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...

Biological sex is increasingly recognized as a critical determinant of health and disease, particularly relevant to the topical COVID-19 pandemic caused by the SARS-CoV-2 coronavirus. Epidemiological data and observational reports from both the original SARS epidemic and the most recent COVID-19 pandemic have a common feature: males are more likely to exhibit enhanced disease severity and mortality than females. Sex differences in cardiovascular disease and COVID-19 share mechanistic foundations, namely, the involvement of both the innate immune system and the canonical renin-angiotensin system (RAS). Immunological differences suggest that females mount a rapid and aggressive innate immune response, and the attenuated antiviral response in males may confer enhanced susceptibility to severe disease. Furthermore, the angiotensin-converting enzyme 2 (ACE2) is involved in disease pathogenesis in cardiovascular disease and COVID-19, either to serve as a protective mechanism by deactivating the RAS or as the receptor for viral entry, respectively. Loss of membrane ACE2 and a corresponding increase in plasma ACE2 are associated with worsened cardiovascular disease outcomes, a mechanism attributed to a disintegrin and metalloproteinase (ADAM17). SARS-CoV-2 infection also leads to ADAM17 activation, a positive feedback cycle that exacerbates ACE2 loss. Therefore, the relationship between cardiovascular disease and COVID-19 is critically dependent on the loss of membrane ACE2 by ADAM17-mediated proteolytic cleavage. This article explores potential mechanisms involved in COVID-19 that may contribute to sex-specific susceptibility focusing on the innate immune system and the RAS, namely, genetics and sex hormones. Finally, we highlight here the added challenges of gender in the COVID-19 pandemic.

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

Objective— In calcific aortic valve disease, myofibroblasts and activation of the transforming growth factor-β1 (TGF-β1) and Wnt/β-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of β-catenin and extracellular matrix stiffness in TGF-β1-induced myofibroblast differentiation of valve interstitial cells (VICs). Methods and Results— TGF-β1 induced rapid β-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-β receptor I kinase. Degrading β-catenin pharmacologically or silencing it with small interfering RNA inhibited TGF-β1-induced myofibroblast differentiation without altering Smad2/3 activity. Conversely, increasing β-catenin availability with Wnt3A alone did not induce differentiation. However, combining TGF-β1 and Wnt3A caused greater myofibroblast differentiation than TGF-β1 treatment alone. Notably, in VICs grown on collagen-coated PA gels with physiological stiffnesses, TGF-β1-induced β-catenin nuclear translocation and myofibroblast differentiation occurred only on matrices with fibrosa-like stiffness, but not ventricularis-like stiffness. In diseased aortic valves from pigs fed an atherogenic diet, myofibroblasts colocalized with increased protein expression of Wnt3A, β-catenin, TGF-β1, and phosphorylated Smad2/3 in the fibrosa. Conclusion— Myofibroblast differentiation of VICs involves matrix stiffness–dependent crosstalk between TGF-β1 and Wnt signaling pathways and may explain in part why the stiffer fibrosa is more susceptible to disease.

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