Valvular interstitial cells suppress calcification of valvular endothelial cells.

Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. We previously proposed that valvular endothelial cells (VECs) replenish injured adult valve leaflets via endothelial-to-mesenchymal transformation (EndMT); however, whether EndMT contributes to valvular calcification is unknown. We hypothesized that aortic VECs undergo osteogenic differentiation via an EndMT process that can be inhibited by valvular interstitial cells (VICs). VEC clones underwent TGF-β1-mediated EndMT, shown by significantly increased mRNA expression of the EndMT markers α-SMA (5.3 ± 1.2), MMP-2 (13.5 ± 0.6) and Slug (12 ± 2.1) (p < 0.05), (compared to unstimulated controls). To study the effects of VIC on VEC EndMT, clonal populations of VICs were derived from the same valve leaflets, placed in co-culture with VECs, and grown in control/TGF-β1 supplemented media. In the presence of VICs, EndMT was inhibited, shown by decreased mRNA expression of α-SMA (0.1 ± 0.5), MMP-2 (0.1 ± 0.1), and Slug (0.2 ± 0.2) (p < 0.05). When cultured in osteogenic media, VECs demonstrated osteogenic changes confirmed by increase in mRNA expression of osteocalcin (8.6 ± 1.3), osteopontin (3.7 ± 0.3), and Runx2 (5.5 ± 1.5). The VIC presence inhibited VEC osteogenesis, demonstrated by decreased expression of osteocalcin (0.4 ± 0.1) and osteopontin (0.2 ± 0.1) (p < 0.05). Time course analysis suggested that EndMT precedes osteogenesis, shown by an initial increase of α-SMA and MMP-2 (day 7), followed by an increase of osteopontin and osteocalcin (day 14). The data indicate that EndMT may precede VEC osteogenesis. This study shows that VICs inhibit VEC EndMT and osteogenesis, indicating the importance of VEC-VIC interactions in valve homeostasis.

Hjortnaes J ，Shapero K ，Goettsch C ，Hutcheson JD ，Keegan J ，Kluin J ，Mayer JE ，Bischoff J ，Aikawa E ... -  《-》

Reciprocal interactions between mitral valve endothelial and interstitial cells reduce endothelial-to-mesenchymal transition and myofibroblastic activation.

Thickening of mitral leaflets, endothelial-to-mesenchymal transition (EndMT), and activated myofibroblast-like interstitial cells have been observed in ischemic mitral valve regurgitation. We set out to determine if interactions between mitral valve endothelial cells (VECs) and interstitial cells (VICs) might affect these alterations. We used in vitro co-culture in Transwell™ inserts to test the hypothesis that VICs secrete factors that inhibit EndMT and conversely, that VECs secrete factors that mitigate the activation of VICs to a myofibroblast-like, activated phenotype. Primary cultures and clonal populations of ovine mitral VICs and VECs were used. Western blot, quantitative reverse transcriptase PCR (qPCR) and functional assays were used to assess changes in cell phenotype and behavior. VICs or conditioned media from VICs inhibited transforming growth factor β (TGFβ)-induced EndMT in VECs, as indicated by reduced expression of EndMT markers α-smooth muscle actin (α-SMA), Slug, Snai1 and MMP-2 and maintained the ability of VECs to mediate leukocyte adhesion, an important endothelial function. VECs or conditioned media from VECs reversed the spontaneous cell culture-induced change in VICs to an activated phenotype, as indicated by reduced expression of α-SMA and type I collagen, increased expression chondromodulin-1 (Chm1), and reduced contractile activity. These results demonstrate that mitral VECs and VICs secrete soluble factors that can reduce VIC activation and inhibit TGFβ-driven EndMT, respectively. These findings suggest that the endothelium of the mitral valve is critical for the maintenance of a quiescent VIC phenotype and that, in turn, VICs prevent EndMT. We speculate that the disturbance of the ongoing reciprocal interactions between VECs and VICs in vivo may contribute to the thickened and fibrotic leaflets observed in ischemic mitral regurgitation, and in other types of valve disease.

Shapero K ，Wylie-Sears J ，Levine RA ，Mayer JE Jr ，Bischoff J ... -  《-》

Transforming growth factor-β1 promotes fibrosis but attenuates calcification of valvular tissue applied as a three-dimensional calcific aortic valve disease model.

Jenke A ，Kistner J ，Saradar S ，Chekhoeva A ，Yazdanyar M ，Bergmann AK ，Rötepohl MV ，Lichtenberg A ，Akhyari P ... -  《-》

Activated p300 acetyltransferase activity modulates aortic valvular calcification with osteogenic transdifferentiation and downregulation of Klotho.

The calcific aortic valve (AV) disease is a common disease with the unclear mechanism, and optimal pharmacological treatment remains unavailable. Epigenetic modulation by histone acetyltransferase (HAT) plays a critical role in osteogenic transdifferentiation and atherosclerosis. The purposes of this study were to investigate whether HAT contributes to the pathophysiology of AV calcification and assess the therapeutic potential of HAT inhibition. Porcine valvular interstitial cells (VICs) were treated with osteogenic medium (10ng/mL of tumor necrosis factor-α and 4mmol/L of high phosphate) for 7days. We analyzed the RNA and protein expression of myofibroblastic (α-SMA, vimentin, collagen 1A1, collagen 3, Egr-1, MMP2, MMP9) and osteoblastic markers (osteocalcin and alkaline phosphatase) in VICs, and studied the effects of a p300 inhibitor (C646, 10μmol/L) on calcification (Alizarin Red S staining), osteogenesis, HAT activity, the mitogen-activated protein kinase (MAPK) and Akt pathway, and Klotho expression on VICs. Osteogenic medium treated VICs had higher expressions of osteocalcin, alkaline phosphatase and acetylated lysine-9 of histone H3 (ac-H3K9) than control cells. C646 attenuated osteogenesis of VICs with simultaneous inhibition of the HAT activity of p300. There was neither significant increase of p300 protein nor p300 transcript during the osteogenesis process. Additionally, osteogenic medium treated VICs decreased the expression of Klotho, which is attenuated by C646. Activated HAT activity of p300 modulates AV calcification through osteogenic transdifferentiation of VICs with Klotho modulation. P300 inhibition is a potential therapeutic target for AV calcification.

Li SJ ，Kao YH ，Chung CC ，Chen WY ，Cheng WL ，Chen YJ ... -  《-》

Simulation of early calcific aortic valve disease in a 3D platform: A role for myofibroblast differentiation.

Calcific aortic valve disease (CAVD) is the most prevalent valve disease in the Western world. Recent difficulty in translating experimental results on statins to beneficial clinical effects warrants the need for understanding the role of valvular interstitial cells (VICs) in CAVD. In two-dimensional culture conditions, VICs undergo spontaneous activation similar to pathological differentiation, which intrinsically limits the use of in vitro models to study CAVD. Here, we hypothesized that a three-dimensional (3D) culture system based on naturally derived extracellular matrix polymers, mimicking the microenvironment of native valve tissue, could serve as a physiologically relevant platform to study the osteogenic differentiation of VICs. Aortic VICs loaded into 3D hydrogel constructs maintained a quiescent phenotype, similar to healthy human valves. In contrast, osteogenic environment induced an initial myofibroblast differentiation (hallmarked by increased alpha smooth muscle actin [α-SMA] expression), followed by an osteoblastic differentiation, characterized by elevated Runx2 expression, and subsequent calcific nodule formation recapitulating CAVD conditions. Silencing of α-SMA under osteogenic conditions diminished VIC osteoblast-like differentiation and calcification, indicating that a VIC myofibroblast-like phenotype may precede osteogenic differentiation in CAVD. Using a 3D hydrogel model, we simulated events that occur during early CAVD in vivo and provided a platform to investigate mechanisms of CAVD. Differentiation of valvular interstitial cells to myofibroblasts was a key mechanistic step in the process of early mineralization. This novel approach can provide important insight into valve pathobiology and serve as a promising tool for drug screening.

Hjortnaes J ，Goettsch C ，Hutcheson JD ，Camci-Unal G ，Lax L ，Scherer K ，Body S ，Schoen FJ ，Kluin J ，Khademhosseini A ，Aikawa E ... -  《-》