Abstract
BackgroundHypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).MethodsChronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O2 for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca2+ concentration ([Ca2+]i) and store-operated Ca2+ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.ResultsHypoxia increased the basal [Ca2+]i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O2, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.ConclusionsHypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca2+]i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.
Highlights
Pulmonary hypertension may occur either as a primary disease or as a complication of some pulmonary disorders such as chronic obstructive pulmonary disease (COPD), and progressively leads to heart failure and increased mortality
To confirm the presence of voltage-dependent calcium channels (VDCCs), which is another characteristic of smooth muscle cells, we measured the effect of 60 mM KCl on [Ca2+]i in the cultured pulmonary venous smooth muscle cells (PVSMCs) and all cells in visual fields exhibited clear increased [Ca2+]i (Figure 1C)
Expression of transient receptor potential canonical (TRPC) in PVSMCs was detected and the results showed that TRPC6, but not TRPC1, was significantly increased at both mRNA and protein levels in hypoxic PVSMCs, compared with that in normoxic controls (Figure 4A–E)
Summary
Pulmonary hypertension may occur either as a primary disease or as a complication of some pulmonary disorders such as chronic obstructive pulmonary disease (COPD), and progressively leads to heart failure and increased mortality. Exposure to hypoxia is associated with vasoconstriction and vasculature remodeling which contributes to pulmonary hypertension [1]. Calcium entry from the extracellular space is a critical step during the hypoxia induced pulmonary vascular smooth muscle contraction. TRPC1, TRPC4, and TRPC6 have been demonstrated to be present in rat pulmonary vasculature and are associated with increased store-operated calcium entry (SOCE), elevated basal intracellular Ca2+ concentration ([Ca2+]i) and the proliferation and migration of PASMCs [5,6]. TRPC1 and TRPC6 have been demonstrated to be up-regulated by hypoxia and are associated with the SOCE enhancement in PASMCs [7]. We explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs)
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