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 Ca 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.

The thermal stability of crystallite powder and bulk single crystals of relaxor 0.91Pb(Zn1/3Nb2/3)O3–0.09PbTiO3 (0.91PZN–0.09PT) solid solution in air and an PbO‐rich environment has been investigated. At 700°C, perovskite PZN–PT decomposes only slightly to the pyrochlore phase. At 800°C, the rate of decomposition is accelerated, promoted by a surface breakaway decomposition process. This process occurs via the inward growth of faceted pyrochlore grains from the particle surface. At a certain point of the growth process, they will break away and detach themselves from the perovskite phase. The transformation stress also causes the adjacent perovskite phase to fracture concurrently, contributing to the breakaway event. At higher annealing temperatures (i.e., 900°–1000°C) and/or in the PbO‐rich environment, a layer of PbO‐rich liquid phase is formed on the surface of the particle. Because of limited wetting between the PbO‐rich liquid phase and the earlier‐formed pyrochlore grains, the latter detach themselves from the perovskite substrate. This action frees the substrate from the pyrochlore nuclei to initiate the breakaway decomposition event, which causes the rate of decomposition to slow substantially. At 1100°C both in air and the PbO‐rich environment, the PbO‐rich liquid formed vaporizes readily, which causes the decomposition rate to accelerate again. The present work shows that (i) the decomposition of perovskite PZN–PT to the pyrochlore phase during high‐temperature annealing is a surface phenomenon and (ii) its rate is controlled by the relative rate of formation and vaporization of the protective liquid layer that is present under the annealing conditions.

Objectives Although both calcium‐sensing receptor (CaSR) and canonical transient receptor potential (TRPC) proteins contribute to chronic hypoxia (CH)‐induced pulmonary arterial smooth muscle cells (PASMCs) proliferation, the relationship between CaSR and TRPC in hypoxic PASMCs proliferation remains poorly understood. The goal of this study was to identify that CH promotes PASMCs proliferation through CaSR‐TRPC pathway. Methods Rat PASMCs were isolated and treated with CH. Cell proliferation was assessed by cell counting, CCK‐8 assay, and EdU incorporation. CaSR and TRPC expressions were determined by qPCR and Western blotting. Store‐operated Ca2+ entry (SOCE) was assessed by extracellular Ca2+ restoration. Results In PASMCs, CH enhanced the cell number, cell viability and DNA synthesis, which is accompanied by upregulated expression of CaSR, TRPC1 and TRPC6. Negative CaSR modulators (NPS2143, NPS2390) inhibited, whereas positive modulators (spermine, R568) enhanced, the CH‐induced increases in cell number, cell viability and DNA synthesis in PASMCs. Knockdown of CaSR by siRNA inhibited the CH‐induced upregulation of TRPC1 and TRPC6 and enhancement of SOCE and attenuated the CH‐induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. However, neither siTRPC1 nor siTRPC6 had an effect on the CH‐induced CaSR upregulation, although both significantly attenuated the CH‐induced enhancements of cell number, cell viability and DNA synthesis in PASMCs. Conclusion These results demonstrate that upregulated CaSR‐TRPC1/6 pathway mediating PASMCs proliferation is an important pathogenic mechanism under hypoxic conditions.

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