NO-dependent Endothelial Function Research Articles

Renin-Angiotensin System Blockade Is Associated with Exercise Capacity, Sympathetic Activity, and Endothelial Function in Patients with Chronic Kidney Disease

Background: Chronic kidney disease (CKD) patients have exercise intolerance and exaggerated blood pressure reactivity during exercise that are mediated by sympathetic nervous system (SNS) overactivation and decreased nitric oxide (NO) bioavailability. The activation of the renin-angiotensin system (RAS) increases SNS activation and reduces NO synthesis, and prior studies suggest that RAS blockade attenuates declines in physical function. We hypothesized that RAS inhibitor (RASi) use is associated with higher exercise capacity mediated by decreased SNS activity and increased NO-dependent endothelial function in CKD. Method: In 35 CKD patients (57 ± 7 years) and 20 controls (CONs) (53 ± 8 years), we measured exercise capacity (peak oxygen consumption [VO_2peak]), muscle sympathetic nervous activity (MSNA), and flow-mediated dilation (FMD) for NO-dependent endothelial function. Results: CKD patients treated with RASi (CKD + RASi, n = 25) had greater VO_2peak than CKD patients not treated with RASi (CKD no RASi, n = 10), but lower VO_2peak than CONs (23.3 ± 5.8 vs. 16.4 ± 2.9, p = 0.007; vs. 30.0 ± 7.7, p = 0.016 mL/min/kg, respectively). CKD + RASi had lower resting MSNA and greater FMD than CKD no RASi. Compared to CONs, CKD + RASi had similar MSNA but lower FMD. VO_2peak was positively associated with FMD (r = 0.417, p = 0.038) and was predicted by the combination of FMD and RASi status (r² = 0.344, p = 0.01) and MSNA and RASi status (r² = 0.575, p = 0.040) in CKD patients. Conclusion: In summary, CKD patients with RASi have higher exercise capacity than those not on RASi. Higher exercise capacity in the RASi-treated group was associated with lower resting SNS activity and higher NO-dependent vascular endothelial function.

Effect of ACEI and ARB treatment on nitric oxide-dependent endothelial function.

Background: Angiotensin-converting-enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) are widely used as a first-line therapy for the treatment of cardiovascular disease. Here, ACEI modulate the bradykinin receptor (BDKRB1 and BDKRB2) system and NO-dependent endothelial function, thus determining cardiovascular health and regenerative arteriogenesis. The current study aims at evaluating nitric oxide-dependent endothelial function, and gene expression of bradykinin receptors in peripheral blood mononuclear cells (PBMC) from patients with ACEI or ARB treatment. Patients and methods: The WalkByLab has been established to screen cardiovascular patients for peripheral artery disease and coronary artery disease. In total 177 patients from WalkByLab with heterogenous disease and risk status were randomly selected, divided according to their medication history into the following groups: 1. ACEI group, 2. ARB group or 3. non-ACE/ARB group. Total plasma nitrite/nitrate (NO) levels were measured, endothelial function was evaluated by assessing flow meditated dilation (FMD). PBMC were isolated from peripheral whole blood, and gene expression (qRT-PCR) of bradykinin receptors and angiotensin converting enzyme were assessed. Results: Plasma total NO concentration in the ACEI group (24.66±16.28, µmol/l) was increased as compared to the ARB group (18.57±11.58, µmol/l, P=0.0046) and non-ACE/ARB group (16.83±8.64, µmol/l, P=0.0127) in patients between 40 to 90 years of age. However, FMD values (%) in the ACEI group (7.07±2.40, %) were similar as compared to the ARB (6.35±2.13, %) and non-ACE/ARB group (6.51±2.15, %), but significantly negatively correlated with age. Interestingly, BDKRB1 mRNA level was significantly higher and BDKRB2 mRNA level lower in the ACEI group (BDKRB1 3.88-fold±1.05, BDKRB2 0.22-fold±0.04) as compared to the non-ACE/ARB group (BDKRB1 1.00-fold±0.39, P<0.0001, BDKRB2 1.00-fold±0.45, P=0.0136). Conclusions: ACEI treatment enhances total nitrite/nitrate concentration, furthermore, upregulates BDKRB1 in PBMC, but downregulates BDKRB2 mRNA expression. FMD is a strong determinant of vascular aging and is sensitive to underlying heterogenous cardiovascular diseases.

Systemic Administration of Insulin Receptor Antagonist Results in Endothelial and Perivascular Adipose Tissue Dysfunction in Mice.

Hyperglycemia linked to diabetes results in endothelial dysfunction. In the present work, we comprehensively characterized effects of short-term hyperglycemia induced by administration of an insulin receptor antagonist, the S961 peptide, on endothelium and perivascular adipose tissue (PVAT) in mice. Endothelial function of the thoracic and abdominal aorta in 12-week-old male C57Bl/6Jrj mice treated for two weeks with S961 infusion via osmotic pumps was assessed in vivo using magnetic resonance imaging and ex vivo by detection of nitric oxide (NO) production using electron paramagnetic resonance spectroscopy. Additional methods were used to analyze PVAT, aortic segments and endothelial-specific plasma biomarkers. Systemic disruption of insulin signaling resulted in severe impairment of NO-dependent endothelial function and a loss of vasoprotective function of PVAT affecting the thoracic as well as abdominal parts of the aorta, however a fall in adiponectin expression and decreased uncoupling protein 1-positive area were more pronounced in the thoracic aorta. Results suggest that dysfunctional PVAT contributes to vascular pathology induced by altered insulin signaling in diabetes, in the absence of fat overload and obesity.

Renin‐Angiotensin System Blockade is Associated with Exercise Capacity, Sympathetic Activity and Endothelial Function in Patients with Chronic Kidney Disease

Chronic kidney disease (CKD) patients have exercise intolerance that is associated with cardiovascular mortality. CKD patients have exaggerated increases in blood pressure (BP) reactivity and impaired ability to maintain skeletal muscle perfusion during exercise mediated by sympathetic nervous system (SNS) overactivation and decreased nitric oxide (NO) bioavailability. Activation of the renin-angiotensin system (RAS) increases central SNS activation and reduces NO synthesis, and prior studies have suggested that RAS blockade may modulate physical function declines. The aim of this study was to investigate whether use of RAS inhibitors (RASi) is associated with greater exercise capacity in CKD, and whether greater exercise capacity with RASi is linked to lower SNS activity and increased NO-dependent endothelial function. 35 hypertensive CKD patients (56.7 ± 7.0 yr) and 20 age-matched controls without CKD (CON, 52.5 ± 8.2 yr) were recruited. Exercise capacity was measured via VO2 peak from a maximal treadmill test, muscle sympathetic nervous activity (MSNA) was measured via microneurography, and vascular endothelial function was measured by flow-mediated dilation (FMD). Participants with CKD were further grouped by current usage of RASi including angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB) for group difference analyses. There were no baseline differences in demographics or hemodynamics between CKD patients with and without RASi. VO2 peak was greater in CKD patients with RASi compared to CKD patients not on RASi and lower compared to CON (23.3 ± 5.8 vs.16.4 ± 2.9, p=0.007; vs.30.0 ± 7.7, p=0.005 ml/min/kg respectively). MSNA in CKD patients with RASi was lower compared to CKD patients not on RASi but similar with CON (43.9 ± 7.1 vs. 53.8 ± 8.2; p=0.009, vs.38.0 ± 20.3; p=0.103 bursts/min respectively). FMD in CKD patients with RASi was greater compared to CKD patients not on RASi and lower compared to CON (3.2 ± 2.6 vs. 1.3 ± 1.6; p=0.007, vs. 5.0 ± 3.0; p=0.045 % respectively). VO2 peak was positively associated with FMD (r=0.417, p=0.038) and was predicted by the combination of FMD and RASi status (r2 =0.344, p=0.01) and MSNA and RASi status (r2 =0.575, p=0.040) in CKD patients. In summary, hypertensive CKD patients on RASi have better exercise capacity compared to those not on RASi. Furthermore, greater exercise capacity in RASi-treated group was associated with lower resting SNS activity and better NO-dependent vascular endothelial function. Long-term randomized trials examining the effects of RASi on physical function and underlying autonomic and vascular mechanisms are needed to determine its causal effect and its responsible mechanisms in CKD.

ATTENUATION OF PLACENTAL LIPID DEPOSITION BY GLUTAMINE SUPPLEMENTATION IMPROVES FETAL OUTCOME IN INSULIN-RESISTANT PREGNANT RATS

Objective: Maternal metabolic adaptation is critical for placental and fetal development, growth and survival. However, evidence exists that indiscriminate consumption of fructose-enrich drink (FRD) during pregnancy disrupts maternal-fetal metabolic tolerance and/or glucose homeostasis with consequent adverse pregnancy outcomes. Glutamine supplementation (GLN) has been shown to exert a modulatory effect in metabolic pathologies. Nevertheless, the effects of GLN on FRD-induced placental lipotoxicity during pregnancy are unknown. The present study was conducted to investigate the protective effects of GLN on pregnancy outcomes of female rats exposed to gestational FRD. Design and method: Pregnant Wistar rats weighing 160–180 g were randomly allotted into control, GLN, FRD and FRD+GLU groups (6 rats per group). The groups received vehicle (p.o.), glutamine (1 g/kg), fructose (10%; w/v) and fructose plus glutamine respectively throughout the period of gestation. Insulin resistance (IR) and insulin sensitivity were estimated by homeostatic model of assessment (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) respectively. Plasma and tissue biochemical analysis were determined using enzyme-linked immunosorbent assay kits or fluorescence assay as appropriate. Results: Data showed that FRD increased placental weight and decreased fetal length and weight. It also caused maternal insulin resistance with correspondent increase in blood glucose, plasma insulin and decreased insulin sensitivity. In addition, FRD increased placental triglyceride, total cholesterol, low density lipoprotein-cholesterol, free fatty acid, atherogenic lipid, lipid peroxidation (malondialdehyde), lactate and uric acid synthesis, and decreased nitric oxide, adenosine and glucose-6-phosphate dehydrogenase-dependent antioxidant defense. Conclusions: The present study demonstrates that high FRD consumption during pregnancy causes placental lipotoxicity-driven defects with consequent fetal growth restriction. The findings also suggest that glutamine supplementation improves fetal outcome by ameliorating placental defects through suppression of uric acid synthesis and lipid peroxidation, as well as enhancement of NO-dependent endothelial function and antioxidant defense (Figure 1). Similarly, the study also suggests that glutamine supplementation in pregnancy could be useful as a reprogramming intervention to prevent impaired placental-fetal development and subsequent newborn and adulthood metabolic-related syndromes (Figure 1).

Protective role of glutamine against cadmium-induced testicular dysfunction in Wistar rats: Involvement of G6PD activity

BackgroundEndocrine disruptor such as cadmium has been widely reported to cause testicular toxicity, which contributes to recent decline in male fertility worldwide. Glutamine, the most abundant amino acid in the body has been demonstrated to exert protective effects in cellular toxicity. However, its role in testicular toxicity is unknown. The present study is therefore aimed at investigating the effects of glutamine supplementation on cadmium-induced testicular toxicity, and the possible involvement of glucose-6-phosphate dehydrogenase (G6PD) activity. Materials and methodMale Wistar rats weighing 160–190 g were allotted into 4 groups (n = 5/group): The groups received vehicle (distilled water; p.o.), glutamine (1gkg−1; p.o.), cadmium chloride (5mgkg−1p.o.) and Cadmium chloride plus glutamine respectively, daily for 30 days. Biochemical and histological analyses were performed with appropriate method. ResultsAdministration of cadmium significantly decreased body weight, sperm count, motility and viability, as well as altered sperm morphology and progressivity. Cadmium also caused atrophy of the seminiferous tubule in addition to disrupted testicular architecture, lumen, Sertoli cells and spermatogonia. Similarly, serum and testicular aspartate transaminase, and malondialdehyde significantly increased, and G6PD, glutathione, nicotinamide adenine dinucleotide phosphate and nitric oxide significantly decreased with corresponding decrease in follicle stimulating hormone, luteinizing hormone and testosterone in cadmium-treated animals compared with control groups. However, supplementation with glutamine attenuated these alterations. ConclusionThe present study demonstrates that cadmium induces testicular dysfunction that is attributable to defective G6PD and accompanied by increased lipid peroxidation and impaired NO-dependent endothelial function. Interestingly, glutamine supplementation ameliorates cadmium-induced testicular dysfunction through enhancement of G6PD activity.

Vitamin K2-MK-7 improves nitric oxide-dependent endothelial function in ApoE/LDLR−/− mice

Although, vitamin K2 displays vasoprotective effects, it is still not known whether K2 treatment improves endothelial function.In ApoE/LDLR−/− mice at the stage prior to atherosclerosis development, four-week treatment with K2-MK-7, given at a low dose (0.05 mg/kg), improved acetylcholine- and flow-induced, endothelium-dependent vasodilation in aorta or in femoral artery, as assessed by MRI in vivo. This effect was associated with an increased NO production, as evidenced by EPR measurements in ex vivo aorta. Treatment with higher doses of K2-MK-7 (0.5; 5 mg/kg) resulted in a dose-dependent increase in plasma K2-MK-7 and K2-MK-4 concentration, without further improvement in endothelial function. In ApoE/LDLR−/− mice with developed atherosclerotic plaques, treatment with a low (0.03 mg/kg) or high (10 mg/kg) dose of K2-MK-7 resulted in a similar degree of endothelium-dependent vasodilation improvement and increase in plasma nitrate concentration, what was not associated with changes in thrombin generation as measured by CAT. Both doses of K2-MK-7 also reduced media thickness in the brachiocephalic artery, but did not modify atherosclerotic plaque size.In conclusion, K2-MK-7 improves NO-dependent endothelial function in ApoE/LDLR−/− mice. This study, identifies the endothelial profile of the pharmacological activity of vitamin K2, which has not been previously described.

152 - Pulmonary and systemic nitric oxide deficiency in the early phase of lung metastasis in 4T1 metastatic breast cancer murine model

The role of nitric oxide (•NO) in metastatic cancer progression has not been fully explained. In the present work we characterized the development of •NO deficiency in the lung and examined changes in •NO and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in Balb/c mice. Furthermore, orthotopic and intravenous models of 4T1 metastatic cancer in mice were used to study the differential effects of L-NAME-induced •NO deficiency on the primary tumor and the metastatic burden. •NO was assessed in isolated aorta and lungs using EPR spin-trapping and chemiluminescence, while NOx in blood were measured using either EPR, chemiluminescence or Griess reaction. Early pulmonary metastasis was correlated with lung inflammation. Pulmonary endothelial function and systemic •NO availability in 4T1 breast cancer-bearing mice were compromised prior to the development of pulmonary metastasis,preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT). Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway. L-NAME-induced •NO-deficiency showed that •NO plays a major role in the primary tumor development, but it is not the key mediator responsible for the regulation of cancer cell extravasation to the lungs in our model, despite clear-cut •NO-deficiency in the lung, reflected by a fall in systemic •NO bioavailability, both occurring early in the pre-metastatic phase.

Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo.

Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood ex vivo. However, the sources of the nitrosylating species in vivo and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO2; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors ex vivo, while it was enhanced upon caveolin-1 deletion in vivo, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function.

Alterations in NO- and PGI2- dependent function in aorta in the orthotopic murine model of metastatic 4T1 breast cancer: relationship with pulmonary endothelial dysfunction and systemic inflammation

BackgroundPatients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer.MethodsBALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI2-dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation.ResultsAs early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI2 production.ConclusionsIn 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway.

Alterations in plasma L-arginine and methylarginines in heart failure and after heart transplantation

Objective. Endothelial function, including the nitric oxide (NO)-pathway, has previously been extensively investigated in heart failure (HF). In contrast, studies are lacking on the NO pathway after heart transplantation (HT). We therefore investigated substances in the NO pathway prior to and after HT in relation to hemodynamic parameters. Design. 12 patients (median age 50.0 yrs, 2 females), heart transplanted between June 2012 and February 2014, evaluated at our hemodynamic lab, at rest, prior to HT, as well as four weeks and six months after HT were included. All patients had normal left ventricular function post-operatively and none had post-operative pulmonary hypertension or acute cellular rejection requiring therapy at the evaluations. Plasma concentrations of ADMA, SDMA, L-Arginine, L-Ornithine and L-Citrulline were analyzed at each evaluation. Results. In comparison to controls, the plasma L-Arginine concentration was low and ADMA high in HF patients, resulting in low L-Arginine/ADMA-ratio pre-HT. Already four weeks after HT L-Arginine was normalized whereas ADMA remained high. Consequently the L-Arginine/ADMA-ratio improved, but did not normalize. The biomarkers remained unchanged at the six-month evaluation and the L-Arginine/ADMA-ratio correlated inversely to pulmonary vascular resistance (PVR) six months post-HT. Conclusions. Plasma L-Arginine concentrations normalize after HT. However, as ADMA is unchanged, the L-Arginine/ADMA-ratio remained low and correlated inversely to PVR. Together these findings suggest that (i) the L-Arginine/ADMA-ratio may be an indicator of pulmonary vascular tone after HT, and that (ii) NO-dependent endothelial function is partly restored after HT. Considering the good postoperative outcome, the biomarker levels may be considered “normal” after HT.

Indispensably evil! The role of oxygen in nitric‐oxide dependent endothelial function

BackgroundEndothelium derived nitric oxide (NO) is crucial in preserving vascular tone. Several evidence shows that NO bioavailability and utilization is regulated by oxygen (O2) concentration. However, there are still not sufficient experimental data to show how O2 concentration affects NO‐dependent endothelial function in vasculature.AimTo investigate the role of O2 concentration on NO‐dependent endothelial function in vasculature. We hypothesized that different O2 concentration would have elicited different responses during single passive limb movement (sPLM) test, which is known to be an index of NO‐dependent systemic endothelial function.MethodsEndothelium‐dependent dilation was measured in 7 active men (age, 34±12 years; weight, 70±8 kg, height, 173±4 cm, VO2max 55.9±10.5 ml/min/kg) by means of sPLM test performed in a normobaric‐hypoxic chamber in two conditions: Normoxic (N; FiO2 ~20.9%), and Hypoxic (H; FiO2 ~16.25%). For the two conditions femoral artery diameter, as well as basal blood flow (BF), Peak BF, ΔPeak BF, area under the curve (AUC) were calculated.ResultsIn H, femoral artery diameter did not change (−2.2±3.7%, p=0.675), basal BF significantly increased (+24±9%, p=0.043), as well as peak BF (+32±10%, p=0.034), ΔPeak BF (+34±15%, p=0.040), and AUC (+44±16%, p=0.018).ConclusionAs hypotized, different O2 concentrations provoked different responses during the sPLM test. These results suggest an O2‐dependent NO availability and utilization, which may play an essential role in endothelial function during hypoxia.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nitric Oxide-Dependent Endothelial Dysfunction and Reduced Arginine Bioavailability in Plasmodium vivax Malaria but No Greater Increase in Intravascular Hemolysis in Severe Disease.

Pathogenesis of severe Plasmodium vivax malaria is poorly understood. Endothelial dysfunction and reduced nitric oxide (NO) bioavailability characterize severe falciparum malaria, but have not been assessed in severe vivax malaria. In patients with severe vivax malaria (n = 9), patients with nonsevere vivax malaria (n = 58), and healthy controls (n = 79), we measured NO-dependent endothelial function by using reactive hyperemia-peripheral arterial tonometry (RH-PAT) and assessed associations with arginine, asymmetric dimethylarginine (ADMA), and hemolysis. The L-arginine level and the L-arginine to ADMA ratio (a measure of L-arginine bioavailability) were reduced in patients with severe vivax malaria and those with nonsevere vivax malaria, compared with healthy controls (median L-arginine level, 65, 66, and 98 µmol/mL, respectively [P = .0001]; median L-arginine to ADMA ratio, 115, 125, and 187, respectively [P = .0001]). Endothelial function was impaired in proportion to disease severity (median RH-PAT index, 1.49, 1.73, and 1.97 in patients with severe vivax malaria, those with nonsevere vivax malaria, and healthy controls, respectively; P = .018) and was associated with the L-arginine to ADMA ratio. While the posttreatment fall in hemoglobin level was greater in severe vivax malaria as compared to nonsevere vivax malaria (2.5 vs 1 g/dL; P = .0001), markers of intravascular hemolysis were not higher in severe disease. Endothelial function is impaired in nonsevere and severe vivax malaria, is associated with reduced L-arginine bioavailability, and may contribute to microvascular pathogenesis. Severe disease appears to be more associated with extravascular hemolysis than with intravascular hemolysis.

Abstract 089: CD36 Genetic Variant Impacts Nitric Oxide Regulation of Endothelial Function: Response to Chronic Treatment with Phosphodiesterase 5 Inhibition

CD36, a scavenger receptor expressed on endothelial cells, interacts with thrombospodin-1, a matrix protein that modulates nitric oxide-soluble guanylate cyclase (NO-sGC) signaling. CD36 genetic variants associate with endothelial dysfunction, atherosclerosis, hypertension and insulin resistance. A coding variant of CD36 (rs3211938, G/T genotype) that causes partial CD36 deficiency (50% reduction) is common (~18%) in African Americans (AA); however, it is unknown, if this genotype influences NO-dependent endothelial function. This study examined whether potentiating NO-sGC pathways with the phosphodiesterase 5 inhibitor, sildenafil citrate, improves endothelial function and insulin sensitivity in AA women with or without the G/T genotype. Forty-six AA women with metabolic syndrome (MetS) participated in a 4-week, parallel-arm, double-blind, and placebo-controlled study. Carefully matched subjects were randomly assigned to sildenafil citrate 20 mg TID versus placebo; sildenafil (n= 23, 42±10 years old, BMI 39±5 kg/m2, fasting insulin 15±8 uU/ml) and placebo (n=23, age 43±10, BMI 39±6 kg/m2, fasting insulin 14±10 uU/ml). Primary endpoints were insulin sensitivity and endothelial function measured by intravenous glucose tolerance test and flow mediated dilation, respectively. Treatment compliance was documented with plasma sildenafil levels (mean 57±50 ng/ml). There was no difference in insulin sensitivity (p=0.676) or flow-mediated dilation (p=0.649) between intervention groups. However, subgroup analyses showed a significant interaction between sildenafil citrate treatment and G/T genotype (p=0.018). Sildenafil citrate improved endothelial function in G/T carriers (the mean difference: 2.9, the 95% CI: -0.90 to 6.8, p = 0.126) and decreased endothelial function in T/T carriers (the mean difference: -2.6, the 95% CI: -5.1 to -0.1, p = 0.040). We conclude that the rs3211938 common CD36 genetic variant influences NO-dependent endothelial function in response to chronic treatment with phosphodiesterase 5 inhibition. Further studies are needed to determine if rs3211938 and other common CD36 genotypes influence endothelial function and the inter-individual variability in response to the drug.

Decreased endothelial nitric oxide bioavailability, impaired microvascular function, and increased tissue oxygen consumption in children with falciparum malaria.

Endothelial nitric oxide (NO) bioavailability, microvascular function, and host oxygen consumption have not been assessed in pediatric malaria. We measured NO-dependent endothelial function by using peripheral artery tonometry to determine the reactive hyperemia index (RHI), and microvascular function and oxygen consumption (VO2) using near infrared resonance spectroscopy in 13 Indonesian children with severe falciparum malaria and 15 with moderately severe falciparum malaria. Compared with 19 controls, children with severe malaria and those with moderately severe malaria had lower RHIs (P = .03); 12% and 8% lower microvascular function, respectively (P = .03); and 29% and 25% higher VO2, respectively. RHIs correlated with microvascular function in all children with malaria (P < .001) and all with severe malaria (P < .001). Children with malaria have decreased endothelial and microvascular function and increased oxygen consumption, likely contributing to the pathogenesis of the disease.

Vascular effects and safety of dalcetrapib in patients with or at risk of coronary heart disease: the dal-VESSEL randomized clinical trial

High-density lipoprotein cholesterol (HDL-C) is inversely associated with cardiovascular (CV) events and thus an attractive therapeutic target. However, in spite of marked elevations in HDL-C, the first cholesterol transport protein (CETP) inhibitor torcetrapib raised blood pressure (BP), impaired endothelial function, and increased CV mortality and morbidity. Dalcetrapib is a novel molecule acting on CETP with a different chemical structure to torcetrapib. As HDL stimulates nitric oxide (NO), suppresses inflammation, and exerts protective CV effects, we investigated the effects of dalcetrapib on endothelial function, blood pressure, inflammatory markers, and lipids in patients with, or at risk of, coronary heart disease (CHD) in a double-blind randomized placebo-controlled trial (clinicaltrials.gov number NCT00655538). Patients with target low-density lipoprotein cholesterol (LDL-C) levels received dalcetrapib 600 mg/day or placebo for 36 weeks on top of standard therapy (including statins). The primary outcome measures were the change from baseline of flow-mediated dilatation (%FMD) of the right brachial artery after 5 min of cuff occlusion at 12 weeks and the 24 h ambulatory blood pressure monitoring (ABPM) at week 4. Secondary outcomes included change from baseline in FMD after 36 weeks and the change in ABPM at 12 and 36 weeks, changes in HDL-C, LDL-C, triglycerides, CETP activity, as well as standard safety parameters. Four hundred seventy-six patients were randomized. Baseline FMD was 4.1 ± 2.2 and 4.0 ± 2.4% with placebo or dalcetrapib, respectively and did not change significantly from placebo after 12 and 36 weeks (P = 0.1764 and 0.9515, respectively). After 4, 24, and 36 weeks of treatment with dalcetrapib, CETP activity decreased by 51, 53, and 56% (placebo corrected, all P < 0.0001), while at weeks 4, 12, and 36 HDL-C increased by 25, 27, and 31% (placebo corrected, all P < 0.0001). Low-density lipoprotein cholesterol levels did not change. At baseline, ABPM was 125 ± 12/74 ± 8mmHg in the placebo and 128 ± 11/75 ± 7mmHg in the dalcetrapib group (P = 0.3372 and 0.1248, respectively, placebo-corrected change from baseline) and did not change for up to 36 weeks. Biomarkers of inflammation, oxidative stress, and coagulation did not change during follow-up except for Lp-PLA(2) mass levels which increased by 17% (placebo corrected). Overall 7 patients given dalcetrapib and 8 patients given placebo experienced at least one pre-specified adjudicated event (11 events with dalcetrapib and 12 events with placebo). The dal-VESSEL trial has established the tolerability and safety of CETP-inhibition with dalcetrapib in patients with or at risk of CHD. Dalcetrapib reduced CETP activity and increased HDL-C levels without affecting NO-dependent endothelial function, blood pressure, or markers of inflammation and oxidative stress. The dal-OUTCOMES trial (NCT00658515) will show whether dalcetrapib improves outcomes in spite of a lack of effect on endothelial function.

Novel strategies to ameliorate radiation injury: a possible role for tetrahydrobiopterin.

Novel pharmacological strategies are urgently needed to prevent or reduce radiation-induced tissue injury. Microvascular injury is a prominent feature of both early and delayed radiation injury. Radiation-induced endothelial dysfunction is believed to play a key role in the pathogenesis of post-irradiation tissue injury. Hence, strategies that could prevent or improve endothelial malfunction are expected to ameliorate the severity of radiation injury. This review focuses on the therapeutic potential of the nitric oxide synthase (NOS) cofactor 5,6,7,8-tetrahydrobiopterin (BH4) as an agent to reduce radiation toxicity. BH4 is an essential cofactor for all NOS enzymes and a critical determinant of NOS function. Inadequate availability of BH4 leads to uncoupling of the NOS enzyme. In an uncoupled state, NOS produces the highly oxidative radicals superoxide and peroxynitrite at the cost of NO. Under conditions of oxidative stress, such as after radiation exposure, BH4 availability might be reduced due to the rapid oxidation of BH4 to 7,8-dihydrobiopterin (7,8-BH2). As a result, free radical-induced BH4 insufficiency may increase the oxidative burden and hamper NO-dependent endothelial function. Given the growing evidence that BH4 depletion and subsequent endothelial NOS uncoupling play a major role in the pathogenesis of endothelial dysfunction in various diseases, there is substantial reason to believe that improving post-irradiation BH4 availability, by either supplementation with it or modulation of its metabolism, might be a novel strategy to reduce radiation-induced endothelial dysfunction and subsequent tissue injury.

Electrophilic nitro-fatty acids: anti-inflammatory mediators in the vascular compartment

Vascular inflammatory disorders are often associated with both decreased NO bioavailability and a lack of responsiveness to NO, a consequence of impaired NO biosynthesis, dysregulated l-arginine metabolism, endothelial nitric oxide synthase (eNOS) uncoupling and NO consumption induced by redox reactions of NO. The latter is mediated via oxidative inflammatory conditions altering NO-dependent endothelial function, including vascular tone and cell proliferation. The redox reactions of NO and byproducts such as nitrite can react to yield electrophilic nitro-fatty acid derivatives (NO(2)-FAs) and exemplify a biochemical convergence of reactions participating in NO and lipid signaling. NO(2)-FAs represent a novel therapeutic strategy to treat vascular disorders by improving endothelial dysfunction through enhancing NO signaling and blocking vascular smooth muscle proliferation, inflammation, and maladaptive remodeling.

Regulation of arginase pathway in response to wall shear stress

ObjectiveAlterations of wall shear stress can predispose the endothelium to the development of atherosclerotic plaques. Ample evidence indicates that arginase expression and/or activity correlates with several risk factors for cardiovascular disease including atherosclerosis. We investigated the regulation of arginase pathway in response to distinct patterns of wall shear stress. MethodsIsolated porcine endothelial cells and carotid arterial segments were perfused under unidirectional high shear stress (HSS) or oscillatory shear stress (OSS) for 1 and 3 days. Arginase I and II expression, cellular localization and enzyme activity were, respectively, assessed by Western blot, immunohistochemistry and colorimetric determination of urea. The contribution of arginase to the processes of endothelial dysfunction, cell proliferation and arterial remodeling induced by OSS was evaluated by administration of the arginase inhibitor N-ω-hydroxy-nor-l-arginine (nor-Noha). ResultsOnly arginase II isoform was detected on porcine carotid endothelial cells and on carotid artery. Exposure of arteries to OSS increased arginase II expression and activity as compared to HSS. Inhibition of arginase by nor-Noha improved NO-dependent endothelial function and decreased total vascular ROS formation in arteries submitted to OSS. In addition, inhibition of arginase activity decreased smooth muscle cell proliferation rate with no effect on collagen content after OSS. ConclusionsExposure of carotid artery to oscillatory flow induced a more pronounced activation of arginase as compared to HSS. Inhibition of arginase in arteries exposed to OSS improved NO-dependent endothelial function and decrease smooth muscle cell proliferation rate, both processes are important for the focal development of atherosclerotic plaque.

Anti-diabetic effects of 1-methylnicotinamide (MNA) in streptozocin-induced diabetes in rats

1-Methylnicotinamide (MNA), a major endogenous metabolite of nicotinamide, possesses anti-thrombotic and anti-inflammatory activity, and reverses endothelial dysfunction. In the present work, we investigated whether such a vasoprotective profile of MNA activity affords anti-diabetic action in rats.Diabetes was induced by streptozotocin (STZ) in Sprague-Dawley rats. Eight weeks after STZ injection in untreated or MNAtreated rats (100mg kg–1 daily), development of diabetes (plasma concentrations of fasting and non-fasting glucose, HbA1c, peptide C), development of oxidant stress (lipid peroxidation, carbonylation of plasma proteins), as well as NO-dependent endothelial function in aorta, coronary and mesenteric vessels were analyzed. Finally, the effect of chronic treatment with MNAon long-term survival of diabetic rats was determined.Chronic treatment withMNAprofoundly lowered fasting glucose concentrations in plasma, displayed mild effects on plasma HbA1c and peptide C concentrations, while having no effects on non-fasting glucose. On the other hand, MNAtreatment considerably lowered lipid peroxidation, protein carbonylation, completely prevented impairment of endothelium-dependent vasodilatation in the aorta that was mediated entirely by NO, but failed to affect endothelial function in resistant vessels, which was mediated only partially by NO. Most importantly, chronic treatment with MNAprolonged the long-term survival of diabetic rats. In conclusion, MNA displayed a significant anti-diabetic effect that may be linked to its vasoprotective activity.