Enhanced angiotensin converting enzyme 2 regulates the insulin/Akt signalling pathway by blockade of macrophage migration inhibitory factor expression.
ABSTRACT: BACKGROUND AND PURPOSE: Macrophage migration inhibitory factor (MIF) is now known to be a pro-inflammatory cytokine associated with insulin resistance. Our aim was to investigate whether angiotensin converting enzyme 2 (ACE2) could modulate the expression of MIF and the insulin/Akt-endothelial nitric oxide (NO) synthase (eNOS) signalling in a human endothelial cell line (EAhy926). EXPERIMENTAL APPROACH: A recombinant plasmid encompassing human ACE2 gene was constructed and transfected into the EAhy926 cells. The mRNA, phosphorylation and protein levels of p22phox, MIF, Akt and eNOS in endothelial cells were determined by real-time PCR and Western blot analysis, respectively. KEY RESULTS: Gene transfer of ACE2 suppressed the expression of p22phox and MIF induced by angiotensin (Ang) II and Ang IV, accompanied by a decreased level of malondialdehyde in cells. In addition, Ang II diminished insulin-stimulated phosphorylation of Akt (at Ser(473)) and eNOS (at Ser(1177)) and NO generation, effects which were reversed by ACE2 gene transfer and anti-MIF treatment in endothelial cells. CONCLUSIONS AND IMPLICATIONS:The results reveal that gene transfer of ACE2 regulated Ang II-mediated impairment of insulin signalling and involved the Akt-eNOS phosphorylation pathway. These beneficial effects of ACE2 overexpression appear to result mainly from blocking MIF expression in endothelial cells, suggesting that the ACE2 gene may be a novel therapeutic target for diseases related to inflammation and insulin resistance.
Project description:Angiotensin II (AngII), a vasoactive peptide that elevates arterial blood pressure and results in hypertension, has been reported to directly induce vascular endothelial cell apoptosis. Recent work has demonstrated that propofol pre-treatment attenuates angiotensin II-induced apoptosis in human coronary artery endothelial cells. However, the underlying mechanism remains largely unknown. Here, we investigated human umbilical vein endothelial cells (HUVECs) subjected to angiotensin II-induced apoptosis in the presence or absence of propofol treatment and found that angiotensin II-induced apoptosis was attenuated by propofol in a dose-dependent manner. Furthermore, ELISA assays demonstrated that the ratio of angiotensin (1-7) (Ang (1-7)) to Ang II was increased after propofol treatment. We examined the expression of ACE2, Ang (1-7) and Mas and found that the ACE2-Ang (1-7)-Mas axis was up-regulated by propofol, while ACE2 overexpression increased phosphorylated endothelial nitric oxide synthase (phosphorylated eNOS) expression and siACE2 resulted in the repression of endothelial nitric oxide synthase (eNOS) phosphorylation. In conclusion, our study revealed that propofol can inhibit endothelial cell apoptosis induced by Ang II by activating the ACE2-Ang (1-7)-Mas axis and further up-regulating the expression and phosphorylation of eNOS.
Project description:Activation of the angiotensin 1-7/Mas receptor (MasR) axis counteracts angiotensin II (Ang II)-mediated cardiovascular disease. Recombinant human angiotensin-converting enzyme 2 (rhACE2) generates Ang 1-7 from Ang II. We hypothesized that the therapeutic effects of rhACE2 are dependent on Ang 1-7 action. Wild type male C57BL/6 mice (10-12 weeks old) were infused with Ang II (1.5 mg/kg/d) and treated with rhACE2 (2 mg/kg/d). The Ang 1-7 antagonist, A779 (200 ng/kg/min), was administered to a parallel group of mice. rhACE2 prevented Ang II-induced hypertrophy and diastolic dysfunction while A779 prevented these beneficial effects and precipitated systolic dysfunction. rhACE2 effectively antagonized Ang II-mediated myocardial fibrosis which was dependent on the action of Ang 1-7. Myocardial oxidative stress and matrix metalloproteinase 2 activity was further increased by Ang 1-7 inhibition even in the presence of rhACE2. Activation of Akt and endothelial nitric oxide synthase (eNOS) by rhACE2 were suppressed by the antagonism of Ang 1-7 while the activation of pathological signaling pathways was maintained. Blocking Ang 1-7 action prevents the therapeutic effects of rhACE2 in the setting of elevated Ang II culminating in systolic dysfunction. These results highlight a key cardioprotective role of Ang 1-7, and increased Ang 1-7 action represents a potential therapeutic strategy for cardiovascular diseases.Activation of the renin-angiotensin system (RAS) plays a key pathogenic role in cardiovascular disease. ACE2, a monocarboxypeptidase, negatively regulates pathological effects of Ang II. Antagonizing Ang 1-7 prevents the therapeutic effects of recombinant human ACE2. Our results highlight a key protective role of Ang 1-7 in cardiovascular disease.
Project description:Despite evidence that hyperactivity of the vasodeleterious axis (ACE/angiotensin II (Ang II)/AT1 receptor) of the renin-angiotensin system (RAS) is associated with the pathogenesis of diabetic retinopathy (DR) use of the inhibitors of this axis has met with limited success in the control of this pathophysiology. We investigated the hypothesis that enhancing the local activity of the recently established protective axis of the RAS, ACE2/Ang-(1-7), using adeno-associated virus (AAV)-mediated gene delivery of ACE2 or Ang-(1-7) would confer protection against diabetes-induced retinopathy. Genes expressing ACE2 and Ang-(1-7) were cloned in AAV vector. The effects of ocular AAV-ACE2/Ang-(1-7) gene transfer on DR in diabetic eNOS(-/-) mice and Sprague-Dawley (SD) rats were examined. Diabetes was associated with approximately tenfold and greater than threefold increases in the ratios of ACE/ACE2 and AT1R/Mas mRNA levels in the retina respectively. Intraocular administration of AAV-ACE2/Ang-(1-7) resulted in significant reduction in diabetes-induced retinal vascular leakage, acellular capillaries, infiltrating inflammatory cells and oxidative damage in both diabetic mice and rats. Our results demonstrate that DR is associated with impaired balance of retinal RAS. Increased expression of ACE2/Ang-(1-7) overcomes this imbalance and confers protection against DR. Thus, strategies enhancing the protective ACE2/Ang-(1-7) axis of RAS in the eye could serve as a novel therapeutic target for DR.
Project description:Dietary administration of 0.30% indole-3-carbinol (I3C) to Cyp1a1-Ren2 transgenic rats (TGRs) generates angiotensin II (ANG II)-dependent malignant hypertension (HTN) and increased renal vascular resistance. However, TGRs with HTN maintain a normal or slightly reduced glomerular filtration rate. We tested the hypothesis that maintenance of renal function in hypertensive Cyp1a1-Ren2 TGRs is due to preservation of the intrarenal nitric oxide (NO) and antioxidant systems.Kidney cortex, kidney medulla, aortic endothelial (e) and neuronal (n) nitric oxide synthase (NOS), superoxide dismutases (SODs), and p22phox (nicotinamide adenine dinucleotide phosphate-oxidase subunit) protein abundances were measured along with kidney cortex total antioxidant capacity (TAC) and NOx. TGRs were fed a normal diet that contained 0.3% I3C or 0.3% I3C + candesartan (AT1 receptor antagonist; 25mg/L in drinking water) (n = 5-6 per group) for 10 days.Blood pressure increased and body weight decreased in I3C-induced TGRs, while candesartan blunted these responses. Abundances of NOS, SOD, and p22phox as well as TAC were maintained in the kidney cortex of I3C-induced TGRs with and without candesartan, while kidney cortex NOx production increased in both groups. Kidney medulla eNOS and extracellular (EC) SOD decreased and nNOS were unchanged in both groups of I3C-induced TGRs. In addition, a compensatory increase occurred in kidney medulla Mn SOD in I3C-induced TGRs + candesartan. Aortic eNOS and nNOS? fell and p22phox and Mn SOD increased in hypertensive I3C-induced TGRs; all changes were reversed with candesartan.The preservation of renal cortical NO and antioxidant capacity is associated with preserved renal function in Cyp1a1-Ren2 TGRs with ANG II-dependent malignant HTN.
Project description:Impaired insulin signaling via phosphatidylinositol 3-kinase/Akt to endothelial nitric oxide synthase (eNOS) in the vasculature has been postulated to lead to arterial dysfunction and hypertension in obesity and other insulin resistant states. To investigate this, we compared insulin signaling in the vasculature, endothelial function, and systemic blood pressure in mice fed a high-fat (HF) diet to mice with genetic ablation of insulin receptors in all vascular tissues (TTr-IR(-/-)) or mice with genetic ablation of Akt1 (Akt1-/-). HF mice developed obesity, impaired glucose tolerance, and elevated free fatty acids that was associated with endothelial dysfunction and hypertension. Basal and insulin-mediated phosphorylation of extracellular signal-regulated kinase 1/2 and Akt in the vasculature was preserved, but basal and insulin-stimulated eNOS phosphorylation was abolished in vessels from HF versus lean mice. In contrast, basal vascular eNOS phosphorylation, endothelial function, and blood pressure were normal despite absent insulin-mediated eNOS phosphorylation in TTr-IR(-/-) mice and absent insulin-mediated eNOS phosphorylation via Akt1 in Akt1-/- mice. In cultured endothelial cells, 6 hours of incubation with palmitate attenuated basal and insulin-stimulated eNOS phosphorylation and NO production despite normal activation of extracellular signal-regulated kinase 1/2 and Akt. Moreover, incubation of isolated arteries with palmitate impaired endothelium-dependent but not vascular smooth muscle function. Collectively, these results indicate that lower arterial eNOS phosphorylation, hypertension, and vascular dysfunction following HF feeding do not result from defective upstream signaling via Akt, but from free fatty acid-mediated impairment of eNOS phosphorylation.
Project description:Microvascular insufficiency represents a major cause of end-organ failure among diabetics. The current studies were undertaken to determine whether dysregulation of the angiopoietins/Tie-2 system would result in an impairment of smooth muscle cell (SMC) recruitment and vascular maturation, which contributes to impaired angiogenesis in diabetes.Tie-2 expression was significantly attenuated, whereas angiopoietin-2 (Ang-2) was increased in db/db mice subjected to myocardial ischemia. Our morphological analysis showed that the number of SMC coverage area per neovessel was significantly reduced in db/db mice. This was accompanied by a significant reduction of myocardial capillary density and arteriole formation. Interestingly, Angiopoietin-1(Ang-1)-induced SMC recruitment and vessel outgrowth were severely impaired in db/db mice. Our in vitro studies further demonstrated that exposure of mouse heart endothelial cells to high glucose resulted in a significant upregulation of Ang-2 and a downregulation of Tie-2 expression. These alterations led to a significant impairment of Ang-1-induced Akt and eNOS phosphorylation, along with a remarkable impairment of Ang-1-induced endothelial cell migration and endothelial cell spheroid sprouting. Ang-1 gene transfer restored Tie-2 expression and rescued these abnormalities in diabetes.Our findings underscore the important role of Ang-1-Tie-2 signaling in the diabetes-induced impairment of vascular maturation and angiogenesis.
Project description:20-Hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser616; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr632 and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser1177 site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser616 is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.
Project description:Albuminuria is an early marker of renovascular damage associated to an increase in oxidative stress. The Munich Wistar Frömter (MWF) rat is a model of chronic kidney disease (CKD), which exhibits endothelial dysfunction associated to low nitric oxide availability. We hypothesize that the new highly selective, non-steroidal mineralocorticoid receptor (MR) antagonist, finerenone, reverses both endothelial dysfunction and microalbuminuria. Twelve-week-old MWF (MWF-C; MWF-FIN) and aged-matched normoalbuminuric Wistar (W-C; W-FIN) rats were treated with finerenone (FIN, 10 mg/kg/day p.o.) or vehicle (C) for 4-week. Systolic blood pressure (SBP) and albuminuria were determined the last day of treatment. Finerenone lowered albuminuria by >40% and significantly reduced SBP in MWF. Aortic rings of MWF-C showed higher contractions to either noradrenaline (NA) or angiotensin II (Ang II), and lower relaxation to acetylcholine (Ach) than W-C rings. These alterations were reversed by finerenone to W-C control levels due to an upregulation in phosphorylated Akt and eNOS, and an increase in NO availability. Apocynin and 3-amino-1,2,4-triazole significantly reduced contractions to NA or Ang II in MWF-C, but not in MWF-FIN rings. Accordingly, a significant increase of Mn-superoxide dismutase (SOD) and Cu/Zn-SOD protein levels were observed in rings of MWF-FIN, without differences in p22phox, p47phox or catalase levels. Total SOD activity was increased in kidneys from MWF-FIN rats. In conclusion, finerenone improves endothelial dysfunction through an enhancement in NO bioavailability and a decrease in superoxide anion levels due to an upregulation in SOD activity. This is associated with an increase in renal SOD activity and a reduction of albuminuria.
Project description:Angiotensin-converting enzyme 2 (ACE2)-angiotensin (1-7) [Ang (1-7)]-Mas constitutes the vasoprotective axis and is demonstrated to antagonize the vascular pathophysiological effects of the classical renin-angiotensin system. We sought to study the hypothesis that upregulation of ACE2-Ang (1-7) signaling protects endothelial function through reducing oxidative stress that would result in beneficial outcome in diabetes.Ex vivo treatment with Ang (1-7) enhanced endothelium-dependent relaxation (EDR) in renal arteries from diabetic patients. Both Ang (1-7) infusion via osmotic pump (500?ng/kg/min) for 2 weeks and exogenous ACE2 overexpression mediated by adenoviral ACE2 via tail vein injection (10(9) pfu/mouse) rescued the impaired EDR and flow-mediated dilatation (FMD) in db/db mice. Diminazene aceturate treatment (15?mg/kg/day) activated ACE2, increased the circulating Ang (1-7) level, and augmented EDR and FMD in db/db mouse arteries. In addition, activation of the ACE2-Ang (1-7) axis reduced reactive oxygen species (ROS) overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortas and also in high-glucose-treated endothelial cells. Pharmacological benefits of ACE2-Ang (1-7) upregulation on endothelial function were confirmed in ACE2 knockout (ACE2 KO) mice both ex vivo and in vitro.We elucidate that the ACE2-Ang (1-7)-Mas axis serves as an important signal pathway in endothelial cell protection in diabetic mice, especially in diabetic human arteries.Endogenous ACE2-Ang (1-7) activation or ACE2 overexpression preserves endothelial function in diabetic mice through increasing nitric oxide bioavailability and inhibiting oxidative stress, suggesting the therapeutic potential of ACE2-Ang(1-7) axis activation against diabetic vasculopathy. Antioxid.
Project description:Generation of physiologically active vascular beds by delivery of combinations of growth factors offers promise for vascular gene therapy.In a mesenteric model of physiological angiogenesis, combining endothelial nitric oxide synthase (eNOS) (and hence NO production) with VEGF and angiopoietin-1 overexpression resulted in a more functional vascular phenotype than growth factor administration alone. eNOS gene delivery upregulated eNOS, VEGF, and Ang-1 to similar levels as gene transfer with VEGF or Ang-1. eNOS overexpression resulted in neovascularization to a similar extent as VEGF and Ang-1 combined, but not by sprouting angiogenesis. Whereas combining Ang-1 and VEGF increased both exchange vessels and conduit vessels, neither growth factor nor eNOS alone resulted in vessels with smooth muscle cell (SMC) coverage. In contrast, combining all three generated microvessels with SMCs (arteriolar genesis) and further increased functional vessels. Use of a vasodilator, prazosin, in combination with Ang1 and VEGF, but not alone, also generated SMC-positive vessels.Coexpression of eNOS, VEGF, and Ang-1 results in a more mature vascularization of connective tissue, and generates new arterioles as well as new capillaries, and provides a more physiological therapeutic approach than single growth factor administration, by combining hemodynamic forces with growth factors.