Intermedin 1–53 attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase A pathway

Abstract

We previously reported that endoplasmic reticulum (ER) stress-mediated apoptosis participated in vascular calcification. Importantly, a novel paracrine/autocrine peptide intermedin_1–53 (IMD_1–53) in the vasculature inhibited vascular calcification in rats. But the mechanisms needed to be fully elucidated. Vascular smooth muscle cells (VSMCs) calcification was induced by CaCl₂ and β-glycerophosphate. Tunicamycin (Tm) or dithiothreitol (DTT) was used to induce ER stress. We found that IMD_1–53 (10⁻⁷mol/L) treatment significantly alleviated the protein expression of ER stress hallmarks activating transcription factor 4 (ATF4), ATF6, glucose-regulated protein 78 (GRP78) and GRP94 induced by Tm or DTT. ER stress occurred in early and late calcification of VSMCs but was inhibited by IMD_1–53. These inhibitory effects of IMD_1–53 were abolished by treatment with the protein kinase A (PKA) inhibitor H89. Pretreatment with IMD_1–53 decreased the number of apoptotic VSMCs and downregulated protein expression of cleaved caspase 12 and C/EBP homologous protein (CHOP) in calcified VSMCs. Concurrently, IMD_1–53 restored the loss of VSMC lineage markers and ameliorated calcium deposition and alkaline phosphatase activity in calcified VSMCs as well. The observation was further verified by Alizarin Red S staining, which showed that IMD_1–53 reduced positive red nodules among calcified VSMCs. In conclusion, IMD_1–53 attenuated VSMC calcification by inhibiting ER stress through cAMP/PKA signalling.