Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

Background

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. 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.

Methods

Human 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.

Results

Hypoxia 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.

Conclusion

The 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.