Increased eNOS Expression Research Articles

Immediate Remote Ischemic Postconditioning Reduces Brain Nitrotyrosine Formation in a Piglet Asphyxia Model

Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention that could be administered as an alternative to cooling in cases of perinatal hypoxia-ischemia (HI). In the current study we hypothesized that RIPostC in the piglet model of birth asphyxia confers protection by reducing nitrosative stress and subsequent nitrotyrosine formation, as well as having an effect on glial immunoreactivity. Postnatal day 1 (P1) piglets underwent HI brain injury and were randomised to HI (control) or HI + RIPostC. Immunohistochemistry assessment 48 hours after HI revealed a significant decrease in brain nitrotyrosine deposits in the RIPostC-treated group (p = 0.02). This was accompanied by a significant increase in eNOS expression (p < 0.0001) and decrease in iNOS (p = 0.010), with no alteration in nNOS activity. Interestingly, RIPostC treatment was associated with a significant increase in GFAP (p = 0.002) and IBA1 (p = 0.006), markers of astroglial and microglial activity, respectively. The current study demonstrates a beneficial effect of RIPostC therapy in the preclinical piglet model of neonatal asphyxia, which appears to be mediated by modulation of nitrosative stress, despite glial activation.

Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

BackgroundTocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established.Aims1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells.MethodsHuman umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB.Resultsδ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations.Conclusionδ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a great potential for TCT isomers as anti-atherosclerotic agents.

Decreased reactive oxygen species production and NOX1, NOX2, NOX4 expressions contribute to hyporeactivity to phenylephrine in aortas of pregnant SHR.

We determined whether decreased reactive oxygen species (ROS) production in the aorta of pregnant spontaneously hypertensive rats (SHR) resulted in increased nitric oxide (NO) bioavailability and hyporeactivity to phenylephrine (PE). Systemic and aortic oxidative stress were measured in pregnant and non-pregnant Wistar rats and SHR. Furthermore, the hypotensive effects of apocynin (30 mg/kg) and Tempol (30 mg/kg) were analyzed. Intact aortic rings of pregnant and non-pregnant rats were stimulated with PE in the absence of or after incubation (30 min) with apocynin (100 μmol/L). The effect of apocynin on the concentrations of NO and ROS were measured in aortic endothelial cells (AEC) using DAF-2DA (10 mmol/L) and DHE (2.5 mmol/L), respectively. Western blotting was performed to analyze eNOS, NOX1, NOX2, NOX4 and SOD expression. ROS production was analyzed by the lucigenin chemiluminescence method. Aortic oxidative stress and ROS concentration in AEC were reduced in pregnant Wistar rats and SHR, when compared to non-pregnant rats. ROS production and NOX1, NOX2 and NOX4 expression in the aortas were decreased in pregnant SHR, but not in pregnant Wistar rats. Increased eNOS expression in aortas and NO concentration in AEC were observed in pregnant Wistar rats and SHR. Apocynin reduced PE-induced vasoconstriction in the aortas of non-pregnant Wistar rats and SHR, and pregnant Wistar rats, but not in the aortas of pregnant SHR. Taken together, these results suggest that ROS production was decreased in the aortas of pregnant SHR and could contribute to higher NO bioavailability and hyporeactivity to PE in the aortas of pregnant SHR.

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.

Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis.

IntroductionMetabolic syndrome causes insulin resistance and is associated with risk factor clustering, thereby increasing the risk of atherosclerosis. Recently, endothelial nitric oxide synthase deficient (eNOS-/-) mice have been reported to show metabolic disorders. Interestingly, eNOS has also been reported to be expressed in non-endothelial cells including adipocytes, but the functions of eNOS in adipocytes remain unclear.Methods and ResultsThe eNOS expression was induced with adipocyte differentiation and inhibition of eNOS/NO enhanced lipolysis in vitro and in vivo. Furthermore, the administration of a high fat diet (HFD) was able to induce non-alcoholic steatohepatitis (NASH) in eNOS-/- mice but not in wild type mice. A PPARγ antagonist increased eNOS expression in adipocytes and suppressed HFD-induced fatty liver changes.ConclusionseNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders.

T3 and T4 decrease ROS levels and increase endothelial nitric oxide synthase expression in the myocardium of infarcted rats.

Myocardial infarction leads to a reduction in nitric oxide (NO) bioavailability and an increase in reactive oxygen species (ROS) levels. This scenario has been shown to be detrimental to the heart. Recent studies have shown that thyroid hormone (TH) administration presents positive effects after ischaemic injury. Based on this, the aim of this study was to evaluate the effect of TH on NO bioavailability as well as on endothelial nitric oxide synthase (eNOS) expression after myocardial infarction. Male Wistar rats were divided into three groups: Sham-operated (SHAM), infarcted (AMI) and infarcted + TH (AMIT). During 26 days, the AMIT group received T3 and T4 (2 and 8 µg/100 g/day, respectively) by gavage, while SHAM and AMI rats received saline. After this, the rats underwent echocardiographic analysis were sacrificed, and the left ventricle was collected for biochemical and molecular analysis. one-way ANOVA with Student-Newman-Keuls post test. AMI rats presented a 38% increase in ROS levels. TH administration prevented these alterations in AMIT rats. The AMIT group presented an increase in eNOS expression, in NOS activity and in nitrite levels. TH administration also increased PGC-1α expression in the AMIT group. In conclusion, TH effects seem to involve a modulation of eNOS expression and an improvement in NO bioavailability in the infarcted heart.

Protective role of oleic acid against cardiovascular insulin resistance and in the early and late cellular atherosclerotic process.

BackgroundSeveral translational studies have identified the differential role between saturated and unsaturated fatty acids at cardiovascular level. However, the molecular mechanisms that support the protective role of oleate in cardiovascular cells are poorly known. For these reasons, we studied the protective role of oleate in the insulin resistance and in the atherosclerotic process at cellular level such as in cardiomyocytes (CMs), vascular smooth muscle cells (VSMCs) and endothelial cells (ECs).MethodsThe effect of oleate in the cardiovascular insulin resistance, vascular dysfunction, inflammation, proliferation and apoptosis of VSMCs were analyzed by Western blot, qRT-PCR, BrdU incorporation and cell cycle analysis.ResultsPalmitate induced insulin resistance. However, oleate not only did not induce cardiovascular insulin resistance but also had a protective effect against insulin resistance induced by palmitate or TNFα. One mechanism involved might be the prevention by oleate of JNK-1/2 or NF-κB activation in response to TNF-α or palmitate. Oleate reduced MCP-1 and ICAM-1 and increased eNOS expression induced by proinflammatory cytokines in ECs. Furthermore, oleate impaired the proliferation induced by TNF-α, angiotensin II or palmitate and the apoptosis induced by TNF-α or thapsigargin in VSMCs.ConclusionsOur data suggest a differential role between oleate and palmitate and support the concept of the cardioprotector role of oleate as the main lipid component of virgin olive oil. Thus, oleate protects against cardiovascular insulin resistance, improves endothelial dysfunction in response to proinflammatory signals and finally, reduces proliferation and apoptosis in VSMCs that may contribute to an ameliorated atherosclerotic process and plaque stability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0237-9) contains supplementary material, which is available to authorized users.

129. Vector and Cell Mediated BMPR2 Therapy Ameliorates PAH

Inherited pulmonary arterial hypertension is a fatal disease caused by mutations in, and reduced expression of, the bone morphogenetic type 2 receptor (BMPR2), leading to abnormal proliferation of pulmonary vascular cells and obliteration of small pulmonary vessels. BMPR2 expression is also reduced in secondary forms of PAH. We previously showed that upregulation of BMPR2 expression in the pulmonary vascular endothelium achieved by an endothelialtargeted adenoviral vector ameliorated PAH in rat models. In the current work, we evaluated a similar vector approach in a transgenic, BMPR2-mutation related PAH model, further characterised signaling mechanisms involved in the rat model, and extended the approach to the use of ex vivo transduced endothelial progenitor cells (EPCs) as a cell therapy approach to BMPR2 upregulation.Methods: SM22-tet / R899x mice were fed doxycycline-containing chow to induce expression of BMPR2 dominant negative protein. Mice were then injected via the tail vein with a pulmonary vascular targeted AdBMPR2-conjugate complex (or control vector), and right ventricular pressure measured (RVSP) by transthoracic puncture. For the EPC approach, rat bone marrow-derived EPCs were cultured in endothelial selective media then transduced ex-vivo with AdBMPR2 for 48 hours. The transduced cells (or untransduced controls) were then injected via tail vein into rats that had induced PAH via subcutaneous monocrotaline injection (60mg/kg) 10 days previously.Results: In the murine experiment, BMPR2 delivery led to a 24% reduction of RVSP compared to disease and vector control animals. Analysis of whole lung lysate using RT-PCR revealed an increase in eNOS expression in the AdBMPR2 treated group. This effect was confirmed in vitro by transduction of cultured endothelial cells, and further confirmed by demonstration of increased NO production. The Ad vector approach in the rat MCT model ameliorated PAH as previously described, an effect associated with an increase in Smad1/5/8 phosphorylation and decreased Smad3 phosphorylation. In the EPC approach, PAH was also ameliorated following EPC+AdBMPR2 treatment, demonstrated by significant reduction compared to MCT only and EPC only for Fulton Index (26% and 16% respectively), RVSP (34% and 36%) and mPAP (25% and 16%), while analysis of downstream signaling is ongoing.Conclusion: These findings indicate that gene delivery strategies via direct vector approaches or cell therapy to upregulate in BMPR2 in the pulmonary vasculature have therapeutic potential in PAH.

Arg⁹⁷² insulin receptor substrate-1 inhibits endothelial nitric oxide synthase expression in human endothelial cells by upregulating microRNA-155.

The dysregulation of nitric oxide (NO) synthesis attributable to the abnormal expression/activity of endothelial NO synthase (eNOS) is considered to be a major characteristic of insulin-resistant states, as well as an essential contributor to the pathogenesis of cardiovascular diseases. The Arg972 insulin receptor substrate-1 (IRS-1) is associated with insulin resistance. In the present study, we investigated the association between Arg972 IRS-1 and eNOS expression/activity in human subjects and in primary cultures of human endothelial cells. Data from 832 human subjects revealed that heterozygous and homozygous Arg972 IRS-1 carriers had significantly lower levels of plasma eNOS and nitrite/nitrate than the homozygous wild-type (WT) IRS-1 carriers. Human umbilical vein endothelial cells (HUVECs) established from delivering mothers expressing heterozygous Arg972 IRS-1 had significantly lower eNOS expression/activity and higher miR-155 levels than those expressing WT homozygous IRS-1. The overexpression of IRS-1 and Arg972 IRS-1 in the HUVECs, respectively, decreased and increased the miR-155 expression level. In addition, the overexpression of IRS-1 in the HUVECs significantly increased eNOS expression; this effect was reversed by transfection with mature miR-155 mimic or treatment with the selective phosphatidylinositol-3 kinase (PI3K) inhibitor, BKM120. On the other hand, the overexpression of Arg972 IRS-1 markedly decreased eNOS expression and this effect was reversed by transfection with antagomir-155. On the whole, our in vivo data demonstrate that Arg972 IRS-1 is associated with decreased plasma eNOS and nitrite/nitrate levels in human subjects. Our in vitro data demonstrate that Arg972 IRS-1 inhibits eNOS expression in human endothelial cells by upregulating miR-155 expression through the impairment of PI3K signaling. The present study provides new insight into the pathophysiological role of Arg972 IRS-1 in cardiovascular diseases.

Efficacy of statins on sirtuin 1 and endothelial nitric oxide synthase expression: the role of sirtuin 1 gene variants in human coronary atherosclerosis

Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and are used to reduce the risk of coronary artery disease (CAD) due to their pleiotropic effects. Recently, greater focus has been placed on the role of sirtuin 1 (SIRT1) in cardiovascular disease research. However, insufficient data exist on the relationships between statins, SIRT1 protein levels, and SIRT1 gene variants. In the present study, we investigated the effects of statins, atorvastatin and rosuvastatin, in CAD patients by analysing the associations between SIRT1 gene variants, rs7069102C>G and rs2273773C>T, and SIRT1/endothelial nitric oxide (eNOS) expression, as well as total antioxidant and oxidant status, and the oxidative stress index. SIRT1 expression was significantly higher, and eNOS expression was significantly lower in CAD patients when compared with controls. Statin treatment reduced SIRT1 expression and increased eNOS expression, similar to the levels found in the control population, independent from the studied SIRT1 gene variants. Oxidative stress parameters were significantly increased in CAD patients, and were decreased by statin treatment, demonstrating the antioxidative effects of statins on atherosclerosis. These results indicate that statin treatment could produce its protective effect on cardiovascular disease through the inhibition of SIRT1 expression. This is the first study reporting on the effect of statins, specifically atorvastatin and rosuvastatin, on SIRT1 expression in CAD patients.

Effect of Momordica charantia fruit extract on vascular complication in type 1 diabetic rats.

Diabetes mellitus is one of the risk factors in the development of vascular complications. Decreased nitric oxide (NO) production and increased lipid peroxidation in diabetes mellitus are the dominant exaggerating factors. Mormodica charantia (MC) was proven to be useful in improving diabetes mellitus and its complications. In the present study, a total of 40 male Sprague-Dawley rats were used. Diabetes was induced by a single dose (50 mg/kg) of streptozotocin (STZ), intramuscularly. Following 4 weeks of STZ induction, the animals were equally divided into five groups (n = 8); Control group (Ctrl), control group treated with MC (Ctrl-MC), diabetic untreated group (DM-Ctrl), diabetic group treated with MC (DM-MC) and diabetic group treated with metformin 150 g/kg (DM-Met). Oral administration of the MC fruit extract (1.5 g/kg) was continued for 28 days. DM-MC group showed a significant decrease (P < 0.05) in blood pressure, total cholesterol and triglyceride levels compared to the DM-Ctrl group. Aortic tissue NO level was significantly increased and malondialdehyde level was decreased in the DM-MC group. Immunohistochemical staining showed an increase in eNOS expression in the endothelial lining of the DM-MC group. Similarly, morphological deterioration of the aortic tissues was reverted to normal. In summary, treatment with the MC fruit extract exerted the significant vasculoprotective effect in the type 1 diabetic rat model.

Mechanisms of protection against diabetes-induced impairment of endothelium-dependent vasorelaxation by Tanshinone IIA

BackgroundImpairment of endothelium-dependent vasorelaxation has been suggested to play a principle role of endothelial dysfunction in the development of cardiovascular complications of diabetes. Recent studies have demonstrated a protective effect of Tanshinone IIA (Tan) on endothelial nitric oxide synthase (eNOS)–NO pathway. However, its role in endothelium-dependent vasorelaxation in diabetes and precise mechanisms remain elusive. MethodsSprague–Dawley rats were injected intraperitoneally with streptozotocin (STZ) to induce diabetes and then administered orally with Tan for 2weeks. For the in vitro study, human umbilical vein endothelial cells (HUVECs) were co-incubated with Tan and high glucose for 48h. ResultseNOS expression and NO generation were significantly decreased in diabetic rats. These decreases were accompanied by an impairment of endothelium-dependent relaxation. Administration of Tan ameliorated the aberrant changes in eNOS expression, NO generation and endothelium-dependent relaxation in diabetic rats. Expectedly, Tan also inhibited high glucose-induced decrease of eNOS expression and NO generation in a concentration-dependent manner in HUVECs. Mechanistically, high glucose attenuated eNOS transcriptional activity through inhibiting the binding activity and nuclear translocation of Sp1 and AP-1. However, Tan did not prevent these effects. At post-transcriptional level, Tan increased eNOS expression and activity through multiple mechanisms including regulation of mRNA and protein half-life, degradation, coupling and serine 1177 phosphorylation. Rather than affecting protein phosphatase 2A (PP2A) expression and activity, Tan markedly inhibited the translocation of PP2A-A from cytosol to membrane and subsequently impaired PP2A-A/eNOS interaction, leading to prevent eNOS dephosphorylation. All these alterations underlie the protective role of Tan on eNOS expression following high glucose stimulation. ConclusionsOur data demonstrate that high glucose decreases eNOS expression initiating at a transcriptional level, whereas Tan prevents such effect through multiple ways of post-transcriptional mechanism. General significanceOur work provided novel mechanisms for Tan in regulating vasorelaxation and may help to better understand the cardiovascular protective action of Tan.

Establishment of a rabbit model of coronary artery bypass graft and endothelial nitric oxide synthase gene transfection.

This study established an animal model of coronary artery bypass graft (CABG) surgery. The human endothelial nitric oxide synthase (eNOS) gene was transfected into grafted arterial walls to verify transfection efficiency. Forty rabbits were randomized into the following 4 equal groups: 1) eNOS gene transfection group (eNOS group); 2) empty eNOS gene transfection group (empty gene group); 3) control group; 4) normal femoral artery group. Grafted arteries, and normal carotid and femoral artery specimens were obtained 3 weeks later. Immunohistochemistry and analyses of tissue nitric oxide (NO) levels, eNOS activity, and eNOS protein western blotting were performed. The effectiveness and efficiency of transfection were observed and confirmed. All rabbits survived. The grafted arteries retained patency. Varying degrees of adaptability changes were observed in grafted arteries in each group. The eNOS group exhibited vascular wall thickening and significantly increased eNOS protein expression. The control and empty gene groups exhibited vessel wall degeneration, and eNOS protein was weakly or not expressed (P < 0.05). The arterial wall NO concentration and total eNOS activity in the eNOS group were significantly higher than those in the other groups (P < 0.05). Western blotting demonstrated that the vascular wall eNOS protein concentration was significantly greater than that in the other groups (P < 0.05). Furthermore, the eNOS gene transfection can increase eNOS expression and activity in vessel walls, increasing local NO concentration and expression.

Abstract 11455: Mechanism Underlying Increased eNOS Expression and Neuroprotection in Mice With Targeted Deletion of ROCK2

Background: The Rho kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton. We have recently shown that ROCK activity is increased during the onset of ischemic stroke and that the ROCK inhibitor, fasudil, decreased the severity of focal cerebral ischemia, in part, through the upregulation of endothelial nitric oxide synthase (eNOS). However, the precise mechanism by which ROCK regulates eNOS expression is unknown. Methods and Results: Using RNA affinity chromatography with 3’UTR of eNOS mRNA followed by MALDI-TOF-MS analysis, we have identified eukaryotic elongation factor 1 alpha 1 (eEF1A1) as an actin-binding protein, which directly binds to eNOS mRNA and regulates eNOS mRNA stability. Overexpression of eEF1A1 decreased, and knock-down of eEF1A1 by siRNA increased, eNOS expression. Protein kinase assays using eEF1A1 with purified ROCK1 and ROCK2 showed that eEF1A1 is strongly phosphorylated by ROCK2, but not ROCK1, and that eEF1A1 phosphorylation by ROCK2 is required for binding to and destabilization of eNOS mRNA. Indeed, treatment of human endothelial cells (EC) with fasudil decreased eEF1A1 phosphorylation, but not expression. This was associated with increased eNOS mRNA stability and expression, and NO production. Furthermore, compared to that of WT and ROCK1+/- mice, eNOS mRNA stability and expression were increased in ECs from ROCK2+/- mice and endothelium-dependent relaxation was enhanced in both ROCK2+/- and EC-specific ROCK2 KO mice (EC-ROCK2-/-). These results correlated with decreased cerebral infarct size and neurological deficits in both ROCK2+/- and EC-ROCK2-/- mice subjected to transient focal cerebral ischemia. When ROCK2+/- mice were place on eNOS-/- background (double mutant ROCK2+/-/eNOS-/- mice), the neuroprotective effects observed in ROCK2+/- mice were abolished, indicating that eNOS mediates neuroprotective effects in ROCK2-deficient mice. Conclusion: ROCK2 negatively regulates eNOS expression by phosphorylating eEF1A1, which is required for eEF1A1 to bind to eNOS mRNA. The binding of eEF1A1 decreases eNOS mRNA stability and expression. These findings suggest a novel mechanism by which ROCK2 could impair endothelial function and worsen cerebral ischemia.

Intravenous transfusion of endothelial colony-forming cells attenuates vascular degeneration after cerebral aneurysm induction

Cerebral aneurysm (CA) rupture is a major cause of subarachnoid hemorrhage with high morbidity and mortality. Using an animal model, we examined the potential of endothelial colony-forming cells (ECFCs) transfusion on vascular degeneration after CA induction and underlying mechanisms. CA was induced in the right anterior cerebral artery–olfactory artery (ACA/OA) bifurcations in Sprague–Dawley rats with or without ECFCs transfusion. The degeneration of internal elastic lamina (IEL), media thickness and CA size were evaluated. Expression of matrix metalloproteinase-2 and 9 (MMP-2 and 9), tissue inhibitor of metalloproteinase-1 (TIMP-1), macrophage chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), nuclear factor κB (NF-κB), endothelial nitric oxide synthase (eNOS), B-cell leukemia/lymphoma-2 (Bcl-2), and inducible nitric oxide synthase (iNOS) were analyzed by quantitative real-time polymerase chain reaction. The macrophages infiltration and apoptosis of smooth muscle cells (SMCs) were examined immunohistologically. Rats in CA+ECFCs transfusion group showed a notable reduction in IEL degeneration, media thinning and CA size compared with those in CA+saline group. ECFCs transfusion inhibited the MMP-driven wall destruction by downregulating MMP-2, MMP-9 expression and upregulating TIMP-1. ECFCs transfusion dramatically decreased VCAM-1 and NF-κB expression, increased eNOS expression and caused no change in MCP-1 expression, which was accompanied by reduced macrophages infiltration. Moreover, ECFCs transfusion reversed downregulation of Bcl-2 expression and upregulation of iNOS expression, and decreased SMCs apoptosis. Collectively, these findings suggest that ECFCs transfusion confers protection against degeneration of aneurysmal wall by inhibiting inflammatory cascades and SMCs apoptosis.

Arginase inhibition restores endothelial function in diet-induced obesity

Arginase may play a major role in the regulation of vascular function in various cardiovascular disorders by impairing nitric oxide (NO) production. In the current study, we investigated whether supplementation of the arginase inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA) could restore endothelial function in an animal model of diet-induced obesity. Arginase 1 expression was significantly lower in the aorta of C57BL/6J mice fed a high-fat diet (HFD) supplemented with nor-NOHA (40mgkg-1/day) than in mice fed HFD without nor-NOHA. Arginase inhibition led to considerable increases in eNOS expression and NO levels and significant decreases in the levels of circulating ICAM-1. These findings were further confirmed by the results of siRNA-mediated knockdown of Arg in human umbilical vein endothelial cells. In conclusion, arginase inhibition can help restore dysregulated endothelial function by increasing the eNOS-dependent NO production in the endothelium, indicating that arginase could be a therapeutic target for correcting obesity-induced vascular endothelial dysfunction.

Functional Role of Connexins and Pannexins in the Interaction Between Vascular and Nervous System

The microvascular network of the microcirculation works in tight communication with surrounding tissues to control blood supply and exchange of solutes. In cerebral circulation, microvascular endothelial cells constitute a selective permeability barrier that controls the environment of parenchymal brain tissue, which is known as the blood-brain barrier (BBB). Connexin- and pannexin-formed channels (gap junctions and hemichannels) play a central role in the coordination of endothelial and smooth muscle cell function and connexin-mediated signaling in endothelial cells is essential in the regulation of BBB permeability. Likewise, gap junction communication between astrocyte end-feet also contributes to maintain the BBB integrity, but the participation of hemichannels in this process cannot be discarded. Sympathetic and sensory perivascular nerves are also involved in the control and coordination of vascular function through the release of vasoconstrictor or vasodilator signals and by the regulation of gap junction communication in the vessel wall. Conversely, ATP release through pannexin-1-formed channels mediates the α1-adrenergic signaling. Furthermore, here we show that capsaicin-induced CGRP release from mesenteric perivascular sensory nerves induces pannexin-1-formed channel opening, which in turn leads to reduction of pannexin-1 and endothelial nitric oxide synthase (eNOS) expression along the time. Interestingly, blockade of CGRP receptors with CGRP8-37 increased eNOS expression by ∼5-fold, suggesting that capsaicin-sensitive sensory nerves are involved in the control of key signaling proteins for vascular function. In this review, we discuss the importance of connexin-based channels in the control of BBB integrity and the functional interaction of vascular connexins and pannexins with the peripheral nervous system.

Nicorandil suppresses urinary protein excretion and activates eNOS in Dahl salt-sensitive hypertensive rats

Hypertension is a risk factor common to both chronic kidney disease and cardiovascular disease. Nicorandil is widely used for the treatment of angina. We investigated the benefits of nicorandil with respect to renal dysfunction in Dahl salt-sensitive hypertensive (DS) rats. DS rats were fed a high-salt (HS) diet and nicorandil was administered via the drinking water. Blood pressure and renal function were measured for 4 weeks after starting the rats on the HS diet. In rats fed the HS diet, renal dysfunction was manifested by an increase in urinary protein and N-acetyl-β-D-glucosaminidase excretion. Nicorandil ameliorated renal function with a concomitant reduction in urinary 8-hydroxy-2'-deoxyguanosine and an increase in urinary NOx. Significant upregulation of endothelial nitric oxide synthase (eNOS) expression and an increase in the eNOS dimer/monomer ratio (reduction of eNOS uncoupling) was demonstrated in glomeruli following nicorandil treatment. The blood pressure of DS rats was increased by salt loading; however, no significant change in blood pressure was observed with nicorandil treatment. In DS rats fed a HS diet, nicorandil prevented the development of renal dysfunction, which was accompanied by an increase in eNOS expression in the kidneys.

Abstract 640: Role of MicroRNA-146a in Vascular Inflammation and Atherosclerosis

Prolonged activation of inflammatory pathways in the endothelium leads to vascular diseases, including sepsis and atherosclerosis. During this process, activated endothelial cells recruit circulating leukocytes by expressing adhesion molecules and chemoattractants, such as vascular cellular adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1), respectively. Down-regulation of the anti-adhesion molecule, nitric oxide, which is synthesized by endothelial nitric oxide synthase (eNOS), is also a critical step during endothelial activation. Here we demonstrate that microRNA-146a and microRNA-146b are upregulated in endothelial cells upon exposure to the pro-inflammatory cytokine, interleukin-1-beta (IL-1β). Over expression of miR-146 in endothelial cells dampens the inflammatory response induced by IL-1β and conversely inhibition of endogenous miR-146 in vitro or deletion of miR-146a in vivo exacerbates the inflammatory response to IL-1β. We found that miR-146 represses VCAM-1 and MCP-1 expression by impinging on the canonical NF-kB pathway by targeting the upstream adaptor proteins TRAF6 and IRAK1. In addition, we identified the RNA-binding protein, HuR, as a novel miR-146 target. Knock-down of HuR results in an increase in eNOS expression and a corresponding decrease in leukocyte adhesion, which parallels with the miR-146 over expression phenotype. Considering the involvement of miR-146a in regulating vascular inflammation, miR-146a-/-; Ldlr-/- mice (DKO) were generated to assess atherogenesis. After 12 weeks on high cholesterol diet, the DKO mice developed more lipid-rich plaques in the lesser curvature of the aortic arch compared to Ldlr-/- mice. This suggests that miR-146a may have atheroprotective properties in addition to its anti-inflammatory role in acute inflammation.

Abstract 660: Local Hemodynamic Forces and Aqueous Cigarette Smoke Extract Affect Development of Endothelial Dysfunction

Endothelial dysfunction is one of the first steps in the development of atherosclerosis. This proinflammatory phenotype is associated with decreased bioavailability of nitric oxide and a corresponding expression profile in the endothelial cells. Tobacco smoking promotes development of atherosclerotic plaques and local hemodynamic forces are key stimuli in this process. Low laminar flow is involved in the development of an unstable plaque phenotype, while high laminar flow has atheroprotective role. The molecular mechanisms controlling plaque stability in response to tobacco smoking remain largely unknown so far. Therefore, we exposed human endothelial cells to cigarette smoke extract (CSEaq) under disturbed flow conditions. Primary human endothelial cells were stimulated with increasing dosages of CSEaq for 24h. Cell viability was reduced by CSEaq in a dose-dependent manner. The impact of specific flow conditions and different doses of CSEaq on the expression of atherosclerosis-related genes was investigated using a cone-and-plate viscometer. High laminar flow induced elongation of endothelial cells in the direction of flow, increased eNOS expression and NO release in a time-dependent manner. This increase was inhibited by CSEaq. Low laminar flow showed no effect on eNOS expression and NO release. The NRF2 antioxidative defense system was also induced by high laminar flow. NRF2 and NRF2 target genes HMOX1 and NQO1 were strongly activated by CSEaq. Furthermore, we monitored the expression of proinflammatory genes. CSEaq strongly induced adhesion molecule ICAM-1. Interestingly, VCAM-1 was unaffected by CSEaq. Induction of endothelial NADPH oxidase isoform 4 by CSEaq was prevented by high laminar flow. Catalase expression was not affected by flow and CSEaq, whereas CSEaq transiently increased SOD1 expression. Endothelial wound healing was improved by atheroprotective high laminar flow. Low flow did not affect wound healing. Furthermore, high laminar flow decreased adhesion of monocytes to endothelial cells, compared to low flow. We suggest novel molecular mechanisms how tobacco smoking promotes the development of endothelial dysfunction. This can contribute to the formation of an unstable atherosclerotic plaque phenotype.