Chronic hypoxia promotes pulmonary venous smooth muscle cell proliferation through the CaSR-TRPC6/ROCE pathway

Abstract

The mechanism underlying chronic hypoxia (CH)-induced pulmonary venous remodeling remains unclear. Cell proliferation is key in vascular remodeling, and the calcium-sensing receptor (CaSR) protein contributes to CH-induced pulmonary venous smooth muscle cell (PVSMC) proliferation. In pulmonary arterial smooth muscle cells, CaSR and transient receptor potential canonical (TRPC) proteins interact, contributing to CH-induced cell proliferation via CaSR-TRPC1/6 signaling. We investigated whether a similar pathway exists in PVSMCs. Rat PVSMCs were isolated and subjected to CH. Cell proliferation was assessed by cell counting, CCK-8, and BrdU incorporation assays. Expression of CaSR and TRPC was analyzed by qPCR and western blotting, while interactions between CaSR and TRPC were detected by co-immunoprecipitation assay. Extracellular Ca2+ restoration was evaluated, to assess store- and receptor-operated Ca2+ entry (SOCE and ROCE, respectively). CH enhanced PVSMC numbers, viability, and DNA synthesis, and upregulated CaSR and TRPC6 expression. Further, CaSR and TRPC6 interacted with one another. CaSR inhibitors (NPS2143, NPS2390) reduced, whereas activators (spermine, R568) enhanced, CH-induced increases in PVSMC numbers, viability, DNA synthesis, and TRPC6 expression. CaSR knockdown using siRNA inhibited CH-induced TRPC6 upregulation and attenuated CH-induced increases in PVSMC numbers, viability, and DNA synthesis. TRPC6 knockdown had no significant effect on CH-induced CaSR upregulation, but significantly attenuated CH-induced increases in PVSMC number, viability, and DNA synthesis. CaSR knockdown reduced ROCE, but not SOCE, enhancement. Overall, CH promotes PVSMC proliferation through the CaSR-TRPC6/ROCE pathway.