Phenotypic Switching of Atherosclerotic Smooth Muscle Cells is Regulated by Activated PARP1-Dependent TET1 Expression.

Abstract

Aim: During the development of atherosclerosis, the vascular smooth muscle cells (SMCs) undergo phenotypic switching from contractile phenotype to synthetic phenotype. This study aimed at examining the role of DNA modification mediated by the oxidative stress dependent ten eleven translocation enzymes (TETs) expression at early stage of phenotypic switching. Methods: Based on the in vitro SMCs calcification model, DNA damage, phenotypic switching and 5-hydroxymethylcytosine (5hmC) were examined by comet assay, alkaline DNA unwinding assay, immunofluorescence staining, Dot blotting and Western blotting. Then Western blotting and qRT-PCR were performed to analyze the TETs expression and the relationship between the activity of poly(ADP-ribose) polymerase 1 (PARP1) and TETs expression. We further alter 5hmC modification by inhibition of TET1 or PARP1 to rescue the phenotypic switching of SMCs using immunofluorescence staining, Dot blotting and qRT-PCR. We performed immunochemistry staining to examine the activated PARP1-TET1 pathway in vivo . Results: The phenotypic switching was observed in the SMCs cultured with calcification medium as the expression of the cell markers of contractile SMCs decreased and cell proliferation increased. In contrast, PAR and 5hmC were markedly increased in SMCs with calcification due to DNA damage. Our study further demonstrated that oxidative stress-activated PARP1, promotes TET1 expression and 5hmC increase during the phenotypic switching. Inhibition of TET1 or PARP1 can rescue the phenotypic switching of SMCs with calcification. Conclusion: Our study demonstrated the important role of PARylation dependent 5hmC, in SMCs phenotypic switching. It raises the possibility to target TET1 and PARP1 for atherosclerosis treatment.