significantly impact stress magnitude and distribution along the surface of the cusps We have recently demonstrated the role of the endothelium in regulating the mechanical properties of the aortic valve in vitro using a biaxial micromechanical testing system [52] In that study, aortic valve cusps were stimulated with endothelin-1 (ET-1), a potent vasoconstrictor peptide which is released by endothe- lial cell or serotonin (5-HT), an agent which mediates the release of nitric oxide by the endothelium Addition of ET-1 resulted in a 25% increase in the elastic modulus (stiffness) of aortic valve cusps, whereas addition of 5-HT induced a 30% decrease in valve stiffness Interestingly, addition of cytochalasin D, an inhibitor of actin polymerization reversed the increase in valve stiffness in response to ET-1, highlighting the role of the contractile elements in the VIC population and the communication between VECs and VICs in the valve These findings suggest that the aortic valve is capable of auto-regulation, but that it is also subject to overall systemic conditions such as hypertension or diabetes which are often accompanied by dysregulated concentrations of circulating vasoactive agents As previously stated, hypertension, smoking and diabetes are among the risk factors associated with aortic valve calcification In addition to their direct metabolic effects, this could be secondary to their effect in modulating valve stiffness which could lead to abnormal stress distribution along the cusps [53] Is the aortic valve endothelium involved in valve calcification? To date, no definitive link between VECs and valve calcification has been directly established Nevertheless, a number of studies have recently demonstrated a potential causal relationship Taken together, this cumulative weight of evidence strongly suggests that the endothelium plays an active role in the cellular and molecular events involved in aortic valve calcification As mentioned earlier, Simmons et al showed a different pattern of gene expression between VECs on either side of the valve characterized by a higher expression of pro-calcific genes and on the aortic side of the valve [42] In addition, an 10 in vitro study examining the role of strain on VEC activation in vitro showed that overstretching of the cells induces the expression of adhesion molecules, an important initial event leading to local inflammation which is characterized by monocyte chemotaxis and infiltration [54] Physiological strain which was evaluated as a cyclical stretch of 10% did not affect VECs, whereas 20% cyclical strain on resulted in expression of VCAM-1, ICAM-1 and E-selectin [54], similar to what is commonly observed in calcified human valves In addition, a recent study by Kennedy et al using an in vitro model of VIC calcification such as described by us and others [55,56], demonstrated that addition of nitric oxide donors or agents raising intracellular cyclic guanosine monophosphate (cGMP) levels led to a decrease in the formation of calcified nodules in response to stimulation with osteogenic medium and transforming growth factor b (TGFb), an important pathogenic element in vascular and valvular calcification [57] Furthermore, in our laboratory, we developed an in vitro model of aortic valve calcification by exposing whole cusp tissue to osteogenic medium for a period of 10–14 days Preliminary data show that similar to what is observed in vivo, calcium nodules (characterized by positive Alizarin-red staining) were present on the aortic side of the valve More importantly, endothelial denudation of the cusp surface using a cell scraper or addition of L-NAME (an inhibitor of nitric oxide synthase) led to a significant increase in the number of Alizarin-red positive nodules (data not published) Taken together, these results strongly suggest that a healthy and functional valve endothelium acts an important first-line barrier against valve calcification The endothelium appears to exert its effect by a combination of effects on overall valve structure and function It can modify the mechanical properties of the cusps which might have an important effect in protecting the leaflets from high mechanical stresses These elevated stresses on the cusps can eventually lead to areas of ‘‘micro-tears’’ or interruption of the endothelial barrier As a consequence, the endothelium which appears to protect against calcification by communicating with the VICs in a paracrine way loses this capacity and exposes the VICs to osteogenic stimuli VICs have been shown to respond to direct osteogenic stimuli by expressing a high number of activated fibroblasts, the myofibroblasts These cells have the capacity to transdifferentiate into osteogenic-like cells and produce hydroxyapatite Clinical implications The importance of the living cellular environment in the valve is aptly illustrated in patients requiring aortic valve replacement surgery [58] Most aortic valve prostheses are acellular, including tissue valve prostheses and even aortic homografts which become rapidly decellularized These different valve substitutes are hampered by their limited durability due to structural valve deterioration [59] However, one operation consists of replacing the diseased aortic root with the patient’s pulmonary root: the Ross procedure It is the only operation which guarantees long-term viability of the neo-aortic valve This in turn directly translates into a significant benefit to the patients in terms of survival, freedom from reoperation, freedom from valve-related complications and quality of life [60] We believe that this observed benefit is in large part due I El-Hamamsy et al to the ability of the neo-aortic root to adapt to its hemodynamic environment, to respond to various stimuli and to continuously repair in a similar fashion to normal valves [61] Nevertheless, the Ross procedure is a technically complex operation which results in uneven results among different centers around the world Therefore, a more reproducible approach is necessary This will be best addressed by progresses in heart valve tissue engineering Although some promising first steps were made, more is required both in understanding normal valve physiology and combining biological, biochemical, engineering, physics and nanotechnology expertise to develop a biocompatible tri-layered aortic valve substitute that can reproduce the sophisticated functions of the normal aortic valve We and others are making big strides towards reaching that goal and are optimistic that a fully tissue-engineered heart valve will eventually become a viable option for patients undergoing valve replacement surgery Finally, therapeutic strategies targeting the endothelium could have a major impact on the treatment of aortic valve disease Various agents have shown promise in reducing, reversing or slowing the progression of vascular atherosclerotic disease, including statins [55] In addition to their lipid-lowering effect, statins exhibit a range of pleiotropic effects which can have a direct impact on endothelial function and address the different pathological mechanisms associated with hypercholesterolemia which lead to valve calcification, such as apoptosis [62] Thus far, most clinical trials have shown mixed results for the use of statins in patients with aortic valve disease However, it is possible that statin administration needs to be initiated as primary prevention in order to show a reduction in the incidence of aortic valve disease In the mean time, search for other more endothelial-specific compounds should be undertaken and will only be possible following a thorough understanding of the specific biology of VECs Conclusion The aortic valve lies at a critical junction in the circulatory system and is subjected to extremes of mechanical and hemodynamic forces with every cardiac cycle Yet, in the majority of people, these thin structures never fail This is a result of the highly sophisticated cellular and molecular functions of aortic valves We have overviewed the contribution of the endothelium to the structure and function of aortic valves in health and disease As our knowledge of these cells both in vitro and in vivo increases, understanding of the pathophysiological mechanisms of aortic valve disease will become clearer It is hoped that this will open opportunities towards targeted therapeutic approaches to aortic valve disease, starting with tissue engineering of heart valves References [1] Higashidate M, Tamiya K, Beppu T, Imai Y Regulation of the 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