Search

20-Hydroxyeicosatetraenoic acid (20-HETE) was recently identified as a novel contributor of ischemia-induced neovascularization based on the key observation that pharmacological interferences of CYP4A/20-HETE decrease ischemic neovascularization. The objective of the present study is to examine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPCs) and preexisting endothelial cells (ECs). We found that ischemia leads to a time-dependent increase of cyp4a12 expression and 20-HETE production, which are endothelial in origin, using immunofluorescent microscopy, Western blot analysis, and LC-MS/MS. This is accompanied by increases in the tissue stromal cell-derived factor-1α (SDF-1α) expressions as well as SDF-1α plasma levels, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of CYP4A/20-HETE with a 20-HETE synthesis inhibitor, dibromo-dodecenyl-methylsulfimide (DDMS), or a 20-HETE antagonist, N -(20-hydroxyeicosa-6( Z ), 15( Z )-dienoyl) glycine (6, 15-20-HEDGE), significantly attenuated these increases. Importantly, we also determined that 20-HETE plays a novel role in maintaining EPC functions and increasing the expression of Oct4 , Sox2 , and Nanog , which are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of CYP4A/20-HETE decrease the EPC population in culture, whereas 20-HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM-1 expression in the preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15-20-HEDGE. Taken together, CYP4A/20-HETE regulates ischemia-induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization. NEW & NOTEWORTHY CYP4A/20-hydroxyeicosatetraenoic acid (20-HETE) was recently discovered as a novel contributor of ischemia-induced neovascularization. However, the underlying molecular and cellular mechanisms are completely unknown. Here, we show that CYP4A/20-HETE regulates the ischemic neovascularization process via its combined actions on both endothelial progenitor cells (EPCs) and preexisting endothelial cells. Moreover, this is the first study, to the best of our knowledge, that associates CYP4A/20-HETE with EPC differentiation and stemness.

Coronary collateral growth (CCG) is impaired in metabolic syndrome (MetS). microRNA-145 (miR-145-Adv) delivery to our rat model of MetS (JCR) completely restored and neutrophil depletion significantly improved CCG. We determined whether low endogenous levels of miR-145 in MetS allowed for elevated production of 20-hydroxyeicosatetraenoic acid (20-HETE), which, in turn, resulted in excessive neutrophil accumulation and endothelial dysfunction leading to impaired CCG. Rats underwent 0-9 days of repetitive ischemia (RI). RI-induced cardiac CYP4F (neutrophil-specific 20-HETE synthase) expression and 20-HETE levels were increased (4-fold) in JCR vs. normal rats. miR-145-Adv and 20-HETE antagonists abolished and neutrophil depletion (blocking antibodies) reduced (~60%) RI-induced increases in CYP4F expression and 20-HETE production in JCR rats. Impaired CCG in JCR rats (collateral-dependent blood flow using microspheres) was completely restored by 20-HETE antagonists [collateral-dependent zone (CZ)/normal zone (NZ) flow ratio was 0.76 ± 0.07 in JCR + 20-SOLA, 0.84 ± 0.05 in JCR + 20-HEDGE vs. 0.11 ± 0.02 in JCR vs. 0.84 ± 0.03 in normal rats]. In JCR rats, elevated 20-HETE was associated with excessive expression of endothelial adhesion molecules and neutrophil infiltration, which were reversed by miR-145-Adv. Endothelium-dependent vasodilation of coronary arteries, endothelial nitric oxide synthase (eNOS) Ser1179 phosphorylation, eNOS-dependent NO ·- production and endothelial cell survival were compromised in JCR rats. These parameters of endothelial dysfunction were completely reversed by 20-HETE antagonism or miR-145-Adv delivery, whereas neutrophil depletion resulted in partial reversal (~70%). We conclude that low miR-145 in MetS allows for increased 20-HETE, mainly from neutrophils, which compromises endothelial cell survival and function leading to impaired CCG. 20-HETE antagonists could provide viable therapy for restoration of CCG in MetS. NEW & NOTEWORTHY Elevated 20-hydroxyeicosatetraenoic acid (20-HETE) impairs coronary collateral growth (CCG) in metabolic syndrome by eliciting endothelial dysfunction and apoptosis via excessive neutrophil infiltration. 20-HETE antagonists completely restore coronary collateral growth in metabolic syndrome. microRNA-145 (miR-145) is an upstream regulator of 20-HETE production in metabolic syndrome; low expression of miR-145 in metabolic syndrome promotes elevated production of 20-HETE., (Copyright © 2017 the American Physiological Society.)

Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling., (Copyright © 2016 the American Physiological Society.)

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD., (Copyright © 2016 the American Physiological Society.)

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid via cytochrome P450 (CYP)/epoxygenases. EETs possess cardioprotective properties and are catalyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) that lack vasoactive property. To date, the role of sEH in the regulation of myogenic response of resistant arteries, a key player in the control of blood pressure, remains unknown. To this end, experiments were conducted on sEH-knockout (KO) mice, wild-type (WT) mice, and endothelial nitric oxide synthase (eNOS)-KO mice treated with t-TUCB, a sEH inhibitor, for 4 wk. sEH-KO and t-TUCB-treated mice displayed significantly lower blood pressure, associated with significantly increased vascular EETs and ratio of EETs/DHETs. Pressure-diameter relationships were assessed in isolated and cannulated gracilis muscle arterioles. All arterioles constricted in response to increases in transmural pressure from 60 to 140 mmHg. The myogenic constriction was significantly reduced, expressed as an upward shift of pressure-diameter curve, in arterioles of sEH-KO and t-TUCB-treated eNOS-KO mice compared with their controls. Removal of the endothelium, or treatment of the vessels with PPOH, an inhibitor of EET synthase, restored the attenuated pressure-induced constriction to the levels similar to those observed in their controls but had no effects on control vessels. No difference was observed in the myogenic index, or in the vascular expression of eNOS, CYP2C29 (EET synthase), and CYP4A (20-HETE synthase) among these groups of mice. In conclusion, the increased EET bioavailability, as a function of deficiency/inhibition of sEH, potentiates vasodilator responses that counteract pressure-induced vasoconstriction to lower blood pressure.

Hypertension in the elderly substantially increases the risk of stroke and vascular cognitive impairment in part due to an impaired functional adaptation of aged cerebral arteries to high blood pressure. To elucidate the mechanisms underlying impaired autoregulatory protection in aging, hypertension was induced in young (3 mo) and aged (24 mo) C57BL/6 mice by chronic infusion of angiotensin II and pressure-induced changes in smooth muscle cell (SMC) intracellular Ca(2+) concentration ([Ca(2+)]i) and myogenic constriction of middle cerebral arteries (MCA) were assessed. In MCAs from young hypertensive mice, pressure-induced increases in vascular SMC [Ca(2+)]i and myogenic tone were increased, and these adaptive responses were inhibited by the cytochrome P-450 ω-hydroxylase inhibitor HET0016 and the transient receptor potential (TRP) channel blocker SKF96365. Administration of 20- hydroxyeicosatetraenoic acid (HETE) increased SMC [Ca(2+)]i and constricted MCAs, and these responses were inhibited by SKF96365. MCAs from aged hypertensive mice did not show adaptive increases in pressure-induced calcium signal and myogenic tone and responses to HET0016 and SKF96365 were blunted. Inhibition of large-conductance Ca(2+)-activated K(+) (BK) channels by iberiotoxin enhanced SMC [Ca(2+)]i and myogenic constriction in MCAs of young normotensive animals, whereas it was without effect in MCAs of young hypertensive mice. Iberiotoxin did not restore myogenic adaptation in MCAs of aged hypertensive mice. Thus functional maladaptation of aged cerebral arteries to hypertension is due to the dysregulation of pressure-induced 20-HETE and TRP channel-mediated SMC calcium signaling, whereas overactivation of BK channels is unlikely to play a role in this phenomenon.

We have previously shown (Ref. 2) that endothelium-derived hyperpolarizing factor (EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450 (CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for omega-hydroxylation (CYP4A31) and 12R-hydroxylation (CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids (EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and omega-hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-linked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel.

Nitric oxide (NO), generated from L-arginine by endothelial nitric oxide synthase (eNOS), is a key endothelial-derived factor whose bioavailability is essential to the normal function of the endothelium. Endothelium dysfunction is characterized by loss of NO bioavailability because of either reduced formation or accelerated degradation of NO. We have recently reported that overexpression of vascular cytochrome P-450 (CYP) 4A in rats caused hypertension and endothelial dysfunction driven by increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a major vasoconstrictor eicosanoid in the microcirculation. To further explore cellular mechanisms underlying CYP4A-20-HETE-driven endothelial dysfunction, the interactions between 20-HETE and the eNOS-NO system were examined in vitro. Addition of 20-HETE to endothelial cells at concentrations as low as 1 nM reduced calcium ionophore-stimulated NO release by 50%. This reduction was associated with a significant increase in superoxide production. The increase in superoxide in response to 20-HETE was prevented by N(G)-nitro-L-arginine methyl ester, suggesting that uncoupled eNOS is a source of this superoxide. The response to 20-HETE was specific in that 19-HETE did not affect NO or superoxide production, and, in fact, the response to 20-HETE could be competitively antagonized by 19(R)-HETE. 20-HETE had no effect on phosphorylation of eNOS protein at serine-1179 or threonine-497 following addition of calcium ionophore; however, 20-HETE inhibited association of eNOS with 90-kDa heat shock protein (HSP90). In vivo, impaired acetylcholine-induced relaxation in arteries overexpressing CYP4A was associated with a marked reduction in the levels of phosphorylated vasodilator-stimulated phosphoprotein, an indicator of bioactive NO, that was reversed by inhibition of 20-HETE synthesis or action. Because association of HSP90 with eNOS is critical for eNOS activation and coupled enzyme activity, inhibition of this association by 20-HETE may underlie the mechanism, at least in part, by which increased CYP4A expression and activity cause endothelial dysfunction.

11,12-Epoxyeicosatrienoic acid (11,12-EET), a potent vasodilator produced by the endothelium, acts on calcium-activated potassium channels and shares biological activities with the heme oxygenase/carbon monoxide (HO/CO) system. We examined whether activation of HO mediates the dilator action of 11,12-EET, and that of the other EETs, on rat mesenteric arteries. Dose-response curves (10(-9) to 10(-6) M) to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and ACh (10(-9) to 10(-4) M) were evaluated in preconstricted (10(-6) mol/l phenylephrine) mesenteric arteries (<350 microm diameter) in the presence or absence of 1) the cyclooxygenase inhibitor indomethacin (2.8 microM), 2) the HO inhibitor chromium mesoporphyrin (CrMP) (15 microM), 3) the soluble guanylyl cyclase (GC) inhibitor ODQ (10 microM), and 4) the calcium-activated potassium channel inhibitor iberiotoxin (25 nM). The vasodilator response to 11,12-EET was abolished by CrMP and iberiotoxin, whereas indomethacin and ODQ had no effect. In contrast, the effect of ACh was attenuated by ODQ but not by CrMP. The vasodilator effect of 8,9-EET, like that of 11,12-EET, was greatly attenuated by HO inhibition. In contrast, the mesenteric vasodilator response to 5,6-EET was independent of both HO and GC, whereas that to 14,15-EET demonstrated two components, an HO and a GC, of equal magnitude. Incubation of mesenteric microvessels with 11,12-EET caused a 30% increase in CO release, an effect abolished by inhibition of HO. We conclude that the rat mesenteric vasodilator action of 11,12-EET is mediated via an increase in HO activity and an activation of calcium-activated potassium channels.