Enhanced eNOS Activity Research Articles

Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice

Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2Akita+/−), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2Akita+/−) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2Akita+/− and TgeNOS-Ins2Akita+/− were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2Akita+/− mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2Akita+/− was significantly higher compared with the Ins2Akita+/− and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target.

Enhancing eNOS activity with simultaneous inhibition of IKKβ restores vascular function in Ins2Akita+/− type-1 diabetic mice

The balance of nitric oxide (NO) versus superoxide generation has a major role in the initiation and progression of endothelial dysfunction. Under conditions of high glucose, endothelial nitric oxide synthase (eNOS) functions as a chief source of superoxide rather than NO. In order to improve NO bioavailability within the vessel wall in type-1 diabetes, we investigated treatment strategies that improve eNOS phosphorylation and NO-dependent vasorelaxation. We evaluated methods to increase the eNOS activity by (1) feeding Ins2Akita spontaneously diabetic (type-1) mice with l-arginine in the presence of sepiapterin, a precursor of tetrahydrobiopterin; (2) preventing eNOS/NO deregulation by the inclusion of inhibitor kappa B kinase beta (IKKβ) inhibitor, salsalate, in the diet regimen in combination with l-arginine and sepiapterin; and (3) independently increasing eNOS expression to improve eNOS activity and associated NO production through generating Ins2Akita diabetic mice that overexpress human eNOS predominantly in vascular endothelial cells. Our results clearly demonstrated that diet supplementation with l-arginine, sepiapterin along with salsalate improved phosphorylation of eNOS and enhanced vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice. More interestingly, despite the overexpression of eNOS, the in-house generated transgenic eNOS-GFP (TgeNOS-GFP)-Ins2Akita cross mice showed an unanticipated effect of reduced eNOS phosphorylation and enhanced superoxide production. Our results demonstrate that enhancement of endogenous eNOS activity by nutritional modulation is more beneficial than increasing the endogenous expression of eNOS by gene therapy modalities.

Polydatin Restores Endothelium-Dependent Relaxation in Rat Aorta Rings Impaired by High Glucose: A Novel Insight into the PPARβ-NO Signaling Pathway.

Polydatin, a natural component from Polygonum Cuspidatum, has important therapeutic effects on metabolic syndrome. A novel therapeutic strategy using polydatin to improve vascular function has recently been proposed to treat diabetes-related cardiovascular complications. However, the biological role and molecular basis of polydatin’s action on vascular endothelial cells (VECs)-mediated vasodilatation under diabetes-related hyperglycemia condition remain elusive. The present study aimed to assess the contribution of polydatin in restoring endothelium-dependent relaxation and to determine the details of its underlying mechanism. By measuring endothelium-dependent relaxation, we found that acetylcholine-induced vasodilation was impaired by elevated glucose (55 mmol/L); however, polydatin (1, 3, 10 μmol/L) could restore the relaxation in a dose-dependent manner. Polydatin could also improve the histological damage to endothelial cells in the thoracic aorta. Polydatin’s effects were mediated via promoting the expression of endothelial NO synthase (eNOS), enhancing eNOS activity and decreasing the inducible NOS (iNOS) level, finally resulting in a beneficial increase in NO release, which probably, at least in part, through activation of the PPARβ signaling pathway. The results provided a novel insight into polydatin action, via PPARβ-NO signaling pathways, in restoring endothelial function in high glucose conditions. The results also indicated the potential utility of polydatin to treat diabetes related cardiovascular diseases.

Acute atorvastatin is hepatoprotective against ischaemia-reperfusion injury in mice by modulating eNOS and microparticle formation.

Steatosis accentuates the severity of hepatic ischaemia-reperfusion injury (IRI); 'statins' (HMG-CoA reductase inhibitors) protect the heart and brain against post-ischaemic injury. We tested whether short-term administration of atorvastatin protects fatty livers in obese mice against IRI. Mice with dietary or genetic simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) were subjected to 60 min partial hepatic ischaemia/24 h reperfusion. Atorvastatin was injected intravenously (5 mg/kg) 1 h before IRI. Liver injury, Toll-like receptor-4 (TLR4), cytokines/chemokines, iNOS/eNOS expression, eNOS activity and thromboxane B2 (TXB2) production were determined. Ischaemia-reperfusion injury was exaggerated by two- to five-fold in SS and NASH compared with lean liver. Atorvastatin pretreatment conferred 70-90% hepatic protection in all animals. Atorvastatin increased post-ischaemic eNOS mRNA/protein and strikingly enhanced eNOS activity (by phospho-eNOS). It also attenuated microparticle (MP) production, NF-κB activation, significantly dampened post-ischaemic thromboxane B2 production, induction of TNF-α, IL-6, MIP-1a, MCP-1, GM-CSF and vascular cell adhesion molecule-1 (VCAM), with a resultant reduction on macrophage and polymorphonuclear neutrophil recruitment. Up-regulation of HMGB1 and TLR4 after IRI was marked in fatty livers; 1 h pretreatment with atorvastatin reduced HMGB1 and TLR4 expression in all livers. Acute (1 h) atorvastatin administration is highly hepatoprotective against IRI in NASH, fatty and lean livers. Key mechanisms include suppression of inflammation by prevention of NF-κB activation, microvascular protection via eNOS activation and suppression of TXB2 and MP release. Short-term intravenous statin treatment is a readily available and effective preventive agent against hepatic IRI, irrespective of obesity and fatty liver disease, and merits clinical trials in at-risk patients.

MP8-14 THE BENEFICIAL EFFECT OF RESVERATROL ON BLADDER FUNCTION IN A RAT MODEL OF CHRONIC BLADDER ISCHEMIA

INTRODUCTION AND OBJECTIVES: Resveratrol(3,5,40-trihydroxy-trans-stilbene), a natural phytoalexin present in grapes, peanuts, mulberries and red wine, is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity and by reduction of reacitive oxygen species (ROS) levels. Its effects on the function of the chronic ischemic bladder are not known. The objective of this study is to investigate the possible beneficial activities of resveratrol in a rat model of chronic ischemiarelated bladder dysfunction. METHODS: Male Sprague-Dawley rats were divided into control (n1⁄410), arterial endothelial injury (AI; n1⁄410), and AI with resveratrol treatment (AI-res; n1⁄410). AI and AI-res groups underwent endothelial injury of the iliac arteries and received a 2% cholesterol diet following AI. AI-res rats received resveratrol (1mg/kg/day) orally for 12 weeks. The control group received a regular diet. After 12 weeks, urodynamic investigation was performed in awake animals. RESULTS: Iliac arteries from both AI and AI-resveratrol rats showed neo-intimal formation and luminal occlusion. The body weight of the three groups had no difference. Micturition interval (MI), bladder capacity (Bcap) and voided volume (VV) in theAI groupwere significantly less than those in the control group (p<0.05). In the AI-res group MI, Bcap and VV were significantly larger than in the AI group (p<0.05), however, significantly less than in the control group (p<0.05)). (Fig.1) Contractile responses of bladder strips to electrical field stimulation (EFS) and carbachol (CCH)were lower inAI than incontrols; responses inpreparations from resveratrol-treated animals were similar to those of controls. (Fig.2) CONCLUSIONS: In the chronically ischemic rat bladder, treatment with resveratrol seems to have beneficial effect on bladder function, resulting in reduced bladder hyperactivity and reversed the low contractile responses to CCH and EFS. Source of Funding: none

The role of arginase and rho kinase in cardioprotection from remote ischemic perconditioning in non-diabetic and diabetic rat in vivo.

BackgroundPharmacological inhibition of arginase and remote ischemic perconditioning (RIPerc) are known to protect the heart against ischemia/reperfusion (IR) injury.PurposeThe objective of this study was to investigate whether (1) peroxynitrite-mediated RhoA/Rho associated kinase (ROCK) signaling pathway contributes to arginase upregulation following myocardial IR; (2) the inhibition of this pathway is involved as a cardioprotective mechanism of remote ischemic perconditioning and (3) the influence of diabetes on these mechanisms.MethodsAnesthetized rats were subjected to 30 min left coronary artery ligation followed by 2 h reperfusion and included in two protocols. In protocol 1 rats were randomized to 1) control IR, 2) RIPerc induced by bilateral femoral artery occlusion for 15 min during myocardial ischemia, 3) RIPerc and administration of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), 4) administration of the ROCK inhibitor hydroxyfasudil or 5) the peroxynitrite decomposition catalyst FeTPPS. In protocol 2 non-diabetic and type 1 diabetic rats were randomosed to IR or RIPerc as described above.ResultsInfarct size was significantly reduced in rats treated with FeTPPS, hydroxyfasudil and RIPerc compared to controls (P<0.001). FeTPPS attenuated both ROCK and arginase activity (P<0.001 vs. control). Similarly, RIPerc reduced arginase and ROCK activity, peroxynitrite formation and enhanced phospho-eNOS expression (P<0.05 vs. control). The cardioprotective effect of RIPerc was abolished by L-NMMA. The protective effect of RIPerc and its associated changes in arginase and ROCK activity were abolished in diabetes.ConclusionArginase is activated by peroxynitrite/ROCK signaling cascade in myocardial IR. RIPerc protects against IR injury via a mechanism involving inhibition of this pathway and enhanced eNOS activation. The beneficial effect and associated molecular changes of RIPerc is abolished in type 1 diabetes.

Abstract 208: Improved Vasodilatory Function in Ins2Akita Type 1 Diabetic Mice with Daily Intake of Oral L-Arginine in Combination with Salsalate

Endothelial dysfunction due to deficient nitric oxide (NO) generation cause progressive vascular complications in diabetes. Our earlier findings revealed that Inhibitor kappa B kinase beta (IKK-beta) -an upstream regulator of an inflammatory mediator, nuclear factor kappa-B (NF-kB), competitively inhibits endothelial nitric oxide synthase (eNOS), the chief source of NO in blood vessels. Therefore, we investigated the influence of an inhibitor of IKK beta, salsalate in augmenting eNOS activity. The co-operative effect of L-arginine supplementation (substrate of eNOS) with salsalate intake in enhancing eNOS activity has been evaluated. Both in vitro cell cultures and diabetic/transgenic animal models were used. Human aortic endothelial cells incubated with 25 mM glucose with or without L-arginine (2mM), the cofactor sepiapterin (100uM) and salsalate(50uM) were examined for NO production. Combined L-arginine and salsalate treatment enhanced NO production, phospho eNOS expression and eNOS-dimer vs monomer ratio. For in vivo assays, diabetic Ins2Akita mice were fed with L-arginine and salsalate for 15 weeks. Aortae of these mice exhibited increased phospho eNOS expression and increased eNOS dimer/monomer ratio. Arterial ring assays showed improved acetylcholine mediated vasodialation. Transgenic-Akita mice that overexpress human eNOS in endothelial cells (Tie-2eNOS-Ins2 Akita) generated in-house showed improved vasodialation. In conclusion, enhancing eNOS activity in diabetic mice either by genetic modification as seen in Tie-2 eNOS-Akita mice or by nutritional approach (supplementation of L-arginine with simultaneous inhibition of IKK beta) restores normal vasodialatory function.

Abstract 608: The Mechanisms of Preservation of Vasorelaxation Induced by Protease-Activated Receptor-2 Activation in the Aorta of Metabolic Syndrome Model Rats

Protease-activated receptor-2 (PAR2) is activated by proteases, such as trypsin, human mast cell beta-tryptase, human kallikrein isoforms, coagulation factors VIIa and Xa, etc. PAR2 is believed to have an important role under inflammatory pathological conditions, but its role in metabolic syndrome remains uncertain. We have reported that PAR2-activating peptide, 2-furoyl-LIGRLO-amide (2fly) causes NO-mediated vasorelaxation in rat aortas similar to that observed with acetylcholine (ACh), and that 2fly-induced vasorelaxation is maintained in aortas from SHRSP.Z- Lepr fa /IzmDmcr (SHRSP.ZF) rats with metabolic syndrome, even though ACh- and sodium nitroprusside (SNP)-induced vasodilation is lower than that in control (Wistar-Kyoto rats, WKY). In this study, we assessed the mechanisms involved in the preservation of PAR2-mediated vasorelaxation in metabolic syndrome. Aortas were isolated from male WKY and SHRSP.ZF rats between 18-20 weeks of age. ACh- and 2fly-induced vasorelaxations were measured in the presence or absence of inhibitors (i.e., COX inhibitors, a TXA 2 synthase inhibitor, and a TXA 2 /PGH 2 receptor antagonist) using organ bath methods. Expression of endothelium NO synthase (eNOS) in aortas was measured by western blot analysis. Indomethacin (a non-selective COX inhibitor), SC560 (a selective COX-1 inhibitor), NS398 (a selective COX-2 inhibitor), OKY-046 (a TXA 2 synthase inhibitor), and ONO-3708 (a TXA 2 /PGH 2 receptor antagonist) did not affect both ACh- and 2fly-induced vasorelaxation in aortas of SHRSP.ZF rats as well as WKY. Expression of phosphorylated eNOS at Ser 1177 in 2fly-stimulated aortas was higher than that in ACh-stimulated ones in SHRSP.ZF rats, but there was no significant difference between these levels in WKY. Our results show that the preservation of 2fly-induced vasorelaxation observed in SHRSP.ZF rats with metabolic syndrome may be because of enhanced eNOS activation via PAR2, but not coupled to the COX pathway. Thus, preservation of PAR2-mediated vasorelaxation may be one of the key responses to maintain blood circulation under inflammatory conditions, such as metabolic syndrome.

Comparing the effects of atorvastatin and vitamin E on endothelial function of sildenafil non‐responders (851.8)

ABSTRACT Endothelial dysfunction causes the failure of sildenafil response due to reduced nitric oxide (NO) . This study aimed to compare the biochemical and clinical effects of atorvastatin and vitamin E on endothelial function of sildenafil non‐responders with investigation to the underlying mechanisms.Methods: Sixty participants were randomly divided into three groups receiving atorvastatin 80 mg daily, or vitamin E 400 IU daily, or placebo capsules. Participants were examined both before and after six weeks for these biochemical tests; NO, glutathione peroxidase (GPO), interlukin 6 (IL‐6), testosterone, and endothelial nitric oxide synthase (eNOS) and for erectile function tests; international index of erectile function (IIEF ‐5) scores, rigiscan, and duplex l Results: in comparison to control group, the treated groups showed a significant increase in GPO, and a significant decrease in IL‐6. Atorvastatin only showed a significant increase in NO, eNOS, IIEF‐5 scores and rigiscan rigidity parameters. Other rigiscan parameters, testosterone and duplex ultrasound showed no difference in the three groups Conclusions: Atorvastatin improves endothelial function of sildenafil non‐responders by increasing NO level attributed to enhancing eNOS activity (main mechanism) added to antioxidant and anti‐inflammatory activities. Atorvastatin is a real promising drug for sildenafil non‐responders.

PKA and Epac activation mediates cAMP-induced vasorelaxation by increasing endothelial NO production

Vascular relaxation induced by 3′,5′-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction–relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2′–O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2′–O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.

Alpha-linolenic acid intake prevents endothelial dysfunction in high-fat diet-fed streptozotocin rats and underlying mechanisms

Endothelial dysfunction is an important factor in the pathogenesis of diabetes related vascular complications, and acute alpha-linolenic acid (ALA) intake can increase flow-mediated dilation of the diabetic artery at 4 h postprandially. However, whether chronic ALA supplementation may prevent endothelial dysfunction in the process of diabetes and underlying mechanisms remains largely unknown. The high-fat diet-fed streptozotocin (HFD-STZ) rats provided an animal model for T2DM. Age-matched normal and HFD-STZ rats randomly received normal diet or ALA (500 mg/kg per day). After 5 weeks of feeding, endothelial function was determined. Diabetes caused significant endothelial dysfunction (maximal vasorelaxation responses to ACh) in aortic segments, and ALA intake alleviated endothelial dysfunction. Superoxide production and peroxynitrite (ONOO-) formation were reduced with ALA supplement in diabetic vascular segments. Interestingly, ALA intake enhanced eNOS but inhibited iNOS activity in diabetic vessels. Moreover, ALA intake significantly increased eNOS phosphorylation. On the other hand, gp91phox and iNOS overexpression were reduced moderately with ALA intake in diabetic vessels. We concluded that ALA prevents diabetes-induced endothelial dysfunction by enhancing eNOS activity and attenuates oxidative/nitrative stress by inhibiting iNOS and NADPH oxidase expression and ONOO- production.

The Effects of Amlodipine and S(-)-Amlodipine on Vascular Endothelial Function in Patients With Hypertension

Amlodipine has been shown to improve vascular endothelial function in hypertensive patients, but whether S(-)-amlodipine has a similar effect remains controversial. This study compared the effects of amlodipine and S(-)-amlodipine on vascular endothelial function in hypertensive patients and investigated relevant mechanisms of action in cell culture. Twenty-four patients with essential hypertension received amlodipine and S(-)-amlodipine for 6 weeks in a randomized, crossover study. Associated flow-mediated dilation (FMD), nitric oxide (NO), and endothelial nitric oxide synthase (eNOS) levels were determined. NO levels were measured after exposure of human umbilical vein endothelial cells (HUVECs) to amlodipine, S(-)-amlodipine, the eNOS inhibitor N w-nitro-L-arginine (L-NA), and the Protein Kinase C (PKC) inhibitor Ro 31-8220. Phosphorylation levels of Ser(1177) and Thr(495) in eNOS were determined after exposure to amlodipine, S(-)-amlodipine, and Ro 31-8220. FMD, NO, and eNOS levels significantly improved after treatment with amlodipine and S(-)-amlodipine. The levels were all higher with amlodipine, although the between-treatment difference was not statistically significant. Amlodipine and S(-)-amlodipine significantly increased NO levels in cultured HUVECs, but increases in NO levels were more marked with amlodipine. Western blot assay showed that both amlodipine and Ro31-8220 induced Ser(1177) phosphorylation and weakened Thr(495) phosphorylation in eNOS. S(-)-amlodipine had no similar effects. Amlodipine, but not S(-)-amlodipine, decreased the PKC phosphorylation in a time-dependent manner. Amlodipine and S(-)-amlodipine can both improve endothelial function in hypertensive patients. Amlodipine has greater potential for vascular endothelial protection than S(-)-amlodipine. It affects eNOS phosphorylation at Ser(1177) and Thr(495) by the PKC pathway, further enhancing eNOS activation.

Uncoupling of endothelial NO synthase in atherosclerosis and vascular disease

Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is an antihypertensive, antithrombotic and anti-atherosclerotic molecule. Hypercholesterolemia leads to a reduction in vascular NO bioavailability. This is attributed to a dysfunction of the eNOS enzyme and a reduced eNOS activity. NADPH oxidase-mediated oxidative stress leads to oxidation of tetrahydrobiopterin (BH4), the essential cofactor of eNOS. In BH4 deficiency, oxygen reduction uncouples from NO synthesis, thereby converting eNOS to a superoxide-producing enzyme. As a consequence of eNOS uncoupling, NO production is reduced and the pre-existing oxidative stress is enhanced, which contribute significantly to atherogenesis. Therefore, pharmacological approaches that prevent eNOS uncoupling and enhance eNOS activity are of therapeutic interest. Angiotensin-converting enzyme inhibitors, AT1 receptor blockers, statins, nebivolol and resveratrol have been shown to reverse eNOS uncoupling and to stimulate eNOS activity concurrently. Molecular mechanisms of the aforementioned drugs/compounds on eNOS functionality is summarized and discussed in this review.

Reverse myocardial effects of intermedin in pressure-overloaded hearts: role of endothelial nitric oxide synthase activity.

Intermedin (IMD) is a cardiac peptide synthesized in a prepro form, which undergoes a series of proteolytic cleavages and amidations to yield the active forms of 47 (IMD(1-47)) and 40 amino acids (IMD(8-47)). There are several lines of evidence of increased IMD expression in rat models of cardiac pathologies, including congestive heart failure and ischaemia; however, its myocardial effects upon cardiac disease remain unexplored. With this in mind, we investigated the direct effects of increasing concentrations of IMD(1-47) (10(-10) to10(-6) m) on contraction and relaxation of left ventricular (LV) papillary muscles from two rat models of chronic pressure overload, one induced by transverse aortic constriction (TAC), the other by nitric oxide (NO) deficiency due to chronic NO synthase inhibition (NG-nitro-l-arginine, l-NAME), and respective controls (Sham and Ctrl). In TAC and l-NAME rats, exogenous administration of IMD(1-47) elicited concentration-dependent positive inotropic and lusitropic effects. By contrast, in Sham and Ctrl rats, IMD(1-47) induced a negative inotropic response without a significant effect on relaxation. Both TAC and l-NAME rats presented LV hypertrophy, elevated LV systolic pressures, preserved systolic function and elevated peroxynitrite levels. In the normal myocardium (Ctrl and Sham), IMD(1-47) induced a 3-fold increase of endothelial nitric oxide synthase (eNOS) phosphorylation at Ser(1177), indicating enhanced eNOS activity. In TAC and l-NAME rats, eNOS phosphorylation was increased at baseline, and its response to IMD(1-47) was blunted. In addition, the distinct myocardial response to IMD(1-47) was accompanied by distinct subcellular mechanisms. While in Sham rats the addition of IMD(1-47) induced the phosphorylation of cardiac troponin I due to NO/cGMP activation, in TAC rats IMD(1-47) induced phospholamban phosphorylation possibly associated with cAMP/protein kinase A activation. Therefore, we demonstrated for the first time a reversed myocardial response to IMD(1-47) neurohumoral stimulation due to impairment of eNOS activation in TAC and l-NAME rats. These results not only reveal the distinct myocardial effects and subcellular mechanisms for IMD(1-47) in normal and hypertrophic hearts, but also highlight the potential pathophysiological relevance of cardiac endothelial dysfunction in neurohumoral myocardial action.

Combined Treatment of Hydroxytyrosol with Carbon Monoxide-Releasing Molecule-2 Prevents TNFα-Induced Vascular Endothelial Cell Dysfunction through NO Production with Subsequent NFκB Inactivation

This study investigated the atheroprotective properties of olive oil polyphenol, hydroxytyrosol (HT), in combination with carbon monoxide-releasing molecule-2 (CORM-2) that acts as a carbon monoxide donor using vascular endothelial cells (VECs). Our results showed that CORM-2 could strengthen the cytoprotective and anti-apoptotic effects of HT against TNFα-induced cellular damage by enhancing cell survival and the suppression of caspase-3 activation. While HT alone attenuated NFκBp65 phosphorylation and IκBα degradation triggered by TNFα in a dose-dependent manner, combined treatment of HT with CORM-2 but not iCORM-2 nearly completely blocked these TNFα effects. Furthermore, combined action of both compounds results in the inhibition of NFκB nuclear translocation. Results also indicate that both compounds time-dependently increased eNOS phosphorylation levels and the combination of HT with CORM-2 was more effective in enhancing eNOS activation and NO production in VECs. The NOS inhibitor, L-NMMA, significantly suppressed the combined effects of HT and CORM-2 on TNFα-triggered NFκBp65 and IκBα phosphorylation as well as decreased cell viability. Together, these data suggest that carbon monoxide-dependent regulation of NO production by the combination of HT with CORM-2 may provide a therapeutic benefit in the treatment of endothelial dysfunction and atherosclerosis.

Analysing calcium dependent and independent regulation of eNOS in endothelium triggered by extracellular signalling events

The vascular endothelium, the intima of blood vessels, coordinately interacts with several biochemical factors expressing endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO), a potent endogenous vasodilator. The present study investigated the regulation of eNOS by multiple molecular signal transduction pathways, namely vascular endothelial growth factor (VEGF-A) and shear stress which are implicated in the process of angiogenesis and vascular remodelling respectively. In response to signal transduction upstream by VEGF-A and shear stress, different signalling pathways mediated by kinases and intracellular calcium potentiates eNOS activation leading to nitric oxide release. Our study revealed a distinct pattern of eNOS activation driven by VEGF-A and shear stress, maintaining the signalling specificity of the respective pathways. A transient response to eNOS activation was observed under VEGF-A and shear stress stimulus when mediated by calcium dependent cascades, whereas a sustained response was produced by calcium independent vascular signalling kinases. Furthermore, we found that the basal arterial shear stress enhanced eNOS activity when stimulated synergistically even at low VEGF-A levels which might be utilized to facilitate specific endothelial cell functions. Moreover, our study revealed that the presence of PI3K imparted transient behaviour to PLCγ1 supporting the hypothesis that regression and formation of tube structures are mediated by PLCγ1 and PI3K respectively in endothelial cells. This fact is corroborated by the absence of transient behaviour when PI3K is inhibited. We therefore obtained subtle insights into the control mechanism governing the role of specific signalling proteins which are obligate for the regulation of endothelial cell function and the consequent modulation of the nitric oxide release pattern.

AVE3085 Protects Coronary Endothelium from the Impairment of Asymmetric Dimethylarginine by Activation and Recoupling of eNOS

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of eNOS and it is recognized as a risk factor for endothelial dysfunction in cardiovascular diseases. We investigated the effect of AVE3085, a newly developed transcription enhancer of eNOS, on ADMA-induced endothelial dysfunction in coronary arteries with underlying mechanisms explored. Porcine coronary small arteries (diameter 600-800 μm) were studied in a myograph for endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to sodium nitroprusside. Protein expressions of eNOS and phosphorylated-eNOS (p-eNOS(Ser1177) and p-eNOS(Thr495)), and nitrotyrosine formation were determined by Western blot. NO release was directly measured with a NO microsensor. Productions of O(2) (.-) and peroxynitrite (ONOO(-)) were determined by lucigenin- and luminol- enhanced chemiluminescence respectively. Exposure to ADMA significantly decreased the bradykinin-induced vasorelaxation and reduced the protein expression of p-eNOS(Ser1177) whereas increased the expression of p-eNOS(Thr495) and nitrotyrosine. Pre-incubation with AVE3085 restored the bradykinin-induced relaxation, reversed the decrease of p-eNOS(Ser1177), and lowered the level of p-eNOS(Thr495) and nitrotyrosine. NO release in response to bradykinin was significantly reduced by ADMA and such reduction was restored by AVE3085. AVE3085 also prevented the elevation of O (2) (.-) and ONOO(-) levels in coronary arteries exposed to ADMA. AVE3085 prevents ADMA-induced endothelial dysfunction in coronary arteries. The protective effect of AVE3085 may be attributed to increased NO production resulting from enhanced eNOS activation, and decreased oxidative stress that involves inhibition of O (2) (.-) generation by eNOS recoupling. The present study suggested the therapeutic potential of AVE3085 in endothelial dysfunction in cardiovascular disorders.

Voltage-Dependent Anion Channel-2 Interaction with Nitric Oxide Synthase Enhances Pulmonary Artery Endothelial Cell Nitric Oxide Production

Increased pulmonary artery endothelial cell (PAEC) endothelium-dependent nitric oxide synthase (eNOS) activity mediates perinatal pulmonary vasodilation. Compromised eNOS activity is central to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Voltage-derived anion channel (VDAC)-1 was recently demonstrated to bind eNOS in the systemic circulation. We hypothesized that VDAC isoforms modulate eNOS activity in the pulmonary circulation, and that decreased VDAC expression contributes to PPHN. In PAECs derived from an ovine model of PPHN: (1) there is eNOS activity, but not expression; and (2) VDAC1 and -2 proteins are decreased. Immunocytochemistry, coimmunoprecipitation, and in situ proximity ligation assays in human PAECs (hPAECs) demonstrate binding between eNOS and both VDAC1 and -2, which increased upon stimulation with NO agonists. The ability of agonists to increase the eNOS/VDAC interaction was significantly blunted in hypertensive, compared with normotensive, ovine PAECs. Depletion of VDAC2, but not VDAC1, blocked the agonist-induced increase in eNOS activity in hPAECs. Overexpression of VDAC2 in hypertensive PAECs increased eNOS activity. Binding of VDAC2 enhances eNOS activity in the pulmonary circulation, and diminished VDAC2 constrains eNOS in PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension. We speculate that decreases in VDAC2 may contribute to the limited eNOS activity that characterizes pulmonary hypertension.

Effects of Simulated Microgravity on Human Umbilical Vein Endothelial Cell Angiogenesis and Role of the PI3K-Akt-eNOS Signal Pathway

Endothelial cells are very sensitive to microgravity and the morphological and functional changes in endothelial cells are believed to be at the basis of weightlessness-induced cardiovascular deconditioning. It has been shown that the proliferation, migration, and morphological differentiation of endothelial cells play critical roles in angiogenesis. However, the influence of microgravity on the ability of endothelial cells to foster angiogenesis remains to be explored in detail. In the present study, we used a clinostat to simulate microgravity, and we observed tube formation, migration, and expression of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC-C). Specific inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) were added to the culture medium and gravity-induced changes in the pathways that mediate angiogenesis were investigated. After 24 h of exposure to simulated microgravity, HUVEC-C tube formation and migration were significantly promoted.This was reversed by co-incubation with the specific inhibitor of N-nitro-L-arginine methyl ester hydrochloride (eNOS). Immunofluorescence assay, RT-PCR, and Western blot analysis demonstrated that eNOS expression in the HUVEC-C was significantly elevated after simulated microgravity exhibition. Ultrastructure observation via transmission electron microscope showed the number of caveolae organelles in the membrane of HUVEC-C to be significantly reduced. This was correlated with enhanced eNOS activity. Western blot analysis then showed that phosphorylation of eNOS and serine/threonine kinase (Akt) were both up-regulated after exposure to simulated microgravity. However, the specific inhibitor of PI3K not only significantly downregulated the expression of phosphorylated Akt, but also downregulated the phosphorylation of eNOS. This suggested that the PI3K-Akt signal pathway might participate in modulating the activity of eNOS. In conclusion, the present study indicates that 24 h of exposure to simulated microgravity promote angiogenesis among HUVEC-C and that this process is mediated through the PI3K-Akt-eNOS signal pathway.

2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran promotes endothelial nitric oxide synthase activity in human endothelial cells

Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide (NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity.DPPB (1–10μM) treatment for 24h induced a significant and dose-dependent increase in eNOS activity as determined by the [14C]l-arginine/[14C]l-citrulline conversion assay. Immunoblotting studies further revealed a time-dependent DPPB-induced increase in eNOS-Ser1177 and decrease in eNOS-Thr495 phosphorylation, as well as increased AMPK phosphorylation at Thr172, whereas Akt phosphorylation at Ser473 was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKβ by STO 609, as well as intracellular Ca2+ chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-Ser1177 and AMPK-Thr172 phosphorylation. Furthermore, we could show that DPPB increases intracellular Ca2+ concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS activity and endothelial NO release by raising intracellular Ca2+ levels and increases signaling through a CaMKKβ–AMPK dependent pathway.