Abstract
BackgroundHypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.MethodsWe investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.ResultsAlthough severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.ConclusionOur study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.
Highlights
Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC
Effects of Beraprost sodium (BPS) on human pulmonary arterial smooth muscle cell (HPASMC) proliferation during hypoxia Moderate hypoxia (2% oxygen) promoted, whereas severe hypoxia (0.1% oxygen) suppressed DNA synthesis in serum-stimulated HPASMC (Fig. 1a)
Cultured HPASMC were exposed to various concentrations of oxygen and BPS in the presence of BrdU for 24 hours. (a) Severe hypoxia (0.1% oxygen) suppressed, whereas moderate hypoxia (2% oxygen) significantly enhanced BrdU incorporation. *P < 0.05 versus 21% oxygen. (b) BrdU incorporation was dose-dependently suppressed by BPS under both normoxic and hypoxic conditions
Summary
Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Prolonged exposure to hypoxia is associated with cellular and histological changes in vascular remodeling, and the key pathological findings of pulmonary vascular remodeling are increased wall thickening of pulmonary vessels and the muscularization of small arteries. Several studies in vitro have shown that exposure to hypoxia stimulates pulmonary arterial smooth muscle cell (PASMC) proliferation, which might be a key component of pulmonary vascular remodeling [9,10,11,12]. TORAY Industries Inc. developed Beraprost sodium (BPS), which was the first chemically stable and orally active PGI2 analogue to increase intracellular cAMP levels via adenylate cyclase activation [16]. Owing to its chemical characteristics, BPS is more stable and persistent than natural PGI2 and has higher affinity for the PGI2 receptor [19]
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