We previously showed that NGF plays a pivotal role in pulmonary hypertension (PH), contributing to pulmonary arterial inflammation, remodelling and altered reactivity. Others and we have also shown a role of connexin-based GAP junctions in PH.

Since NGF modulates connexin-43 (Cx43) expression/activity in neuronal cells, we wondered whether NGF may alter Cx43 expression in pulmonary arteries (PA) and whether these alterations may contribute to NGF pathophysiological effects in PH.

Control rat PA and control human pulmonary arterial smooth muscle (hPASMC) or endothelial cells (hPAEC) were treated with NGF (0–100ng/ml, 24h). In vivo, experimental PH in the rat was induced by chronic hypoxia (CH, 0.5atm, 28 days), with anti-NGF blocking antibodies (10μg/kg ip) administered as a preventive treatment. Cx43 expression in rat PA and human cells was assessed by Western blotting. Contractions of control rat PA were induced ex vivo by phenylephrine (PHE, 10-10-10-4M) in absence or presence of NGF (100ng/ml, 24h).

Cx43 expression was significantly increased by NGF ex vivo in rat PA (P<0.05) and in vitro in both hPASMC (P<0.05) and hPAEC (P<0.05). This increase was abolished after treatment with K252a (TrkA kinase inhibitor, 300nM) or in cells transfected with a TrkA siRNA (1nM). Wortmannin (PI3K inhibitor, 100nM) and PD98059 (ERK pathway inhibitor, 10μM) also totally blocked this increase. In vivo, anti-NGF blocking antibodies prevented Cx43 increased expression in PA from CH rats. Ex vivo, NGF significantly increased rat PA reactivity to PHE (P<0.01). K252a, wortmannin, PD98059 and 43Gap26 (Cx43 blocking peptide, 300μM) abolished this effect.

Our results show that NGF increases Cx43 expression in PA through activation of its TrkA receptor and a PI3K/ERK-dependent signalling pathway. This mechanism seems to participate in NGF-induced PA hyperreactivity and may thus contribute to PH pathophysiology.