Overexpression Of eNOS Research Articles

Role of iNOS-NO-cGMP signaling in modulation of inflammatory and myelination processes.

Nitric oxide (NO) is the main activator of the soluble guanylate cyclase (sGC)-guanosine 3'5' cyclic monophosphate (cGMP) pathway. The level of cGMP is regulated by phosphodiesterases (PDEs), which break down cGMP. It has been reported that levels of NO in the central nervous system (CNS) can greatly increase during demyelination and/or neuroinflammation. Controversially, in demyelination models, mice without iNOS may develop more severe cases of disease. Furthermore, cGMP accumulation caused by PDE inhibitors has an anti-inflammatory/neuroprotective effect in MS-models. The role of the NO-cGMP pathway in the nervous tissue is, therefore, complex and not fully understood. The aim of the present study was to contribute to existing knowledge of the role of this pathway in the CNS. Wild type (WT - C57BL/6) and iNOS(-/-) animals were treated with sildenafil (25mg/kg) for 8 weeks. Control animals were not treated. VCAM and ICAM (adhesion proteins), GFAP and Iba-1 (astrocyte and microglia markers, respectively), PKG (cGMP-dependent protein kinase), sGC, eNOS (constitutive endothelial NO sinthase) and GSTpi (a marker of mature oligodendrocytes) were evaluated in the cerebellum using immunohistochemistry or western blotting. Myelin was assessed by luxol fast blue staining and electron transmission microscopy. Treatment with sildenafil reduced ICAM and VCAM levels (anti-inflammatory effect) and increased GFAP and Iba-1 expression (clearance phenotype) in WT animals. The expression of VCAM, ICAM, GFAP, PKG and sGC was lower in iNOS(-/-) mice than in WT control animals. The treatment of iNOS(-/-) animals with sildenafil resulted in an increase of all proteins (pro-inflammatory effect). There was overexpression of eNOS in untreated iNOS(-/-) mice. The myelin structure of iNOS(-/-) animals was damaged in comparison with WT control. Sildenafil increased GSTpi and resulted in an improved myelin structure in iNOS(-/-) mice. In conclusion, NO-cGMP signaling plays a role in the regulation of inflammation and myelination processes. The accumulation of cGMP produced opposite effects in WT and iNOS(-/-) mice. This can be explained by the overexpression of eNOS in iNOS(-/-) mice, unbalancing cGMP signaling, or cGMP has a dual role in inflammation. Drugs that modulate the NO-sGC-cGMP pathway may be clinically beneficial in the treatment of neuroinflammatory/demyelinating disorders, but further studies of the regulation of this pathway are required.

Adenovirus-Mediated eNOS Expression Augments Liver Injury after Ischemia/Reperfusion in Mice

Hepatic ischemia/reperfusion (l/R) injury continues to be a critical problem. The role of nitric oxide in liver I/R injury is still controversial. This study examines the effect of endothelial nitric oxide synthase (eNOS) over-expression on hepatic function following I/R. Adenovirus expressing human eNOS (Ad-eNOS) was administered by tail vein injection into C57BL/6 mice. Control mice received either adenovirus expressing LacZ or vehicle only. Sixty minutes of total hepatic ischemia was performed 3 days after adenovirus treatment, and mice were sacrificed after 6 or 24 hrs of reperfusion to assess hepatic injury. eNOS over expression caused increased liver injury as evidenced by elevated AST and ALT levels and decreased hepatic ATP content. While necrosis was not pervasive in any group, TUNEL demonstrated significantly increased apoptosis in Ad-eNOS infected livers. Western blotting demonstrated increased levels of protein nitration and upregulation of the pro-apoptotic proteins bax and p53. Our data suggest that over-expression of eNOS is detrimental in the setting of hepatic I/R.

Exercise training in adverse cardiac remodeling

Cardiac remodeling in response to a myocardial infarction or chronic pressure-overload is an independent risk factor for the development of heart failure. In contrast, cardiac remodeling produced by regular physical exercise is associated with a decreased risk for heart failure. There is evidence that exercise training has a beneficial effect on disease progression and survival in patients with cardiac remodeling and dysfunction, but concern has also been expressed that exercise training may aggravate pathological remodeling and dysfunction. Here we present studies from our laboratory into the effects of exercise training on pathological cardiac remodeling and dysfunction in mice. The results indicate that even in the presence of a large infarct, exercise training exerts beneficial effects on the heart. These effects were mimicked in part by endothelial nitric oxide synthase (eNOS) overexpression and abrogated by eNOS deficiency, demonstrating the importance of nitric oxide signaling in mediating the cardiac effects of exercise. Exercise prior to a myocardial infarction was also cardioprotective. In contrast, exercise tended to aggravate pathological cardiac remodeling and dysfunction in the setting of pressure-overload produced by an aortic stenosis. These observations emphasize the critical importance of the underlying pathological stimulus for cardiac hypertrophy and remodeling, in determining the effects of exercise training. Future studies are needed to define the influence of exercise type, intensity and duration in different models and severities of pathological cardiac remodeling. Together such studies will aid in optimizing the therapy of exercise training in the setting of cardiovascular disease.

Endothelial Nitric Oxide Synthase Protects Neurons against Ischemic Injury through Regulation of Brain‐Derived Neurotrophic Factor Expression

Several lines of evidence demonstrated that endothelial nitric oxide synthase (eNOS) confers protective effects during cerebral ischemia. In this study, we explored the underlying cellular and molecular mechanisms of neuroprotection by eNOS. A series of in vivo and in vitro ischemic models were employed to study the role of eNOS in maintaining neuronal survival and to identify the downstream factors. The current data showed that pretreatment with a specific eNOS inhibitor, L-N5-(1-iminoethyl) ornithine (L-NIO), aggravated the neuronal loss in the rat cerebral ischemic model, accompanied by reduction in brain-derived neurotrophic factor (BDNF) level, which was consistent with the findings in an oxygen-glucose deprivation model (OGD) with two neuronal cells: primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Furthermore, the extensive neuronal loss induced by L-NIO was totally abolished by exogenous BDNF in both in vitro and in vivo models. On the other hand, eNOS overexpression through an adenoviral vector exerted a prominent protective effect on the neuronal cells subject to OGD, and the protective effect was totally abrogated by a neutralizing anti-BDNF antibody. Collectively, our results indicate that the neuroprotection of neuron-derived eNOS against the cerebral ischemia was mediated through the regulation of BDNF secretion. In conclusion, our discovery provides a novel explanation for the neuroprotective effect of eNOS under pathological ischemic conditions such as stroke.

ENOS Overexpressing Bone Marrow Cells are Safe and Effective in a Porcine Model of Myocardial Regeneration Following Acute Myocardial Infarction

Cell therapy has been shown to be effective in improving LV function postmyocardial infarction (MI). We hypothesized that eNOS-transfected bone marrow cells (BMCs) are safe in a swine model of myocardial infarction (MI). We also hypothesized that endothelial nitric oxide synthase (eNOS) transfection would enhance cell function, as assessed by myocardial functional recovery post-MI. Fifteen female Yorkshire pigs underwent bone marrow aspiration and creation of MI. Bone marrow cells were cultured for 7days, and each pig received either autologous BMCs transiently transfected with eNOS plasmid (eNOS-BMC, n=5), nontransfected BMCs (nt-BMC, n=4), or phosphate-buffered saline (PBS) control (n=6). Cardiac MRI was performed at baseline (1week post-MI) and 6weeks post-MI. There was no difference in safety outcomes between groups. Absolute left ventricular ejection fraction (LVEF) at 6weeks showed a trend toward improvement in both cell therapy groups compared with baseline but worsened in the PBS control group. The absolute improvement in LVEF was significantly greater in both cell therapy groups compared with PBS control. Infarct mass was significantly lower in the eNOS-BMC group between baseline and 6weeks, but the absolute change in infarct mass was not different between groups. Finally, there was a trend toward reduced LV mass in the eNOS-BMC group. Bone marrow cell delivery, with and without eNOS overexpression, is safe and leads to improvement in LVEF when administered in the coronary circulation 7days following acute MI in swine. Transfection of healthy BMCs with eNOS resulted in some improvement in left ventricular remodeling. Further study is warranted in a preclinical model that approximates the impact of cardiovascular risk factors on BMC function.

Increased sympathetic drive during the onset of hypertension in conscious Cyp1a1-Ren2 rats

Despite advances in our understanding concerning the pathology of hypertension, the mechanisms that underpin the origin of hypertension remain to be fully elucidated. This enigma is, at least in part, due to inherent limitations of various animal models of hypertension. Here, we show the genetically modified Cyp1a1-Ren2 rat model, in which the onset and severity of angiotensin II-dependent hypertension can be tightly controlled, as an effective model for investigating increased sympathetic drive for the onset of hypertension. Cyp1a1-Ren2 rats were surgically prepared with radiotelemetric transmitters for the continuous measurement of arterial blood pressure (ABP). ABP was recorded in freely moving rats that were fed with either normal rat chow or a diet containing indole-3-carbinol (0.225% w/w) for 7 days to induce hypertension. Structural morphology of and endothelial NO synthase (eNOS) protein expression in heart and/or vascular tissue were analyzed. Sympathetic tone was estimated using spectral analysis of heart rate variability. The progressive induction of hypertension over 7 days was matched with a parallel increase in sympathetic tone. By day 7 of hypertension, eNOS expression in the mesenteric artery was elevated. However, the elevated ABP, sympathetic tone, and eNOS had not elicited gross morphological remodeling of the heart or vasculature. Importantly, both the increase in sympathetic tone and overexpression of eNOS within the vasculature were reversed when ABP was returned to normal. We conclude that the Cyp1a1-Ren2 rat provides an effective model for investigating specific adverse and transient changes in central sympathetic modulation of arterial blood pressure during the early onset of angiotensin-dependent hypertension.

Can moderate training reverse hypertrophic cardiomyopathy in a mouse model of vascular dysfunctional eNOS?

Purpose: Heart failure is one of the most prevalent causes of death in the western world. Caveolin1 knockout mice (cav1-/-) develop a severe hypertrophic cardiomyopathy with functional impairment especially in cardiac output and ejection fraction. Behavior observation during the breeding of this mouse line revealed, that the animals showed a markedly reduced activity. Physiological inactivity can also contribute to heart diseases. The aim of the current study was to test, if moderate physical activity (swim training) can improve heart function and reverse cardiac morphological alterations in this particular model. The efficiency of training was unclear, because caveolin-1 deficiency is associated with eNOS hyperactivation. Hyperactivation of eNOS can lead to massive radical production. Overexpression of eNOS has been described to be counteracting the beneficial effects of training with respect to improved cardiovascular function. Methods and results: Echocardiographic measurements of 8 week old animals show that cav1-/- mice had an increased left ventricular posterior wall thickness resulting in a reduced left ventricular volume. Previous data from our group have demonstrated that this left ventricular hypertrophy leads to a reduced cardiac function (reduced stroke volume and cardiac output) in older mice combined with reduced physical ability in swimming tests compared to wild type mice. Six weeks of moderate training (2 weeks 5 min/day, 2 weeks 10 min/day and 2 weeks 15 min/day) resulted in markedly prolonged swim duration (untrained: 11±4 min vs. training: 50±12 min) in cav1-/- mice. In line with this, the functional parameters following training showed that stroke volume and cardiac output (measured by echocardiography) were comparable to wild type littermates. Furthermore, the left ventricular wall thickness was reduced in during the training period leading to a significantly higher intraventricular volume. None the less, heart and lung weight of these animals were still significantly elevated. Conclusions: In conclusion the present study documents that moderate training can inhibit the progression of heart failure in a hypertrophic mouse model with elevated eNOS activity. It remains to be established, if the beneficial effects are mediated by direct cardiac mechanisms, or if alterations in the peripheral circulation e.g. in blood viscosity or in peripheral resistance are involved here.

Effects of NO on the expression and function of bradykinin type 2 receptors. Role for angioedema?

Purpose: The pathophysiology of angioedema induced by angiotensin-converting enzyme inhibitors (ACEi) is not well understood. While activation of bradykinin (BK) type 2 receptors (B2) has been established, the triggering events remain obscure. In this study we investigated effect of NO as one product of B2 stimulation. Methods: Brain (bEND3), human umbilical vein (HUVEC) and porcine aortic endothelial cells (PAEC) were incubated with the NO-donors DEA/NO (10μM), DETA/NO (100μM) and SNAP (1-100μM) for 3, 6 and 12 h. B2 expression was assessed by RT-PCR and westernblot using monoclonal B2 antibodies staining either glycosylated or unglycosylated B2. Function of B2 was determined by monitoring iCa2+ upon activation with BK. Likewise, B2 protein levels were evaluated in aorta, heart and lung of (1) two different eNOS-/- strains, (2) mice with endothelial-specific overexpression of eNOS (eNOS-tg), (3) in C57Bl/6 mice treated with NOS inhibitor L-nitroarginine (L-NA). Here, B2 function was evaluated in aortic rings. Results: After 3h incubation with DEA/NO of bEND3, B2-mRNA was unchanged (160±32%, n=6, P>0.05). Likewise, B2 protein levels remained stable (98±13.6%, n=8, P>0.05). Similar results were obtained in PAECs and HUVECs and after extended incubation time and increased concentration. There was also a similar rise of iCa2+ in response to 0.01-100 μM BK in bEND3 treated with DEA/NO or vehicle as demonstrated by almost identical flourescence signals. These results obtained in cell lines and primary cultured PAEC were confirmed using transgenic mice with different levels of vascular NO-bioavailability. In aorta, neither the complete lack of endothelial NO in eNOS-/- (93±26%, n=5, P>0.05), nor overexpression in eNOS-tg (112±13.7%, n=10, P>0.05) changed B2 protein expression and a similar observation was made in C56Bl/6 treated with L-NA (110±19.4%, n=5, P>0.05). Aortic constrictor responses to 10 μM BK (related to maximal constriction to KCl) were identical in C57Bl/6 (24.3±2.7%, n=7) and eNOS-tg (30±11.8%, n=4, P>0.05). However, B2 signaling involved generation of NO as constrictor responses were significantly increased following L-NA in both C57BL/6 (64±18.3%, n=4) and eNOS-tg (67±3.6%, n=4) and a similar constriction was observed in eNOS-/- without (61±10.9%, n=7) and with L-NA (61±14.6%, n=6). None of these responses differed significantly. Conclusion: These data demonstrate that NO does not regulate vascular B2 gene expression and function suggesting that changes of B2 expression by this product of B2 activation are unlikely involved in triggering ACEi-induced angioedema.

Regulation of the sympathetic nervous system by nitric oxide and oxidative stress in the rostral ventrolateral medulla: 2012 Academic Conference Award from the Japanese Society of Hypertension

Sympathoexcitation has an important role in the pathogenesis of hypertension. Previous studies have demonstrated that nitric oxide (NO) and/or oxidative stress in the brain are important for the regulation of the sympathetic nervous system. We have investigated the role of NO derived from an overexpression of endothelial NO synthase (eNOS) or oxidative stress in the rostral ventrolateral medulla (RVLM), which is known as a vasomotor center in the brainstem, on the regulation of the sympathetic nervous system. Our results indicated that NO derived from an overexpression of eNOS in the RVLM caused sympathoinhibition via an increase in γ-amino butyric acid and that angiotensin II type 1 receptor (AT1R)-induced oxidative stress in the RVLM caused sympathoexcitation. We also demonstrated that oxidative stress in the RVLM caused sympathoexcitation via interactions with NO, effects on the signal transduction or apoptosis of the astrocytes. Furthermore, several orally administered AT1R blockers have been found to cause sympathoinhibition via a reduction in oxidative stress through the blockade of AT1R in the RVLM of hypertensive rats. In conclusion, our studies suggest that the increase in AT1R-induced oxidative stress and/or the decrease in NO in the RVLM mainly cause sympathoexcitation in hypertension.

Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Hydrogen sulfide (H2S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S-induced angiogenesis.

Requirement of phosphorylatable endothelial nitric oxide synthase at Ser-1177 for vasoinhibin-mediated inhibition of endothelial cell migration and proliferation in vitro

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide is a major vasorelaxing factor and a mediator of vasopermeability and angiogenesis. Vasoinhibins, a family of antiangiogenic prolactin fragments that include 16K prolactin, block most eNOS-mediated vascular effects. Vasoinhibins activate protein phosphatase 2A, causing eNOS inactivation through dephosphorylationof eNOS at serine residue 1179 in bovine endothelial cells and thereby blocking vascular permeability. In this study, we examined whether human eNOS phosphorylation at S1177 (analogous to bovine S1179) influences other actions of vasoinhibins. Bovine umbilical vein endothelial cells were stably transfected with human wild-type eNOS (WT) or with phospho-mimetic (S1177D) or non-phosphorylatable (S1177A) eNOS mutants. Vasoinhibins inhibited the increases in eNOS activity, migration, and proliferation following the overexpression of WT eNOS but did not affect these responses in cells expressing S1177D and S1177A eNOS mutants. We conclude that eNOS inhibition by dephosphorylation of S1177 is fundamental for the inhibition of endothelial cell migration and proliferation by vasoinhibins.

The 10 Most Read Articles Published in Circulation Research in 2012

The 10 Most Read Articles Published in <i>Circulation Research</i> in 2012

ENOS Overexpression Exacerbates Vascular Closure in the Obliterative Phase of OIR and Increases Angiogenic Drive in the Subsequent Proliferative Stage

In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O(2)(-)), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina. Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA. In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH₄) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels. In hyperoxia, reduced BH₄ bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia.

Nitric oxide as a mediator of fructose 1,6-bisphosphate protection in galactosamine-induced hepatotoxicity in rats

Fructose 1,6-bisphosphate (F1,6BP) has been widely used as a therapeutic agent for different harmful conditions in a variety of tissues. The hypothesis of the present work was that the increase in nitric oxide production and the prevention of oxidative stress induced by exogenous F1,6BP mediate its protective effect against the hepatotoxic action of GalN.Experimental groups used were sham, F1,6BP (2g/kg bw i.p.), GalN (0.4g/kg bw i.p), l-NAME (10mg/kg bw i.v.), F1,6BP+GalN, l-NAME+GalN and l-NAME+F1,6BP+GalN. Animals were killed after 24h of bolus administration.F1,6BP induced an increase in NO and the redox ratio (GSH/GSSG) in liver. Western blot assays pointed to overexpression of liver eNOS in F1,6BP-treated rats. The hepatic injury induced by GalN increased transaminases in plasma and decreased the reduced/oxidized glutathione ratio in liver. The concomitant administration of F1,6BP reversed this damage, while the addition of l-NAME worsened the liver injury. We provided evidence that this F1,6BP-induced protection may be related to the increase in NO production through the positive modulation of eNOS, and the increase in intracellular reduced glutathione, thus providing a higher reducing capacity.

Overexpression of Endothelial Nitric Oxide Synthase Improves Endothelium-Dependent Vasodilation in Arteries Infused with Helper-Dependent Adenovirus

Adenoviral vectors (Ad) are useful tools for in vivo gene transfer into endothelial cells. However, endothelium-dependent vasodilation is impaired after Ad infusion, and this impairment is not prevented by use of advanced-generation "helper-dependent" (HD) Ad that lack all viral genes. We hypothesized that endothelium-dependent vasodilation could be improved in Ad-infused arteries by overexpression of endothelial nitric oxide synthase (eNOS). We tested this hypothesis in hyperlipidemic, atherosclerosis-prone rabbits because HDAd will likely be used for treating and preventing atherosclerosis. Moreover, the consequences of eNOS overexpression might differ in normal and atherosclerosis-prone arteries and could include atherogenic effects, as reported in transgenic mice. We cloned rabbit eNOS and constructed an HDAd that expresses it. HDAdeNOS increased NO production by cultured endothelial cells and increased arterial eNOS mRNA in vivo by ∼10-fold. Compared to arteries infused with a control HDAd, HDAdeNOS-infused arteries of hyperlipidemic rabbits had significantly improved endothelium-dependent vasodilation, and similar responses to phenylephrine and nitroprusside. Moreover, infusion of HDAdeNOS had local atheroprotective effects including large, significant decreases in intimal lipid accumulation and arterial tumor necrosis factor (TNF)-α expression (p≤0.04 for both). HDAdeNOS infusion yields a durable (≥2 weeks) increase in arterial eNOS expression, improves vasomotor function, and reduces artery wall inflammation and lipid accumulation. Addition of an eNOS expression cassette improves the performance of HDAd, has no harmful effects, and may reduce atherosclerotic lesion growth.

Overexpression of Endothelial Nitric Oxide Synthase Prevents Diet-Induced Obesity and Regulates Adipocyte Phenotype

Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high-fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied. The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance. In db/db mice and in high-fat diet-fed wild-type C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS transgenic mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with wild-type mice, high-fat diet-fed eNOS transgenic mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS transgenic mice showed higher levels of PPAR-α and PPAR-γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption. These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high-fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.

Dimethylarginine Dimethylaminohydrolase/Nitric Oxide Synthase Pathway in Liver and Kidney: Protective Effect of Cyanidin 3-O-β-D-Glucoside on Ochratoxin-A Toxicity

The aim of the present study was to evaluate the effect of long-term cyanidin 3-O-β-D-glucoside (C3G) and/or Ochratoxin A (OTA)-exposure on dimethylarginine dimethylamino hydrolase/nitric oxide synthase (DDAH/NOS) pathway in rats. The experiments were performed in rats supplemented with C3G (1 g/kg feed), OTA (200 ppb), and OTA + C3G. After 4 weeks of daily treatment, liver and kidneys were processed for eNOS, iNOS and DDAH-1 Western blotting, nitrite levels evaluation and DDAH activity determination. Results show that OTA is able to induce iNOS both in kidney and liver, whereas OTA is able to induce eNOS and DDAH-1 overexpression and DDAH activation only in kidney, resulting in increased nitrite levels. In kidney of OTA + C3G fed rats, iNOS, eNOS and DDAH-1 expression were less pronounced compared with those observed in the OTA-treated group. Coherent with the decreased iNOS, eNOS and DDAH-1 expression a decrease in nitrite levels and DDAH activity was observed in the OTA + C3G group. Results demonstrate that C3G is able to counteract the deleterious effects of chronic consumption of OTA and also suggest a possible involvement of iNOS-eNOS-DDAH impairment in OTA nephrocarcinogenity.

Gut Region‐Specific Diabetic Damage to the Capillary Endothelium Adjacent to the Myenteric Plexus

Damage in the capillaries supplying the MP has been proposed as a critical factor in the development of diabetic enteric neuropathy. We therefore investigated connections between STZ-induced diabetes and the BM morphology, the size of caveolar compartments, the width of TJs, the transport of albumin, and the quantitative features of Cav-1 and eNOS expression in these microvessels. Gut segments from diabetic rats were compared with those from insulin-treated diabetics and those from controls. The effects of diabetes on the BM, the caveolar compartments, and the TJs were evaluated morphometrically. The quantitative features of the albumin transport were investigated by postembedding immunohistochemistry. The diabetes-related changes in Cav-1 and eNOS expression were assessed by postembedding immunohistochemistry and molecular method. Thickening of the BM, enlargement of the caveolar compartments, opening of the junctions, enhanced transport of albumin, and overexpression of Cav-1 and eNOS were documented in diabetic animals. Insulin replacement in certain gut segments prevented the development of these alterations. These data provide morphological, functional, and molecular evidence that the endothelial cells in capillaries adjacent to the MP is a target of diabetic damage in a regional manner.

Abstract 17307: Endothelial Nitric Oxide Synthase Promotes a Shift in Adipocyte Phenotype in Diet-Induced Obesity

Obesity and diabetes are the most pressing health issues of our time. Current CDC estimates show that over two-thirds of American adults are overweight or obese and nearly 10% display frank type 2 diabetes. Although a decrease in nitric oxide (NO) bioavailability has been linked to insulin resistance, it is not known how increasing NO affects diet-induced obesity and diabetes. We hypothesized that overexpression of endothelial nitric oxide synthase (eNOS) would mitigate diet-induced obesity and insulin resistance. Mice overexpressing eNOS (eNOS TG ) and wild-type C57BL/6 mice (WT) were placed on either 60% high fat or 10% low fat diet for six weeks. The eNOS TG mice showed a striking resistance to diet-induced obesity compared with WT mice, as evidenced by decreased weight gain (4.29±1.04g vs. 9.00±1.52g; n=13, p&lt;0.05), despite nearly identical increases in plasma cholesterol and similar glucose and insulin intolerance. This resistance to obesity was further supported by the differences evident in adipocyte morphology. The mean adipocyte area of the high fat-fed eNOS TG mice was decreased compared with WT mice (1949±461µm 2 vs. 4022±249µm 2 n=7, p&lt;0.05). Adipose tissue of eNOS TG mice also showed increased mitochondrial content compared with WT mice. Protein levels of the mitochondrial proteins cytochrome c oxidase subunit 1 and aldehyde dehydrogenase 2, as well as PGC1α and Sirt3 were increased by two fold or greater (p&lt;0.05). However, no increase in UCP-1 levels was detected. Extracellular flux analysis of differentiated 3T3 adipocytes treated with an NO donor (DETA-NONOate) showed increases in oxidative phosphorylation, glycolysis, and fatty acid oxidation compared with untreated adipocytes (n=5, p&lt;0.05). The triglyceride content of differentiated 3T3 adipocytes was decreased by nearly half after incubation with NO in culture. These data suggest that overexpression of eNOS prevents diet-induced obesity and promotes a shift in the adipocytes to a previously un-described phenotype characterized by increased mitochondria and fat oxidation capacity.

Adipose tissue-derived stem cells embedded with eNOS restore cardiac function in acute myocardial infarction model

ObjectiveThis study assessed the potential therapeutic efficacy of endothelial NO syntheses (eNOS)-expressing adipose tissue-derived stem cells (ADSCs) on infarcted hearts.MethodsWe isolated CD29+, CD44+, CD45 cells from adipose tissue. We hypothesised...