Low-level overexpression of p53 promotes warfarin-induced calcification of porcine aortic valve interstitial cells by activating Slug gene transcription

Abstract

The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), while the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participating in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification.Immunostaining, quantitative PCR and western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. TUNEL staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to lowlevel increase of p53 mRNA and protein both in pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis, but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNAmediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification and increased slug transcription by p53 is required for p53-mediated pAVIC calcification.The tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis, and DNA p53 and aortic valve interstitial cell calcification 2 damage (1), as well as in osteoblastic differentiation and bone development. Deletion of the p53 inhibitor MDM2 in osteoblast lineage cells leads to increased p53 production and suppression of bone organogenesis and homeostasis, whereas deletion of p53 accelerates osteoblast cell differentiation and bone formation (2,3). In addition, using p53 knockout mice with chronic kidney disease, Li, KL, et al. showed that p53 negatively regulates osteogenic differentiation of vascular smooth muscle cells and aortic calcification suggesting the p53 is involved in pathological tissue calcification (4).The vitamin K antagonist, warfarin, is the most frequently used oral anti-coagulant to treat thromboprophylaxis in the presence of atrial fibrillation or a mechanical prosthetic heart valve. Recently, human and animal studies have implicated warfarin use with vascular and valvular calcification. Long-term warfarin treatment impairs vitamin K-dependent modification of many proteins, including matrix gla protein (MGP), inhibits the bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein...

Inhibition of acetylation of histones 3 and 4 attenuates aortic valve calcification

“…In contrast to the role of C646 in cancer cells, our results showed that C646 plays a protective role in high calcium/high phosphate–induced apoptosis and upregulates the level of Bcl-2 in pAVICs. In our previous study, we found higher baseline levels of the tumor suppressor p53 in AVICs than in other cell types 22 . We speculate that C646 may have opposing effect in different cell and chromatin status contexts or that some AVIC-specific expressed proteins may hinder the proapoptotic effect of C646.…”

“…Porcine aortic valve interstitial cells (pAVICs) were isolated by collagenase digestion, as previously described 22 and cultured in Dulbecco’s modified Eagle’s medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (ScienCell Research Laboratories, Inc., San Diego, CA, USA) and 1% penicillin-streptomycin. Normal cells from passages 3 to 5 were used for later experiments.…”

Conserved and Divergent Modulation of Calcification in Atherosclerosis and Aortic Valve Disease by Tissue Extracellular Vesicles

“…Directionality of p53 regulation in valvular calcification remains complicated, as warfarin treatment induces p53 overexpression and calcification in VIC cultures and murine aortic valves via recruitment to the Slug promoter. (27) While further studies will be needed to delineate the mechanistic contributions of valvular EVs to p53 transcriptional regulation, our data determined a putative role for EVs in contributing to this aspect of calcific valve disease.…”

Alcohol‐mediated calcium signals dysregulate pro‐survival Snai2/PUMA/Bcl2 networks to promote p53‐mediated apoptosis in avian neural crest progenitors

“…Unfortunately, attempts to further interrogate this network were stymied because many components of this pathway are not annotated in Galgal5 . However, although co‐expression of Snai2 and p53 was unexpected, it is not without precedent and was recently described for aortic valve interstitial cells in response to warfarin‐induced stress, and it may be noteworthy that these cells are endothelial in origin and similarly emerged from an EMT event (Gao et al, ).…”

“…A similar situation occurs in ribosomopathies, wherein endogenous Snai2 fails to prevent p53‐mediated NC death. Moreover, p53 has been shown to bind and directly stimulate Snai2 transcription (Gao et al, ; Wu et al, ), and it is possible the elevated Snai2 represents a failed attempt to counteract this p53 increase.…”