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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. more...

Chronic hypoxia (CH) causes remodeling not only in pulmonary arteries but also in pulmonary veins. Pulmonary vascular remodeling stems from increased pulmonary vascular myocyte proliferation. However, the pathogenesis of CH-induced proliferation of pulmonary venous smooth muscle cells (PVSMCs) remains unknown. The present study aimed to explore the mechanisms by which CH affects PVSMCs proliferation. PVSMCs were isolated from rat distal pulmonary veins and exposed to CH (4% O2 for 60 h). The expression of calcium sensing receptor (CaSR) was determined by immunofluorescence, real-time quantitative PCR and Western blotting. Cell proliferation was assessed by cell counting, CCK-8 assay, and BrdU incorporation. Apoptosis analysis was examined by flow cytometry. In rat distal PVSMCs, CH increased the cell number and cell viability and enhanced DNA synthesis, which is accompanied by upregulated mRNA and protein expression levels of CaSR. Two negative CaSR modulators (NPS2143, NPS2390) not only attenuated CH-induced CaSR upregulation but also inhibited CH-induced increases in cell number, cell viability and the proliferation index of PVSMCs, whereas two positive modulators (spermine, R568) not only amplified CH-induced CaSR upregulation but also intensified CH-induced increases in cell number, cell viability and the proliferation index of PVSMCs. Silencing CaSR with siRNA similarly attenuated the CH-induced enhancement of cell number, cell viability and DNA synthesis in PVSMCs. Neither CH nor downregulation of CaSR with siRNA had an effect on apoptosis in PVSMCs. These results suggest that CaSR mediating excessive proliferation is a new pathogenic mechanism involved in the initiation and progression of distal PVSMCs proliferation under CH conditions. more...

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. more...

Primary culture of pulmonary arterial smooth muscle cells is used extensively for in vitro studies of the physiology and pathophysiology of numerous lung diseases, including chronic hypoxic pulmonary hypertension (CHPH). Despite the potentially important functions of pulmonary veins in CHPH, primary culturing of pulmonary venous smooth muscle cells (PVSMCs) has received very little attention to date. No efficient and widely accepted methods have been established. Consequently, related studies have been delayed, which inhibits progress in exploring the mechanisms of CHPH and other lung diseases. In this study, we describe a simple and efficient method of obtaining primary cultures of PVSMCs isolated from rat distal pulmonary veins. By following four steps, isolation of pulmonary veins, enzymatic digestion, concentration of resuspended pellets and incubation, we acquired purified PVSMCs (>95%). PVSMCs were characterized by morphological activity and by immunoblotting and immunofluorescence staining for alpha-smooth muscle actin. Furthermore, the response of cells to 60 mM KCl was tested, confirming the presence of functional L-type voltage-dependent Ca(2+) channels that are characteristic of smooth muscle cells. In conclusion, we have established a simple and reliable technique to isolate and culture PVSMCs from rat distal pulmonary veins. These PVSMCs exhibit features consistent with vascular smooth muscle cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary vein. more...