Objective-Extensive remodeling of the valve ECM in calcific aortic valve sclerosis alters its mechanical properties, but little is known about the impact of matrix mechanics on the cells within the valve interstitium. In this study, the influence of matrix stiffness in modulating calcification by valve interstitial cells (VICs), and their differentiation to pathological phenotypes was assessed. Methods and Results-Primary porcine aortic VICs were cultured in standard media or calcifying media on constrained type I fibrillar collagen gels. Matrix stiffness was altered by changing only the thickness of the gels. Calcification did not occur in standard media, regardless of matrix stiffness. However, when VICs were grown in calcifying media on relatively compliant matrices with stiffness similar to that of normal tissue, they readily formed calcified aggregates of viable cells that expressed osteoblast-related transcripts and proteins. In contrast, VICs cultured in calcifying media on stiffer matrices (similar to stenotic tissue) differentiated to myofibroblasts and formed calcified aggregates that contained apoptotic cells. Actin depolymerization reduced aggregation on stiff, but not compliant, matrices. TGF-␤1 potentiated aggregate formation on stiff matrices by enhancing ␣-smooth muscle actin expression and cellular contractility, but not on compliant matrices attributable to downregulation of TGF-␤ receptor I. Cell contraction by VICs inhibited Akt activation and enhanced apoptosis-dependent calcification on stiff matrices. Key Words: aortic valve Ⅲ matrix mechanics Ⅲ mechanobiology Ⅲ collagen Ⅲ sclerosis D ysregulation of normal cellular processes 1,2 leads to aortic valve sclerosis (AS), a common disease 3 that involves chronic inflammation, fibrosis, and calcification. 4,5 The consequences of AS are serious, as even minor valve calcification increases the risk of other cardiovascular disorders by 50%, and the prognosis with progression to sclerosis is poor. 6 Treatment is limited to surgical replacement of the stenotic valves, as effective medical therapies do not exist. Conclusions-Differentiation of VICs to pathological phenotypes in response toThe progression of sclerosis and calcification is mediated primarily by valve interstitial cells (VICs) that populate the interstitial matrix. 1,7 As in the vasculature, 8 calcification of the aortic valve occurs through multiple mechanisms, 9 including apoptosis-related calcification typically associated with myofibrogenic activation of VICs, 10,11 calcium deposition associated with necrotic cells, 12 and bone formation by resident VICs 13 or bone marrow-derived cells. 10 However, details of the cellular mechanisms by which VICs contribute to calcification are not well understood, largely because of the limited number of studies in vitro and difficulties with their interpretation. For example, when VICs are induced to form calcified multicellular aggregates in vitro, the aggregates are associated with the expression of bone-related transcripts and proteins, the expr...

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The hallmarks of calcific aortic valve disease (CAVD) are the significant changes that occur in the organization, composition, and mechanical properties of the extracellular matrix (ECM), ultimately resulting in stiffened stenotic leaflets that obstruct flow and compromise cardiac function. Increasing evidence suggests that ECM maladaptations are not simply a result of valve cell dysfunction; they also contribute to CAVD progression by altering cellular and molecular signaling. In this review, we summarize the ECM changes that occur in CAVD. We also discuss examples of how the ECM influences cellular processes by signaling through adhesion receptors (matricellular signaling), by regulating the presentation and availability of growth factors and cytokines to cells (matricrine signaling), and by transducing externally applied forces and resisting cell-generated tractional forces (mechanical signaling) to regulate a wide range of pathological processes, including differentiation, fibrosis, calcification, and angiogenesis. Finally, we suggest areas for future research that should lead to new insights into bidirectional cell-ECM interactions in the aortic valve, their contributions to homeostasis and pathobiology, and possible targets to slow or prevent the progression of CAVD.

Advanced valvular lesions often contain ectopic mesenchymal tissues, which may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitium. The identity, frequency, and differentiation potential of the putative progenitor subpopulation are unknown. The objectives of this study were to determine whether valve interstitial cells (VICs) contain a subpopulation of multipotent mesenchymal progenitor cells, to measure the frequencies of the mesenchymal progenitors and osteoprogenitors, and to characterize the osteoprogenitor subpopulation because of its potential role in calcific aortic valve disease. The multilineage potential of freshly isolated and subcultured porcine aortic VICs was tested in vitro. Progenitor frequencies and selfrenewal capacity were determined by limiting dilution and colony-forming unit assays. VICs were inducible to osteogenic, adipogenic, chondrogenic, and myofibrogenic lineages. Osteogenic differentiation was also observed in situ in sclerotic porcine leaflets. Primary VICs had strikingly high frequencies of mesenchymal progenitors (48.0 ؎ 5.7%) and osteoprogenitors (44.1 ؎ 12.0%). High frequencies were maintained for up to six population doublings , but decreased after nine population doublings to 28.2 ؎ 9.9% and 5.8 ؎ 1.3% , for mesenchymal progenitors and osteoprogenitors , respectively. We further identified the putative osteoprogenitor subpopulation as morphologically distinct cells that occur at high frequency , self-renew , and elaborate bone matrix from single cells. These findings demonstrate that the aortic valve is rich in a mesenchyma l progenitor cell population that has strong potential to contribute to valve calcification. (Am J Pathol