20-HETE increases survival and decreases apoptosis in pulmonary arteries and pulmonary artery endothelial cells

Abstract

Hydroxyeicosatetraenoic acid (20-HETE) is an endogenous cytochrome P-450 product present in vascular smooth muscle and uniquely located in the vascular endothelium of pulmonary arteries (PAs). 20-HETE enhances reactive oxygen species (ROS) production of bovine PA endothelial cells (BPAECs) in an NADPH oxidase-dependent manner and is postulated to promote angiogenesis via activation of this pathway in systemic vascular beds. We tested the capacity of 20-HETE or a stable analog of this compound, 20-hydroxy-eicosa-5(Z),14(Z)-dienoic acid, to enhance survival and protect against apoptosis in BPAECs stressed with serum starvation. 20-HETE produced a concentration-dependent increase in numbers of starved BPAECs and increased 5-bromo-2Ј-deoxyuridine incorporation. Caspase-3 activity, nuclear fragmentation studies, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays supported protection from apoptosis and enhanced survival of starved BPAECs treated with a single application of 20-HETE. Protection from apoptosis depended on intact NADPH oxidase, phosphatidylinositol 3 (PI3)-kinase, and ROS production. 20-HETE-stimulated ROS generation by BPAECs was blocked by inhibition of PI3-kinase or Akt activity. These data suggest 20-HETEassociated protection from apoptosis in BPAECs required activation of PI3-kinase and Akt and generation of ROS. 20-HETE also protected against apoptosis in BPAECs stressed by lipopolysaccharide, and in mouse PAs exposed to hypoxia reoxygenation ex vivo. In summary, 20-HETE may afford a survival advantage to BPAECs through activation of prosurvival PI3-kinase and Akt pathways, NADPH oxidase activation, and NADPH oxidase-derived superoxide.reduced nicotinamide adenine dinucleotide phosphatase oxidase; reactive oxygen species; phosphatidylinositol 3-kinase; Akt; hypoxia CYTOCHROME P-450 (CYP) ENZYMES can metabolize arachidonic acid into numerous eicosanoids, with the relative abundance dependent on the tissue and species (21). The major products in most tissues are the -hydroxylated metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) and regio-and stereo-specific epoxyeicosatrienoic acids. 20-HETE, a -hydroxylation product of arachidonic acid catalyzed by CYP4A, is a paracrine and autocrine mediator of numerous cellular processes (20,29,35). It is produced in vascular smooth muscle, renal, cerebral, pulmonary, mesenteric, and skeletal muscle beds and acts on the microvasculature and kidney tubules (11,18,22,33,38).Our laboratory has recently reported that 20-HETE/CYP4 enhances reactive oxygen species (ROS) production in bovine pulmonary artery (PA) endothelial cells (BPAECs) in a manner that is associated with activation of NADPH oxidase (24). 20-HETE has been linked to ROS production in other vascular beds (19), with 20-HETE-stimulated production of ROS being reported to exert either positive or negative effects in a tissueand concentration-specific manner (e.g., Refs. 9, 17). In addition, NADPH oxidase is proposed to have a key role in growth and migration of human coronary endot...

20-HETE-induced nitric oxide production in pulmonary artery endothelial cells is mediated by NADPH oxidase, H2O2, and PI3-kinase/Akt

“…First, these data suggest that enhanced H 2 O 2 formation may serve as an important mediator of 20-HETE prosurvival effects (10,23). This speculation is consistent with reports that NO derived from eNOS protects cells from oxidative stress by an antioxidant mechanism (26).…”

“…Our previous reports demonstrated 20-HETE induced increases in superoxide production, activation of NADPH oxidase, and enhanced NO release from BPAECs (9,23). We have also shown that 20-HETE protects against starvation-induced apoptosis in BPAECs and ischemia reoxygenation injury in ex vivo pulmonary arteries in a manner that depends on NADPH oxidase and PI3K and Akt activation (10). Together then, these data suggest that 1) 20-HETE-induced activation of NADPH oxidase promotes the formation of superoxide, which is rapidly dismutated to H 2 O 2 , 2) activation of PI3K/Akt by 20-HETE and phosphorylation of eNOS requires H 2 O 2 , and finally, 3) stimulated NO release in response to 20-HETE or its structural and stable analog, 20 -5,14-HEDE, and/or enhanced survival are the end result of this signaling pathway.…”

“…In BPAECs, 20-HETE stimulates production of superoxide in a manner that can be blocked by inhibitors of NADPH oxidase, and is accompanied by p47 phox translocation and Rac1 activation (23). Activation of NADPH oxidase is required for 20-HETEassociated prosurvival effects in BPAECs (10). The present studies provide new information regarding 20-HETE-induced signaling through NADPH oxidase in that we demonstrate phosphorylation of both eNOS and Akt in BPAECs is suppressed by pretreatment with two mechanistically distinct inhibitors of NADPH oxidase, apocynin or gp91phox ds tat (Fig.…”

20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca2+ channel via a PKC-dependent mechanism in cardiomyocytes

“…For example, in pulmonary artery endothelial cells, 20-HETE has been reported to prevent apoptosis induced by serum deprivation, LPS, and hypoxia-reperfusion (7). In contrast to the salutary effects in pulmonary artery endothelial cells, in cerebral vascular smooth muscle cells of spontaneously hypertensive rats, 20-HETE contributes to the severity of oxidative stress and stroke.…”

Non-classical Signalling Mechanisms in Stem Cells

“…Addition of either exogenous H 2 O 2 or receptor-mediated intracellular H 2 O 2 leads to the phosphoryaltion of Akt (Esposito et al, 2003;Ushio-Fukai et al, 1999). Recently, it has been demonstrated that ROS-mediated activation of the PI3kinase/Akt pathway increased the production of nitric oxide and again promoted survival of endothelial cells Dhanasekaran et al, 2009). Previously, we showed that the proliferative state of endothelial cells exhibits higher ROS production and phosphorylation of Akt compared to quiescent cells, and inhibition of either ROS production or the PI3kinase/Akt pathway reduces endothelial cell proliferation .…”