Restored Nitric Oxide Production Research Articles

TXNIP in liver sinusoidal endothelial cells ameliorates alcohol-associated liver disease via nitric oxide production.

Dysregulation of liver sinusoidal endothelial cell (LSEC) differentiation and function has been reported in alcohol-associated liver disease (ALD). Impaired nitric oxide (NO) production stimulates LSEC capillarization and dysfunction; however, the mechanism underlying NO production remains unclear. Here, we investigated the role of thioredoxin-interacting protein (TXNIP), an important regulator of redox homeostasis, in endothelial cell NO production and its subsequent effects on ALD progression. We found that hepatic TXNIP expression was upregulated in patients with ALD and in ethanol diet-fed mice with high expression in LSECs. Endothelial cell-specific Txnip deficiency (TxnipΔEC) in mice exacerbated alcohol-induced liver injury, inflammation, fibrosis, and hepatocellular carcinoma development. Deletion of Txnip in LSECs led to sinusoidal capillarization, downregulation of NO production, and increased release of proinflammatory cytokines and adhesion molecules, whereas TXNIP overexpression had the opposite effects. Mechanistically, TXNIP interacted with transforming growth factor β-activated kinase 1 (TAK1) and subsequently suppressed the TAK1 pathway. Inhibition of TAK1 activation restored NO production and decreased the levels of proinflammatory cytokines, thereby, blocking liver injury and inflammation in TxnipΔEC mice. Our findings indicate that upregulated TXNIP expression in LSECs serves a protective role in ameliorating ALD. Enhancing TXNIP expression could, therefore, be a potential therapeutic approach for ALD.

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion Injury.

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NO, way to go: critical amino acids to replenish nitric oxide production in treating mucositis

Purpose of review There is still an unmet need for preventive and treatment strategies for chemotherapy-induced and radiotherapy-induced mucositis and its associated systemic inflammatory response (SIR) in cancer patients. Because of citrulline depletion due to cytotoxic therapy, nitric oxide (NO) production can be reduced, limiting its effect in many physiological processes. Restoring NO production could relieve mucositis severity by supporting host damage control mechanisms. Amino acids glutamine, arginine and citrulline are involved in NO production. This review including recent literature of preclinical and clinical studies will discuss the potential benefits of glutamine, arginine and citrulline on mucositis development with focus on NO production. Recent findings Mucositis severity is more defined by host response to DNA damage than by DMA damage itself. Citrulline depletion because of afunctional enterocytes could be responsible for NO depletion during cytotoxic therapy. Restoring NO production during cytotoxic therapy could have a beneficial effect on mucositis development. Citrulline seems a more promising NO donor than glutamine or arginine during cytotoxic therapy, although clinical studies in mucositis patients are currently lacking. Summary Glutamine, arginine and citrulline show in-vitro beneficial effects on inflammatory processes involved in mucositis. Translation to the clinic is difficult as demonstrated with use of glutamine and arginine. Citrulline, being the most potent NO donor with excellent oral bio-availability, is very promising as treatment choice for mucositis and its use deserves to be investigated in clinical trials with mucositis patients.

Hydroxysafflor Yellow A inhibits the viability and migration of vascular smooth muscle cells induced by serum from rats with chronic renal failure via inactivation of the PI3K/Akt signaling pathway.

It has been reported that the viability and migration of vascular smooth muscle cells contributes to arteriovenous fistula stenosis. Hydroxysafflor Yellow A (HSYA) has been demonstrated to inhibit the viability and migration of VSMCs by regulating Akt signaling. The present study aimed to investigate the role of HSYA on the viability and migration of human umbilical vein smooth muscle cells (HUVSMCs) following stimulation using serum from rats with chronic renal failure (CRF), and to determine the effects of HSYA on PI3K/Akt signaling. Wistar rats were randomly divided into two groups, control and CRF groups. Serum from each group was collected to stimulate the HUVSMCs. Cell Counting Kit-8 and wound healing assays were performed to assess cell viability and migration, respectively. Flow cytometry analysis was performed to assess apoptosis, and western blot analysis was performed to detect protein expression levels of PI3K and Akt. Nitric oxide (NO) production was measured using the Nitrate/Nitrite assay kit. The results demonstrated that serum from CRF rats significantly enhanced cell viability, migration and apoptosis, the effects of which were reversed following treatment with HSYA. In addition, CRF serum decreased NO and endothelial NO synthase expression, whilst increasing the protein expression levels of PI3K and phosphorylated-Akt in HUVSMCs. Notably, treatment with HSYA markedly restored NO production and inactivated the PI3K/Akt signaling pathway. Furthermore, the PI3K/Akt inhibitor, AMG511, exerted similar effects to HSYA. Taken together, the results of the present study suggest that HSYA suppresses cell viability and migration in the presence of CRF serum by inactivating the PI3K/Akt signaling pathway.

Nitric oxide and bone: The phoenix rises again.

The involvement of nitric oxide (NO) in preventing bone loss has long been hypothesized, but despite decades of research the mechanisms remain obscure. In this issue of the JCI, Jin et al. explored NO deficiency using human cell and mouse models that lacked argininosuccinate lyase (ASL), the enzyme involved in synthesizing arginine and NO production. Osteoblasts that did not express ASL produced less NO and failed to differentiate. Notably, in the context of Asl deficiency, heterozygous deletion of caveolin 1, which normally inhibits NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass. These experiments suggest that ASL regulates arginine synthesis in osteoblasts, which leads to enhanced NO production and increased glucose metabolism. After a period when research slowed, these studies, like the legendary phoenix, renew the exploration of NO in bone biology, and provide exciting translational potential.

L-Citrulline Supplementation Increases Plasma Nitric Oxide Levels and Reduces Arginase Activity in Patients With Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is becoming a major contributor to cardiovascular disease. One of the early signs of T2DM associated cardiovascular events is the development of vascular dysfunction. This dysfunction has been implicated in increasing the morbidity and mortality of T2DM patients. One of the important characteristics of vascular dysfunction is the impaired ability of endothelial cells to produce nitric oxide (NO). Additionally, decreases in the availability of NO is also a major contributor of this pathology. NO is produced by the activity of endothelial NO synthase (eNOS) on its substrate, L-arginine. Reduced availability of L-arginine to eNOS has been implicated in vascular dysfunction in diabetes. Arginase, which metabolizes L-arginine to urea and ornithine, competes directly with NOS for L-arginine. Hence, increases in arginase activity can decrease arginine levels, reducing its availability to eNOS and decreasing NO production. Diabetes has been linked to elevated arginase and associated vascular endothelial dysfunction. We aimed to determine levels of plasma NO and arginase activity in (T2DM) patients and the effects of L-citrulline supplementation, a natural arginase inhibitor, on inhibiting arginase activity in these patients. Levels of arginase correlated with HbA1c levels in diabetic patients. Twenty-five patients received L-citrulline supplements (2000 mg/day) for 1 month. Arginase activity decreased by 21% in T2DM patients after taking L-citrulline supplements. Additionally, plasma NO levels increased by 38%. There was a modest improvement on H1Ac levels in these patients, though not statistically significant. The effect of L-citrulline on arginase activity was also studied in bovine aortic endothelial cells (BAECs) grown in high glucose (HG) conditions. HG (25 mM, 72 h) caused a 2-fold increase in arginase activity in BAECs and decreased NO production by 30%. L-citrulline (2.5 mM) completely prevented the increase in arginase activity and restored NO production levels. These data indicate that L-citrulline can have therapeutic benefits in diabetic patients through increasing NO levels and thus maintaining vascular function possibly through an arginase inhibition related pathway.

Fumarase Overexpression Abolishes Hypertension Attributable to endothelial NO synthase Haploinsufficiency in Dahl Salt-Sensitive Rats.

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through an arginine regeneration pathway. We examined the role of the fumarase-NO pathway in the development of hypertension using genetically engineered rat models. Dahl salt-sensitive (SS) rats with heterozygous mutation of eNOS (endothelial NO synthase or Nos3; SS-Nos3+/-) were bred with SS rats with a hemizygous Fh transgene. SS-Nos3+/- rats without the Fh transgene (SS-Nos3+/-/Fh0/0) developed substantial hypertension with a mean arterial pressure of 134.2±3.7 mm Hg on a 0.4% NaCl diet and 178.0±3.5 mm Hg after 14 days on a 4% NaCl diet. Mean arterial pressure decreased remarkably to 123.1±1.4 mm Hg on 0.4% NaCl, and 143.3±1.5 mm Hg on 4% NaCl in SS-Nos3+/- rats with a Fh transgene (SS-Nos3+/-/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS-Nos3+/-/Fh0/1 rats compared with SS-Nos3+/-/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression in rats on the 0.4% NaCl diet. However, the decrease in NO metabolite in the renal outer medulla of SS-Nos3+/-/Fh0/0 rats on the 0.4% NaCl diet was reversed in SS-Nos3+/-/Fh0/1 rats, and levels of L-arginine, but not the other 12 amino acids analyzed, were significantly higher in SS-Nos3+/-/Fh0/1 rats than in SS-Nos3+/+/Fh0/0 rats. In conclusion, fumarase has potent effects in restoring NO production and blunting the development of hypertension attributable to eNOS haploinsufficiency.

Fumarase Overexpression Abolishes Hypertension Attributable to eNOS Haploinsufficiency in Dahl Salt‐Sensitive Rats

BackgroundHuman blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through a malate‐aspartate‐arginine pathway.ObjectiveWe examined the role of the fumarase‐NO pathway in the development of hypertension in the genetic background of the Dahl salt‐sensitive (SS) rat, a model of human salt‐sensitive hypertension and renal injury used widely.Methods and ResultsSS rats with heterozygous mutation of endothelial NO synthase (eNOS or Nos3) (SS‐Nos3+/−) were bred with SS rats with a hemizygous Fh transgene. SS‐Nos3+/− rats without the Fh transgene (SS‐Nos3+/−/Fh0/0) developed substantial hypertension with a mean arterial pressure (MAP) of 138.5 ± 3.8 mmHg on a 0.4% NaCl diet, and 183.0 ± 3.7 mmHg after 14 days on a 4% NaCl diet. MAP decreased remarkably to 123.1 ± 1.4 mmHg on 0.4% NaCl, and 143.3 ± 1.5 mmHg on 4% NaCl in SS‐Nos3+/− rats with a Fh transgene (SS‐Nos3+/−/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS‐Nos3+/−/Fh0/1 rats compared to SS‐Nos3+/−/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression. However, the decrease in NO metabolite in the renal outer medulla of SS‐Nos3+/−/Fh0/0 rats was reversed in SS‐Nos3+/−/Fh0/1 rats. Levels of L‐arginine, but not the other 12 amino acids analyzed, were significantly higher in SS‐Nos3+/−/Fh0/1 rats than in SS‐Nos3+/+/Fh0/0 rats.ConclusionsFumarase has potent effects in restoring NO production in renal outer medulla and blunting the development of hypertension attributable to eNOS haploinsufficiency.Support or Funding InformationThis work was supported by US National Institutes of Health grant HL116264‐5244 (M.L.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

G protein-coupled receptor kinase 2 inhibition improves erectile function through amelioration of endothelial dysfunction and oxidative stress in a rat model of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a common cause of erectile dysfunction (ED). It has been demonstrated that G protein-coupled receptor kinase 2 (GRK2) overexpression contributes to diabetic endothelial dysfunction and oxidative stress, which also underlies ED in T2DM. We hypothesized that GRK2 overexpressed and attenuated endothelial function of the cavernosal tissue in a rat model of T2DM. T2DM rats were established by feeding with a high-fat diet (HFD) for 2 weeks and then administering two intraperitoneal (IP) injections of a low dose of streptozotocin (STZ), followed by continuous feeding with a HFD for 6 weeks. GRK2 was inhibited by IP injection of paroxetine, a selective GRK2 inhibitor, after STZ injection. Insulin challenge tests, intracavernous pressure (ICP), GRK2 expression, the protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit gp91phox, nitric oxide (NO), reactive oxygen species (ROS) production, and apoptosis in cavernosal tissue were examined. Less response to insulin injection was observed in T2DM rats 2 weeks after HFD. Markedly increased GRK2 expression, along with impaired Akt/eNOS pathway, reduced NO production, increased gp91phox expression and ROS generation, increased apoptosis and impaired erectile function were found in T2DM rats. Inhibition of GRK2 with paroxetine ameliorated Akt/eNOS signaling, restored NO production, downregulated NADPH oxidase, subsequently inhibited ROS generation and apoptosis, and ultimately preserved erectile function. These results indicated that GRK2 upregulation may be an important mechanism underlying T2DM ED, and GRK2 inhibition may be a potential therapeutic strategy for T2DM ED.

Arginase inhibition improves vascular function and restores NO production in diabetic conditions

Cardiovascular complications of diabetes are a leading cause of morbidity and mortality. Vascular endothelial dysfunction (VED) is strongly implicated in the pathogenesis of diabetic vascular complications. Diabetes has been linked to elevated arginase and associated with vascular endothelial dysfunction. The pathological process is characterized by impaired endothelial cell production of nitric oxide (NO) and/or decreased NO bioavailability. NO is produced by activity of endothelial NO synthase (eNOS) on its substrate L‐arginine. Arginase in ECs also uses L‐arginine as a substrate to produce urea and ornithine. Thus, elevated levels of arginase will limit availability of L‐arginine to eNOS and decrease NO production.We performed analysis of arginase activity in type 2 diabetes mellitus (T2DM). Arginase activity was elevated in diabetes patients as compared to age‐matched healthy volunteers. Levels of arginase activity had a positive correlation with HbA1c levels in diabetic patients (R2=0.8 Pearson r=0.87). Cell studies also agreed with these findings, as high glucose (25 mmol/L, 72 hrs) treatment to ECs resulted in a significant increase in arginase activity. This increase in arginase activity was concomitant with a 27% drop in NO produced by EC. Inhibition of arginase by ABH (100 uM) restored NO level to normal.The effects of L‐citrulline, a natural arginase inhibitor, on inhibiting arginase activity in T2DM patients was assessed. Twenty five patients received L‐citrulline supplements (2000 mg/day) for 1 month. Arginase activity was decreased 30% in T2DM patients after taking L‐citrulline supplements. There was a modest improvement on HbA1c levels in these patients. The effect on L‐citrulline on arginase activity was also measured in BAECs grown in high glucose conditions. L‐citrulline 100μM completely prevented the increase in arginase activity and restored NO production levels. These data indicate that inhibiting arginase activity can have therapeutic benefits in diabetic patients by preventing vascular dysfunction and maintaining NO levels.Our results indicate that arginase can be regarded as a novel marker for the vascular complications of diabetes. Drugs targeting arginase or its signaling pathway may show benefits in delaying or preventing these vascular complications.Support or Funding InformationThe University of Jordan deanship of researchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Poly(ADP-ribose) polymerase 1 deficiency increases nitric oxide production and attenuates aortic atherogenesis through downregulation of arginase II.

Poly (ADP-ribose) polymerase (PARP) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular-related diseases. However, whether PARP regulates nitric oxide (NO), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP-1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E-deficient (apoE-/- ) mice fed a high-cholesterol diet for 12weeks, PARP-1 inhibition via treatment with 3,4-dihydro-54-(1-piperindinyl) butoxy-1(2H)-isoquinoline (DPQ) or PARP-1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells (MAECs), PARP-1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase (eNOS) was preserved in the aortas and MAECs when PARP-1 was inhibited. Reduced NO production in vitro due to PARP-1 deficiency could be restored by treating the MAECs with oxidized low-density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP-1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity.

319 - Nitric Oxide-Mediated Angiogenesis Is Improved by β3 Adrenergic Receptor Stimulation

Background Angiogenesis is essential for vascular regeneration following ischemic disease and nitric oxide (NO) is a key regulator of this. β3 adrenergic receptor (β3AR) agonists are known to activate endothelial nitric oxide synthase (eNOS) and we have recently demonstrated that this is at least partially mediated by their ability to decrease glutathionylation-mediated uncoupling of the enzyme, restoring NO production, and decreasing superoxide generation. We hypothesized that β3AR stimulation would promote angiogenesis. Methods and Results: Hindlimb ischemia in mice caused a drastic reduction in limb perfusion, with a gradual reperfusion indicative of angiogenesis. Mice treated with a β3AR agonist, CL 316, 243 (1mg/kg/day subcutaneously via osmotic minipump) showed a more rapid improvement, with reperfusion returning to pre-ischemic levels by day 10 post-ischemia compared with only ~30% reperfusion in control mice (P Conclusion β3AR stimulation increased angiogenesis in vitro and in vivo, likely by an NO-dependent mechanism. As β3AR agonists are approved in some countries for treating non-cardiovascular conditions they may prove a novel and imminently translatable therapy for diseases involving impaired angiogenesis, such as peripheral artery disease.

L-citrulline prevents asymmetric dimethylarginine-mediated reductions in nitric oxide and nitrosative stress in primary human airway epithelial cells.

Asthma is associated with reduced systemic levels of l-arginine and increased asymmetric dimethylarginine (ADMA). This imbalance leads to nitric oxide synthase (NOS) uncoupling with reduced nitric oxide (NO) formation and greater oxidative and nitrosative stress. Whether this imbalance also occurs in bronchial epitheliumof asthmatics is unknown. We used primary human bronchial epithelial cells (HBECs) from asthmatics and healthy controls to evaluate: (i) ADMA-mediated NOS uncoupling reduces epithelial production of NO and increases oxygen and nitrogen reactive species, and (ii) l-citrulline can reverse this mechanism by recoupling NOS, restoring NO production and reducing oxidative and nitrosative stress. In HBECsIL-13 and INFγ stimulated NOS2 and increased NOx levels. The addition of ADMA reduced NOx and increased H2 O2 levels (p<0.001). Treatment with l-citrulline (800, 1600 μm) rescued NOx when the l-arginine media concentration was 25 μm but failed to do so with higher concentrations (100 μm). Under reduced l-arginine media conditions, HBECs treated with l-citrulline increased the levels of argininosuccinate, an enzyme that metabolizes l-citrulline to l-arginine. l-citrulline prevented the ADMA-mediated increase in nitrotyrosine in HBECs in cells from asthmatics and controls. Increasing ADMA reduces NO formation and increases oxidative and nitrosative stress in airway epithelial cells. l-citrulline supplementation restores NO formation, while preventing nitrosative stress. These results, suggest that l-citrulline supplementation may indeed be a powerful approach to restore airway NO production and may have a therapeutic potential in diseases in which there is a defective production of NO.

Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension.

The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by β-blocker also restored NO formation by PAH cells, identifying one mechanism by which β-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions.

Abstract 11602: Direct Evidence That Vascular Endothelial Protection Leads to aa Reduction of Heart Failure

Introduction: Heart failure (HF) induces endothelial dysfunction, and especially decreases nitric oxide (NO) production, but the direct link between endothelial dysfunction and aggravation of HF is unknown. We previously reported a new treatment of endothelial dysfunction, based on inhibition of protein tyrosine phosphatase 1B (PTP1B), which both increases NO production and reduces cardiac dysfunction in HF. However the exact role of endothelial PTP1B in these effects is unknown. Hypothesis: Specific endothelial deletion of PTP1B reduces HF by limiting endothelial dysfunction Methods: Endothelial deletion of PTP1B was obtained by crossing LoxP-PTP1B mice with mice expressing Cre recombinase under the control of the endothelial promoter Tie2. Since this Tie2-Cre approach is known to also induce gene extinction in hematopoietic cells, Tie2Cre(+)PTP1Bf/f mice were also lethally irradiated and reinjected with bone marrow from wild type (WT; CD45.1) mice, in order to obtain a specific deletion of endothelial PTP1B (endoPTP1B-/- mice). All mice were then subjected to 4 months of myocardial infarction (MI) in order to induce HF, or to sham surgery Results: Vascular function evaluated ex vivo in mesenteric arteries (arteriograph) showed that in WT mice, HF markedly impaired endothelium-dependent, flow-mediated dilatation (sham: 40±4%; HF: 5±5; p&lt;0.001). This HF-induced endothelial dysfunction was less marked in Tie2Cre(+)PTP1Bf/f (MI: 30±5; p&lt;0.001 vs. WT) and in endoPTP1B-/- mice (35±6%). This improved response in endoPTP1B-/- mice were abolished by a NO-synthase inhibitor, suggesting a restored NO production. Evaluation of cardiac function by echocardiography showed that in WT, MI decreased left ventricular fractional shortening (sham: 49±2; HF: 12±1%; p&lt;0.001) and which was improved in Tie2Cre(+)PTP1Bf/f (26±1%; p&lt;0.01 vs. WT), and in endoPTP1B-/- mice (29±1%; p&lt;0.01 vs. WT). In parallel, endothelial PTP1B deficiency was associated with an increased survival. Conclusions: Endothelial PTP1B deficiency induced an improvement of endothelial function but especially improved cardiac function. These results provide a direct demonstration of the beneficial effect of endothelial protection in HF.

Abstract 15199: L-Citrulline Supplementation Reduces Plasma Arginase Activity in Type 2 Diabetes Patients

Vascular dysfunction is a major cause of morbidity and mortality in diabetic patients. Diabetes has been linked to elevated arginase and associated with vascular endothelial dysfunction. The pathological process is characterized by impaired endothelial cell production of nitric oxide (NO) and/or decreased NO bioavailability. NO is produced by activity of endothelial NO synthase (eNOS) on its substrate L-arginine. Reduced availability of L-arginine to eNOS has been implicated in vascular dysfunction in diabetes. Arginase, which metabolizes L-arginine to urea and ornithine, competes directly with NOS for L-arginine. Hence increases in arginase activity can decrease arginine levels, reducing its availability to eNOS and decreasing NO production. We aimed to determine levels of plasma arginase activity in type 2 diabetic (T2DM) patients and the effects of L-citrulline, a natural arginase inhibitor, on inhibiting arginase activity in T2DM patients. We determined arginase activity levels in 62 T2DM patients and compared it to age matched healthy volunteers (ages 45-65). Our results show that arginase activity is 1.7 fold higher in T2DM patient’s plasma than healthy controls. Levels of arginase correlated with H1AC levels in diabetic patients r=0.81. Twenty five patients received L-citrulline supplements (2000 mg/day) for 1 month. Arginase activity was decreased 30% in T2DM patients after taking L- citrulline supplements. There was a modest improvement on H1Ac levels in these patients. The effect on L-citrulline on arginase activity was also measure in bovine aortic endothelial cells (BAECs) grown in high glucose (HG) conditions. HG (25mM, 72 hrs) caused a 2 fold increase in arginase activity in BAECs and decreased NO production by 40%. L-citrulline 100μM completely prevented the increase in arginase activity and restored NO production levels. These data indicate that inhibiting arginase activity can have therapeutic benefits in diabetic patients by preventing vascular dysfunction and maintaining NO levels.

Vascular and hepatic impact of short-term intermittent hypoxia in a mouse model of metabolic syndrome.

BackgroundExperimental models of intermittent hypoxia (IH) have been developed during the last decade to investigate the consequences of obstructive sleep apnea. IH is usually associated with detrimental metabolic and vascular outcomes. However, paradoxical protective effects have also been described depending of IH patterns and durations applied in studies. We evaluated the impact of short-term IH on vascular and metabolic function in a diet-induced model of metabolic syndrome (MS).MethodsMice were fed either a standard diet or a high fat diet (HFD) for 8 weeks. During the final 14 days of each diet, animals were exposed to either IH (1 min cycle, FiO2 5% for 30s, FiO2 21% for 30s; 8 h/day) or intermittent air (FiO2 21%). Ex-vivo vascular reactivity in response to acetylcholine was assessed in aorta rings by myography. Glucose, insulin and leptin levels were assessed, as well as serum lipid profile, hepatic mitochondrial activity and tissue nitric oxide (NO) release.ResultsMice fed with HFD developed moderate markers of dysmetabolism mimicking MS, including increased epididymal fat, dyslipidemia, hepatic steatosis and endothelial dysfunction. HFD decreased mitochondrial complex I, II and IV activities and increased lactate dehydrogenase (LDH) activity in liver. IH applied to HFD mice induced a major increase in insulin and leptin levels and prevented endothelial dysfunction by restoring NO production. IH also restored mitochondrial complex I and IV activities, moderated the increase in LDH activity and liver triglyceride accumulation in HFD mice.ConclusionIn a mouse model of MS, short-term IH increases insulin and leptin levels, restores endothelial function and mitochondrial activity and limits liver lipid accumulation.

Role of Sigma-1 Receptor in Cocaine-Induced Endothelial Cell Damage

Background :Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We showed that chronic cocaine use is associated with markers of endothelial dysfunction (ED) (Sáez et al. Thromb Res 2011; 128:18). The hallmark of ED is a change to a prothrombotic and proadhesive phenotype associated with a decreased bioavailability of nitric oxide (NO). Several experimental studies have shown that cocaine induces ED in vitro and in vivo; however, the receptor and the signaling pathways involved in the interaction of cocaine with endothelial cells (EC) are unknown. Sigma-1 receptor (sigma-1R) belongs to the family of nonopioid sigma receptors that regulate a variety of cellular functions in neuronal, glial and peripheral cells and are involved in pathological conditions such as neurodegenerative diseases, drug addiction, smooth muscle cell contraction and ischemia. Cocaine binding to sigma-1R has been demonstrated in microglial cell lines, where is located in the lipid raft microdomains of the endoplasmic reticulum being translocated into the plasma membrane upon activation. The effect of cocaine on sigma-1R expression and function in systemic endothelial cells EC are largely unknown. We hypothesize that activation of sigma-1R in EC and its downstream signaling pathway may be involved in the pathogenesis of cocaine-associated endothelial dysfunction, a key phenomenon in the onset and progression of atherosclerosis.Objectives:The main aim of this study was to demonstrate the presence of sigma-1R in EC and its role in cocaine-induced EC damage/activation.Methods:Human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions and exposed to cocaine (10μM) or vehicle in the presence or absence of BD1047 (sigma-1 receptor antagonist). Sigma-1R was demonstrated by western blotting using rabbit-anti-OPRS1 polyclonal antibody. Sub-cellular distribution of sigma-1R and von Willebrand factor (VWF) bound to the membrane were studied by indirect immunofluorescence (IF) microscopy. The production of NO was determined by fluorometric assay using DAF-2DA (5μM) and results expressed as arbitrary fluorescence units (AFU). Platelet adhesion under static conditions was assessed by co-incubation of resting platelets with cultured HUVEC; platelets and EC were identified and quantified by IF microscopy using anti-glycoprotein Ib and anti-VE-cadherin antibodies, respectively.Results:HUVEC expressed sigma-1R protein as demonstrated by Western blot analysis. By IF the receptor showed a perinuclear pattern in untreated HUVEC. Treatment with cocaine resulted in translocation to the cytoplasm and suggest co-localization with actin. These changes were abrogated completely by pre-incubation of the cells with BD1047. HUVEC treated with cocaine exhibited an increase in VWF deposited on the cell membrane with the formation of strings, phenomena that were inhibited by incubation with BD1047. With respect to platelet adhesion, cocaine induced a significant increase in the number of platelets adhered to HUVEC as compared with the controls (46±24 versus 13.3±7 platelets per cell, respectively), which was drastically reduced by BD1047 (10.5± 6.3 platelets per cell). NO production in HUVEC exposed to cocaine was significantly reduced with respect to cells treated with vehicle (10.7±0.67 vs 19.5± 6.4 nM/ml, P=0.002), the use of BD1047 only partially restored NO production (12.1±2.5 nM/ml).Conclusions: Our results show that in cultured HUVEC expressed sigma 1-R with a sub-cellular distribution and changes upon cell activation similar to those observed in other cell systems. The blockade of sigma-1R inhibited significantly the pro-adhesive and pro-thrombotic changes observed in HUVEC exposed to cocaine, which support the notion that the receptor may play an important role in cocaine-induce endothelial cell dysfunction. These findings have implications for the management of drug addiction, since sigma-1R antagonists may provide a novel therapeutic intervention to protect the endothelium from the deleterious effects associated with cocaine abuse. DisclosuresNo relevant conflicts of interest to declare.

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy.

Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca(2+) concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca(2+) content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy.