Increasing Nitric Oxide Bioavailability Research Articles

Interactions between adenosine, angiotensin II and nitric oxide on the afferent arteriole influence sensitivity of the tubuloglomerular feedback

Adenosine, via activation of A1 receptors on the afferent arteriole (AA), mediates the tubuloglomerular feedback (TGF) mechanism. Angiotensin II and nitric oxide (NO) can modulate the sensitivity of the TGF mechanism. However, the interaction among these substances in regulating the TGF resetting phenomenon has been debated. Studies in isolated perfused AA have shown a biphasic response to accumulating doses of adenosine alone. In the nanomolar range adenosine has a weak contractile effect (7%), whereas vasodilatation is observed at high concentrations. However, a synergistic interaction between the contractile response by adenosine and that of angiotensin II has been demonstrated. Adenosine in low concentrations strongly enhances the response to angiotensin II. At the same time, angiotensin II in physiological concentrations increases significantly the contractile response to adenosine. Moreover, addition of a NO donor (spermine NONOate) to increase NO bioavailability abolished the contractile response from combined application of angiotensin II and adenosine. These mutual modulating effects of adenosine and angiotensin II, and the effect of NO on the response of AA can contribute to the resetting of the TGF sensitivity.

Boldine protects endothelial function in hyperglycemia-induced oxidative stress through an antioxidant mechanism

Increased oxidative stress is involved in the pathogenesis and progression of diabetes. Antioxidants are therapeutically beneficial for oxidative stress-associated diseases. Boldine ([s]-2,9-dihydroxy-1,10-dimethoxyaporphine) is a major alkaloid present in the leaves and bark of the boldo tree (Peumus boldus Molina), with known an antioxidant activity. This study examined the protective effects of boldine against high glucose-induced oxidative stress in rat aortic endothelial cells (RAEC) and its mechanisms of vasoprotection related to diabetic endothelial dysfunction. In RAEC exposed to high glucose (30mM) for 48h, pre-treatment with boldine reduced the elevated ROS and nitrotyrosine formation, and preserved nitric oxide (NO) production. Pre-incubation with β-NAPDH reduced the acetylcholine-induced endothelium-dependent relaxation; this attenuation was reversed by boldine. Compared with control, endothelium-dependent relaxation in the aortas of streptozotocin (STZ)-treated diabetic rats was significantly improved by both acute (1μM, 30min) and chronic (20mg/kg/daily, i.p., 7 days) treatment with boldine. Intracellular superoxide and peroxynitrite formation measured by DHE fluorescence or chemiluminescence assay were higher in sections of aortic rings from diabetic rats compared with control. Chronic boldine treatment normalized ROS over-production in the diabetic group and this correlated with reduction of NAD(P)H oxidase subunits, NOX2 and p47phox. The present study shows that boldine reversed the increased ROS formation in high glucose-treated endothelial cells and restored endothelial function in STZ-induced diabetes by inhibiting oxidative stress and thus increasing NO bioavailability.

Abstract 18980: Cardioprotective Role of NO Derived from Sodium Nitrite in Attenuating Severity of Chronic Ischemia-Induced Heart Failure

Background: Nitric oxide (NO) bioavailability is reduced in the setting of congestive heart failure. Nitrite (NO 2 ) is a critically important NO intermediate that is readily metabolized to NO during ischemic conditions. We have previously demonstrated that NO 2 - ameliorates acute myocardial ischemia/reperfusion (MI/R). However, no evidence exists as to whether increasing NO bioavailability through NO 2 - therapy attenuates chronic heart failure following acute myocardial infarction. Methods: Male C57/BL6J Mice (n=13-15 per group) at 12-14 weeks of age underwent 60 min. of myocardial ischemia (MI) induced by transient LCA occlusion followed by 4 weeks of reperfusion. Sodium nitrite (NaNO 2 ) or saline vehicle (VEH) was administered at a dose of 165 µg/kg (intracardiac injection) at reperfusion and then daily for 4 weeks in the drinking water (50 mg/L or 100 mg/L). Two-dimensional echocardiography was performed at baseline and 4 weeks post MI to assess left ventricular (LV) dimensions and ejection fraction (EF). Myocardial and blood NO metabolite levels were measured using HPLC and chemiluminescence methods. Results: Both circulating and cardiac NaNO 2 levels were increased in mice treated with oral NaNO 2 . Furthermore, NaNO 2 (100 mg/L) significantly preserved LVEF (47 ± 4% vs. 32 ± 4%, p < 0.01) and improved left ventricular LV dimensions (LVEDD/LVESD; 4.0/3.1 mm vs. 4.5/3.9 mm, p<0.05) at 4 weeks when compared to VEH. Conclusions : These data demonstrate that sodium nitrite therapy significantly improves LV function during heart failure following acute myocardial infarction. Studies are currently underway to define the cellular and molecular mechanisms by which NO derived from NaNO 2 protects the myocardium in the setting of heart failure.

Protective Effect of Fetal Hemoglobin On Inflammation-Induced Expression of Hypoxia-Inducible Factor-1α (HIF-1α) in Sickle Mice: Microvascular Implications

AbstractAbstract 3250Chronic inflammation is a salient feature of human sickle cell disease (SCD) and transgenic-knockout sickle (BERK) mouse model. Although tissue ischemia is the primary instigator of hypoxia-inducible factor (HIF) activation, a number of inflammatory factors/pathways and oxidative stress can potentially induce expression of HIF-1α. Increased oxidative stress and inflammation are implicated in the activation of HIF-1α under normoxic conditions. HIF can trigger transcription of genes for vasoactive molecules such as vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1) and endothelin, which are implicated in the pathophysiology of SCD. We hypothesize that, in SCD, inflammation coupled with nitric oxide (NO) depletion will induce expression of HIF-1α. To this end, we have examined the expression of HIF-1α in normoxic BERK mice expressing exclusively human α- and βS- globins, and evaluated the effect of HbF in BERK mice (i.e., <1.0%, 20% and 40% HbF). We have previously shown that HbF exerts anti-sickling and anti-inflammatory effects (Kaul et al. J Clin Invest, 2004; Dasgupta et al. Am J Physiol, 2010). Here, we show that HIF-1α is expressed in BERK mice under normoxic conditions (i.e., normal hemoglobin oxygen saturation levels). In BERK mice expressing HbF, HIF-1α expression decreased concomitantly with increasing HbF, commensurately with increased NO bioavailability, and showed a strong inverse correlation with plasma NO metabolites (NOx) levels. Reduced HIF-1α expression in BERK mice expressing HbF was associated with decreased HO-1 and VEGF expression, and reduced serum endothelin-1 (ET-1) levels, which are among the target vasoactive molecules of HIF-1α. Furthermore, the commensurate decrease in HIF-1α expression with increase in HbF levels in BERK mice was accompanied by a distinct decrease in soluble (s) forms of endothelial activation markers such as sP-selectin and vascular cell adhesion molecule-1 (sVCAM-1). Notably, arteriolar dilation, enhanced volumetric blood flow and low blood pressure in normoxic BERK mice all showed a trend toward normalization with the introduction of HbF. Also, arginine treatment reduced HIF-1α expression as well as ET-1 levels in normoxic BERK mice, supporting a role of decreased NO bioavailability in HIF-1α activation. The present in vivo studies show that reduced inflammation and increased NO production in normoxic BERK mice (expressing HbF or treated with arginine) are distinctly associated with suppression of HIF-1α activation and inhibition of vasodilators, resulting in improved microvascular and hemodynamic parameters in the BERK model of sickle cell disease. The unique feature of inflammation in SCD is that it can be ameliorated by increased HbF, thereby coupling HbS polymerization/sickling to NO depletion, HIF-1α expression and inflammation in this disease. Disclosures:No relevant conflicts of interest to declare.

Antisickling fetal hemoglobin reduces hypoxia-inducible factor-1α expression in normoxic sickle mice: microvascular implications

Chronic inflammation is a salient feature of sickle cell disease (SCD) and transgenic-knockout sickle (BERK) mice. Inflammation is implicated in the activation of hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. We hypothesize that, in SCD, inflammation coupled with nitric oxide (NO) depletion will induce expression of HIF-1α, a transcription factor with wide-ranging effects including activation of genes for vasoactive molecules. To this end, we have examined the expression of HIF-1α in normoxic BERK mice expressing exclusively human α- and β(S)- globins, and evaluated the effect of fetal hemoglobin (HbF) in BERK mice (i.e., <1.0%, 20%, and 40% HbF). HbF exerts antisickling and anti-inflammatory effects. Here, we show that HIF-1α is expressed in BERK mice under normoxic conditions, accompanied by increased expression of its vasoactive biomarkers such as VEGF, heme oxygenase-1 (HO-1), and serum ET-1 levels. In BERK mice expressing HbF, HIF-1α expression decreases concomitantly with increasing HbF, commensurately with increased NO bioavailability, and shows a strong inverse correlation with plasma NO metabolites (NOx) levels. Reduced HIF-1α expression is associated with decreased HO-1, VEGF, and ET-1. Notably, arteriolar dilation, enhanced volumetric blood flow, and low blood pressure in normoxic BERK mice all show a trend toward normalization with the introduction of HbF. Also, arginine treatment reduced HIF-1α, as well as VEGF expression in normoxic BERK mice, supporting a role of NO bioavailability in HIF-1α activation. Thus HIF-1α expression in normoxic sickle mice is likely a consequence of chronic inflammation, and HbF exerts an ameliorating effect by decreasing sickling, increasing NO bioavailability, and reducing inflammation.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Enhanced Nitric Oxide Release and Reduced Blood Pressure and sICAM-1 Levels in Hypertensive Rats

Most patients with diabetes also have hypertension, a risk factor associated with atherothrombotic disease and characterized by endothelial cell (EC) dysfunction and loss of nitric oxide (NO) bioavailability. Recent studies suggest a possible antihypertensive effect with dipeptidyl peptidase-4 (DPP4) inhibition; however, the underlying mechanism is not understood. In this study, we tested the effects of the DPP4 inhibitor, saxagliptin, on EC function, blood pressure, and soluble intercellular adhesion molecule 1 (sICAM-1) levels in hypertensive rats. Spontaneously hypertensive rats were treated with vehicle or saxagliptin (10 mg·kg(-1)·day(-1)) for 8 weeks. NO and peroxynitrite (ONOO(-)) release from aortic and glomerular ECs was stimulated with calcium ionophore and measured using electrochemical nanosensor technology. Changes in EC function were correlated with fasting glucose levels. Saxagliptin treatment was observed to increase aortic and glomerular NO release by 22% (P < 0.001) and 23% (P < 0.001), respectively, with comparable reductions in ONOO(-) levels; the NO/ONOO(-) ratio increased by >50% in both EC types (P < 0.001) as compared with vehicle. Saxagliptin also reduced mean arterial pressure from 170 ± 10 to 158 ± 10 mm Hg (P < 0.001) and decreased sICAM-1 levels by 37% (P < 0.01). The results of this study suggest that DPP4 inhibition reduces blood pressure and inflammation in hypertensive rats while increasing NO bioavailability.

PPARα activation in portal hypertension

Abstract Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor activated by ligands that regulates genes related to vascular tone, oxidative stress, and fibrogenesis, pathways implicated in the development of cirrhosis and portal hypertension. This study aims at evaluating the effects of PPARα activation with fenofibrate on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl(4)-cirrhotic rats. Mean arterial pressure (MAP), portal pressure (PP), and portal blood flow (PBF) were measured in cirrhotic rats treated with oral fenofibrate (25mg/kg/day, n=10) or its vehicle (n=12) for 7 days. The liver was then perfused and dose-relaxation curves to acetylcholine (Ach) were performed. We also evaluated Sirius Red staining of liver sections, collagen-I mRNA expression, and smooth muscle actin (α-SMA) protein expression, cyclo-oxygenase-1 (COX-1) protein expression, and cGMP levels in liver homogenates, and TXB(2) production in perfusates. Nitric oxide (NO) bioavailability and eNOS activation were measured in hepatic endothelial cells (HEC) isolated from cirrhotic rat livers. CCl(4) cirrhotic rats treated with fenofibrate had a significantly lower PP (-29%) and higher MAP than those treated with vehicle. These effects were associated with a significant reduction in hepatic fibrosis and improved vasodilatory response to acetylcholine. Moreover, a reduction in COX-1 expression and TXB(2) production in rats receiving fenofibrate and a significant increase in NO bioavailability in HEC with fenofibrate were observed. PPARα activation markedly reduced PP and liver fibrosis and improved hepatic endothelial dysfunction in cirrhotic rats, suggesting it may represent a new therapeutic strategy for portal hypertension in cirrhosis.

Cardiomyocyte-restricted overexpression of extracellular superoxide dismutase increases nitric oxide bioavailability and reduces infarct size after ischemia/reperfusion

Increased levels of extracellular superoxide dismutase (ecSOD) induced by preconditioning or gene therapy protect the heart from ischemia/reperfusion injury. To elucidate the mechanism responsible for this action, we studied the effects of increased superoxide scavenging on nitric oxide (NO) bioavailability in a cardiac myocyte-specific ecSOD transgenic (Tg) mouse. Results indicated that ecSOD overexpression increased cardiac myocyte-specific ecSOD activity 27.5-fold. Transgenic ecSOD was localized to the sarcolemma and, notably, the cytoplasm of cardiac myocytes. Ischemia/reperfusion injury was attenuated in ecSOD Tg hearts, in which infarct size was decreased and LV functional recovery was improved. Using the ROS spin trap, DMPO, electron paramagnetic resonance (EPR) spectroscopy demonstrated a significant decrease in ROS in Tg hearts during the first 20 min of reperfusion. This decrease in ROS was accompanied by an increase in NO production determined by EPR using the NO spin trap, Fe-MGD. Attenuated ROS in ecSOD Tg myocytes was also supported by decreased production of peroxynitrite (ONOO−). Increased NO bioavailability was confirmed by attenuated guanylate cyclase-dependent (p-VASP) signaling. In conclusion, attenuation of ROS levels by cardiac-specific ecSOD overexpression increases NO bioavailability in response to ischemia/reperfusion and protects against reperfusion injury. These findings are the first to demonstrate increased NO bioavailability with attenuation of ROS by direct measurement of these reactive species (EPR, reactive fluorescent dyes) with cardiac-specific ecSOD expression. This is also the first indication that the predominantly extracellular SOD isoform is capable of cytosolic localization that affects myocardial intracellular signal transduction and function.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-012-0305-1) contains supplementary material, which is available to authorized users.

TrACEing Angiotensin II Type 1 to Right Ventricular Hypertrophy

TrACEing Angiotensin II Type 1 to Right Ventricular Hypertrophy

Interaction between NO and COX pathways modulating hepatic endothelial cells from control and cirrhotic rats

Reduced intrahepatic nitric oxide (NO) bioavailability and increased cyclooxygenase-1 (COX-1)-derived vasoconstrictor prostanoids modulate the hepatic vascular tone in cirrhosis. We aimed at investigating the reciprocal interactions between NO and COX in the hepatic endothelium of control and cirrhotic rats. NO bioavailability (DAF-FM-DA staining), superoxide (O2−) content (DHE staining), prostanoid production (PGI2 and TXA2 by enzyme immunoassays) as well as COX expression (Western Blot), were determined in hepatic endothelial cells (HEC) from control and cirrhotic rats submitted to different experimental conditions: COX activation, COX inhibition, NO activation and NO inhibition. In control and cirrhotic HEC, COX activation with arachidonic acid reduced NO bioavailability and increased O2− levels. These effects were abolished by pre-treating HEC with the COX inhibitor indomethacin. In control, but not in cirrhotic HEC, scavenging of O2− by superoxide dismutase (SOD) incubation partially restored the decrease in NO bioavailability promoted by COX activation. NO supplementation produced a significant and parallel reduction in PGI2 and TXA2 production in control HEC, whereas it only reduced TXA2 production in cirrhotic HEC. By contrast, in control and cirrhotic HEC, NO inhibition did not modify COX expression or activity. Our results demonstrate that NO and COX systems are closely interrelated in HEC. This is especially relevant in cirrhotic HEC where COX inhibition increases NO bioavailability and NO supplementation induces a reduction in TXA2. These strategies may have beneficial effects ameliorating the vasoconstrictor/vasodilator imbalance of the intrahepatic circulation of cirrhotic livers.

Sodium nitrite protects against kidney injury induced by brain death and improves post-transplant function

Renal injury induced by brain death is characterized by ischemia and inflammation and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 hours of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 hours of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain dead rats showed significant improvement in function over the first 2 to 4 days post transplantation compared to untreated brain dead animals. Gene microarray analysis after 2 hours of brain death without or with nitrite therapy showed the latter significantly altered the expression of about 400 genes. Ingenuity Pathway analysis indicated multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription and genes related to humoral immune responses. Thus, nitrite-therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

Protective effect of dietary flavonoid quercetin against lipemic-oxidative hepatic injury in hypercholesterolemic rats

Context: Quercetin, a dietary-derived flavonoid, is ubiquitous in fruits and vegetables and plays important roles in human health by virtue of its antioxidant activity.Objective: This study was conducted to investigate the possible modulatory effect of quercetin against hepatic lipemic-oxidative injury in rats fed with a high cholesterol diet (HCD), and to highlight the underlying mechanisms of such effect.Materials and methods: Different groups of male Sprague–Dawley rats were used; one group was treated by gavage with HCD cocktail (1 mL/100 g) whereas another group was orally administered HCD-enriched with quercetin (15 mg/kg). Corresponding control animals were also used.Results: Quercetin administration significantly decreased liver triglycerides (24%), liver total cholesterol (TC) (22%), serum TC (20%), serum low-density lipoprotein cholesterol (31%), and duplicated serum high-density lipoprotein cholesterol (HDL-C). This study also revealed that quercetin administration significantly reduced the activity of serum alanine aminotransferase (41%), aspartate aminotransferase (51%), and γ-glutamyl transpeptidase (G-GT) (35%). Significant inhibition of thiobarbituric acid-reacting substances (40%), together with a valuable enhancement of reduced glutathione (GSH) content (53%) in the liver homogenates, was observed. In addition, quercetin-treated hypercholesterolemic animals exhibited a reasonable improvement of hepatic antioxidant enzymes. Moreover, serum and liver content of nitric oxide (NO) were markedly decreased in this model (26 and 25%, respectively), and were almost normalized following quercetin administration.Discussion and conclusion: These data revealed that quercetin has the ability to ameliorate HCD-induced lipemic-oxidative injury in rat liver possibly through its antioxidant potential and/or increased NO bioavailability.

Adiponectin associates positively with nitrite levels in children and adolescents

To compare the circulating levels of adiponectin and nitric oxide (NO) bioavailability in eutrophic, eutrophic hypertensive, obese, and obese hypertensive children and adolescents, and to assess whether adiponectin is associated with increased NO bioavailability in these children and adolescents. We studied 129 eutrophic, 8 eutrophic hypertensive, 91 obese, and 44 obese hypertensive children and adolescents in this cross-sectional study. Adiponectin concentrations were measured in plasma samples by enzyme-linked immunosorbent assay. To assess NO bioavailability, nitrite concentrations were measured in whole-blood samples by chemiluminescence. Multiple linear regression analysis was carried out to assess the effects of adiponectin on NO bioavailability. We found no significant differences in nitrite levels among groups (P>0.05). The obese hypertensive group had the lowest adiponectin levels among groups (P<0.05). Additionally, obese subjects had lower adiponectin levels than eutrophic individuals (P<0.05). A multiple linear regression analysis showed that NO bioavailability was positively associated with adiponectin concentrations (P<0.05). Our findings suggest that adiponectin increases NO bioavailability in children and adolescents. Further studies are needed to assess the cardiovascular protective role for this adipokine in childhood obesity.

Involvment of nitric oxide and MPTP in heart adaptation to physical training

After physical training of rats (4 weeks swimming) we observed in myocardium and aorta the activation of both salvage and de novo nitric oxide synthesis, an increase in nitric oxide bioavailability and endothelium-dependent vasorelaxation. However, it reminds unclear how NO takes part in myocardial adaptation to workload. Using isolated rat heart by Langendorf preparation we studied myocardial response to gradually increased left ventricular volume (Frank-Starling low) and increasing concentration of Ca2+ in perfusion solution (from 1.7 mM to 12,5 mM) in trained and untrained rats. It was shown that swimming improved heart function: heart rate was decreased, contractile activity and coronary flow were increased by 20% and 33%, respectively. Equal volume stretching of balloon in left ventricle provoked greater contraction in trained comparing to untrained hearts, demonstrating extended functional reserves after swimming course. Furthermore, trainingsignificantly increased mitochondrial membrane potential in isolatedmitochondria (−176.5 ± 8.4 mV vs −56 ± 3.5 mV in control), that points to improved efficiency of oxidative phosphorylation. The opening of mitochondrial permeability transition pore (MPTP) in trained heart was detected at higher concentration of calcium that reveals extended calcium capacity of mitochondria and lesser sensitivity of MPTP to its inductor - calcium. Blockade of NO synthesis with L-NAME application of (10–4 M for 15 min) abolished reaction of trained heart during Frank-Starling and calcium upload. NOS inhibition prevented the increased functional hyperaemia of the hearts from trained rats. Thus, heart adaptation to physical training and extend of functional reserves of heart are provided by increased endogenous NO production.

Effect of dietary nitrate on skeletal muscle energetics in normoxia and hypoxia

Hypoxia is associated with a reduction in the maximal oxidative metabolic rate of skeletal muscle which is reflected in a slowing of muscle phosphocreatine (PCr) recovery kinetics following exercise. For the same metabolic rate, there is a greater muscle metabolic perturbation (e.g., greater fall in [PCr]) during exercise performed in hypoxia compared to normoxia. Nitric oxide (NO) is a key signalling molecule for hypoxic vasodilatation and it also modulates mitochondrial O2 consumption. Dietary nitrate intake, which increases NO bioavailability, might therefore improve O2 delivery to active muscle and reduce O2 demand during hypoxic exercise. It has been shown that nitrate supplementation lessens muscle metabolic perturbation during high-intensity exercise and considerably enhances exercise tolerance in moderate hypoxia. Nitrate supplementation has also been shown to abolish the reduction in the rate of PCr recovery which is typically observed in hypoxia, indicating enhanced muscle oxygenation and a restoration of muscle oxidative function. Further research is warranted to identify to what extent these effects can be attributed to changes in muscle energy metabolism and/or improved O2 delivery. These findings indicate that dietary nitrate supplementation may have important therapeutic applications for improving skeletal muscle energetics and functional capacity in conditions where muscle O2 delivery is compromised.

Endothelial cell expression of hemoglobin a regulates nitric oxide signaling

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signaling in many cell systems. For example, endothelial NOS (eNOS) controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion between endothelial and smooth muscle cells. Here, we report for the first time that hemoglobin (Hb) α is expressed in arteriolar endothelial cells, where it is in complex with eNOS. Moreover, we demonstrate that endothelial Hb α is enriched at the myoendothelial junction (MEJ), where it regulates the effect of eNOS on vascular reactivity. Surprisingly, this function is unique to Hb α and abrogated by its genetic depletion. Mechanistically, Hb α heme iron in the Fe3+ state permits active signaling across the MEJ, and this signaling is shut off when Hb α is reduced to the Fe 2+ state by endothelial cytochrome B5 reductase 3 (CytB5R3). Genetic and pharmacological inhibition of CytB5R3 increases NO bioavailability and decreases the reactivity of resistance vessels. These data reveal a novel paradigm by which the regulation of intracellular Hb oxidation controls NOS signaling in non-erythroid cells. This paradigm may be relevant to a broad range of somatic cells discovered to express Hb and known also to express NOS isoforms.

Investigating the interaction between GTP-cyclohydrolase1 and its feedback regulatory protein

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic amino acid-hydroxylases and all isoforms of nitric oxide-synthase. In the cardiovascular system nitric oxide (NO) elicits potent vasodilatory and antithrombotic effects and maintains endothelial function. Administration of BH4 can improve endothelial function in numerous cardiovascular pathologies, an effect that likely results from increased NO bioavailability and/or local antioxidant effects. GTP cyclohydrolase1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. GCH1 activity has been shown to be modified by both levels of phenylalanine and BH4 through allosteric regulation when bound to GCH1-feedback regulatory protein (GFRP). Studies have shown that phenylalanine binds to the complex and activates GCH1, whilst BH4 binding to the complex inhibits GCH1 activity. Our studies aim to investigate the significance of this protein–protein interaction on BH4 bioavailability. We have shown that oral phenylalanine administration (100 mg/kg) in rodents leads to an increase in BH4 production in aortic tissue, suggesting that targeting the GCH1–GFRP complex may provide a means to regulate vascular BH4 bioavailability. Using recombinantly expressed human proteins; the nature of the GCH1–GFRP interaction has been investigated using biophysical techniques. In addition the activity of purified recombinant enzymes has been measured by two methods. Initial results suggest the importance of the first 42 amino acids of the N-terminus of GCH1, and its potential role in mediating the activity of the enzyme and its interaction with GFRP. A greater understanding of GCH1–GFRP protein–protein interactions and how these translate to BH4 bioavailability may help identify novel therapies for a range of cardiovascular diseases.

Translational evidence that impaired autophagy contributes to arterial ageing

Ageing causes arterial endothelial dysfunction that increases the risk of cardiovascular diseases (CVD), but the underlying mechanisms are incompletely understood. The aim of the present study was to determine the role of autophagy, the cellular process of recycling damaged biomolecules, in endothelial dysfunction with ageing. In older humans, expression of autophagy markers in arterial endothelial cells was impaired by ∼50% (P <0.05) and was associated with an ∼30% (P <0.05) reduction in arterial endothelium-dependent dilatation (EDD). Similarly, in C57BL/6 control mice ageing was associated with an ∼40% decrease (P <0.05) in arterial markers of autophagy and an ∼25% reduction (P <0.05) in EDD. In both humans and mice, impaired EDD was mediated by reduced nitric oxide (NO) bioavailability and was associated with increased oxidative stress and inflammation (P <0.05). In old mice, treatment with the autophagy-enhancing agent trehalose restored expression of autophagy markers, rescued NO-mediated EDD by reducing oxidative stress, and normalized inflammatory cytokine expression. In cultured endothelial cells, inhibition of autophagy increased oxidative stress and reduced NO production, whereas trehalose enhanced NO production via an autophagy-dependent mechanism. These results provide the first evidence that autophagy is impaired with ageing in vascular tissues. Our findings also suggest that autophagy preserves arterial endothelial function by reducing oxidative stress and inflammation and increasing NO bioavailability. Autophagy-enhancing strategies may therefore have therapeutic efficacy for ameliorating age-associated arterial dysfunction and preventing CVD.

Uncoupling Endothelial Nitric Oxide Synthase Is Ameliorated by Green Tea in Experimental Diabetes by Re-establishing Tetrahydrobiopterin Levels

The current study investigated the potential of green tea (GT) to improve uncoupling of endothelial nitric oxide synthase (eNOS) in diabetic conditions. In rats with streptozotocin-induced diabetes, nitric oxide (NO) bioavailability was reduced by uncoupling eNOS, characterized by a reduction in tetrahydrobiopterin (BH4) levels and a decrease in the eNOS dimer-to-monomer ratio. GT treatment ameliorated these abnormalities. Moreover, immortalized human mesangial cells (ihMCs) exposed to high glucose (HG) levels exhibited a rise in reactive oxygen species (ROS) and a decline in NO levels, which were reversed with GT. BH4 and the activity of guanosine triphosphate cyclohydrolase I decreased in ihMCs exposed to HG and was normalized by GT. Exogenous administration of BH4 in ihMCs reversed the HG-induced rise in ROS and the decline in NO production. However, coadministration of GT with BH4 did not result in a further reduction in ROS production, suggesting that reduced ROS with GT was indeed secondary to uncoupled eNOS. In summary, GT reversed the diabetes-induced reduction of BH4 levels, ameliorating uncoupling eNOS, and thus increasing NO bioavailability and reducing oxidative stress, two abnormalities that are involved in the pathogenesis of diabetic nephropathy.

Activation of peroxisome proliferator-activated receptor-β/-δ (PPARβ/δ) prevents endothelial dysfunction in type 1 diabetic rats

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. Herein, we have analyzed if the peroxisome proliferator-activated receptor-β/-δ (PPARβ/δ) agonist GW0742 exerts protective effects on endothelial function in type 1 diabetic rats. The rats were divided into 4 groups: control, control-treated (GW0742, 5mgkg−1day−1 for 5 weeks), diabetic (streptozotocin injection), and diabetic-treated. GW0742 administration in diabetic rats did not alter plasma glucose, systolic blood pressure, or heart rate, but reduced plasma triglyceride levels. The vasodilatation induced by acetylcholine was decreased in aortas from diabetic rats. GW0742 restored endothelial function, increasing eNOS phosphorylation. Superoxide production, NADPH oxidase activity, and mRNA expression of prepro endothelin-1, p22phox, p47phox, and NOX-1 were significantly higher in diabetic aortas, and GW0742 treatment prevented these changes. In addition, GW0742 prevented the endothelial dysfunction and the upregulation of prepro endothelin-1and p47phox after the in vitro incubation of aortic rings with high glucose and these effects were prevented by the PPARβ/δ antagonist GSK0660. PPARβ/δ activation restores endothelial function in type 1 diabetic rats. This effect seems to be related to an increase in nitric oxide bioavailability as a result of reduced NADPH oxidase-driven superoxide production and downregulation of prepro endothelin-1.