Hypoxia Promotes Proliferation and Inhibits Apoptosis of Pulmonary Arterial Smooth Muscle Cells via Modulating TRPC6.

Ascites syndrome (AS) is a common nutritional metabolic disease in broilers that results in major loss to the breeding industry. The occurrence of AS is closely related to the abnormal proliferation of pulmonary artery smooth muscles cells (PASMCs) caused by hypoxia. The transient receptor potential cation channel subfamily C member 6 (TRPC6) is a Ca2+ channel situated on cell membranes, and belongs to the proliferation of PASMCs caused by hypoxia in mammals. However, its role in hypoxic PASMCs in broilers remains unclear. Here, we investigated the effects of TRPC6 on the proliferation and apoptosis of primary chicken PASMCs under hypoxic conditions using an in vitro model. Primary chicken PASMCs were cultured under normoxic or hypoxic conditions (3% O2). The hypoxic cells were treated with 10 μM of SKF 96365 or 50 μM of fluofenamic acid (FFA) to inhibit or activate TRPC6, respectively. Cell viability was detected by CCK-8, intracellular Ca2+ levels, cell cycle and cell apoptosis were assayed by a flow cytometric assay, the mRNA levels of TRPC6 were measured by digital-droplet PCR (ddPCR), the protein levels of TRPC6 were tested by immunoblotting, and the mRNA levels of caspase3 were detected by RT-PCR. Our results revealed that exposing PASMCs to hypoxic conditions for 48h increased cell viability and intracellular Ca2+ levels. Additionally, hypoxic conditions increased the expression of TRPC6 and promoted cell cycle progression, but decreased cell apoptosis and caspase 3 mRNA levels. When the hypoxic PASMCs were treated with SKF 96365, inhibition of TRPC6 and cell proliferation and promotion of apoptosis were observed only in the first 24h of treatment. Treatment with FFA for 24h had the opposite effects. These results suggested that TRPC6-mediated Ca2+ entry contributed to hypoxia-induced PASMCs proliferation and apoptosis resistance, which identified TRPC6 as a possible key target in vascular remodeling in chicken. [ABSTRACT FROM AUTHOR]