We have previously shown that the nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity.

To characterize mechanisms activated by NGF to induce PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity.

Expression of NGF and its TrkA receptor were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from controls or from patients suffering from idiopathic pulmonary arterial hypertension (IPAH). In cells treated or not with NGF (0.1-100 ng/ml, 24h), Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and cell surface biotinylation. Gap junction activity was assessed by Lucifer yellow (LY) dye transfer assay. Contractions of control rat PA were induced by phenylephrine (PHE, 10-10-10-4M) in absence or presence of NGF (100ng/ml, 24h). The role of Cx43 in NGF-induced rat PA reactivity was evaluated by use of 43Gap26 (Cx43 blocking peptide, 300μM), or an anti-Cx43 siRNA (10nmol IV).

NGF and TrkA expression were increased in hPASMC from IPAH patients compared to controls. NGF increased Cx43 expression in hPASMC from both controls and IPAH patients, but with a higher sensitivity in IPAH cells (maximum at 1ng/ml vs. 100ng/ml). This increase was abolished after treatment with K252a (TrkA kinase inhibitor). In control hPASMC, NGF increased Cx43 plasma membrane localisation and LY diffusion speed through Gap junctions. NGF-induced PA hyperreactivity to PHE was inhibited ex vivo by treatment with 43Gap26 and in vivo after administration of Cx43 siRNA.

NGF pathway is unregulated in IPAH cells. NGF increases Cx43 PA expression through activation of its TrkA receptor. Cx43 increased expression at hPASMC plasma membrane upregulates gap junction activity, thus promoting NGF-induced PA hyperreactivity.