Objective-Our laboratory has previously demonstrated that 15-lipoxygenase (15-LO)/15-hydroxyeicosatetraenoic acid (15-HETE) is involved in hypoxic pulmonary arterial hypertension. Chronic hypoxia-induced vascular inflammation has been considered as an important stage in the development of pulmonary arterial hypertension. Here, we determined the contribution of 15-HETE in the hypoxia-induced pulmonary vascular inflammation. Approach and Results-Chronic hypoxia-induced monocyte/macrophage infiltration and the expressions of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were analyzed in hypoxic rat model and cultured pulmonary arterial endothelium cells using immunochemistry methods. We found that monocyte/macrophage infiltration and the expressions of intercellular adhesion molecules under hypoxia were markedly inhibited by 15-HETE inhibitors or 15-LO1/2 small interfering RNA. In addition, exogenous 15-HETE enhanced the expression of both adhesion molecules in pulmonary arterial endothelium cells in a time-dependent manner. Hypoxia-induced 15-LO1/2 expression in rat pulmonary arterial endothelium cells was significantly abolished by nuclear factor-κB inhibitors. Meanwhile, nuclear factor-κB activity was enhanced prominently by the 15-LO1/2 product, 15-HETE, suggesting a positive feedback mechanism. Conclusions-Taken together, our results suggest that chronic hypoxia promotes monocyte infiltration into the vasculature and adhesion molecules upregulation in pulmonary arterial endothelium cells via a positive interaction between 15-LO/15-HETE and nuclear factor-κB. Our study revealed a novel mechanism underlying hypoxia-induced pulmonary arterial inflammation and suggested new therapeutic strategies targeting 15-LO/15-HETE and nuclear factor-κB in the treatment of pulmonary arterial hypertension. (Arterioscler Thromb Vasc Biol. 2013;33:971-979.)

Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid, have been reported to increase intracellular calcium concentration in aortic vascular smooth muscle cells (SMCs). As EETs are labile, we synthesized a new stable urea EET analog with agonist and soluble epoxide hydrolase (sEH) inhibitor properties. We refer to this analog, 12-(3-hexylureido)dodec-8-enoic acid, as 8-HUDE. Measuring tension of vascular rings, intracellular calcium signaling by confocal laser scanning microscopy and gene expression by reverse-transcription-PCR and western blots, we examined the effects of 8-HUDE on pulmonary vascular tone and calcium signaling in rat pulmonary artery (PA) SMCs (PASMCs). 8-HUDE increased the tension of rat PAs to 145% baseline, whereas it had no effect on the tension of mesenteric arteries (MAs). The 8-HUDE-induced increase in vascular tone was abolished by removal of extracellular Ca2+ or by pretreatment with either La3+ or SKF96365, which are inhibitors of canonical transient receptor potential channels (TRPCs). Furthermore, 8-HUDE-evoked increases in [Ca2+]i in PASMCs could be blunted by inhibition of TRPC with SKF96365, removal of extracellular calcium or depletion of intracellular calcium stores with caffeine, cyclopiazonic acid or 2-aminoethoxydiphenyl borate, but not by the voltage-activated calcium channel blocker nifedipine. In addition to immediate effects on calcium signaling, 8-HUDE upregulated the expression of TRPC1 and TRPC6 at both mRNA and protein levels in rat PASMCs, whereas it suppressed the expression of sEH. Our observations suggest that 8-HUDE increases PA vascular tone through increased release of calcium from intracellular stores, enhanced [Ca2+]i influx in PASMCs through store-operated Ca2+ channels and modulated the expression of TRPC and sEH proteins in a proconstrictive manner.

Peroxisome proliferator activated receptor gamma coactivator 1α (PGC-1α) induced by hypoxia regulates mitochondrial biogenesis and oxidative stress. However, the potential role of PGC-1α in hypoxia-promoted proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is completely unknown. In this study, we found that hypoxia significantly induced the expression of PGC-1α in cultured PASMCs and activated mitochondrial biogenesis through upregulation of nuclear respiratory factor-1 and mitochondria transcription factor A in a time-dependent manner. Knockdown of PGC-1α by siRNA abrogated hypoxia-induced PASMCs proliferation via the downregulation of PCNA, cyclinA, and cyclinE. Furthermore, we observed that PI3K/Akt signaling pathway was involved in hypoxia induced PGC-1α expression and PASMCs proliferation. Taken together, these datas reveal PGC-1α as the key regulator to mediate mitochondrial biogenesis and the proliferation of PASMCs at an early stage of hypoxic exposure. This process might bring to light a potential adaptive mechanism for PASMCs to minimize hypoxic damage and our novel findings provide new insight into the development of hypoxic pulmonary hypertension.

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