Marker Of Nitric Oxide Bioavailability Research Articles

Pharmacological mechanisms involved in the diuretic activity of the ethanol-soluble fraction of Baccharis milleflora (Less.) DC. - An ethnopharmacological investigation

Baccharis milleflora (Less.) DC. is a plant native to Brazil that is frequently used in traditional medicine as a diuretic and antihypertensive. However, even though it is traditionally used for these purposes, its diuretic and hypotensive effects have not been fully elucidated. Investigate the cardiorenal effects of the ethanol-soluble fraction (ESBM) of Baccharis milleflora in normotensive rats. Cladodes of B. milleflora were analyzed using light and scanning electron microscopy to provide anatomical data to support quality control. Subsequently, the ESBM was obtained and analyzed using LC-DAD-MS, and its components were annotated. The acute toxicity of ESBM was assessed in female Wistar rats. The acute and prolonged diuretic and hypotensive effects were then studied in Wistar rats. Finally, we assessed the mechanisms responsible for the diuretic effects of ESBM, including the activity of renal Na+/K+/ATPase, angiotensin-converting enzyme, and erythrocyte carbonic anhydrase. Additionally, we also investigated the involvement of bradykinin, prostaglandins, and nitric oxide. From LC-DAD-MS data, thirty-three metabolites were identified from ESBM, including chlorogenic acids, glycosylated phenolic derivatives, C-glycosylated flavones, and O-glycosylated flavonols. No signs of acute toxicity were observed in female rats. The findings showed that ESBM had significant diuretic and natriuretic effects, as well as a potassium-sparing effect. The treatment with ESBM was able to significantly decrease serum levels of creatinine and malondialdehyde, and also significantly increase levels of nitrite, an indirect marker of nitric oxide bioavailability. Furthermore, pre-treatment with L-NAME abolished all diuretic effects induced by ESBM. This study presented important morpho-anatomical and phytochemical data that support the quality control of Baccharis milleflora. The ESBM exhibited a significant diuretic and natriuretic effect following acute and seven-days repeated treatment in Wistar rats, without affecting renal potassium elimination. These effects appear to be dependent on the activation of the nitric oxide-cyclic GMP pathway. This study suggests the potential use of B. milleflora preparations in clinical situations where a diuretic effect is needed.

5-MTHF enhances the portal pressure reduction achieved with propranolol in patients with cirrhosis: A randomized placebo-controlled trial

β-blockers reduce hepatic venous pressure gradient (HVPG) by decreasing portal inflow, with no reduction in intrahepatic vascular resistance. 5-Methyltetrahydrofolate (5-MTHF) can prevent oxidative loss of tetrahydrobiopterin (BH4), a cofactor for endothelial nitric oxide synthase coupling. It also converts homocysteine (tHcy) into methionine and enables the degradation of asymmetric dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase. The aim of this study was to evaluate the effects of 5-MTHF in combination with propranolol on HVPG and nitric oxide bioavailability markers in patients with cirrhosis and portal hypertension. Sixty patients with cirrhosis and HVPG ≥12mmHg were randomized 1:1 to receive treatment with 5-MTHF+propranolol or placebo+propranolol for 90 days under double-blind conditions. HVPG and markers of nitric oxide bioavailability (BH4, ADMA and tHcy) were measured again at the end of treatment. Groups were similar in terms of baseline clinical and hemodynamic data and nitric oxide bioavailability markers. HVPG decreased in both groups, but the magnitude of the change was significantly greater in the group treated with 5-MTHF+propranolol compared to placebo+propranolol (percentage decrease, 20 [29-9] vs. 12.5 [22-0], p=0.028), without differences in hepatic blood flow. At the end of treatment, 5-MTHF+propranolol (vs. placebo+propranolol) was associated with higher BH4 (1,101.4 ± 1,413.3 vs. 517.1 ± 242.8pg/ml, p<0.001), lower ADMA (109.3 ± 52.7 vs. 139.9 ± 46.7μmol/L, p=0.027) and lower tHcy (μmol/L, 11.0 ± 4.6 vs. 15.4 ± 7.2μmol/L, p=0.010) plasma levels. In patients with cirrhosis and portal hypertension, 5-MTHF administration significantly enhanced the HVPG reduction achieved with propranolol. This effect appears to be mediated by improved nitric oxide bioavailability in the hepatic microcirculation. 2014-002018-21. Currently, the pharmacological prevention of cirrhosis complications due to portal hypertension, such as esophageal varices rupture, is based on the use of β-blockers, but some patients still present with acute variceal bleeding, mainly due to an insufficient reduction of portal pressure. In this study, we sought to demonstrate that the addition of folic acid to β-blockers is more effective in reducing portal pressure than β-blockers alone. This finding could represent the basis for validation studies in larger cohorts, which could impact the future prophylactic management of variceal bleeding in cirrhosis. Enhancing the benefit of β-blockers with a safe, accessible, cost-effective drug could improve clinical outcomes in cirrhosis, which in turn could translate into a reduction in the rates and costs of hospitalization, and ultimately into improved survival.

Seasonal Variation In Sunlight Exposure, Nitric Oxide Metabolites, And Blood Pressure

A reduced exposure to ultraviolet (UV) radiation in winter can decrease 25-hydroxyvitamin D (25(OH)D) levels, increase cardiovascular risk factors, and inhibit athletic performance. Exposing skin to UV-A light in the laboratory has also been shown to release nitric oxide (NO) metabolites into the circulation; molecules known to regulate multiple cardiovascular and metabolic processes. However, it is presently unclear whether NO availability is influenced by the seasonal variation in UV exposure. PURPOSE: To compare UV exposure, serum 25(OH)D, plasma nitrate and nitrite, and blood pressure between the summer and winter months. METHODS: Thirty-four healthy adults (21 males) residing in Scotland (~55.8o N, 4.1o W) were monitored for 7 days in the summer (June - August) and winter (December - February) in a randomised order. Participants wore a personal UV monitor on the wrist throughout each monitoring phase before visiting the laboratory to provide a venous blood sample and to have blood pressure measured. Serum levels of 25(OH)D were measured using an enzyme-linked immunosorbent assay and plasma nitrate and nitrite analysed using gas-phase chemiluminescence. RESULTS: Total UV-A exposure was higher in summer (17 ± 21 J.cm2) compared to winter (2.5 ± 3.0 J.cm2, P<0.001). Plasma nitrate did not differ between seasons (P=0.57) but nitrite (137 ± 31 nM) and serum 25(OH)D (22 ± 8 ng/ml) were lower (both P<0.001) in the winter compared to summer (200 ± 56 nM and 35 ± 13 ng/ml, respectively). Blood pressure was higher in winter (systolic 126 ± 13 mmHg; diastolic 76 ± 9 mmHg) than in summer (systolic 119 ± 11 mmHg; diastolic 67 ± 8 mmHg; both P<0.001). UV-A exposure was positively associated with plasma nitrite (R=0.41, P<0.01) and 25(OH)D (R=0.43, P<0.01). Plasma nitrite was negatively associated with systolic (R=−0.5, P<0.01) and diastolic blood pressure (R=−0.4, P<0.01). CONCLUSIONS: In a similar fashion to 25(OH)D, circulating levels of plasma nitrite, a marker of NO bioavailability, appear to be influenced by seasonal variations in UV exposure. The negative association between nitrite and blood pressure suggest that a reduced level of NO may increase cardiovascular risk factors in the winter months.

Short-term supplement of virgin coconut oil improves endothelial-dependent dilation but not exercise-mediated hyperemia in young adults

Virgin coconut oil (VCO) is high in antioxidants, which reduce reactive oxygen species–induced conversion of vascular endothelial-derived nitric oxide (NO) to toxic peroxynitrite. As such, flow-mediated dilation (FMD, a surrogate marker of NO bioavailability) and exercise-mediated hyperemia may be enhanced following VCO treatment. Animal research supports these findings, but direct assessments of FMD after short-term VCO use in humans are unknown. We tested the hypotheses that a 4-week VCO supplement (30 mL·d−1) would improve popliteal artery (PA) FMD and the hyperemic response to aerobic exercise. Thirty-four young adults were divided into VCO (n = 19, 9 women, 22 ± 2 years, 24 ± 3 kg·m−2) and control (CON: n = 15, 7 women, 24 ± 2 years, 24 ± 3 kg·m−2) groups. PA-FMD and blood flow were assessed via high-resolution duplex ultrasonography (Vivid i, GE Healthcare, Mississauga, Ontario, Canada). PA blood flow was measured at rest and for 5 minutes following a 10-minute bout of moderate-intensity (60% heart rate reserve) cycling exercise. Total PA blood volume was calculated as the integral of the 5-minute postexercise PA blood flow response. After 4 weeks, PA-FMD increased (P = .04) following VCO supplementation (4.9% ± 0.9% to 5.5% ± 1.2%) with no change (P > .9) in the CON group (5.7% ± 2.1% to 5.8% ± 1.9%). There were no differences (both P > .28) in the postexercise total PA blood volume response in either group (VCO: 495 ± 355 to 598 ± 384 mL; CON: 562 ± 362 to 488 ± 229 mL). Short-term VCO supplementation does not alter aerobic exercise–mediated blood flow responses in young adults. However, the augmented popliteal FMD response observed in the VCO supplement group indicates that short-term VCO supplementation improves vascular endothelial function in young, healthy adults.

Short‐Term Ingestion of Virgin Coconut Oil Improves Endothelial‐Dependent Dilation but not Exercise‐Mediated Hyperemia in Healthy Young Adults

Virgin coconut oil (VCO) is high in antioxidants (e.g., polyphenols) that may reduce reactive oxygen species‐induced conversion of vascular endothelial‐derived nitric oxide (NO) to toxic peroxynitrite. As such, flow‐mediated dilation (FMD, a surrogate marker of NO bioavailability) and exercise‐mediated hyperemia may be enhanced following short‐term VCO supplementation. However, these potential beneficial effects have never been assessed in humans. We tested the hypotheses that a 4 week VCO supplement (30 ml·day−1) would improve resting popliteal artery (PA) FMD and the hyperemic response to aerobic exercise. Thirty‐four young, healthy participants were divided into VCO (n=19, 10♂, 22±2 years, 24±3 kg·m−2), and Control (n=15, 10♂, 24±2 years, 24±3 kg·m−2) groups. PA‐FMD and PA blood flow were assessed via high‐resolution duplex ultrasonography (Vivid i, GE Healthcare) and analyzed using commercial software (Cardiovascular Suite, Quipu). After resting measurements, a 10‐minute bout of moderate‐intensity (60% heart rate reserve) cycling exercise was completed. PA blood flow was measured for 5‐minutes post‐exercise and total PA blood volume was calculated from the area under the curve. After 4 weeks, PA‐FMD increased (p &lt; 0.05) following VCO supplementation (4.9±0.9% to 5.5±1.2%) with no change (p &gt; 0.9) in the Control group (5.7±2.1% to 5.8±1.9%). There were no differences (both, p &gt; 0.28) in post‐exercise total PA blood volumes in either group (VCO: 495±355 ml to 598±384 ml; Control: 562±362 ml to 488±229 ml). The increased FMD response observed in our young healthy adults suggests that short‐term VCO supplementation has potential as a dietary treatment for populations with elevated ROS levels and reduced vascular endothelial (e.g., older adults, smokers, etc.). However, additional research is required to test this hypothesis.Support or Funding InformationSupport provided by: Canadian Foundation for Innovation: Leaders Opportunity Fund, and a Faculty of Health Professions Research Development grant. SAR and MWO were recipients of a NS Graduate Scholarship. MWO was awarded a NSHRF Scotia Scholar Award and a Heart &amp; Stroke BrightRed Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Salivary nitrite production is elevated in individuals with a higher abundance of oral nitrate-reducing bacteria.

Nitric oxide (NO) can be generated endogenously via NO synthases or via the diet following the action of symbiotic nitrate-reducing bacteria in the oral cavity. Given the important role of NO in smooth muscle control there is an intriguing suggestion that cardiovascular homeostasis may be intertwined with the presence of these bacteria. Here, we measured the abundance of nitrate-reducing bacteria in the oral cavity of 25 healthy humans using 16S rRNA sequencing and observed, for 3.5 h, the physiological responses to dietary nitrate ingestion via measurement of blood pressure, and salivary and plasma NO metabolites. We identified 7 species of bacteria previously known to contribute to nitrate-reduction, the most prevalent of which were Prevotella melaninogenica and Veillonella dispar. Following dietary nitrate supplementation, blood pressure was reduced and salivary and plasma nitrate and nitrite increased substantially. We found that the abundance of nitrate-reducing bacteria was associated with the generation of salivary nitrite but not with any other measured variable. To examine the impact of bacterial abundance on pharmacokinetics we also categorised our participants into two groups; those with a higher abundance of nitrate reducing bacteria (> 50%), and those with a lower abundance (< 50%). Salivary nitrite production was lower in participants with lower abundance of bacteria and these individuals also exhibited slower salivary nitrite pharmacokinetics. We therefore show that the rate of nitrate to nitrite reduction in the oral cavity is associated with the abundance of nitrate-reducing bacteria. Nevertheless, higher abundance of these bacteria did not result in an exaggerated plasma nitrite response, the best known marker of NO bioavailability. These data from healthy young adults suggest that the abundance of oral nitrate-reducing bacteria does not influence the generation of NO through the diet, at least when the host has a functional minimum threshold of these microorganisms.

Effects of Dietary Supplementation with Brazil Nuts on Microvascular Endothelial Function in Hypertensive and Dyslipidemic Patients: A Randomized Crossover Placebo-Controlled Trial.

To investigate the effects of dietary supplementation with GBNs on microvascular endothelial function in hypertensive and dyslipidemic patients. Ninety-one patients of both sexes aged 62.1 ± 9.3 years received 13 g/day of GBNs or a placebo for three months with a washout period of one month between treatments. Microvascular endothelial function was assessed using LSCI coupled with iontophoresis of ACh and PORH. We also used skin video capillaroscopy to measure capillary density and recruitment at rest and during PORH. Plasma concentrations of NOx were also measured as a marker of nitric oxide bioavailability. Supplementation with GBNs significantly increased the plasma levels of Se (p < 0.05) and NOx (p < 0.05). However, we did not observe any effects of GBN consumption on microvascular vasodilator responses to ACh or PORH (p > 0.05), and GBNs did not improve capillary density at baseline or recruitment during PORH (p > 0.05). Supplementation with GBNs induced significant increases in the plasma Se concentration and systemic bioavailability of nitric oxide. Nevertheless, GBN supplementation did not lead to any improvement in systemic microvascular reactivity or density in patients with arterial hypertension and dyslipidemia who were undergoing multiple drug therapies.

Loss of function in heparan sulfate elongation genes EXT1 and EXT 2 results in improved nitric oxide bioavailability and endothelial function.

BackgroundHeparanase is the major enzyme involved in degradation of endothelial heparan sulfates, which is associated with impaired endothelial nitric oxide synthesis. However, the effect of heparan sulfate chain length in relation to endothelial function and nitric oxide availability has never been investigated. We studied the effect of heterozygous mutations in heparan sulfate elongation genes EXT1 and EXT2 on endothelial function in vitro as well as in vivo.Methods and ResultFlow‐mediated dilation, a marker of nitric oxide bioavailability, was studied in Ext1+/− and Ext2+/− mice versus controls (n=7 per group), as well as in human subjects with heterozygous loss of function mutations in EXT1 and EXT2 (n=13 hereditary multiple exostoses and n=13 controls). Endothelial function was measured in microvascular endothelial cells under laminar flow with or without siRNA targeting EXT1 or EXT2. Endothelial glycocalyx and maximal arteriolar dilatation were significantly altered in Ext1+/− and Ext2+/− mice compared to wild‐type littermates (glycocalyx: wild‐type 0.67±0.1 μm, Ext1+/− 0.28±0.1 μm and Ext2+/− 0.25±0.1 μm, P<0.01, maximal arteriolar dilation during reperfusion: wild‐type 11.3±1.0%), Ext1+/− 15.2±1.4% and Ext2+/− 13.8±1.6% P<0.05). In humans, brachial artery flow‐mediated dilation was significantly increased in hereditary multiple exostoses patients (hereditary multiple exostoses 8.1±0.8% versus control 5.6±0.7%, P<0.05). In line, silencing of microvascular endothelial cell EXT1 and EXT2 under flow led to significant upregulation of endothelial nitric oxide synthesis and phospho‐endothelial nitric oxide synthesis protein expression.ConclusionsOur data implicate that heparan sulfate elongation genes EXT1 and EXT2 are involved in maintaining endothelial homeostasis, presumably via increased nitric oxide bioavailability.

Time lasting S-nitrosoglutathione polymeric nanoparticles delay cellular protein S-nitrosation

Physiological S-nitrosothiols (RSNO), such as S-nitrosoglutathione (GSNO), can be used as nitric oxide (NO) donor for the treatment of vascular diseases. However, despite a half-life measured in hours, the stability of RSNO, limited by enzymatic and non-enzymatic degradations, is too low for clinical application. So, to provide a long-lasting effect and to deliver appropriate NO concentrations to target tissues, RSNO have to be protected. RSNO encapsulation is an interesting response to overcome degradation and provide protection. However, RSNO such as GSNO raise difficulties for encapsulation due to its hydrophilic nature and the instability of the S-NO bound during the formulation process. To our knowledge, the present study is the first description of the direct encapsulation of GSNO within polymeric nanoparticles (NP). The GSNO-loaded NP (GSNO-NP) formulated by a double emulsion process, presented a mean diameter of 289±7nm. They were positively charged (+40mV) due to the methacrylic acid and ethylacrylate polymer (Eudragit® RL) used and encapsulated GSNO with a satisfactory efficiency (i.e. 54% or 40mM GSNO loaded in the NP). In phosphate buffer (37°C; pH 7.4), GSNO-NP released 100% of encapsulated GSNO within 3h and remained stable still 6h. However, in contact with smooth muscle cells, maximum protein nitrosation (a marker of NO bioavailability) was delayed from 1h for free GSNO to 18h for GSNO-NP. Therefore, protection and sustained release of NO were achieved by the association of a NO donor with a drug delivery system (such as polymeric NP), providing opportunities for vascular diseases treatment.

P159 - Vascular xanthine oxidoreductase contributes to the antihypertensive effects of sodium nitrite in L-NAME hypertension

Nitrate and nitrite have emerged as an important novel source of nitric oxide (NO). We have previously demonstrated that sodium nitrite is an antihypertensive compound that exerts antioxidant effects in experimental hypertension. These unpredicted antioxidant effects of nitrite raised the question whether the beneficial effects found were caused by its conversion to NO or simply due to reversal of endothelial dysfunction as a consequence of its antioxidant effects. Here, we evaluated the antihypertensive effects of a daily dose of sodium nitrite for 4 weeks in l-NAME-induced hypertension in rats. We studied the effects of nitrite on markers of NO bioavailability, vascular oxidative stress, and expression of xanthine oxidoreductase. Moreover, we tested if xanthine oxidoreductase inhibition could attenuate the acute hypotensive effects of sodium nitrite in l-NAME hypertensive rats. We found that a single pharmacological dose of sodium nitrite exerts antihypertensive effects in l-NAME-induced hypertension. While the beneficial antihypertensive properties of nitrite were associated with increased levels of NO metabolites, hypertension increased vascular xanthine oxidoreductase expression by approximately 40 %, with minor increases in vascular superoxide production. The inhibition of xanthine oxidoreductase by oxypurinol attenuated the acute hypotensive effects of nitrite. Taken together, our results show that nitrite exerts antihypertensive effects in l-NAME hypertensive rats and provide evidence that xanthine oxidoreductase plays an important role in this antihypertensive effect.

Vascular xanthine oxidoreductase contributes to the antihypertensive effects of sodium nitrite in l-NAME hypertension

Nitrate and nitrite have emerged as an important novel source of nitric oxide (NO). We have previously demonstrated that sodium nitrite is an antihypertensive compound that exerts antioxidant effects in experimental hypertension. These unpredicted antioxidant effects of nitrite raised the question whether the beneficial effects found were caused by its conversion to NO or simply due to reversal of endothelial dysfunction as a consequence of its antioxidant effects. Here, we evaluated the antihypertensive effects of a daily dose of sodium nitrite for 4 weeks in L-NAME-induced hypertension in rats. We studied the effects of nitrite on markers of NO bioavailability, vascular oxidative stress, and expression of xanthine oxidoreductase. Moreover, we tested if xanthine oxidoreductase inhibition could attenuate the acute hypotensive effects of sodium nitrite in L-NAME hypertensive rats. We found that a single pharmacological dose of sodium nitrite exerts antihypertensive effects in L-NAME-induced hypertension. While the beneficial antihypertensive properties of nitrite were associated with increased levels of NO metabolites, hypertension increased vascular xanthine oxidoreductase expression by approximately 40%, with minor increases in vascular superoxide production. The inhibition of xanthine oxidoreductase by oxypurinol attenuated the acute hypotensive effects of nitrite. Taken together, our results show that nitrite exerts antihypertensive effects in L-NAME hypertensive rats and provide evidence that xanthine oxidoreductase plays an important role in this antihypertensive effect.

Diabetes status differentiates endothelial function and plasma nitrite response to exercise stress in peripheral arterial disease following supervised training

AimsTo determine if type 2 diabetes mellitus (T2D) differentiates endothelial function and plasma nitrite response (a marker of nitric oxide bioavailability) during exercise in peripheral arterial disease (PAD) subjects prior to and following 3months supervised exercise training (SET). MethodsIn subjects with T2D+PAD (n=13) and PAD-only (n=14), endothelial function was measured using brachial artery flow-mediated dilation. On a separate day, venous blood draws were performed at rest and 10min following a symptom-limited graded treadmill test (SL-GXT). Plasma samples were snap-frozen for analysis of nitrite by reductive chemiluminescence. All testing was repeated following 3months of SET. ResultsPrior to training both groups demonstrated endothelial dysfunction, which was correlated with a net decrease in plasma nitrite following a SL-GXT (p≤0.05). Following SET, the PAD-only group demonstrated an improvement in endothelial function (p≤0.05) and COT (p≤0.05), which was related to a net increase in plasma nitrite following the SL-GXT (both p≤0.05). The T2D+PAD group had none of these increases. ConclusionsT2D in the presence of PAD attenuated improvements in endothelial function, net plasma nitrite, and COT following SET. This suggests that T2D maybe associated with an inability to endogenously increase vascular NO bioavailability to SET.

Helium Breathing Provides Modest Antiinflammatory, but No Endothelial Protection Against Ischemia-Reperfusion Injury in Humans In Vivo

The noble gas helium is devoid of anesthetic effects, and it elicits cardiac preconditioning. We hypothesized that inhalation of helium provides protection against postocclusive endothelial dysfunction after ischemia-reperfusion of the forearm in humans. Eight healthy male subjects were enrolled in this study with a crossover design. Each volunteer was randomly exposed to 15 min of forearm ischemia in the presence or absence of helium inhalation. Helium was inhaled at an end-tidal concentration of 50 vol% from 15 min before ischemia until 5 min after the onset of reperfusion ("helium conditioning"). Hyperemic reaction, a marker of nitric oxide bioavailability and endothelial function, was determined at 15 and 30 min of reperfusion on the forearm using venous occlusion plethysmography. Expression of the proinflammatory markers CD11b, ICAM-1, PSGL-1, and L-selectin (CD62L) on leukocytes and P-selectin (CD62P), PSGL-1, and CD42b on platelets were measured by flow cytometry during reperfusion. Ischemia-reperfusion consistently reduced the postocclusive endothelium-dependent hyperemic reaction at 15 and 30 min of reperfusion. Periischemic inhalation of helium at 50 vol% did not improve postocclusive hyperemic reaction. Helium decreased expression of the proinflammatory marker CD11b and ICAM-1 on leukocytes and attenuated the expression of the procoagulant markers CD42b and PSGL-1 on platelets. Although inhalation of helium diminished the postischemic inflammatory reaction, our data indicate that human endothelium, which is a component of all vital organs, is not amenable to protection by helium at 50 vol% in vivo. This is in contrast to sevoflurane, which protects human endothelium at low subanesthetic concentrations.

Androgen-Androgen Receptor System Protects against Angiotensin II-Induced Vascular Remodeling

Age-related andropause promotes cardiovascular disease in males. Although we had previously reported that the androgen-androgen receptor (AR) system plays important roles in cardiac growth and remodeling, the system's involvement in vascular remodeling remains unclear. To clarify this role, 25-wk-old male AR knockout (ARKO) mice and littermate male wild-type (WT) mice were divided into two groups with and without angiotensin II (Ang II) administration (2.0 mg/kg . d) for 14 d, respectively. No morphological differences in the coronary artery and thoracic aorta were observed between the groups without Ang II. Ang II stimulation markedly increased medial thickness and perivascular fibrosis in ARKO mice, with enhanced TGF-beta1, collagen type I, and collagen type III gene expression in the aorta. Ang II stimulation also prominently increased superoxide production, lipid peroxidation, and gene expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components in ARKO mice compared with WT mice. In addition, phosphorylation of c-Jun N-terminal kinase (JNK) and phosphorylated (Smad2/3) was remarkably enhanced in Ang II-treated ARKO mice compared with Ang II-treated WT mice. Notably, daily urinary nitric oxide (NO) metabolites excretion as a marker of NO bioavailability, aortic endothelial NO synthase expression and phosphorylation, and Akt phosphorylation were significantly reduced in ARKO mice compared with WT mice, regardless of Ang II stimulation. In conclusion, the androgen-AR system is required for the preservation of NO bioavailability through Akt-endothelial NO synthase system activation and exerts protective effects against Ang II-induced vascular remodeling by regulating oxidative stress, c-Jun N-terminal kinase (JNK) signaling, and the TGF-beta-phosphorylated Smad pathway.

Inverse relationship between markers of nitric oxide formation and plasma matrix metalloproteinase-9 levels in healthy volunteers

Background Nitric oxide (NO) is a major regulator of cardiovascular homeostasis and has anti-atherogenic properties. Reduced NO formation is associated with endothelial dysfunction and with cardiovascular risk factors. Although NO downregulates the expression and activity of the pro-atherogenic enzyme matrix metalloproteinase-9 (MMP-9), no previous clinical study has examined whether endogenous NO formation is inversely associated with the circulating levels of pro-MMP-9, which are associated with cardiovascular events. We examined this hypothesis in 175 healthy male subjects who were non-smokers. Methods To assess NO bioavailability, the plasma concentrations of nitrite, nitrate, and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Pro-MMP-9 and pro-MMP-2 levels were measured in plasma samples by gelatin zymography. Results We found significant negative correlations between pro-MMP-9 levels and plasma nitrite ( P = 0.035, rs = − 0.159), nitrate ( P = 0.040, rs = − 0.158), and cGMP ( P = 0.011, rs = − 0.189) concentrations. However, no significant correlations were found between pro-MMP-2 levels and the plasma concentrations of markers of NO bioavailability (all P > 0.05). Conclusions There is an inverse relationship between markers of NO formation and plasma MMP-9 levels. This finding may shed some light on the possible mechanisms involved in the increased cardiovascular risk of apparently healthy subjects with low NO bioavailability or high circulating levels of pro-MMP-9.

Growth Hormone Treatment Improves Markers of Systemic Nitric Oxide Bioavailability via Insulin-Like Growth Factor-I

Impaired nitric oxide (NO) bioavailability and low levels of circulating endothelial progenitor cells (EPC) are correlated to an increased risk for development of cardiovascular diseases. We investigated whether improved systemic NO bioavailability and increased levels of EPC after GH treatment are related and mediated by the IGF-I. Healthy middle-aged volunteers (n = 16) were treated for 10 d with recombinant human GH. Before and after GH treatment, we analyzed markers of NO bioavailability and EPC levels. GH treatment was responded by significant increases in plasma IGF-I levels. Urinary cGMP levels were increased and diastolic blood pressure reduced after GH treatment (P < 0.05). Likewise, plasma nitrate and nitrite levels were increased, whereas the NO synthase inhibitor asymmetric dimethylarginine was reduced. Correspondingly, IGF-I treatment increased expression of the asymmetric dimethylarginine-metabolizing enzyme dimethylarginie dimethylaminohydrolase-1 and dimethylarginie dimethylaminohydrolase-2 in cultured human endothelial cells. IGF-I levels correlated with cGMP concentrations (r = 0.51; P < 0.05). EPC numbers were increased after GH treatment and correlated with markers for NO bioavailability. These findings were also observed in mice treated with GH for 7 d. GH treatment additionally increased aortic endothelial NO synthase expression of mice. Importantly, blocking of the IGF-I receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability. GH treatment induced markers of increased NO bioavailability and enhanced circulating EPC numbers in healthy volunteers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-I plasma levels. Thus, GH treatment enhances systemic NO bioavailability via IGF-I and may be beneficial in certain cardiovascular diseases.

Redox Regulation of Circulating NO Bioavailability

PURPOSE: The present study sought to determine free radical-mediated regulation of circulating Nitric Oxide (NO) bioavailability during a sustained period of post-exercise hypotension (PEH) in hypertensive men. METHODS: Nine borderline hypertensive males, MAP = 106 ± 5 mmHg (50 ± 10 yr), not on medication, were studied following 30-minutes of cycle exercise at 70% maximal oxygen consumption. The subjects were followed post-exercise for 2-hours. Peripheral venous blood was sampled from an antecubital vein pre-, immediately post-, 1-hour post- (P1) and 2-hours post- (P2) exercise and corrected for plasma volume shifts. Plasma nitrate/nitrite was determined utilising a fluorometric assay, whilst S-Nitrosothiol (RSNO) concentrations were assayed by the Saville reaction and ELISA technique. Indirect markers of free radical generation were determined via the FOX1 assay detecting lipid hydroperoxides (LHP). RESULTS: Exercise decreased systemic mean arterial pressure by ∼5mmHg and elevated LHP (Table 1) whereas exercise failed to modify nitrite or RSNO concentration. However nitrate fell significantly below baseline following exercise.Table 1: Blood Pressure and Metabolic Responses to Sub-maximal Cycle ExerciseCONCLUSION: The results of the present study indicate that despite increases in oxidative stress circulating markers of NO bioavailability had no effect on the observed hypotension. An intriguing antagonism exists whereby elevated oxidative stress reduces the bioavailability of NO but a sustained hypotension remains. This suggests that PEH is resultant from a metabolic pathway independent of NO.

Plasma Nitroso Compounds Are Decreased in Patients With Endothelial Dysfunction

We investigated whether plasma nitros(yl)ated species (RXNOs) that mediate systemic nitric oxide (NO) bioactivity are depleted in individuals with cardiovascular risk factors and endothelial dysfunction. Endothelium-derived NO acts not only as a regional messenger but exerts significant systemic effects via formation of circulating RXNOs delivering NO to sites of impaired production. Endothelial function was assessed in 68 patients with one to four major cardiovascular risk factors (RF) and 39 healthy control subjects (C) by measurement of flow-mediated dilation (FMD) of the brachial artery using high-resolution ultrasound. In parallel, plasma RXNOs were determined by reductive gas phase chemiluminescence. Increasing numbers of risk factors were accompanied by a progressive decrease in FMD: 6.5 +/- 0.4% (C); 4.7 +/- 0.5% (one RF); 2.8 +/- 0.4% (two RF); 2.2 +/- 0.4% (three RF); and 1.0 +/- 0.3% (four RF). Progressively impaired vascular function was associated with a concomitant decrease in plasma RXNOs (p < 0.01): 39 +/- 2 nmol/l (C); 30 +/- 2 nmol/l (one RF); 24 +/- 3 nmol/l (two RF); 22 +/- 3 nmol/l (three RF); and 15 +/- 2 nmol/l (four RF), with univariate correlation between FMD and RXNO (r = 0.41, p < 0.001). In a multivariate regression model, RXNO was an independent predictor of endothelial function. Endothelial dysfunction in patients with cardiovascular risk factors is associated with decreased levels of circulating RXNOs. Plasma RXNOs may be diagnostically useful markers of NO bioavailability and a surrogate index of endothelial function. Whether the observed decrease in concentration reflects impaired NO formation, accelerated decomposition, and/or consumption of RXNOs and whether these processes play a causal role in the pathophysiology of arteriosclerosis remain to be investigated.

Effect of diet and exercise intervention on blood pressure, insulin, oxidative stress, and nitric oxide availability.

Diet and exercise can affect blood pressure and atherosclerotic risk. The present study was designed to examine the effects of a short-term, rigorous diet and exercise intervention on blood pressure, hyperinsulinemia, and nitric oxide (NO) availability. Men (n=11) were placed on a low-fat, high-fiber diet combined with daily exercise for 45 to 60 minutes for 3 weeks. Pre- and post fasting blood was drawn for serum lipid, insulin, 8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)), and glucose measurements. Anthropometric parameters, blood pressure (BP), and 24-hour urinary NO metabolite excretion (NO(X)), a marker of NO bioavailability, were measured. Systolic (P<0.01) and diastolic BP (P<0.01) and 8-iso-PGF(2alpha) decreased (P<0.05), whereas urinary NO(X) increased (P<0.05). There was a significant reduction in fasting insulin (P<0.01) and a significant correlation between the decrease in serum insulin and the increase in urinary NO(X) (r2=0.68, P<0.05). All fasting lipids decreased significantly, and the total cholesterol to high-density lipoprotein cholesterol ratio improved. Although body weight and body mass index (P<0.01) decreased, obesity was still present and there were no correlations between the change in body mass index and the change in insulin, BP, or urinary NO(X). This intervention resulted in dramatic improvements in BP, oxidative stress, NO availability, and the metabolic profile within 3 weeks, mitigating the risk for atherosclerosis progression and its clinical sequelae.