ROCK pathway participates in the processes that 15‐hydroxyeicosatetraenoic acid (15‐HETE) mediated the pulmonary vascular remodeling induced by hypoxia in rat

Abstract

15-Hydroxyeicosatetraenoic acid (15-HETE), a product of arachidonic acid (AA) catalyzed by 15-lipoxygenase (15-LO), plays an essential role in hypoxic pulmonary arterial hypertension. We have previously shown that 15-HETE inhibits apoptosis in pulmonary artery smooth muscle cells (PASMCs). To test the hypothesis that such an effect is attributable to the hypoxia-induced pulmonary vascular remodeling (PVR), we performed these studies. We found subtle thickening of proximal media/adventitia of the pulmonary arteries (PA) in rats that had been exposed to hypoxia. This was associated with an up-regulation of the anti-apoptotic Bcl-2 expression and down-regulation of pro-apoptotic caspase-3 and Bax expression in PA homogenates. Nordihydroguaiaretic acid (NDGA), which inhibits the generation of endogenous 15-HETE, reversed all the alterations following hypoxia. In situ hybridization histochemistry and immunocytochemistry showed that the 15-LO-1 mRNA and protein were localized in pulmonary artery endothelial cells (PAECs), while the 15-LO-2 mRNA and protein were localized in both PAECs and PASMCs. Furthermore, the Rho-kinase (ROCK) pathway was activated by both endogenous and exogenous 15-HETE, alleviating the serum deprivation (SD)-induced PASMC apoptosis. Thus, these findings indicate that 15-HETE protects PASMC from apoptosis, contributing to pulmonary vascular medial thickening, and the effect is, at least in part, mediated via the ROCK pathway.

Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells

“…Cleavage of Ac-DEVD-pNA (acetyl-Asp-Glu-Val-Asp p-nitroanilide), a caspase-3 substrate, was examined for caspase-3 activity according to the manufacturer's protocol ( 12 ). The specifi c caspase-3 activity was normalized for total protein and then expressed as the fold of the baseline caspase-3 activity of control cells cultured in DMEM with 20% FBS.…”

“…The proteins were extracted from PAECs as previously reported ( 12 ). The protein samples (20-50 µg) were fractionated by SDS-PAGE (12% polyacrylamide gels) and transferred onto nitrocellulose membrane.…”

“…We applied serum deprivation (SD) for making a cell apoptotic model ( 12 ). The mitochondrial membrane potential and caspase-3 activity were examined in the starved PAECs.…”

Key Role of 15-Lipoxygenase/15-Hydroxyeicosatetraenoic Acid in Pulmonary Vascular Remodeling and Vascular Angiogenesis Associated With Hypoxic Pulmonary Hypertension

“…13 15-HETE inhibits pulmonary artery smooth muscle cell (PASMC) apoptosis, 14 contributing to pulmonary vascular medial thickening. 15 These results raise the possibility of 15-HETE contributing to hypoxia-induced pulmonary vascular remodeling.…”

“…13 15-HETE inhibits pulmonary artery smooth muscle cell (PASMC) apoptosis, 14 contributing to pulmonary vascular medial thickening. 15 These results raise the possibility of 15-HETE contributing to hypoxia-induced pulmonary vascular remodeling. Because both endogenous and exogenous 15-HETE increase the mRNA and protein expression of ROCK in PASMCs, 15 we hypothesize that 15-HETE mediates hypoxia-induced pulmonary vascular remodeling and angiogenesis through activation of the ROCK pathway.…”