CYP4A/20-HETE regulates ischemia-induced neovascularization via its actions on endothelial progenitor and preexisting endothelial cells.

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.

PGF2α regulates the basolateral K channels in the distal convoluted tubule.

Our aim is to examine the role of PGF2α receptor (FP), a highly expressed prostaglandin receptor in the distal convoluted tubule (DCT) in regulating the basolateral 40-pS K channel. The single-channel studies demonstrated that PGF2α had a biphasic effect on the 40-pS K channel in the DCT-PGF2α stimulated at low concentrations (less than 500 nM), while at high concentrations (above 1 µM), it inhibited the 40-pS K channels. Moreover, neither 13,14-dihydro-15-keto-PGF2α (a metabolite of PGF2α) nor PGE2 was able to mimic the effect of PGF2α on the 40-pS K channel in the DCT. The inhibition of PKC had no significant effect on the 40-pS K channel; however, it abrogated the inhibitory effect of 5 µM PGF2α on the K channel. Moreover, stimulation of PKC inhibited the 40-pS K channel in the DCT, suggesting that PKC mediates the inhibitory effect of PGF2α on the 40-pS K channel. Conversely, the stimulatory effect of PGF2α on the 40-pS K channel was absent in the DCT treated with DPI, a NADPH oxidase (NOX) inhibitor. Also, adding 100 µM H2O2 mimicked the stimulatory effect of PGF2α and increased the 40-pS K channel activity in DCT. Moreover, the stimulatory effect of 500 nM PGF2α and H2O2 was not additive, suggesting the role of superoxide-related species in mediating the stimulatory effect of PGF2α on the 40-pS K channel. The inhibition of Src family tyrosine protein kinase (SFK) not only inhibited the 40-pS K channel in the DCT but also completely abolished the stimulatory effects of PGF2α and H2O2 on the 40-pS K channel. We conclude that PGF2α at low doses stimulates the basolateral 40-pS K channel by a NOX- and SFK-dependent mechanism, while at high concentrations, it inhibits the K channel by a PKC-dependent pathway.

Recent developments on the vascular effects of 20-hydroxyeicosatetraenoic acid.

PURPOSE OF REVIEW: 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoactive eicosanoid and a key constituent of the microcirculation. Its effects on vascular function are multifaceted and include stimulation of smooth muscle, contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Such effects have significant implications with regard to the control of vascular homeostasis and pathophysiology. The clinical relevance of 20-HETE is highlighted by recent studies linking 20-HETE and its biosynthetic enzymes to the development of hypertension, stroke, and myocardial infarction. RECENT FINDINGS: This article presents past and recent findings that focus on the role of 20-HETE in the regulation of the vasculature in health and disease and the implication of its actions on endothelial and vascular smooth muscle cells to the pathogenesis of hypertension and stroke. SUMMARY: To date clinical studies corroborated animal studies in that they place 20-HETE as a significant contributor to the pathogenesis of cardiovascular diseases. Consequently, uncovering 20-HETE effects in the vasculature along with understanding its mechanism of action provide a strong basis for the development of novel therapeutic strategies to prevent vascular/end organ damage in these diseases.

Involvement of gap junctions between smooth muscle cells in sustained hypoxic pulmonary vasoconstriction development: a potential role for 15-HETE and 20-HETE.

In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18ß-glycyrrhetinic acid (18ß-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18ß-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18ß-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18ß-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition.

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.

Androgen-sensitive hypertension associated with soluble guanylate cyclase-α1 deficiency is mediated by 20-HETE.

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.

Heme oxygenase-2 deletion impairs macrophage function: implication in wound healing.

Heme oxygenase (HO)-2 deficiency impairs wound healing and exacerbates inflammation following injury. We examine the impact of HO-2 deficiency on macrophage function and the contribution of macrophage HO-2 to inflammatory and repair responses to injury. Corneal epithelial debridement was performed in control and macrophage-depleted HO-2(-/-) and wild-type (WT) mice and in bone marrow chimeras. Peritoneal macrophages were collected for determination of phagocytic activity and classically activated macrophage (M1)-alternatively activated macrophage (M2) polarization. Depletion of macrophages delayed corneal healing (13.2%) and increased neutrophil infiltration (54.1%) by day 4 in WT mice, whereas in HO-2(-/-) mice, it did not worsen the already impaired wound healing and exacerbated inflammation. HO-2(-/-) macrophages displayed an altered M1 phenotype with no significant expression of M2 or M2-like activated cells and a 31.3% reduction in phagocytic capacity that was restored by inducing HO-1 activity or supplementing biliverdin. Macrophage depletion had no effect, whereas adoptive transfer of WT bone marrow improved wound healing (34% on day 4) but did not resolve the exaggerated inflammatory response in HO-2(-/-) mice. These findings indicate that HO-2-deficient macrophages are dysfunctional and that macrophage HO-2 is required for proper macrophage function but is insufficient to correct the impaired healing of the HO-2(-/-) cornea, suggesting that corneal epithelial expression of HO-2 is a key to resolution and repair in wound healing.

Hypertension is a major contributor to 20-hydroxyeicosatetraenoic acid-mediated kidney injury in diabetic nephropathy.

In the kidney, 20-hydroxyeicosatetraenoic acid (20-HETE) is a primary cytochrome P450 4 (Cyp4)-derived eicosanoid that enhances vasoconstriction of renal vessels and induces hypertension, renal tubular cell hypertrophy, and podocyte apoptosis. Hypertension and podocyte injury contribute to diabetic nephropathy and are strong predictors of disease progression. In this study, we defined the mechanisms whereby 20-HETE affects the progression of diabetic nephropathy. We used Cyp4a14KO male mice that exhibit androgen-sensitive hypertension due to increased Cyp4a12-mediated 20-HETE production. We show that, upon induction of diabetes type 1 via streptozotocin injection, Cyp4a14KO male mice developed worse renal disease than streptozotocin-treated wild-type mice, characterized by increased albuminuria, mesangial expansion, glomerular matrix deposition, and thickness of the glomerular basement membranes. Castration blunted androgen-mediated Cyp4a12 synthesis and 20-HETE production, normalized BP, and ameliorated renal damage in diabetic Cyp4a14KO mice. Notably, treatment with a 20-HETE antagonist or agents that normalized BP without affecting Cyp4a12 expression and 20-HETE biosynthesis also ameliorated diabetes-mediated renal damage and albuminuria in Cyp4a14KO male mice. Taken together, these results suggest that hypertension is the major contributor to 20-HETE-driven diabetes-mediated kidney injury.

Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.

Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived 20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the role of CYP4F2 in vascular disease, we generated mice with endothelial expression of human CYP4F2 (Tie2-CYP4F2-Tr). LC/MS/MS analysis revealed 2-foldincreases in 20-HETE levels in tissues and endothelial cells (ECs), relative to wild-type (WT) controls. Tie2-CYP4F2-Tr ECs demonstrated increases in growth (267.1 ± 33.4 vs. 205.0 ± 13% at 48 h) and tube formation (7.7 ± 1.1 vs. 1.6 ± 0.5 tubes/field) that were 20-HETE dependent and associated with up-regulation of prooxidant NADPH oxidase and proangiogenic VEGF. Increases in VEGF and NADPH oxidase levels were abrogated by inhibitors of NADPH oxidase and MAPK, respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-CYP4F2-Tr mice (18.6 ± 2.7 vs. 7.9 ± 2.7 pg/ml) were up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and blood pressure were unchanged. Our findings indicate that human CYP4F2 significantly increases 20-HETE production, CYP4F2-derived 20-HETE mediates EC proliferation and angiogenesis via VEGF- and NADPH oxidase-dependent manners, and the Tie2-CYP4F2-Tr mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the vasculature.-Cheng, J., Edin, M. L., Hoopes, S. L., Li, H., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Lih, F. B., Garcia, V., Shaik, J. S. B., Tomer, K. B., Flake, G. P., Falck, J. R., Lee, C. R., Poloyac, S. M., Schwartzman, M. L., Zeldin, D. C. Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.

Soluble epoxide hydrolase null mice exhibit female and male differences in regulation of vascular homeostasis.

Increased CYP epoxygenase activity and consequently up regulation of epoxyeicosatrienoic acids (EETs) levels provides protection against metabolic syndrome and cardiovascular diseases. Conversion of arachidonic acid epoxides to diols by soluble epoxide hydrolase (sEH) diminishes the beneficial cardiovascular properties of these epoxyeicosanoids. We therefore examined the possible biochemical consequences of sEH deletion on vascular responses in male and female mice. Through the use of the sEH KO mouse, we provide evidence of differences in the compensatory response in the balance between nitric oxide (NO), carbon monoxide (CO), EETs and the vasoconstrictor 20-HETE in male and female KO mice. Serum levels of adiponectin, TNFα, IL-1b and MCP1 and protein expression in vascular tissue of p-AMPK, p-AKT and p-eNOS were measured. Deletion of sEH caused a significant (p<0.05) decrease in body weight, and an increase in adiponectin, pAMPK and pAKT levels in female KO mice compared to male KO mice. Gene deletion resulted in a higher production of renal EETs in female KO compared to male KO mice and, concomitantly, we observed an increase in renal 20-HETEs levels and superoxide anion production only in male KO mice. sEH deletion increased p-AKT and p-eNOS protein expression but decreased p-AMPK levels in female KO mice. Increased levels of p-eNOS at Thr-495 were observed only in KO male mice. While p-eNOS at 1177 were not significantly different between male and female. Nitric oxide production was unaltered in male KO mice. These results provide evidence of gender differences in the preservation of vascular homeostasis in response to sEH deletion which involves regulation of phosphorylation of eNOS at the 495 site.

Vascular actions of 20-HETE.

20-hydroxyeicosatetraenoic acid (20-HETE) is a metabolite of arachidonic acid that exhibits a myriad of biological effects in the vascular system. This review discusses the current knowledge related to the effects of 20-HETE on vascular reactivity, activation, and remodeling, as well as its role in vascular inflammation and angiogenesis. The information explaining how 20-HETE and the renin-angiotensin system interact to promote hypertension, vasoconstriction, and vascular dysfunction is summarized in this article. 20-HETE enhances vascular inflammation and injury in models of diabetes, ischemia/reperfusion, and cerebrovascular oxidative stress. Recent studies also established a role for 20-HETE in normal and pathological angiogenesis conditions. This review will also discuss the molecular mechanisms through which 20-HETE induces these vascular actions. Potential additional studies are suggested to address shortcomings in the current knowledge of 20-HETE in the vascular system.

Androgen-induced hypertension in angiotensinogen deficient mice: role of 20-HETE and EETS.

20-HETE is a potent inducer of endothelial ACE in vitro and administration of lisinopril or losartan attenuates blood pressure in models of 20-HETE-dependent hypertension. The present study was undertaken to further define the relationship between 20-HETE and the renin-angiotensin system in hypertension using an angiotensinogen-deficient mouse (Agt+/-). Treatment of male AGT+/- with 5α-dihydrotestosterone (DHT) increased systolic BP from 102±2 to 125±3mmHg; in comparison, the same treatment raised BP in wild type (WT) from 110±2 to 138±2mmHg. DHT increased vascular 20-HETE levels in AGT+/- and WT from 1.5±0.7 and 2.1±0.6 to 13.0±2.0 and 15.8±4.0ng/mg, respectively. Concurrent treatment with the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented the increases in BP in both AGT+/- and WT mice. Administration of 20-HEDE at the peak of the DHT-induced BP increase (12 days) reduced BP to basal levels after 48h. Interestingly, basal levels of renal microvascular EETs were higher in AGT+/- compared to WT (55.2±9.7 vs 20.0±4.1ng/mg) and treatment of AGT+/- with DHT decreased the levels of EETs (28.4±5.1ng/mg). DHT-mediated changes in vascular EET level were not observed in WT mice. Vascular Cyp4a12 and ACE protein levels were increased in both AGT+/- and WT by 30-40% and decreased with concomitant administration of 20-HEDE. Lisinopril was as effective as 20-HEDE in preventing DHT-mediated increases in BP in both AGT+/- and WT mice. This study substantiates our previous findings that the RAS plays an important role in 20-HETE-mediated hypertension. It also proposes a novel interaction between 20-HETE and EETs.

Soluble epoxide hydrolase-dependent regulation of myogenic response and blood pressure.

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.

20-HETE regulates the angiogenic functions of human endothelial progenitor cells and contributes to angiogenesis in vivo.

Circulating endothelial progenitor cells (EPC) contribute to postnatal neovascularization. We identified the cytochrome P450 4A/F-20-hydroxyeicosatetraenoic acid (CYP4A/F-20-HETE) system as a novel regulator of EPC functions associated with angiogenesis in vitro. Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis in vivo. Results showed that both hypoxia and vascular endothelial growth factor (VEGF) induce CYP4A11 gene and protein expression (the predominant 20-HETE synthases in human EPC), and this is accompanied by an increase in 20-HETE production by ~1.4- and 1.8-fold, respectively, compared with the control levels. Additional studies demonstrated that 20-HETE and VEGF have a synergistic effect on EPC proliferation, whereas 20-HETE antagonist 20-HEDGE or VEGF-neutralizing antibody negated 20-HETE- or VEGF-induced proliferation, respectively. These findings are consistent with the presence of a positive feedback regulation on EPC proliferation between the 20-HETE and the VEGF pathways. Furthermore, we found that 20-HETE induced EPC adhesion to fibronectin and endothelial cell monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which was accompanied by a rapid induction of very late antigen-4 and chemokine receptor type 4 mRNA and protein expression. Basal and 20-HETE-stimulated increases in adhesion were negated by the inhibition of the CYP4A-20-HETE system. Lastly, EPC increased angiogenesis in vivo by 3.6 ± 0.2-fold using the Matrigel plug angiogenesis assay, and these increases were markedly reduced by the local inhibition of 20-HETE system. These results strengthened the notion that 20-HETE regulates the angiogenic functions of EPC in vitro and EPC-mediated angiogenesis in vivo.

Androgen-sensitive hypertension associates with upregulated vascular CYP4A12-20-HETE synthase.

Although the mechanism underlying the effect of androgen on BP and cardiovascular disease is not well understood, recent studies suggest that 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a primary cytochrome P450 4 (Cyp4)-derived eicosanoid, may mediate androgen-induced hypertension. Here, treatment of normotensive mice with 5α-dihydrotestosterone increased BP and induced both Cyp4a12 expression and 20-HETE levels in preglomerular microvessels. Administration of a 20-HETE antagonist prevented and reversed the effects of dihydrotestosterone on BP. Cyp4a14(-/-) mice, which exhibit androgen-sensitive hypertension in the male mice, produced increased levels of vascular 20-HETE; furthermore, administration of a 20-HETE antagonist normalized BP. To examine whether androgen-independent increases in 20-HETE are sufficient to cause hypertension, we studied Cyp4a12-transgenic mice, which express the CYP4A12-20-HETE synthase under the control of a doxycycline-sensitive promoter. Administration of doxycycline increased BP by 40%, and administration of a 20-HETE antagonist prevented this increase. Levels of CYP4A12 and 20-HETE in preglomerular microvessels of doxycycline-treated transgenic mice approximately doubled, correlating with increased 20-HETE-dependent sensitivity to phenylephrine-mediated vasoconstriction and with decreased acetylcholine-mediated vasodilation in the renal microvasculature. We observed a similar contribution of 20-HETE to myogenic tone in the mesenteric microvasculature. Taken together, these results suggest that 20-HETE both mediates androgen-induced hypertension and can cause hypertension independent of androgen.

Cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress and, in clinical studies, is associated with increased body mass index (BMI) and the metabolic syndrome. This study was designed to examine the effects of exogenous 20-HETE on mesenchymal stem cell (MSC)-derived adipocytes. The expression levels of CYP4A11 and CYP4F2 (major 20-HETE synthases in humans) in MSCs decreased during adipocyte differentiation; however, exogenous administration of 20-HETE (0.1-1 µM) increased adipogenesis in a dose-dependent manner in these cells (P < 0.05). The inability of a 20-HETE analog to reproduce these effects suggested the involvement of a metabolic product of 20-HETE in mediating its pro-adipogenic effects. A cyclooxygenase (COX)-1 selective inhibitor enhanced, whereas a COX-2 selective or a dual COX-1/2 inhibitor attenuated adipogenesis induced by 20-HETE. The COX-derived metabolite of 20-HETE, 20-OH-PGE2, enhanced adipogenesis and lipid accumulation in MSCs. The pro-adipogenic effects of 20-HETE and 20-OH-PGE2 resulted in the increased expression of the adipogenic regulators PPARγ and ß-catenin in MSC-derived adipocytes. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE2 enhances mature inflamed adipocyte hypertrophy in MSC undergoing adipogenic differentiation.

20-HETE induces remodeling of renal resistance arteries independent of blood pressure elevation in hypertension.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 (Cyp)-derived arachidonic acid metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation, and promote hypertension. We examined if 20-HETE contributes to microvascular remodeling in hypertension. In Sprague-Dawley rats, administration of the 20-HETE biosynthesis inhibitor HET0016 or the 20-HETE antagonist N-20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented 5α-dihydrotestosterone (DHT)-induced increases in blood pressure as well as abrogated DHT-induced increases in the media-to-lumen ratio (M/L), media thickness, and collagen IV deposition in renal interlobar arteries. Reserpine prevented blood pressure elevation in DHT-treated rats but did not affect microvascular remodeling (M/L, media thickness, and collagen deposition); under these conditions, treatment with the 20-HETE antagonist attenuated microvascular remodeling, suggesting that 20-HETE contributes to DHT-induced vascular remodeling independent of blood pressure elevation. In Cyp4a14(-/-) mice, which display androgen-driven and 20-HETE-dependent hypertension, treatment with the 20-HETE antagonist abolished remodeling of renal resistance arteries measured as media thickness (24 ± 1 vs. 15 ± 1 µm) and M/L (0.29 ± 0.03 vs. 0.17 ± 0.01). Moreover, in Cyp4a12 transgenic mice in which the expression of Cyp4a12-20-HETE synthase is driven by a tetracycline-sensitive promoter, treatment with doxycycline resulted in blood pressure elevation (140 ± 4 vs. 92 ± 5 mmHg) and a significant increase in remodeling of renal resistance arteries (media thickness: 23 ± 1 vs. 16 ± 1 µm; M/L: 0.39 ± 0.04 vs. 0.23 ± 0.02); these increases were abrogated by cotreatment with 20-HEDE. This study demonstrated that 20-HETE is a key regulator of microvascular remodeling in hypertension; its effect is independent of blood pressure elevation and androgen levels.

Heme oxygenase-2/adiponectin protein-protein interaction in metabolic syndrome.

Insulin resistance with adipose tissue dysfunction and dysregulation in the production and secretion of adipokines is one of the hallmarks of metabolic syndrome. We have previously reported that increased levels of the heme oxygenase (HO) system, HO-1/HO-2 results in increased levels of adiponectin. Despite documentation of the existence of the anti-inflammatory axis HO-adiponectin, a possible protein-protein interaction between HO and adiponectin has not been examined. Here, we investigated the existence of protein interactions between HO-2 and adiponectin in the maintenance of adipocyte function during metabolic syndrome by integrating phenotypic and in silico studies. Compared to WT animals, HO-2 null mice displayed an increase in both visceral and subcutaneous fat content and reduced circulating adiponectin levels. The decrease in adiponectin was reversed by upregulation of HO-1. HO-2 depletion was associated with increased adipogenesis in cultured mesenchymal stem cells (MSCs) and decreased adiponectin levels in the culture media. In addition, HO-1 siRNA decreased adiponectin release. HO-2 was found to bind to the monomeric form of adiponectin, according to poses and calculated energies. HO-2-adiponectin interactions were validated by the two-hybrid system assay. In conclusion, protein-protein interactions between HO-2 and adiponectin highlight the role of HO-2 as a molecular chaperone for adiponectin assembly, while HO-1 increases adiponectin levels. Thus, crosstalk between HO-2 and HO-1 could be manipulated in a therapeutic approach to ameliorate the deleterious effects of obesity and the metabolic syndrome.

Global and endothelial G-protein coupled receptor 75 (GPR75) knockout relaxes pulmonary artery and mitigates hypoxia-induced pulmonary hypertension.

RATIONALE: Pulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH. METHODS: To test this hypothesis, we exposed global (Gpr75-/-) and endothelial cell (EC) GPR75 knockout (EC-Gpr75-/-) mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations. RESULTS: Chronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75-/- or EC-Gpr75-/- mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (CC motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75-/-, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner. CONCLUSION: These results suggest GPR75 has a significant role in the development of hypoxia-induced PH.

20-Hydroxyeicosatetraenoic acid (20-HETE): Bioactions, receptors, vascular function, cardiometabolic disease and beyond.

Vascular function is dynamically regulated and dependent on a bevy of cell types and factors that work in concert across the vasculature. The vasoactive eicosanoid, 20-Hydroxyeicosatetraenoic acid (20-HETE) is a key player in this system influencing the sensitivity of the vasculature to constrictor stimuli, regulating endothelial function, and influencing the renin angiotensin system (RAS), as well as being a driver of vascular remodeling independent of blood pressure elevations. Several of these bioactions are accomplished through the ligand-receptor pairing between 20-HETE and its high-affinity receptor, GPR75. This 20-HETE axis is at the root of various vascular pathologies and processes including ischemia induced angiogenesis, arteriogenesis, septic shock, hypertension, atherosclerosis, myocardial infarction and cardiometabolic diseases including diabetes and insulin resistance. Pharmacologically, several preclinical tools have been developed to disrupt the 20-HETE axis including 20-HETE synthesis inhibitors (DDMS and HET0016), synthetic 20-HETE agonist analogues (20-5,14-HEDE and 20-5,14-HEDGE) and 20-HETE receptor blockers (AAA and 20-SOLA). Systemic or cell-specific therapeutic targeting of the 20-HETE-GPR75 axis continues to be an invaluable approach as studies examine the molecular underpinnings activated by 20-HETE under various physiological settings. In particular, the development and characterization of 20-HETE receptor blockers look to be a promising new class of compounds that can provide a considerable benefit to patients suffering from these cardiovascular pathologies.