Pulmonary hypertension (PH) is a severe disease leading to right heart failure and death. We have previously shown that blockade of the nerve growth factor NGF may be of therapeutic interest in PH [1Freund-Michel V., Cardoso Dos Santos M., Guignabert C., et al. Role of nerve growth factor in development and persistence of experimental pulmonary hypertension Am J Respir Crit Care Med 2015 ;  192 : 342-355 [cross-ref]

Cliquez ici pour aller à la section Références]. Indeed, NGF plays a significant role in PH pathophysiology, particularly in pulmonary arterial hyperreactivity. Our experiments have suggested that NGF can activate different mechanisms to induce pulmonary arterial hyperreactivity depending on a short- or a long-term treatment. The aim of the present study was to determine the mechanisms activated by NGF to induce pulmonary arterial hyperreactivity after a short-term treatment (1h).

Contractions of rat pulmonary arteries (PA) were induced ex vivo by endothelin-1 (ET-1, 10-12-10-6M) in the absence or presence of NGF (100ng/mL, 1h), with or without K252a (TrkA kinase inhibitor, 300nM), anti-p75NTR blocking antibodies (10μg/mL) or Y-27632 (inhibitor of the Rho-associated protein kinase ROCK, 10μM). NGF-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1) was evaluated in vitro by Western blotting in both rat and human pulmonary arterial smooth muscle cells (PASMC) in primary culture. Basal and ET-1-induced (10-7M) calcium responses were evaluated in vitro in PASMC using calcium imaging (Fura2, Fluo-4), in the absence or presence of NGF (100ng/mL, 1h). Calcium sources involved in NGF-dependent increase in calcium levels and response to ET-1 were determined using inhibitors of the sarco/endoplasmic reticulum calcium ATPase SERCA (thapsigargin, 1μM or cyclopiazonic acid, 10μM) or calcium L-Type channels blockers (verapamil or nicardipine, 2μM for both).

Ex vivo, NGF (1h) induced rat pulmonary arterial hyperreactivity to ET-1 in a TrkA- and ROCK-dependent manner. In vitro, in both rat and human PASMC, NGF (1h) enhanced basal calcium levels through activation of calcium L-type channels. NGF also enhanced ET-1-induced increase in intracellular calcium levels, with endoplasmic reticulum (ER) being the main calcium source contributing to these effects. Finally, in both rat and human PASMC, NGF also induced phosphorylation of MYPT1 in a TrkA- and ROCK-dependent manner.

Altogether, our results show that NGF induces pulmonary arterial hyperreactivity to ET-1 through activation of its TrkA receptor, leading to both increased calcium intracellular levels and increased ER calcium loading. NGF also activates ROCK-dependent calcium sensitization mechanisms. Such mechanisms may therefore contribute to NGF-induced pulmonary arterial hyperreactivity in pulmonary hypertension.