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 ... -  《-》

Bone Morphogenetic Protein Signaling Is Required for Aortic Valve Calcification.

Calcific aortic valve disease (CAVD) is the most prevalent type of heart valve disease, affecting ≈2% of the US population. CAVD is characterized by the presence of calcific nodules, resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that bone morphogenetic protein (BMP) signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane coreceptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho(-/-) mice. We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho(-/-) mice. In addition, cellular and extracellular matrix changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho(-/-) AoV. Likewise, osteogenic media treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction, and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the BMP type IA receptor in Klotho(-/-) aortic VICs, as well as BMP pathway inhibition of osteogenic media-treated aortic VICs in vitro, results in the inhibition of AoV calcification. BMP signaling and osteochondrogenic gene induction are active in calcified Klotho(-/-) AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.

Gomez-Stallons MV ，Wirrig-Schwendeman EE ，Hassel KR ，Conway SJ ，Yutzey KE ... -  《-》

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 ... -  《-》

MicroRNA-22 promoted osteogenic differentiation of valvular interstitial cells by inhibiting CAB39 expression during aortic valve calcification.

Calcific aortic valve disease (CAVD) is a common valve disease characterized by the fibro-calcific remodeling of the aortic valves, which is an actively regulated process involving osteogenic differentiation of valvular interstitial cells (VICs). MicroRNA (miRNA) is an essential regulator in diverse biological processes in cells. The present study aimed to explore the role and mechanism of miR-22 in the osteogenic differentiation of VICs. The expression profile of osteogenesis-related miRNAs was first detected in aortic valve tissue from CAVD patients (n = 33) and healthy controls (n = 12). miR-22 was highly expressed in calcified valve tissues (P < 0.01), and the expression was positively correlated with the expression of OPN (rs = 0.820, P < 0.01) and Runx2 (rs = 0.563, P < 0.01) in VICs isolated from mild or moderately calcified valves. The sustained high expression of miR-22 was also validated in an in-vitro VICs osteogenic model. Adenovirus-mediated gain-of-function and loss-of-function experiments were then performed. Overexpression of miR-22 significantly accelerated the calcification process of VICs, manifested by significant increases in calcium deposition, alkaline phosphate activity, and expression of osteoblastic differentiation markers. Conversely, inhibition of miR-22 significantly negated the calcification process. Subsequently, calcium-binding protein 39 (CAB39) was identified as a target of miR-22. Overexpression of miR-22 significantly reduced the expression of CAB39 in VICs, leading to decreased catalytic activity of the CAB39-LKB1-STRAD complex, which, in turn, exacerbated changes in the AMPK-mTOR signaling pathway, and ultimately accelerated the calcification process. In addition, ROS generation and autophagic activity during VIC calcification were also regulated by miR-22/CAB39 pathway. These results indicate that miR-22 is an important accelerator of the osteogenic differentiation of VICs, and a potential therapeutic target in CAVD.

Yang F ，Liu S ，Gu Y ，Yan Y ，Ding X ，Zou L ，Xu Z ，Wang G ... -  《-》

Endoplasmic reticulum stress participates in aortic valve calcification in hypercholesterolemic animals.

Cai Z ，Li F ，Gong W ，Liu W ，Duan Q ，Chen C ，Ni L ，Xia Y ，Cianflone K ，Dong N ，Wang DW ... -  《-》