De novo lipogenesis maintains vascular homeostasis through endothelial nitric-oxide synthase (eNOS) palmitoylation

Endothelial dysfunction leads to lethal vascular complications in diabetes and related metabolic disorders. Here, we demonstrate that de novo lipogenesis, an insulin-dependent process driven by the multifunctional enzyme fatty-acid synthase (FAS), maintains endothelial function by targeting endothelial nitric-oxide synthase (eNOS) to the plasma membrane. In mice with endothelial inactivation of FAS (FASTie mice), eNOS membrane content and activity were decreased. eNOS and FAS were physically associated; eNOS palmitoylation was decreased in FAS-deficient cells, and incorporation of labeled carbon into eNOS-associated palmitate was FAS-dependent. FASTie mice manifested a proinflammatory state reflected as increases in vascular permeability, endothelial inflammatory markers, leukocyte migration, and susceptibility to LPS-induced death that was reversed with an NO donor. FAS-deficient endothelial cells showed deficient migratory capacity, and angiogenesis was decreased in FASTie mice subjected to hindlimb ischemia. Insulin induced FAS in endothelial cells freshly isolated from humans, and eNOS palmitoylation was decreased in mice with insulin-deficient or insulin-resistant diabetes. Thus, disrupting eNOS bioavailability through impaired lipogenesis identifies a novel mechanism coordinating nutritional status and tissue repair that may contribute to diabetic vascular disease.     

Alpha-mangostin improves endothelial dysfunction in db/db mice through inhibition of aSMase/ceramide pathway

Diabetic vascular complications are the leading causes of death and disability in patients with diabetes. Alpha-mangostin has been reported to have anti-diabetic capacity in recent years. Here, we investigated the protective function of alpha-mangostin on endothelium in vitro and in vivo experiments. We also observed that alpha-mangostin improved impaired endothelium-dependent vasodilation (EDV) of diabetic animals while it limited the aSMase/ceramide pathway and up-regulated eNOS/NO pathway in aortas from diabetic mice. Meanwhile, alpha-mangostin inhibited elevated aSMase/ceramide pathway and reversed impaired EDV induced by high glucose in isolated mouse aortas. In addition, alpha-mangostin increased phosphorylation of eNOS and NO production in high glucose-treated aortas. Alpha-mangostin normalized high glucose-induced activation of aSMase/ceramide pathway and improved eNOS/NO pathway in endothelial cells with high glucose. In conclusion, alpha-mangostin regulates eNOS/NO pathway and improves EDV in aortas of diabetic mice through inhibiting aSMase activity and endogenous ceramide accumulation.     

Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice

We tested the hypothesis that endothelial nitric oxide (NO) synthase (eNOS)-derived NO modulates rho-kinase-mediated vascular contraction. Because 3-hydroxy-3-methylglutaryl (HMG)-CoA-reductase inhibition can both upregulate eNOS expression and inhibit rhoA/rho-kinase function, a second hypothesis tested was that statin treatment modulates rho-kinase-mediated contraction and that this can occur independently of eNOS. Contractile responses to the receptor-dependent agonists serotonin and phenylephrine but not to the receptor-independent agent KCl were greater in aortic rings from eNOS-null (eNOS(-/-)) vs. wild-type (eNOS(+/+)) mice. Similarly enhanced responses were seen in eNOS(+/+) rings after acute NOS inhibition. The rho-kinase inhibitor Y-27632 abolished or profoundly attenuated responses to receptor agonists in both eNOS(+/+) and eNOS(-/-) rings, but responses in eNOS(+/+) were more sensitive to Y-27632. Mevastatin treatment (20 mg/kg sc per day, 14 days) reduced responses to serotonin and phenylephrine in female mice of both strains. KCl-induced contractions were slightly smaller in eNOS(+/+)-derived aortic rings only. Levels of plasma cholesterol, and aortic expression of rhoA and rho-kinase, did not differ between groups. Thus eNOS-derived NO suppresses rhoA/rho-kinase-mediated vascular contraction. Moreover, a similar suppressive effect on rho-kinase-mediated vasoconstriction by statin therapy occurs independently of effects on eNOS or plasma cholesterol.     

Blockade of geranylgeranylation by rosuvastatin upregulates eNOS expression in human venous endothelial cells

Endothelial dysfunction is associated with a reduction in nitric oxide (NO) bioavailability. Positive effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on the improvement of endothelial dysfunction have been shown. We investigated the effects of rosuvastatin and isoprenoid metabolites on endothelial NO synthase (eNOS) mRNA and protein expression in human umbilical venous endothelial cells after exposure to 10(-8)-10(-5) mol/l rosuvastatin for 8 and 12 h. Cell viability was not significantly altered after exposure to the statin for 12h. In a concentration-dependent manner, rosuvastatin upregulated eNOS mRNA and protein expression. The effects on eNOS expression mediated through rosuvastatin could be reversed by treatment with mevalonate indicating inhibition of HMG-CoA reductase as the underlying mechanism. Treatment with geranylgeranylpyrophosphate, but not farnesylpyrophosphate, reversed the increase of eNOS expression induced by rosuvastatin. Rosuvastatin may have beneficial effects on endothelial dysfunction associated with cardiovascular diseases beyond its effects on lowering cholesterol.     

Sildenafil-mediated acute cardioprotection is independent of the NO/cGMP pathway

Sildenafil, a potent inhibitor of phosphodiesterase type 5, has recently been investigated in animal models of myocardial ischemia-reperfusion (MI/R) injury. Previous studies have suggested that the protective effects of sildenafil are mediated via activation of endothelial nitric oxide (NO) synthesis (eNOS) and inducible NOS (iNOS). To further investigate the protective mechanism of sildenafil, we subjected wild-type, eNOS, and iNOS null animals to 30 min of myocardial ischemia and 24 h of reperfusion. Treatment with 0.06 mg/kg sildenafil 5 min before reperfusion significantly reduced myocardial infarct size in wild-type, eNOS null mice (eNOS(-/-)), and iNOS(-/-) animals. Additionally, the low dose utilized in this study did not alter myocardial cGMP. These results suggest that acute low-dose sildenafil-mediated cardioprotection is independent of eNOS, iNOS, and cGMP. In a second series of experiments, we investigated sildenafil in db/db diabetic mice subjected to MI/R. We found that sildenafil failed to protect diabetic mice against MI/R. However, NO(.) donor therapy was found to significantly protect against MI/R injury in both nondiabetic and diabetic mice, suggesting that protection could be conferred in diabetic mice and that the upstream modulator of soluble guanylyl cyclase, NO(.), may mediate protection independent of cGMP signaling. The present study suggests that further research is needed to delineate the precise mechanisms by which sildenafil exerts cardioprotection.     

Isoprenoid depletion by statins antagonizes cytokine-induced down-regulation of endothelial nitric oxide expression and increases NO synthase activity in human umbilical vein endothelial cells.

Endothelial dysfunction and atherosclerosis are associated with an inflammation-induced decrease in endothelial nitric oxide synthase (eNOS) expression. Based on the differences between hydrophobic and hydrophilic statins in their reduction of cardiac events, we analyzed the effects of rosuvastatin and cerivastatin on eNOS and inducible NO synthase (iNOS) expression and NOS activity in TNF-alpha-stimulated human umbilical vein endothelial cells (HUVEC). Both statins reversed down-regulation of eNOS mRNA and protein expression by inhibiting HMG-CoA reductase and isoprenoid synthesis. Cerivastatin tended to a more pronounced effect on eNOS expression compared to rosuvastatin. NOS activity - measured by conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline - was enhanced under treatment with both drugs due to inhibition of HMG-CoA reductase. Statin-treatment reduced iNOS mRNA expression under normal conditions, but had no relevant effects on iNOS mRNA expression in cytokine-treated cells. Rosuvastatin and cerivastatin reverse the detrimental effects of TNF-alpha-induced down-regulation in eNOS protein expression and increase NO synthase activity by inhibiting HMG-CoA reductase and subsequent blocking of isoprenoid synthesis. These results provide evidence that statins have beneficial effects by increasing eNOS expression and activity during the atherosclerotic process.     

Ischemia/Reperfusion-Induced MKP-3 Impairs Endothelial NO Formation via Inactivation of ERK1/2 Pathway

Mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases. Endothelial cells express multiple MKP family members, such as MKP-3. However, the effects of MKP-3 on endothelial biological processes have not yet been fully elucidated. Here, we address the association between MKP-3 and endothelial Nitric oxide (NO) formation under ischemia/reperfusion (IS/RP) condition. Human umbilical vein endothelial cells (HUVECs) were subjected to IS/RP treatment. The MKP-3 expression and NO formation were examined. IS/RP induced endothelial MKP-3 expression and inhibited eNOS expression and NO formation, accompanied by an increase of endothelial apoptosis. The siRNA experiments showed that MKP-3 was an important mediator in impairing eNOS expression and NO production in endothelial cells. Transfection of HUVECs with constitutively active ERK plasmids suggested that the above mentioned effect of MKP-3 was via inactivation of ERK1/2 pathway. Furthermore, impairment of eNOS expression was restored by treatment of histone deacetylase (HDAC) inhibitor and related to histone deacetylation and recruitment of HDAC1 to the eNOS promoter. Finally, Salvianolic acid A (SalA) markedly attenuated induction of MKP-3 and inhibition of eNOS expression and NO formation under endothelial IS/RP condition. Overall, these results for the first time demonstrated that IS/RP inhibited eNOS expression by inactivation of ERK1/2 and recruitment of HDAC1 to the gene promoter, leading to decreased NO formation through a MKP-3-dependent mechanism in endothelial cells, and SalA has therapeutic significance in protecting endothelial cells from impaired NO formation in response to IS/RP.     

Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase

Ischemia induces angiogenesis as a compensatory response. Although ischemia is known to causes synthesis and release of calcitonin gene-related peptide (CGRP), it is not clear whether CGRP regulates angiogenesis under ischemia and how does it function. Thus we investigated the role of CGRP in angiogenesis and the involved mechanisms. We found that CGRP level was increased in the rat hindlimb ischemic tissue. The expression of exogenous CGRP by adenovirus vectors enhanced blood flow recovery and increased capillary density in ischemic hindlimbs. In vitro, CGRP promoted human umbilical vein endothelial cell (HUVEC) tube formation and migration. Further more, CGRP activated AMP-activated protein kinase (AMPK) both in vivo and in vitro, and pharmacological inhibition of CGRP and cAMP attenuated the CGRP-activated AMPK in vitro. CGRP also induced endothelial nitric oxide synthase (eNOS) phosphorylation in HUVECs at Ser1177 and Ser633 in a time-dependent manner, and such effects were abolished by AMPK inhibitor Compound C. As well, Compound C blocked CGRP-enhanced HUVEC tube formation and migration. These findings indicate that CGRP promotes angiogenesis by activating the AMPK-eNOS pathway in endothelial cells.     

Far infra-red therapy promotes ischemia-induced angiogenesis in diabetic mice and restores high glucose-suppressed endothelial progenitor cell functions

ABSTRACT: BACKGROUND: Far infra-red (IFR) therapy was shown to exert beneficial effects in cardiovascular system, but effects of IFR on endothelial progenitor cell (EPC) and EPC-related vasculogenesis remain unclear. We hypothesized that IFR radiation can restore blood flow recovery in ischemic hindlimb in diabetic mice by enhancement of EPCs functions and homing process.Materials and methodsStarting at 4 weeks after the onset of diabetes, unilateral hindlimb ischemia was induced in streptozotocine (STZ)-induced diabetic mice, which were divided into control and IFR therapy groups (n = 6 per group). The latter mice were placed in an IFR dry sauna at 34[DEGREE SIGN]C for 30 min once per day for 5 weeks. RESULTS: Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio in the thermal therapy group was significantly increased beyond that in controls, and significantly greater capillary density was seen in the IFR therapy group. Flow cytometry analysis showed impaired EPCs (Sca-1+/Flk-1+) mobilization after ischemia surgery in diabetic mice with or without IFR therapy (n = 6 per group). However, as compared to those in the control group, bone marrow-derived EPCs differentiated into endothelial cells defined as GFP+/CD31+ double-positive cells were significantly increased in ischemic tissue around the vessels in diabetic mice that received IFR radiation. In in-vitro studies, cultured EPCs treated with IFR radiation markedly augmented high glucose-impaired EPC functions, inhibited high glucose-induced EPC senescence and reduced H2O2 production. Nude mice received human EPCs treated with IFR in high glucose medium showed a significant improvement in blood flow recovery in ischemic limb compared to those without IFR therapy. IFR therapy promoted blood flow recovery and new vessel formation in STZ-induced diabetic mice. CONCLUSIONS: Administration of IFR therapy promoted collateral flow recovery and new vessel formation in STZ-induced diabetic mice, and these beneficial effects may derive from enhancement of EPC functions and homing process.     

Acute activation and phosphorylation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, statins, provide beneficial effects independent of their lipid-lowering effects. One beneficial effect appears to involve acute activation of endothelial nitric oxide (NO) synthase (eNOS) and increased NO release. However, the mechanism of acute statin-stimulated eNOS activation is unknown. Therefore, we hypothesized that eNOS activation may be coupled to altered eNOS phosphorylation. Bovine aortic endothelial cells (BAECs), passages 2-6, were treated with either lovastatin or pravastatin from 0 to 30 min. eNOS phosphorylation was examined by Western blot by use of phosphospecific antibodies for Ser-1179, Ser-635, Ser-617, Thr-497, and Ser-116. Statin stimulation of BAECs increased eNOS phosphorylation at Ser-1179 and Ser-617, which was blocked by the phosphatidylinositol 3-kinase (PI3-kinase)/Akt inhibitor wortmannin, and at Ser-635, which was blocked by the protein kinase A (PKA) inhibitor KT-5720. Statin treatment of BAECs transiently increased NO release by fourfold, measured by cGMP accumulation, and was attenuated by N-nitro-l-arginine methyl ester, wortmannin, and KT-5720 but not by mevalonate. In conclusion, these data demonstrate that eNOS is acutely activated by statins independent of HMG-CoA reductase inhibition and that in addition to Ser-1179, eNOS phosphorylation at Ser-635 and Ser-617 through PKA and Akt, respectively, may explain, in part, a mechanism by which eNOS is activated in response to acute statin treatment.     

Tetrahydrobiopterin, L-arginine and vitamin C actsynergistically to decrease oxidative stress, increase nitricoxide and improve blood flow after induction of hindlimbischemia in the rat

Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is a potent vasodilator and signaling molecule that plays an essential role in vascular remodeling of collateral arteries and perfusion recovery in response to hindlimb ischemia. In ischemic conditions, decreased NO bioavailability was observed because of increased oxidative stress, decreased l-arginine and tetrahy-drobiopterin. This study tested the hypothesis that dietary cosupplementation with tetrahydrobiopterin (BH4), l-arginine, and vitamin C acts synergistically to decrease oxidative stress, increase nitric oxide and improve blood flow in response to acute hindlimb ischemia. Rats were fed normal chow, chow supplemented with BH4 or l-arginine (alone or in combination) or chow supplemented with BH4 + l-arginine + vitamin C for 1 wk before induction of unilateral hindlimb ischemia. Cosupplementation with BH4 + l-arginine resulted in greater eNOS expression, Ca²⁺-dependent NOS activity and NO concentration in gastrocnemius from the is-chemic hindlimb, as well as greater recovery of foot perfusion and more collateral artery enlargement than did rats receiving either agent separately. The addition of vitamin C to the BH4 + l-arginine regimen did further increase these dependent variables, although only the increase in eNOS expression reached statistical significances. In addition, rats given all three supplements demonstrated significantly less Ca²⁺-independent activity, less nitrotyrosine accumulation, greater glutathione:glutathione disulfide (GSH:GSSG) ratio and less gastrocnemius muscle necrosis, on both macroscopic and microscopic levels. In conclusion, cosupplementation with BH4 + l-arginine + vitamin C significantly increased vascular perfusion after hindlimb ischemia by increasing eNOS activity and reducing oxidative stress and tissue necrosis. Oral cosupplementation of l-arginine, BH4 and vitamin C holds promise as a biological therapy to induce collateral artery enlargement.     

LOX-1 Plays an Important Role in Ischemia-Induced Angiogenesis of Limbs

LOX-1, lectin-like oxidized low-density lipoprotein (LDL) receptor-1, is a single transmembrane receptor mainly expressed on endothelial cells. LOX-1 mediates the uptake of oxidized LDL, an early step in atherosclerosis; however, little is known about whether LOX-1 is involved in angiogenesis during tissue ischemia. Therefore, we examined the role of LOX-1 in ischemia-induced angiogenesis in the hindlimbs of LOX-1 knockout (KO) mice. Angiogenesis was evaluated in a surgically induced hindlimb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD) and arteriole density (AD). After right hindlimb ischemia, the ischemic/nonischemic hindlimb blood flow ratio was persistently lower in LOX-1 KO mice than in wild-type (WT) mice. CD and AD were significantly smaller in LOX-1 KO mice than in WT mice on postoperative day 14. Immunohistochemical analysis revealed that the number of macrophages infiltrating ischemic tissues was significantly smaller in LOX-1 KO mice than in WT mice. The number of infiltrated macrophages expressing VEGF was also significantly smaller in LOX-1 KO mice than in WT mice. Western blot analysis and ROS production assay revealed that LOX- KO mice show significant decrease in Nox2 expression, ROS production and HIF-1α expression, the phosphorylation of p38 MAPK and NF-κB p65 subunit as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and LOX-1 itself in ischemic muscles, which is supposed to be required for macrophage infiltration expressing angiogenic factor VEGF. Reduction of VEGF expression successively suppressed the phosphorylation of Akt and eNOS, which accelerated angiogenesis, in the ischemic leg of LOX-1 KO mice. Our findings indicate that LOX-1 plays an important role in ischemia-induced angiogenesis by 1) Nox2-ROS-NF-κB activation, 2) upregulated expression of adhesion molecules: VCAM-1 and LOX-1 and 3) promoting macrophage infiltration, which expresses angiogenic factor VEGF.     

Relative contributions of NOS isoforms during experimental colitis: endothelial-derived NOS maintains mucosal integrity

The role of nitric oxide (NO) in inflammatory bowel diseases has traditionally focused on the inducible form of NO synthase (iNOS). However, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms may also impact on colitis, either by contributing to the inflammation or by regulating mucosal integrity in response to noxious stimuli. To date, studies examining the roles of the NOS isoforms in experimental colitis have been conflicting, and the mechanisms by which these enzymes exert their effects remain unclear. To investigate and clarify the roles of the NOS isoforms in gut inflammation, we induced trinitrobenzenesulfonic acid colitis in eNOS, nNOS, and iNOS knockout (KO) mice, assessing the course of colitis at early and late times. Both eNOS and iNOS KO mice developed a more severe colitis compared with wild-type mice. During colitis, iNOS expression dramatically increased on epithelial and lamina propria mononuclear cells, whereas eNOS expression remained localized to endothelial cells. Electron and fluorescence microscopy identified bacteria in the ulcerated colonic mucosa of eNOS KO mice, but not in wild-type, iNOS, or nNOS KO mice. Furthermore, eNOS KO mice had fewer colonic goblet cells, impaired mucin production, and exhibited increased susceptibility to an inflammatory stimulus that was subthreshold to other mice. This susceptibility was reversible, because the NO donor isosorbide dinitrate normalized goblet cell numbers and ameliorated subsequent colitis in eNOS KO mice. These results identify a protective role for both iNOS and eNOS during colitis, with eNOS deficiency resulting in impaired intestinal defense against lumenal bacteria and increased susceptibility to colitis.     

Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia

Blood flow restoration to ischemic tissue is affected by various risk factors. The aim of this study was to examine gender effects on arteriogenesis and angiogenesis in a mouse ischemic hindlimb model. C57BL/6J mice were subjected to unilateral hindlimb ischemia. Flow recovery was less and hindlimb use impairment was greater in females. No gender difference in vessel number was found at baseline, although 7 days postsurgery females had fewer α-smooth muscle actin-positive vessels in the midpoint of the adductor region. Females had higher hindlimb vascular resistance, were less responsive to vasodilators, and were more sensitive to vasoconstrictors postligation. Western blotting showed that females had higher baseline levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the calf, while 7 days postligation males had higher levels of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein. Females had less angiogenesis in a Matrigel plug assay and less endothelial cell proliferation in vitro. Females have impaired recovery of flow, a finding presumably caused by multiple factors including decreased collateral remodeling, less angiogenesis, impaired vasodilator response, and increased vasoconstrictor activity; our results also suggest the possibility that new collateral formation, from capillaries, is impaired in females.     

The role of nitric oxide on rosuvastatin-mediated S-nitrosylation and translational proteomes in human umbilical vein endothelial cells

ABSTRACT: BACKGROUND: The pleiotropic effects of 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), which are independent from their cholesterol-lowering action, have been widely recognized in various biological systems. Statins can affect endothelial homeostasis, which is partly modulated by the production of nitric oxide (NO). However, it is unclear how statin/NO-mediated posttranslational S-nitrosylation of endothelial proteins and changes in translational profiles may benefit endothelial integrity. Therefore, it is important to understand the statin/NO-mediated S-nitrosylation in endothelial cells. RESULTS: Rosuvastatin treatment of human umbilical vein endothelial cells (ECs) enhanced the enzymatic activity of endothelial nitric oxide synthase (eNOS) and the expression of 78 S-nitrosoproteins. Among these S-nitrosoproteins, we identified 17 proteins, including protein disulfide bond isomerase, phospholipase C, transaldolase and heat shock proteins. Furthermore, a hydrophobic Cys66 was determined as the S-nitrosylation site of the mitochondrial HSP70. In addition to the statin-modulated posttranslational S-nitrosylation, changes in the NO-mediated translational proteome were also observed. Seventeen major proteins were significantly upregulated after rosuvastatin treatment. However, 12 of these proteins were downregulated after pretreating ECs with an eNOS inhibitor (L-NAME), which indicated that their expression was modulated by NO. CONCLUSIONS: ECs treated with rosuvastatin increase eNOS activation. The increased NO production is involved in modulating S-nitrosylation and translation of proteins. We provide further evidence of the pleiotropic effect of rosuvastatin on endothelial physiology.     

Caloric restriction stimulates revascularization in response to ischemia via adiponectin-mediated activation of endothelial nitric-oxide synthase

Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, the functional roles of CR in regulation of revascularization in response to ischemia have not been examined. Here we investigated whether CR modulates vascular response by employing a murine hindlimb ischemia model. Wild-type (WT) mice were randomly divided into two groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb ischemic surgery. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared with WT mice fed AL (AL-WT). Revascularization of ischemic hindlimb relative to the contralateral limb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow and capillary density analyses. CR-WT mice had significantly higher plasma levels of the fat-derived hormone adiponectin compared with AL-WT mice. In contrast to WT mice, CR did not affect the revascularization of ischemic limbs of adiponectin-deficient (APN-KO) mice. CR stimulated the phosphorylation of endothelial nitric-oxide synthase (eNOS) in the ischemic limbs of WT mice. CR increased plasma adiponectin levels in eNOS-KO mice but did not stimulate limb perfusion in this strain. CR-WT mice showed enhanced phosphorylation of AMP-activated protein kinase (AMPK) in ischemic muscle, and administration of AMPK inhibitor compound C abolished CR-induced increase in limb perfusion and eNOS phosphorylation in WT mice. Our observations indicate that CR can promote revascularization in response to tissue ischemia via an AMPK-eNOS-dependent mechanism that is mediated by adiponectin.     

Cardiac malformations are associated with altered expression of vascular endothelial growth factor and endothelial nitric oxide synthase genes in embryos of diabetic mice

The aim of this study was to investigate the role of nitric oxide (NO), and the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) genes in developing hearts at embryonic day 13.5 of embryos from diabetic mice. The protein and mRNA expression levels of eNOS and VEGF were significantly altered in the developing hearts of embryos from diabetic mice. The NO level was significantly decreased, whereas the VEGF concentration was significantly increased in the developing hearts of the embryos from diabetic mice. In vitro study showed a significant reduction in eNOS expression and cell proliferation in cardiac myoblast cells exposed to high glucose concentrations. Further, high glucose induced apoptosis in myoblast cells. Ultrastructural changes characteristics of apoptosis, including cell blebbing, aggregation of ribosomes and vacuoles in the cytoplasm were also evident in myoblast cells exposed to high glucose. It is suggested that hyperglycemia alters the expression of eNOS and VEGF genes that are involved in the regulation of cell growth and vasculogenesis, thereby contributing to the cardiac malformations seen in embryos from diabetic mice.     

IGF-I alleviates diabetes-induced RhoA activation, eNOS uncoupling, and myocardial dysfunction

IGF-I rescues diabetic heart defects and oxidative stress, although the underlying mechanism of action remains poorly understood. This study was designed to delineate the beneficial effects of IGF-I with a focus on RhoA, Akt, and eNOS coupling. Echocardiography was performed in normal or diabetic Friend Virus-B type (FVB) and IGF-I transgenic mice. Cardiomyocyte contractile properties were evaluated using peak shortening (PS), time-to-90% relengthening (TR90), and intracellular Ca2+ rise and decay. Diabetes reduced fraction shortening, PS, and intracellular Ca2+; it increased chamber size, prolonged TR90, and intracellular Ca2+ decay. Levels of RhoA mRNA, active RhoA, and O2(-) were elevated, whereas nitric oxide (NO) levels were reduced in diabetes. Diabetes-induced O2(-) accumulation was ablated by the NO synthase (NOS) inhibitor nitro-L-arginine methyl ester (L-NAME), indicating endothelial NOS (eNOS) uncoupling, all of which except heart size were negated by IGF-I. The IGF-I-elicited beneficial effects were mimicked by the Rho kinase inhibitor Y27632 and BH4. Diabetes depressed expression of Kv1.2 and dihydrofolate reductase (DHFR), increased beta-myosin heavy-chain expression, stimulated p38 MAPK, and reduced levels of total Akt and phosphorylated Akt/eNOS, all of which with the exception of myosin heavy chain were attenuated by IGF-I. In addition, Y27632 and the eNOS coupler folate abrogated glucose toxicity-induced PS decline, TR90 prolongation, while it increased O2(-) and decreased NO and Kv1.2 levels. The DHFR inhibitor methotrexate impaired myocyte function, NO/O2(-) balance, and rescued Y27632-induced cardiac protection. These results revealed that IGF-I benefits diabetic hearts via Rho inhibition and antagonism of diabetes-induced decrease in pAkt, eNOS uncoupling, and K+ channel expression.     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

ETV-2 activated proliferation of endothelial cells and attenuated acute hindlimb ischemia in mice

Ischemia is the reduction of blood flow to tissues by injury of blood vessels. Depending on the sites of tissues and grade of ischemia, ischemia can cause many serious complications. This study aimed to evaluate the effects of the E-twenty six (ETS) factor Ets variant 2 (ETV2) gene expression in angiogenesis and the effect of ETV2 gene therapy in a mouse model of hindlimb ischemia. The role of ETV2 on endothelial cell proliferation was evaluated in vitro. Knockdown of ETV2 expression was done using short hairpin RNA (shRNA) lentiviral viral particles. The ETV2 viral vector was injected into the skeletal muscles at the ligated and burned sites of the hindlimb and evaluated for its efficacy as a gene therapy modality for ischemia. Vascular regeneration in mice was indirectly evaluated by changes in mouse survival, necrotic grades of the leg, normal blood oxygen saturation level (SpO₂), and blood flow by trypan blue injection assay. Preliminary data showed that ETV2 expression played a role in angiogenesis of endothelial cells. ETV2 overexpression could trigger and stimulate proliferation of skeletal endothelial cells. In vivo knockdown of ETV2 expression inhibited the auto-recovery of ischemic hindlimb, while overexpression of ETV2 helped to rescue leg loss and reduce necrosis, significantly improving angiogenesis in hindlimb ischemia. Our findings demonstrate that ETV2 gene therapy is a potentially effective modality for vascular regeneration.