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Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, which exists in both pulmonary arteries and pulmonary veins. Pulmonary vascular remodeling stems from excessive proliferation of pulmonary vascular myocytes. Platelet-derived growth factor-BB (PDGF-BB) is a vital vascular regulator whose level increases in PH human lungs. Although the mechanisms by which pulmonary arterial smooth muscle cells respond to PDGF-BB have been studied extensively, the effects of PDGF-BB on pulmonary venous smooth muscle cells (PVSMCs) remain unknown. We herein examined the involvement of calcium sensing receptor (CaSR) in PDGF-BB-induced PVSMCs proliferation under hypoxic conditions. In PVSMCs isolated from rat intrapulmonary veins, PDGF-BB increased the cell number and DNA synthesis under normoxic and hypoxic conditions, which was accompanied by upregulated CaSR expression. The influences of PDGF-BB on proliferation and CaSR expression in hypoxic PVSMCs were greater than that in normoxic PVSMCs. In hypoxic PVSMCs superfused with Ca(2+)-free solution, restoration of extracellular Ca(2+) induced an increase of [Ca(2+)](i), which was significantly smaller than that in PDGF-BB-treated hypoxic PVSMCs. The positive CaSR modulator spermine enhanced, whereas the negative CaSR modulator NPS2143 attenuated, the extracellular Ca(2+)-induced [Ca(2+)](i) increase in PDGF-BB-treated hypoxic PVSMCs. Furthermore, the spermine enhanced, whereas the NPS2143 inhibited, PDGF-BB-induced proliferation in hypoxic PVSMCs. Silencing CaSR with siRNA attenuated the extracellular Ca(2+)-induced [Ca(2+)](i) increase in PDGF-BB-treated hypoxic PVSMCs and inhibited PDGF-BB-induced proliferation in hypoxic PVSMCs. In conclusion, these results demonstrated that CaSR mediating PDGF-BB-induced excessive PVSMCs proliferation is an important mechanism involved in the initiation and progression of PVSMCs proliferation under hypoxic conditions.

The culture of pulmonary arterial smooth muscle cells (PASMCs) is one of the most powerful tools for exploring the mechanisms of pulmonary hypertension (PH). Both pulmonary vasoconstriction and remodeling occur predominantly in distal pulmonary arteries (PA). In this study, we provide our detailed and standardized protocol for easy isolation and culture of PASMCs from rat distal PA to supply every investigator with a simple, economical and useful method in studying PH. The protocol can be divided into four stages: isolation of distal PA, isolation of cells, growth in culture and passage of cells. Rat distal PASMCs were characterized by morphological activity and by immunostaining for smooth muscle α-actin and smooth muscle myosin heavy chain, but not for CD90/Thy-1 or von Willebrand factor. Furthermore, functional assessments were performed, confirming the presence of voltage-dependent Ca2+ channels and physiological characteristic of response to hypoxia. In conclusion, we have developed a detailed and simple protocol for obtaining rat distal PASMCs. These PASMCs exhibit features consistent with vascular smooth muscle cells, and they could subsequently be used to further explore the pathophysiological mechanisms of PH.