Effect of HIF-PHI on Vascular Calcification and Apoptosis in High Phosphate-Induced VSMCs

Abstract

Background: Vascular calcification (VC) is a significant risk factor for cardiovascular disease. The pathogenesis involves various factors such as hyperphosphatemia, oxidative stress, inflammation, and apoptotic cell death, leading to phenotypic changes in vascular smooth muscle cells (VSMCs) and subsequent calcification. This study aims to investigate the impact of roxadustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), on the development of calcification in VSMCs induced by hyperphosphatemia in mice. Methods: Aortic VSMCs were cultured and exposed to high phosphate conditions to induce calcification. The cells were divided into control, high phosphate, and HIF-PHI plus high phosphate groups. Cell viability was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, and calcium content was measured through Von Kossa staining and the bicinchoninic acid (BCA) assay. The apoptotic rate was determined by flow cytometry, alkaline phosphatase concentration was determined by enzyme-linked immunosorbent assay (ELISA), and mitochondrial reactive oxygen species (ROS) levels were determined by Dichlorodihydrofluorescein acetoacetate (DCFH-DA). Protein expression levels were evaluated using immunoblotting techniques. Results: Compared to the control group, calcium deposition, calcium content, alkaline phosphatase, Runt-related Transcription Factor 2 (RUNX2) expression and apoptotic rate were significantly higher, while Alpha Smooth Muscle Actin (α-SMA) expression was significantly lower in the high phosphate group (p < 0.05). The HIF-PHI group showed an opposite trend to the high phosphate group in a dose-dependent manner (p < 0.05). The mitochondrial ROS and BCL2/adenovirus E1B 19kDa protein-interacting protein 3-like (BNIP3L) expression were significantly higher in the high phosphate group than in the control group (p < 0.05). Compared to the high phosphate group, the mitochondrial ROS was lower, while the protein expression levels of BNIP3L and Bcl-2 Interacting Protein 1 (Beclin-1) were higher in the HIF-PHI group (p < 0.05). HIF-1α protein expression was significantly higher in the high phosphate group than in the control group, which was increased by the treatment of HIF-PHI (p < 0.05). Conclusions: HIF-PHI ameliorates vascular calcification and apoptosis in high-phosphate induced viability of VSMCs by upregulation of HIF-1α expression, reduced mitochondrial ROS production and increased expression of autophagy-related proteins.