Ngf Induces Pulmonary Arterial Hyperreactivity Through Connexin 43 Increased Expression

Introduction Nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. Aim To determine the mechanisms of NGF-induced PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Methods Expression of NGF, its TrkA receptor and Cx43 were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from control donors or from patients suffering from idiopathic pulmonary arterial hypertension (iPAH). Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and surface biotinylation, after NGF treatment. Gap junction activity was assessed by FRAP microscopy. Contractions of rat PA were induced by phenylephrine (PHE), with or without NGF pre-treatment. The role of Cx43 in NGF-induced rat PA hyperreactivity was evaluated ex vivo by use of 43Gap26, a Cx43 blocking peptide, or in vivo by use of an anti-Cx43 siRNA. Results TrkA expression was increased in hPASMC from iPAH patients compared to control hPASMC. NGF increased Cx43 expression, plasma membrane localisation and phosphorylation, with a higher sensitivity in iPAH cells compared to controls. In both cell types, this increase was abolished with K252a, a TrkA kinase inhibitor. Activity of Cx43-dependent GAP junctions was also increased after NGF treatment, with iPAH hPASMC being more sensitive to NGF than control cells. NGF-induced PA hyperreactivity to PHE was inhibited after treatment with 43Gap26 or with anti-Cx43 siRNA. Conclusion NGF, through activation of its TrkA receptor, increases Cx43 expression and plasma membrane localisation in hPASMC. This mechanism enhances Cx43-dependent GAP junction activity and contributes to PA hyperreactivity. Moreover, NGF pathway is upregulated in iPAH hPASMC compared to control hPASMC, and iPAH cells are more sensitive to NGF than control cells regarding Cx43 increased expression and Cx43-dependent GAP junction increased activity.