Calcific and Degenerative Heart Valve Disease

Aortic Valve Cusps

Aortic valve (AV) cusps are attached to the aortic wall in a crescentic/semilunar manner, each ascending to the commissures and descending to its basal attachment to the aortic wall. Coaptation of the cusps during valve closure (diastole) occurs along a surface near the free edge of the cusps. The non-coapting portion of the cusp, which separates aortic from ventricular blood, is called the belly. During diastole, when the valve is closed, the cusps are stretched via backpressure of 80 mm Hg (the cuspal area increases by about 30%), filling the orifice; during the opened phase (systole), the cuspal tissue becomes relaxed and moves out of the central flow stream. Within the normal aortic root and behind the cusps are three bulging aortic sinuses of Valsalva. Pulmonary valve structure is analogous to that of the AV, but the sinuses are not prominent, and the cusps are thinner (than in the left ventricle and aorta), consistent with the lower blood pressure in the right ventricle and pulmonary circulation.

The structural architecture, composition and integrity of valve ECM (composed of collagen, elastin, and glycosaminoglycans) are the major determinants of valve durability. For the AV, the mechanism of function is largely mediated by the intrinsic architecture. Immediately below the endothelium on the outflow (aortic) surface is the fibrosa layer, composed predominantly of densely packed and microscopically crimped collagen fibers arranged largely parallel to the free edge of the leaflet. Collagen, the major stress-bearing component of the cusps, is abundant in the fibrosa, and is essential for the maintenance of valve durability. Individual collagen fibers can withstand high tensile forces but cannot be compressed. The ventricularis layer, near to the ventricular face, is rich in elastin, and provides tissue elasticity. When the valve is closed during cardiac diastole, the collagen is pulled taut, permitting maximum coaptation of the cusps without prolapse and shifting the load from the cusps to the aortic wall. During systole, the cusps, which were stretched during diastole, become relaxed due to elastin recoil. With its high compliance and bonds that link it to the adjacent fibrous layers, the proteoglycan-rich, central spongiosa layer facilitates the rearrangements of the fibrosa and thereby provides cushioning and shear absorbing function during the cardiac cycle. The predominant type of collagen in the valves, type I collagen, and elastin comprise ~80% of total valvular protein. AV cusps have anisotropic mechanical properties (i.e., different in the radial and circumferential directions), with compliance and therefore stretch greater in the radial direction. In addition, AV demonstrate (1) layer-specific directionality (i.e., the stiffer fibrosa dominates in the circumferential direction, and more compliant ventricularis dominates in the redial direction) and (2) regional heterogeneity (i.e., the cuspal belly is stiffer than the commissural region).

Mitral Valve Leaflets

In cardiac systole, the back pressure on the closed mitral valve (MV) is approximately 120 mmHg, thereby engendering substantial forces tending to push the leaflets backward into the left atrium. Thus, normal MV function requires assistance from structures extrinsic to and largely below the valve leaflets, specifically a complex and coordinated interaction of leaflets, annulus, chordae tendineae, and papillary muscle (collectively, the MV apparatus). The MV has two leaflets, the anterior below the aortic valve and the posterior embedded into the posterolateral valve annulus, and composed of three scallops. At the leaflet edges of the MV, collagen-rich chordae tendineae extend from the fibrosa to link the leaflets to the papillary muscles.

Similar to the AV cusps, normal MV leaflets have well-defined tissue layers: fibrosa (closest to the ventricle), spongiosa and atrialis (near the atrium). As with the AV discussed above, the anatomy and ECM composition of the MV are critical to understanding its material behavior. The anterior leaflet has heterogeneous anatomy in which the tissue layers vary in thickness from the mitral annulus to the free edge. The thickest region, located close to the annulus, is called the ‘clear zone’ owing to the lack of chordal basal attachments. This region withstands high load when the valve is closed. Toward the middle of the leaflet the relative thickness of the fibrosa is diminished, contributing to the radial extensibility. The thickness of the spongiosa also increases, thereby promoting compressive load-bearing in the region of coaptation. The complex microstructure of the mitral valve leaflets results in marked non-linear anisotropic mechanical properties within leaflets and throughout individual layers, similar to AV cusps.

Valvular Endothelial and Interstitial Cells

A continuous endothelial cell layer covers the cusps and leaflets on both inflow and outflow aspects. While valvular endothelial cells (VECs) resemble endothelial cells (ECs) elsewhere in the circulation, they have distinctive phenotypic features. For example, in response to fluid shear stress in vitro, VECs align perpendicularly to flow in contrast to vascular ECs, which align parallel to flow. In addition, VECs from the AV express different transcriptional profiles on the arterial vs. ventricular sides. Indeed, VECs covering the fibrosa have elevated expression of proteins expected to promote calcification, while VECs subjected to the ventricular hemodynamic waveform (i.e., experienced in vivo by the VEC lining the ventricularis) have increased expression of the ‘atheroprotective’ transcription factor Kruppel-like factor2 (KLF2), expected to inhibit calcification. These valve-side-specific VEC phenotypic differences may contribute to the typical pattern of initiation and predominance of calcification near the AV outflow surface.

Moreover, a key property of VECs is the ability to undergo an epithelial to mesenchymal transition (EMT). Emerging evidence suggests that VEC mesenchymal differentiation can be specifically directed toward osteogenic, chondrogenic, and adipogenic phenotypes. This multi-lineage differentiation potential of VEC combined with a robust self-renewal capacity supports the hypothesis that VECs are capable of serving as progenitor cells.

Deep to the VECs, heart valve cusps and leaflets contain valvular interstitial cells (VICs), a heterogeneous, dynamic and highly plastic population of resident cells. Adult VICs are predominantly quiescent non-contractile, alpha-smooth muscle actin-negative, vimentin-positive fibroblast-like cells; few are activated. In response to a need for either adaptation to changing conditions or repair of ECM functional damage (e.g., valve development, disease, abrupt changes in the mechanical stress, or surgical intervention), large numbers of VICs transition to an activated myofibroblast-like phenotype. In these situations, VIC express smooth muscle contractile proteins, matrix remodeling enzymes (e.g., matrix metalloproteinases [MMP-1, MMP-2, MMP-9, MMP-13] and their inhibitors [tissue inhibitors of metalloproteinases]), cathepsins (cathepsin K, cathepsin S) and cytokines (IL-1, TGFβ) . Five specialized VIC phenotypes have been described: embryonic progenitor (eVICs), quiescent (qVICs), activated (aVICs), progenitor (pVICs), and osteoblastic (oVICs) ( Figure 9.3 ). The transition from a quiescent to an activated VIC phenotype is thought to be induced by changes in the mechanical environment of the VICs and/or TGF-β signaling. In healthy valves, VIC plasticity contributes to adaptation to dynamic changes in the valve and the surrounding milieu. Following adaptation or repair; this transition may be reversible and VICs may revert to a quiescent phenotype if equilibrium is established. In contrast, when adaptation to a new environmental condition is not possible, e.g., with ongoing, progressive and excessive mechanical stress, valve disease can result.

Development and Maturation

There is increasing evidence that the regulatory mechanisms involved in normal valve morphogenesis and development are relevant to the pathobiology of valvular diseases. However, it is unknown to what extent and under what circumstances reactivation (or potentially failure) of these developmental mechanisms mediates repair or contributes to disease progression.

Valve formation is a complex and highly regulated process. Valve development begins with the formation of the endocardial cushions, the precursors of the cardiac valves, which occurs by the separation of the endocardium and overlying myocardium in the outflow tract and in the atrioventricular canal by expansion of the acellular ECM called cardiac jelly. Subsets of endothelial cells change their phenotype into mesenchymal cells expressing alpha-smooth-muscle cell actin; they lose cell–cell contacts, and migrate into the cardiac jelly via the process of EMT discussed above. Migration of neural crest cells to the primordial aortic valve in the outflow tract occurs later. Swelling of the endocardial cushions and induction of EMT also contribute to the morphogenesis of the semilunar valve leaflets. EMT is induced by signaling molecules, including TGFβ1–3, BMP 2–4, and Notch 1–4, originating from the underlying myocardium. Additionally, several transcription factors are important in endocardial cushion formation and EMT. Stimulation of the Notch pathway depends on the transcription factor RBPJ, which activates transcription factor snail1 (Snai1). Loss of Snai1 in endothelial cells inhibits endocardial cushion formation. Gain- and loss-of-function studies have demonstrated critical roles for Twist1, Tbx20, and Sox9 in proliferation of mesenchymal cells in the endocardial cushions. After endocardial cushion formation, Twist1 is down-regulated and cell proliferation is decreased. Moreover, in normal adult valves, cell proliferation is very low, and expression of valve developmental transcription factors such as Twist1 and Sox9 is negligible. Also, emerging evidence suggests that VICs in adult valves are continuously replenished via EMT, with this process potentially playing an important role in valve healing and remodeling under physiological and pathological conditions.

The later stages of valve development and maturation are characterized by gradual thinning and elongation of pre-valvular tissue; this process is concurrent with the differentiation of the cushion mesenchymal cells into VEC and VIC, with concomitant ECM remodeling. Post-EMT valve development involves cell migration and proliferation associated with RANKL, cathepsin K, NFAT, periostin, cadherin, Notch, MMP-2, MMP-13, and VEGF expression. Cathepsin K, a matrix remodeling enzyme expressed during heart valve elongation, is regulated by the RANKL/NFATc1 pathway, and involved in differentiation and function of osteoclasts.

Beyond calcification-promoting proteins discussed earlier, some signaling molecules and pathways important in valve pathophysiology are expressed differentially on the two sides of the valves. Notch signaling is localized on the surface adjacent to the AV ventricularis, while Wnt/β-catenin signaling is found throughout the valve interstitium in late gestation. Periostin, required for collagen remodeling, is expressed in the fibrosa layer. Loss of periostin in mice leads to valve malformation and cardiac dysfunction. Mutations in Collagen 1a2 or elastin insufficiency affect aortic valve morphogenesis leading to valve disease. MMP-1, MMP-2, MMP-9, and MMP-13 are also expressed during late valve morphogenesis and involved in ECM maturation and organization. In certain disease conditions these transcriptional regulatory and signaling mechanisms are reactivated and have been implicated in aortic and mitral valve diseases.

Dynamic changes in ECM architecture and VEC and VIC phenotypes in response to environmental stimuli continue throughout fetal and postnatal development and throughout life. Studies of human valves obtained from second- and third-trimester fetuses, neonates, children, and adults have shown that valve structural architecture evolves over a lifetime ( Figure 9.4 ). This likely effects a progressive adaptation of valvular cells to continuous hemodynamic changes and potential injury. Embryonic VECs possess an activated phenotype throughout fetal development (i.e., expression of VCAM-1, ICAM-1), and VICs concurrently show a myofibroblast-like phenotype (alpha-smooth muscle actin, vimentin, desmin), abundant embryonic myosin (SMemb) and MMP-1, MMP-2, MMP-9, and MMP-13 expression, indicating an immature/activated phenotype engaged in rapid matrix remodeling. This compares with the quiescent fibroblast-like VICs usually present in adults. In addition, VIC density, proliferation and apoptosis are significantly higher in fetal than adult valves. Indeed, cell density in adult valves is reduced to ∼10% and cell proliferation is below 2% of that in utero. At birth, after the single fetal circulation separates into pulmonary and systemic circulations, pulmonary pressure decreases and aortic pressure increases. This abrupt hemodynamic change is accompanied by increased aortic VIC activation and increased alpha-smooth-muscle actin expression consistent with increased mechanical stress. Moreover, a tri-laminar architecture evolves in human valves over time and becomes well-defined histologically only after 36 weeks of gestation, but the structure at that stage is less well demarcated than that of adult valves. Collagen content increases from early to late fetal stages, but is subsequently stable, while mature elastic fibers significantly increase only post-natally.

Post-Natal Adaptation

Under physiologic conditions of valve repair, adaptation, remodeling, and in a pathological environment, cardiac valves respond to changes in mechanical stress by cell activation leading to matrix remodeling. Accumulating evidence suggests that during adaptation (as evidenced by the pulmonary-to-aortic allograft surgical procedure), disease (CAVD, DMVD), mechanical stretching (mitral valve chordae displacement), or remodeling (tissue-engineered valves), many VICs acquire an activated myofibroblast-like phenotype. Nevertheless, as stated above, owing to high plasticity, these changes in VIC phenotype are potentially reversible, and VICs can return to a quiescent state after mechanical equilibrium is achieved by adaptive ECM remodeling. Thus, cardiac valves can adapt to pathological conditions by reversible phenotypic modulation of quiescent VICs to activated, myofibroblast-like VICs; moreover, analogous molecular mechanisms may direct both physiological and pathological cell activation.

Aging

Progressive age-associated decrease in cell density, cell turnover and reduction in the active capacity for remodeling, as described above, accompanies aging. In some AVs after the age of 30 years, histological analysis demonstrates atheromatous changes (lipid association with foamy macrophages) and increased extracellular matrix. Although these lipid-rich lesions are similar in some respects to fatty streaks and early atherosclerotic plaques found in the vasculature, they do not develop complications of thrombosis or ulceration/rupture. Moreover, it is uncertain whether these lesions are precursors of CAVD. Also potentially important to the development of valve disease is the fact that the valve assumes a progressively more stretched configuration and collagen fibers become increasingly more aligned with increasing age, correlating with decreasing valve mechanical compliance.