Pharmacological Blockade of the 20-HETE Receptor Lowers Blood Pressure and Alters Vascular Function in Mice with Vascular Smooth Muscle- Specific Overexpression of CYP4a12-20-HETE Synthase

20-hydroxyeicosatetraenoic acid (20-HETE) is the ω-hydroxylation product of arachidonic acid catalyzed by CYP4A and 4F enzymes. 20-HETE is a vasoactive eicosanoid of the microcirculation exhibiting effects on both vascular smooth muscle cells (VSMC) and endothelial cells (EC). In VSMCs, 20-HETE’s bioactions include the stimulation of contraction, migration, and growth. In ECs, elevated 20-HETE is associated with reduced nitric oxide (NO) bioavailability, increased angiotensin converting enzyme (ACE) expression, and the promotion of inflammation. Recently in our laboratory, we identified GPR75 as a novel target of 20-HETE that promotes changes in blood pressure and vascular function. The aim of this study is to assess the consequences of VSMC-targeted overexpression of Cyp4a12, the primary 20-HETE producing enzyme in mice, on blood pressure, vascular function, and vascular remodeling. Moreover, we looked to examine whether the administration of a 20-HETE receptor antagonist reverses the vascular phenotype associated with elevations in 20-HETE. Mice with VSMC-specific overexpression of Cyp4a12 (Myh11-4a12) and their littermate controls (WT) were generated by crossbreeding Cyp4a12-flox mice (gifted by Dr. Schunck) with Myh11-Cre mice. Myh11-4a12 were administered AAA (10 mg.kg-1.day-1), a 20-HETE receptor antagonist, in the drinking water (vehicle). At the end of the experiments (12 days), renal interlobar arteries (RIA) and mesenteric arteries (MA) were harvested for the assessment of 20-HETE levels by LC-MS/MS, and vascular contractility, vasodilation, and remodeling using wire and pressure myography. The Myh11-4a12 mice showed higher Cyp4a levels in MA compared to WT mice (6.5±0.71 vs 3.4±0.70; Cyp4a/β-actin p increased 20-HETE levels in the MA (3334±891 vs. 545±197 pg/mg protein; p HETE levels were not different (117±9 vs. 93±2 pg/mL; p=0.18) when compared to WT. Myh11-4a12 mice displayed higher SBP compared to WT mice (145±2 vs. 127±2 mmHg; Myh11-4a12 mice (124±2 mmHg vs. 147±4 mmHg, treated Myh11-4a12 mice displayed a higher media to lumen (M:L) ratio (0.277±0.025 vs 0.163 ± 0.009; (15114±1871 vs 10560±641 μm2; Moreover, RIA from Myh11-4a12 mice on AAA exhibit a lower M:L ratio compared to Myh11-4a12 mice on vehicle (0.215 ± 0.013, not different (14268 ± 1259 μm2). Higher constrictor responsiveness to phenylephrine (EC50: 1.63x10-7 ± 3.75x10-8 vs 5.00x10-7 ± 0.7.85x10-8 M, response at 10-4 M: 65 ± 1 vs 83 ± 2 % relaxation, from Myh11-4a12 mice compared to WT. Treatment of Myh11-4a12 mice with AAA diminished the constrictor responsiveness to phenylephrine (EC50 of 4.22x10-7± 4.55x10-8 M, acetylcholine (92 ± 2 % relaxation, phosphorylation of myosin light chain compared to WT mice (1.60 ± 0.23 vs 1.00 ± 0.06, Ser19 p-MLC/total MLC fold change, exhibited a decrease in myosin light chain phosphorylation compared to Myh11-4a12 mice on vehicle (0.94 ± 0.11, Ser19 p-MLC/total MLC fold change, p\u3c0.05). Upstream of myosin light chain is Rho kinase (ROCK). Expression of ROCK1 was assessed in RIA and Myh11-4a12 mice had increased ROCK1 expression compared to WT mice (1.60 ± 0.21 vs 1.00 ± 0.18, ROCK1/β-tubulin fold change, ROCK1/β-tubulin fold change). Pressure myography was performed to assess 20-HETE receptor antagonism on ROCK activity. In RIA from WT mice, 20-HETE administration promoted a greater myogenic response and both AAA and ROCK inhibitor (Y-27632) inhibited the 20-HETE response in relation to the myogenic tone. The results of this study suggest that VSMC specific overproduction of Cyp4a12 and 20-HETE promotes increase in blood pressure and changes in vascular reactivity. 20-HETE receptor antagonism was capable of reversing the vascular pathology in mice with elevated 20-HETE presumably through inhibition of ROCK activity

Metformin mitigates SASP secretion and LPS-triggered hyper-inflammation in Doxorubicin-induced senescent endothelial cells

Introduction: Doxorubicin (DOX), a chemotherapeutic drug, induces senescence and increases the secretion of senescence-associated secretory phenotype (SASP) in endothelial cells (ECs), which contributes to DOX-induced inflammaging. Metformin, an anti-diabetic drug, demonstrates senomorphic effects on different models of senescence. However, the effects of metformin on DOX-induced endothelial senescence have not been reported before. Senescent ECs exhibit a hyper-inflammatory response to lipopolysachharide (LPS). Therefore, in our current work, we identified the effects of metformin on DOX-induced endothelial senescence and LPS-induced hyper-inflammation in senescent ECs.Methods: ECs were treated with DOX ± metformin for 24 h followed by 72 h incubation without DOX to establish senescence. Effects of metformin on senescence markers expression, SA-β-gal activity, and SASP secretion were assessed. To delineate the molecular mechanisms, the effects of metformin on major signaling pathways were determined. The effect of LPS ± metformin was determined by stimulating both senescent and non-senescent ECs with LPS for an additional 24 h.Results: Metformin corrected DOX-induced upregulation of senescence markers and decreased the secretion of SASP factors and adhesion molecules. These effects were associated with a significant inhibition of the JNK and NF-κB pathway. A significant hyper-inflammatory response to LPS was observed in DOX-induced senescent ECs compared to non-senescent ECs. Metformin blunted LPS-induced upregulation of pro-inflammatory SASP factors.Conclusion: Our study demonstrates that metformin mitigates DOX-induced endothelial senescence phenotype and ameliorates the hyper-inflammatory response to LPS. These findings suggest that metformin may protect against DOX-induced vascular aging and endothelial dysfunction and ameliorate infection-induced hyper-inflammation in DOX-treated cancer survivors

EA.hy926 Cells and HUVECs Share Similar Senescence Phenotypes but Respond Differently to the Senolytic Drug ABT-263

Doxorubicin (DOX) induces endothelial cell (EC) senescence, which contributes to endothelial dysfunction and cardiovascular complications. Senolytic drugs selectively eliminate senescent cells to ameliorate senescence-mediated pathologies. Previous studies have demonstrated differences between immortalized and primary EC models in some characteristics. However, the response of DOX-induced senescent ECs to senolytics has not been determined across these two models. In the present work, we first established a comparative characterization of DOX-induced senescence phenotypes in immortalized EA.hy926 endothelial-derived cells and primary human umbilical vein EC (HUVECs). Thereafter, we evaluated the senolytic activity of four senolytics across both ECs. Following the DOX treatment, both EA.hy926 and HUVECs shared similar senescence phenotypes characterized by upregulated senescence markers, increased SA-β-gal activity, cell cycle arrest, and elevated expression of the senescence-associated secretory phenotype (SASP). The potentially senolytic drugs dasatinib, quercetin, and fisetin demonstrated a lack of selectivity against DOX-induced senescent EA.hy926 cells and HUVECs. However, ABT-263 (Navitoclax) selectively induced the apoptosis of DOX-induced senescent HUVECs but not EA.hy926 cells. Mechanistically, DOX-treated EA.hy926 cells and HUVECs demonstrated differential expression levels of the BCL-2 family proteins. In conclusion, both EA.hy926 cells and HUVECs demonstrate similar DOX-induced senescence phenotypes but they respond differently to ABT-263, presumably due to the different expression levels of BCL-2 family proteins

Proximal Tubular-Targeted Overexpression of the Cyp4a12-20-HETE Synthase Promotes Salt-Sensitive Hypertension in Male Mice

20-Hydroxyeicosatetraenoic acid (20-HETE) has been linked to blood pressure (BP) regulation via actions on the renal microvasculature and tubules. We assessed tubular 20-HETE contribution to hypertension by generating transgenic mice overexpressing the CYP4A12-20-HETE synthase (PT-4a12 mice) under the control of the proximal tubule (PT)-specific promoter, phosphoenolpyruvate carboxykinase (PEPCK). 20-HETE levels in the kidney cortex of male (967±210 vs. 249±69 pg/mg protein), but not female (121±15 vs. 92±11 pg/mg protein) PT-4a12 mice, showed a 2.5-fold increase compared to WT. Renal cortical Cyp4a12 mRNA and CYP4A12 protein in male, but not female PT-4a12 mice increased by 2-3-fold compared to WT. Male PT-4a12 mice displayed elevated BP (142±1 vs. 111±4 mmHg,

20-HETE Interferes with Insulin Signaling and Contributes to Obesity-Driven Insulin Resistance

20-HETE, a metabolite of arachidonic acid produced by Cytochrome P450 (CYP) 4A/4 F, has been implicated in the development of obesity-associated complications such as diabetes and insulin resistance. In this study, we examined whether the acute elevation of 20-HETE levels contributes to the development of diet-driven hyperglycemia and insulin resistance. We employed a conditional transgenic mouse model to overexpress Cyp4a12 (Cyp4a12tg), a murine 20-HETE synthase, together with high fat diet (HFD) feeding. Mice in which Cyp4a12 was induced by doxycycline (DOX) at the onset of HFD feeding gained weight at a greater rate and extent than corresponding DOX-untreated Cyp4a12 mice. Cyp4a12tg mice fed HFD + DOX displayed hyperglycemia and impaired glucose metabolism while corresponding HFD-fed Cyp4a12tg mice (no DOX) did not. Importantly, administration of a 20-HETE antagonist, 20-SOLA, to Cyp4a12tg mice fed HFD + DOX significantly attenuated weight gain and prevented the development of hyperglycemia and impaired glucose metabolism. Levels of insulin receptor (IR) phosphorylation at Tyrosine 972 and insulin receptor substrate-1 (IRS1) phosphorylation at serine 307 were markedly decreased and increased, respectively, in liver, skeletal muscle and adipose tissues from Cyp4a12tg mice fed HFD + DOX; 20-SOLA prevented the IR and IRS1 inactivation, suggesting that 20-HETE interferes with insulin signaling. Additional studies in 3T3-1 differentiated adipocytes confirmed that 20-HETE impairs insulin signaling and that its effect may require activation of its receptor GPR75. Taken together, these results provide strong evidence that 20-HETE interferes with insulin function and contributed to diet-driven insulin resistance

Gpr75-Deficient Mice Are Protected From High-Fat Diet-Induced Obesity

OBJECTIVE: G-protein coupled receptor 75 (GPR75) has been identified as the high-affinity receptor of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive and proinflammatory lipid, and mice overproducing 20-HETE have been shown to develop insulin resistance when fed a high-fat diet (HFD), which was prevented by a 20-HETE receptor blocker. Simultaneously, a large-scale exome sequencing of 640,000 subjects identified an association between loss-of-function GPR75 variants and protection against obesity. METHODS: Wild-type (WT) and Gpr75-deficient mice were placed on HFD for 14 weeks, and their obesity phenotype was examined. RESULTS: Male and female Gpr75 null (knockout [KO]) and heterozygous mice gained less weight than WT mice when placed on HFD. KO mice maintained the same level of energy expenditure during HFD feeding, whereas WT mice showed a significant reduction in energy expenditure. Diet-driven adiposity and adipocyte hypertrophy were greatly lessened in Gpr75-deficient mice. HFD-fed KO mice did not develop insulin resistance. Adipose tissue from Gpr75-deficient mice had increased expression of thermogenic genes and decreased levels of inflammatory markers. Moreover, insulin signaling, which was impaired in HFD-fed WT mice, was unchanged in KO mice. CONCLUSIONS: These findings suggest that GPR75 is an important player in the control of metabolism and glucose homeostasis and a likely novel therapeutic target to combat obesity-driven metabolic disorders

Adipocyte Specific HO-1 Gene Therapy is Effective in Antioxidant Treatment of Insulin Resistance and Vascular Function in an Obese Mice Model

Obesity is a risk factor for vascular dysfunction and insulin resistance. The study aim was to demonstrate that adipocyte-specific HO-1 (heme oxygenase-1) gene therapy is a therapeutic approach for preventing the development of obesity-induced metabolic disease in an obese-mice model. Specific expression of HO-1 in adipose tissue was achieved by using a lentiviral vector expressing HO-1 under the control of the adiponectin vector (Lnv-adipo-HO-1). Mice fed a high-fat diet (HFD) developed adipocyte hypertrophy, fibrosis, decreased mitochondrial respiration, increased levels of inflammatory adipokines, insulin resistance, vascular dysfunction, and impaired heart mitochondrial signaling. These detrimental effects were prevented by the selective expression of HO-1 in adipocytes. Lnv-adipo-HO-1-transfected mice on a HFD display increased cellular respiration, increased oxygen consumption, increased mitochondrial function, and decreased adipocyte size. Moreover, RNA arrays confirmed that targeting adipocytes with HO-1 overrides the genetic susceptibility of adiposopathy and correlated with restoration of the expression of anti-inflammatory, thermogenic, and mitochondrial genes. Our data demonstrate that HO-1 gene therapy improved adipose tissue function and had positive impact on distal organs, suggesting that specific targeting of HO-1 gene therapy is an attractive therapeutic approach for improving insulin sensitivity, metabolic activity, and vascular function in obesity

Blockade of 20-Hydroxyeicosatetraenoic Acid Receptor Lowers Blood Pressure and Alters Vascular Function in Mice With Smooth Muscle-Specific Overexpression of CYP4A12-20-HETE Synthase

OBJECTIVE: 20-Hydroxyeicosatetraenoic acid (20-HETE) is a vasoactive eicosanoid exhibiting effects on vascular smooth muscle cell (VSMC) via G-protein coupled receptor 75 (GPR75) and include stimulation of contractility, migration, and growth. We examined whether VSMC-targeted overexpression of CYP4A12, the primary 20-HETE-producing enzyme in mice, is sufficient to promote hypertension. METHODS: Mice with VSM-specific Cyp4a12 overexpression (Myh11-4a12) and their littermate controls (WT) were generated by crossbreeding Cyp4a12-floxed with Myh11-Cre mice. The 20-HETE receptor blocker, N-disodium succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA), was administered in the drinking water. Experiments were carried out for 12 days. SBP was measured by tail cuff. Renal interlobar and mesenteric arteries were harvested for assessment of gene expression, 20-HETE levels, vascular contractility, vasodilation, and remodeling. RESULTS: Vascular and circulatory levels of 20-HETE were several folds higher in Myh11-4a12 mice compared with WT. The Myh11-4a12 mice compared with WT were hypertensive (145 ± 2 vs. 127 ± 2 mmHg; P \u3c 0.05) and their vasculature displayed a contractile phenotype exemplified by increased contractility, reduced vasodilatory capacity, and increased media to lumen ratio. All these features were reversed by the administration of AAA. The mechanism of increased contractility includes, at least in part, Rho-kinase activation followed by increased myosin light chain phosphorylation and activation of the contractile apparatus. CONCLUSION: VSM-specific Cyp4a12 overexpression is sufficient to alter VSM cell phenotype through changes in contractile markers and enhancement in contractility that promote hypertension and vascular dysfunction in a 20-HETE-dependent manner. The 20-HETE receptor GPR75 may represent a novel target for the treatment of hypertension and associated vascular conditions

Adipocyte Specific HO-1 Gene Therapy Is Effective in Antioxidant Treatment of Insulin Resistance and Vascular Function in an Obese Mice Model

Obesity is a risk factor for vascular dysfunction and insulin resistance. The study aim was to demonstrate that adipocyte-specific HO-1 (heme oxygenase-1) gene therapy is a therapeutic approach for preventing the development of obesity-induced metabolic disease in an obese-mice model. Specific expression of HO-1 in adipose tissue was achieved by using a lentiviral vector expressing HO-1 under the control of the adiponectin vector (Lnv-adipo-HO-1). Mice fed a high-fat diet (HFD) developed adipocyte hypertrophy, fibrosis, decreased mitochondrial respiration, increased levels of inflammatory adipokines, insulin resistance, vascular dysfunction, and impaired heart mitochondrial signaling. These detrimental effects were prevented by the selective expression of HO-1 in adipocytes. Lnv-adipo-HO-1-transfected mice on a HFD display increased cellular respiration, increased oxygen consumption, increased mitochondrial function, and decreased adipocyte size. Moreover, RNA arrays confirmed that targeting adipocytes with HO-1 overrides the genetic susceptibility of adiposopathy and correlated with restoration of the expression of anti-inflammatory, thermogenic, and mitochondrial genes. Our data demonstrate that HO-1 gene therapy improved adipose tissue function and had positive impact on distal organs, suggesting that specific targeting of HO-1 gene therapy is an attractive therapeutic approach for improving insulin sensitivity, metabolic activity, and vascular function in obesity