Nitric oxide in cardiovascular and renal disease : role of organic nitrates, inorganic nitrate and red blood cells

RATIONALE: Cardiovascular and renal disorders are major health problems, which are often co-existing. Mechanistically, these conditions can be attributed to endothelial dysfunction, a process coupled with reduced nitric oxide (NO) bioavailability. In the vasculature, NO is predominantly formed by endothelial NO-synthase (eNOS), which uses L-arginine and oxygen as substrates. Organic nitrates like glyceryl trinitrate (GTN) are clinically used as an exogenous source of NO, with potent vasodilator actions. Despite their long history of use, the undesired side-effects induced by organic nitrates, such as hypotension, headache and development of tolerance, limit their clinical use and demand a need to develop new nitrate preparations. In addition to the classical NOS system, the nitrate-nitrite-NO pathway can serve as additional source of NO generation. Inorganic nitrate is abundant in certain foods, e.g. leafy green vegetables and beetroot. Stimulating this NOS-independent system, via the diet, has been linked with favorable cardiovascular, metabolic and renal effects in several disease models. Moreover, the recent discovery of existing NOS in red blood cells (RBCs) has started a debate regarding the role and interaction between RBCs and endothelial NOSs in regulation of vascular function. AIM: This thesis I) characterizes the therapeutic value of restoring NO bioavailability in cardiovascular and renal disease models by using novel organic mononitrate(s) and inorganic nitrate, and II) investigates the potential role and interaction between NOS in RBCs and the endothelium in regulation or modulation of vascular function. METHODS & RESULTS: Combination of in vivo disease models, ex vivo vessel reactivity as well as in vitro studies were used. Study I-II: A novel organic nitrate 1,3-bis(hexyloxy)propan- 2-yl nitrate (NDHP) was synthesized and functionally characterized. It was found that NDHPderived NO formation was enzymatically mediated by xanthine oxidoreductase (XOR). Moreover, NDHP treatment in contrast to GTN was not subject to tolerance in isolated small arteries and NDHP attenuated angiotensin II-induced hypertension and endothelial dysfunction in rats. Study III: Inorganic nitrate supplementation, increased NO bioactivity, dampened oxidative stress and inflammation and ameliorated mitochondrial abnormalities, which were associated with protection against development of renal ischemia-reperfusion (IR) injuries in mice. This novel approach may have therapeutic value by reducing the risk of acute and chronic kidney disease as well as cardiovascular co-morbidities. Study IV: Using ex vivo co-incubation of isolated RBCs and aortas from eNOS-deficient and control mice, it was shown that RBCs lacking NOS induced oxidative stress and endothelial dysfunction in healthy vessels. This pathological vascular phenotype could be prevented by inhibition of arginase activity and by scavenging of NADPH oxidase-derived reactive oxygen species. CONCLUSION: Restoring NO bioactivity, by using novel organic nitrates or inorganic nitrate, is coupled with favorable effects in models of cardiorenal disease. In addition, arginase and oxidative stress is involved in the interaction between NOS in RBCs and the endothelium. Future studies are needed to further characterize underlying mechanisms and to investigate the potential therapeutic value

AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis

Advanced age and unhealthy dietary habits contribute to the increasing incidence of obesity and type 2 diabetes. These metabolic disorders, which are often accompanied by oxidative stress and compromised nitric oxide (NO) signaling, increase the risk of adverse cardiovascular complications and development of fatty liver disease. Here, we investigated the therapeutic effects of dietary nitrate, which is found in high levels in green leafy vegetables, on liver steatosis associated with metabolic syndrome. Dietary nitrate fuels a nitrate–nitrite–NO signaling pathway, which prevented many features of metabolic syndrome and liver steatosis that developed in mice fed a high-fat diet, with or without combination with an inhibitor of NOS (L-NAME). These favorable effects of nitrate were absent in germ-free mice, demonstrating the central importance of host microbiota in bioactivation of nitrate. In a human liver cell line (HepG2) and in a validated hepatic 3D model with primary human hepatocyte spheroids, nitrite treatment reduced the degree of metabolically induced steatosis (i.e., high glucose, insulin, and free fatty acids), as well as drug-induced steatosis (i.e., amiodarone). Mechanistically, the salutary metabolic effects of nitrate and nitrite can be ascribed to nitrite-derived formation of NO species and activation of soluble guanylyl cyclase, where xanthine oxidoreductase is proposed to mediate the reduction of nitrite. Boosting this nitrate–nitrite–NO pathway results in attenuation of NADPH oxidase-derived oxidative stress and stimulation of AMP-activated protein kinase and downstream signaling pathways regulating lipogenesis, fatty acid oxidation, and glucose homeostasis. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against liver steatosis associated with metabolic dysfunction.</p

Renal denervation attenuates hypertension and renal dysfunction in a model of cardiovascular and renal disease, which is associated with reduced NADPH and xanthine oxidase activity

Oxidative stress is considered a central pathophysiological event in cardiovascular disease, including hypertension. Early age reduction in renal mass is associated with hypertension and oxidative stress in later life, which is aggravated by increased salt intake. The aim of the present study was to examine if renal sympathetic denervation can exert blood pressure lowering effects in uninephrectomized (UNX) rats (3-week old) fed with high salt (HS, 4%; w/w) diet for 4 weeks. Moreover, we investigated if renal denervation is associated with changes in NADPH and xanthine oxidase-derived reactive oxygen species. Rats with UNX + HS had reduced renal function, elevated systolic and diastolic arterial pressures, which was accompanied by increased heart weight, and cardiac superoxide production compared to sham operated Controls. UNX + HS was also associated with higher expression and activity of NADPH and xanthine oxidase in the kidney. Renal denervation in rats with UNX + HS attenuated the development of hypertension and cardiac hypertrophy, but also improved glomerular filtration rate and reduced proteinuria. Mechanistically, renal de nervation was associated with lower expression and activity of both NADPH oxidase and xanthine oxidase in the kidney, but also reduced superoxide production in the heart. In conclusion, our study shows for the first time that renal denervation has anti-hypertensive, cardio- and reno-protective effects in the UNX + HS model, which can be associated with decreased NADPH oxidase- and xanthine oxidase-derived reactive oxygen species (i.e., superoxide and hydrogen peroxide) in the kidney

Synthesis and characterization of a novel organic nitrate NDHP: Role of xanthine oxidoreductase-mediated nitric oxide formation

In this report, we describe the synthesis and characterization of 1,3-bis(hexyloxy)propan-2-yl nitrate (NDHP), a novel organic mono nitrate. Using purified xanthine oxidoreductase (XOR), chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy, we found that XOR catalyzes nitric oxide (NO) generation from NDHP under anaerobic conditions, and that thiols are not involved or required in this process. Further mechanistic studies revealed that NDHP could be reduced to NO at both the FAD and the molybdenum sites of XOR, but that the FAD site required an unoccupied molybdenum site. Conversely, the molybdenum site was able to reduce NDHP independently of an active FAD site. Moreover, using isolated vessels in a myograph, we demonstrate that NDHP dilates pre-constricted mesenteric arteries from rats and mice. These effects were diminished when XOR was blocked using the selective inhibitor febuxostat. Finally, we demonstrate that NDHP, in contrast to glyceryl trinitrate (GTN), is not subject to development of tolerance in isolated mesenteric arteries

Red blood cells from endothelial nitric oxide synthase-deficient mice induce vascular dysfunction involving oxidative stress and endothelial arginase I

Background &amp; aims: Nitric oxide bioactivity (NO) from endothelial NO synthase (eNOS) importantly contributes to the maintenance of vascular homeostasis, and reduced eNOS activity has been associated with cardiovascular disease. Emerging evidence suggests interaction(s) between red blood cells (RBCs) and the endothelium in vascular control; however, the specific role of RBC eNOS is less clear. We aimed to investigate the hypothesis that a lack of RBC eNOS induces endothelial dysfunction. Methods &amp; Results: RBCs from global eNOS knockout (KO) and wildtype (WT) mice were co-incubated ex vivo overnight with healthy mouse aortic rings, followed by functional and mechanistic analyses of endothelium-dependent and independent relaxations. RBCs from eNOS KO mice induced endothelial dysfunction and vascular oxidative stress, whereas WT RBC did not. No differences were observed for endothelium-independent relaxations. This eNOS KO RBC-induced endothelial dysfunctional phenotype was prevented by concomitant co-incubation with reactive oxygen species scavenger (TEMPOL), arginase inhibitor (nor-NOHA), NO donor (detaNONOate) and NADPH oxidase 4 (NOX4) inhibitor. Moreover, vessels from endothelial cell-specific arginase 1 KO mice were resistant to eNOS KO-RBC-induced endothelial dysfunction. Finally, in mice aortae co-incubated with RBCs from women with preeclampsia, we observed a significant reduction in endothelial function compared to when using RBCs from healthy pregnant women or from women with uncomplicated gestational hypertension. Conclusions: RBCs from mice lacking eNOS, and patients with preeclampsia, induce endothelial dysfunction in adjacent blood vessels. Thus, RBC-derived NO bioactivity acts to prevent induction of vascular oxidative stress occurring via RBC NOX4-derived ROS in a vascular arginase-dependent manner. Our data highlight the intrinsic protective role of RBC-derived NO bioactivity in preventing the damaging potential of RBCs. This provides novel insight into the functional relationship between RBCs and the vasculature in health and cardiovascular disease, including preeclampsia

Dietary nitrate attenuates renal ischemia-reperfusion injuries by modulation of immune responses and reduction of oxidative stress

Ischemia-reperfusion (IR) injury involves complex pathological processes in which reduction of nitric oxide (NO) bioavailability is suggested as a key factor. Inorganic nitrate can form NO in vivo via NO synthase-independent pathways and may thus provide beneficial effects during IR. Herein we evaluated the effects of dietary nitrate supplementation in a renal IR model. Male mice (C57BL/6J) were fed nitrate-supplemented chow (1.0 mmol/kg/day) or standard chow for two weeks prior to 30 min ischemia and during the reperfusion period. Unilateral renal IR caused profound tubular and glomerular damage in the ischemic kidney. Renal function, assessed by plasma creatinine levels, glomerular filtration rate and renal plasma flow, was also impaired after IR. All these pathologies were significantly improved by nitrate. Mechanistically, nitrate treatment reduced renal superoxide generation, pro-inflammatory cytokines (IL-1 beta, IL-6 and IL-12 p70) and macrophage infiltration in the kidney. Moreover, nitrate reduced mRNA expression of pro-inflammatory cytokines and chemo attractors, while increasing anti-inflammatory cytokines in the injured kidney. In another cohort of mice, two weeks of nitrate supplementation lowered superoxide generation and IL-6 expression in bone marrow-derived macrophages. Our study demonstrates protective effect of dietary nitrate in renal IR injury that may be mediated via modulation of oxidative stress and inflammatory responses. These novel findings suggest that nitrate supplementation deserve further exploration as a potential treatment in patients at high risk of renal IR injury

Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice

Background/Aims: Canonical Wnt signaling is involved in oxidative stress, vasculopathy and diabetes mellitus but its role in diabetic renal microvascular dysfunction is unclear. We tested the hypothesis that enhanced canonical Wnt signaling in renal afferent arterioles from diabetic mice increases reactive oxygen species (ROS) and contractions to endothelin-1 (ET-1). Methods: Streptozotocin-induced diabetes or control C57Bl/6 mice received vehicle or sulindac (40 mg·kg-1·day-1) to block Wnt signaling for 4 weeks. ET-1 contractions were measured by changes of afferent arteriolar diameter. Arteriolar H2O2, O2 -, protein expression and enzymatic activity were assessed using sensitive fluorescence probes, immunoblotting and colorimetric assay separately. Results: Compared to control, diabetic mouse afferent arteriole had increased O2- (+ 84%) and H2O2 (+ 91%) and enhanced responses to ET-1 at 10-8 mol·l-1 (-72±4% of versus -43±4%, P&#x3c; 0.05) accompanied by reduced protein expressions and activities for catalase and superoxide dismutase 2 (SOD2). Arteriolar O2 - was increased further by ET-1 and contractions to ET-1 reduced by PEG-SOD in both groups whereas H2O2 unchanged by ET-1 and contractions were reduced by PEG-catalase selectively in diabetic mice. The Wnt signaling protein β-catenin was upregulated (3.3-fold decrease in p-β-catenin/β-catenin) while the glycogen synthase kinase-3β (GSK-3β) was downregulated (2.6-fold increase in p-GSK-3β/ GSK-3β) in preglomerular vessels of diabetic mice. Sulindac normalized the Wnt signaling proteins, arteriolar O2 -, H2O2 and ET-1 contractions while doubling microvascular catalase and SOD2 expression in diabetic mice. Conclusion: Increased ROS, notably H2O2 contributes to enhanced afferent arteriolar responses to ET-1 in diabetes, which is closely associated with Wnt signaling. Antioxidant pharmacological strategies targeting Wnt signaling may improve vascular function in diabetic nephropathy

The novel organic mononitrate NDHP attenuates hypertension and endothelial dysfunction in hypertensive rats

Rationale: Development and progression of cardiovascular diseases, including hypertension, are often associated with impaired nitric oxide synthase (NOS) function and nitric oxide (NO) deficiency. Current treatment strategies to restore NO bioavailability with organic nitrates are hampered by undesirable side effects and development of tolerance. In this study, we evaluated NO release capability and cardiovascular effects of the newly synthesized organic nitrate 1, 3-bis (hexyloxy) propan-2-yl nitrate (NDHP). Methods: A combination of in vitro and in vivo approaches was utilized to assess acute effects of NDHP on NO release, vascular reactivity and blood pressure. The therapeutic value of chronic NDHP treatment was assessed in an experimental model of angiotensin II-induced hypertension in combination with NOS inhibition. Results: NDHP mediates NO formation in both cell-free system and small resistance arteries, a process which is catalyzed by xanthine oxidoreductase. NDHP-induced vasorelaxation is endothelium independent and mediated by NO release and modulation of potassium channels. Reduction of blood pressure following acute intravenous infusion of NDHP was more pronounced in hypertensive rats (two-kidney-one-clip model) than in normotensive sham-operated rats. Toxicological tests did not reveal any harmful effects following treatment with high doses of NDHP. Finally, chronic treatment with NDHP significantly attenuated the development of hypertension and endothelial dysfunction in rats with chronic NOS inhibition and angiotensin II infusion. Conclusion: Acute treatment with the novel organic nitrate NDHP increases NO formation, which is associated with vasorelaxation and a significant reduction of blood pressure in hypertensive animals. Chronic NDHP treatment attenuates the progression of hypertension and endothelial dysfunction, suggesting a potential for therapeutic applications in cardiovascular disease

Hemoglobin β93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell

BACKGROUND:Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin β chain (β93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS:To test this hypothesis, we used RBCs from mice in which the β93 cysteine had been replaced with alanine (β93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS:In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (β93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with β93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in β93C and β93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between β93C and β93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in β93C and β93A mice. CONCLUSIONS:RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the β93 cysteine of Hb