Abstract
Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease; however, the role of apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) in inorganic phosphate (Pi)-induced vascular smooth muscle cell (VSMC) calcification remains unknown. In this study, we investigated the possible role of APE1/Ref-1 in Pi-induced VSMC calcification. We observed that Pi decreased endogenous APE1/Ref-1 expression and promoter activity in VSMCs, and that adenoviral overexpression of APE1/Ref-1 inhibited Pi-induced calcification in VSMCs and in an ex vivo organ culture of a rat aorta. However, a redox mutant of APE1/Ref-1(C65A/C93A) did not reduce Pi-induced calcification in VSMCs, suggesting APE1/Ref-1-mediated redox function against vascular calcification. Additionally, APE1/Ref-1 overexpression inhibited Pi-induced intracellular and mitochondrial reactive oxygen species production, and APE1/Ref-1 overexpression resulted in decreased Pi-induced lactate dehydrogenase activity, pro-apoptotic Bax levels, and increased anti-apoptotic Bcl-2 protein levels. Furthermore, APE1/Ref-1 inhibited Pi-induced osteoblastic differentiation associated with alkaline phosphatase activity and inhibited Pi-exposure-induced loss of the smooth muscle phenotype. Our findings provided valuable insights into the redox function of APE1/Ref-1 in preventing Pi-induced VSMC calcification by inhibiting oxidative stress and osteoblastic differentiation, resulting in prevention of altered osteoblastic phenotypes in VSMCs.
Highlights
Vascular calcification described abnormal calcium deposition in vessel walls and is a characteristic feature of atherosclerosis, diabetes mellitus, and end-stage renal disease
We investigated the effect of Pi on endogenous APE1/Ref-1 expression in vascular smooth muscle cell (VSMC)
To determine whether Pi-mediated downregulation of APE1/Ref-1 occurred at the promoter level, cells were transfected with an APE1/Ref-1-promoter–luciferase reporter construct (−4000/+65 APE1/Ref-1–Luc, containing the APE1/Ref-1-promoter sequence comprising an area from −4000 bp to +65 bp of the gene) and incubated with 5 mM Pi for 4 days
Summary
Vascular calcification described abnormal calcium deposition in vessel walls and is a characteristic feature of atherosclerosis, diabetes mellitus, and end-stage renal disease. The contractile phenotype of VSMCs is regulated by a balance of several transcriptional factors and can change to support repair processes or return to a normal phenotype. This phenotype change is referred to as phenotypic plasticity or VSMC transition; inappropriate phenotype changes occurring under certain conditions, such as high inorganic phosphate (Pi) concentrations, induce vascular calcification via modulation of osteogenic transcription factors [10]. Inhibition of APE1/Ref-1-mediated redox signalling increases the osteoblastic differentiation of dental papilla cells [13]. It remains unknown whether APE1/Ref-1 affects vascular calcification and in what context. We tested the hypothesis that APE1/Ref-1 suppresses vascular calcification and investigated the role and mechanism of APE1/Ref-1 in Pi-induced calcification in VSMCs and rat aorta
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