Abstract
BackgroundSildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca2+] ([Ca2+]i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca2+]i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca2+ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca2+ influx and increase in [Ca2+]i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca2+]i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca2+/NFAT pathway.MethodsHuman PASMC were cultured under hypoxia (3% O2) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca2+]i was measured with a dynamic digital Ca2+ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.ResultsHypoxia induced PASMC proliferation with increases in basal [Ca2+]i and Ca2+ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca2+]i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.ConclusionThe SOC/Ca2+/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.
Highlights
Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH)
The effect of store-operated channels (SOC)/[Ca2+]i in this process was studied to clarify the mechanism of hypoxia-induced pulmonary artery smooth muscle cells (PASMC) proliferation
Nifedipine (1 μM, blocker of voltage-dependent calcium channels (VDCC)) had no effect on hypoxia-induced cell proliferation. These data suggested that sustained entry of extracellular Ca2+ via SOC is the main pathway of maintaining the high [Ca2+]i in PASMC
Summary
Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. A few molecular mechanisms such as prostacyclin, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) and endothelin pathways have been shown of pathological importance and involved in the abnormal proliferation and contraction of pulmonary artery smooth muscle cells (PASMC) in PAH patients. Therapies developed towards these targets, such as prostacyclin analogs, endothelin-1 receptor antagonists and phosphodiesterase type-5 (PDE5) inhibitors [1], have been shown of clinical benefit. The anti-proliferative properties of sildenafil may operate through other signaling molecules in addition to the NO/cGMP axis by targeting PKG/PKA [5]
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