Endothelial Nitric Oxide Production Research Articles

Circulating Extracellular Vesicles Are Elevated with Prediabetes and Adversely Affect Cerebral Endothelial Nitric Oxide and Endothelin-1 Production

Experimental and clinical studies indicate that a proatherogenic endothelial phenotype develops in the prediabetic, hyperglycemic state providing the platform for accelerated vascular disease with prediabetes and, in turn, type 2 diabetes. Prediabetes (fasting plasma glucose concentrations: 100-125 mg/dL) is associated with an increased risk of stroke. Circulating endothelial cell-derived microvesicles (EMVs) are now recognized as both a biomarker and mediator of vascular health and disease. In fact, EMVs have been shown to be involved in the pathogenesis of cerebrovascular dysfunction leading to increased stroke risk. Circulating EMVs are elevated in adults with prediabetes; whether EMVs associated with the prediabetic state promote endothelial abnormalities known to increase stroke risk is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with prediabetes on brain endothelial cell nitric oxide (NO) and endothelin (ET)-1 production. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 20 mid-life adults (age:44-69 yr): 10 normoglycemic (NG; 8M/2F; age: 55±2 yr; BMI: 25.7±0.8 kg/m2; fasting plasma glucose: 87±2 mg/dL) and 10 prediabetic (PreD: 8M/2F; 55±2 yr; 27.1±1.5 kg/m2; 107±2 mg/dL). Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Expression of intracellular proteins of interest was determined by capillary electrophoresis immunoassay. Circulating EMV concentrations were significantly higher (~65%) in PreD vs NG (180±25 vs 110±13 EMV/μL) and significantly associated with plasma glucose concentrations (r=0.48; P=0.03). Phosphorylation is the primary posttranslational modification regulating eNOS enzyme activity. Phosphorylation of Ser1177 confers the greatest activation of eNOS; whereas, phosphorylation at Thr495 reduces eNOS activation. Expression of p-eNOS (Ser1177) was lower (47.6±2.6 vs 58.6±3.5 AU; P=0.02) and p-eNOS (Thr495) higher (82.6±4.4 vs 31.1±2.5 AU; p<0.001) in cells treated with EMVs from PreD vs NG. Concordantly, NO production was ~20% lower (6.0±0.2 vs 7.2±0.4 μmol/L; p<0.05) in cells treated with EMVs from PreD adults. PreD-associated EMVs also significantly increased (~25-70%) the expression of Big ET-1 (804.4±47.7 vs 656.7±30.7 AU) and endothelin converting enzyme (290.0±24.6 vs 169.6±19.0 AU) as well as ET-1 production (30.2±4.3 vs 24.2±2.0 pg/mL) in hCMECs. In summary, EMVs harvested from adults with PreD reduced endothelial nitric oxide synthase (eNOS) activation and NO production and increased ET-1 synthesis and release in brain endothelial cells in vitro. Increased risk and incidence of stroke associated with prediabetes may be mediated by circulating EMVs. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of exercise training on endothelial metabolism and skeletal muscle angiogenic potential in type 2 diabetes

Type 2 diabetes (T2D) is associated with vascular endothelial dysfunction and microvascular rarefaction in skeletal muscle tissue (1). Although endothelial dysfunction in metabolic disease is proposed to be associated with altered endothelial cell metabolism, there is to date a paucity of human data to support this notion. The aim of this study was to investigate the metabolism in microvascular endothelial cells derived from skeletal muscle biopsies. Moreover, as exercise training is known to reverse endothelial dysfunction and promote angiogenesis, the effect of a period of exercise training was assessed. Microvascular endothelial cells were isolated from m. vastus lateralis biopsies taken from obese individuals with (n = 7) and without (n = 8) T2D before and after 12 weeks of aerobic exercise training. Mitochondrial respiration, hydrogen peroxide (H2O2) emission, and nitric oxide (NO) formation were determined in the cells by the Oroboros Oxygraph-2K. In addition, angiogenic-, metabolic- and antioxidant enzymes and proteins, cell proliferation, and skeletal muscle capillarization were determined. At baseline, there was no difference between endothelial cells from obese control participants and T2D obese participants for any of the parameters measured: glycolytic capacity, as evidenced by a similar lactate production and PFK-1 protein content, respiratory capacity, H2O2 emission, NO emission, endothelial NO synthase protein (eNOS) content. The training period led to an overall increase in the respiratory capacity in endothelial cells from both groups (main effect, P = 0.021) whereas mitochondrial H2O2 emission remained unaltered. There was no change in glycolytic capacity, superoxide dismutase 2 or eNOS protein contents, or NO emission. Training did not influence the capillary-to-muscle fiber ratio in either group. The results of this study show that aerobic exercise training of obese control and obese diabetic subjects leads to an increase in the respiratory capacity of muscle microvascular endothelial cells. The data also indicate that there are no differences in endothelial metabolism and NO production between obese control and obese diabetic individuals. However, as the study is ongoing and only half of the subjects have been included at this time, power is limited and results may differ upon completion. Data from all subjects will be presented at the conference. Funding: The study is funded by Novo Nordisk A/S. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Circulating Extracellular Vesicles from Adults with Spinal Cord Injury on Pulmonary Artery Endothelial Cell Function

Spinal cord injury (SCI) is associated with an increased risk and prevalence of cardiopulmonary and cerebrovascular disease-related morbidity and mortality. Despite an improved understanding of the neurological and physical consequences associated with SCI, factors that initiate, promote and accelerate cardiopulmonary disease are poorly understood. It has become apparent that the increased incidence of pulmonary disorders in adults with chronic SCI is not solely due to worsening of traditional risk factors, but also involves ill-defined factors. Several studies have implicated circulating endothelial cell-derived microvesicles (EMVs) in the etiology of cardiopulmonary diseases. We have previously reported that circulating EMVs are elevated in adults with chronic SCI and the expression of their microRNA cargo promotes disease. Reduction in pulmonary nitric oxide (NO) bioavailability and increased endothelin (ET)-1 production underly many cardiopulmonary disorders associated with SCI. The experimental aim of this study was to determine the effect of EMVs isolated from adults with chronic tetraplegic SCI on pulmonary endothelial cell NO and ET-1 production. As part of an ongoing study, circulating EMVs (CD144-PE) were isolated (flow cytometry) from 7 non-injured adults (all male; age: 38±4 yr) and 7 motor complete SCI adults with tetraplegic SCI (male; 44±5 yr). All subjects were free of overt cardiometabolic disease. Human pulmonary artery endothelial cells (HPAECs) were cultured and separately treated with EMVs from each subject for 24 hr. Expression of intracellular NO and ET-1 proteins of interest was determined by capillary electrophoresis immunoassay. Circulating EMV concentrations were significantly higher (~200%) in SCI vs non-injured (154±36 vs 54±7 EMV/μL). Expression of p-eNOS (Ser1177), the primary activation site for eNOS, was ~30% lower (96.0±2.3 vs 140.7±8.9 AU; p<0.01) and p-eNOS (Thr495), primary inhibitory site, ~40% higher (40.7±1.7 vs 28.8±3.1 AU; p<0.01) in cells treated with EMVs from SCI vs non-injured. As a result, NO production was ~20% lower (6.5±0.3 vs 8.3±0.7 μmol/L; P=0.03) in HPAECs treated with EMVs from adults with SCI. SCI-associated EMVs also significantly increased (~70%) the expression of Big ET-1 (60.6±3.6 vs 36.1±2.7 AU) resulting in enhanced endothelial ET-1 production (884.0±22.9 vs 778.7±18.0 pg/mL). In conclusion, EMVs harvested from adults with SCI markedly reduced eNOS activation and NO production and increased ET-1 synthesis and release in pulmonary endothelial cells in vitro. Circulating EMVs represent a potential mechanistic factor underlying pulmonary disorders and increased disease risk with SCI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel dual inhibitor of the angiotensin and endothelin systems for the treatment of hypertension

Hypertension, affecting nearly half of the US adult population, is a primary cause of morbidity and mortality worldwide. Despite the availability of several classes of antihypertensive medications, only 25% of patients have their blood pressure under control, highlighting the need for novel antihypertensive therapeutics with better effcacy. Among hypertensive cases, salt-sensitive and drug-resistant hypertension are particularly concerning, as they are unresponsive to a combined regimen of three or more classes of medications, including angiotensin II (Ang II) receptor blockers (ARBs), angiotensin-converting enzyme inhibitors (ACEis), and diuretics. Salt-sensitive, observed in 50% of the general and 73% of black hypertensive patients, disproportionately affects the latter, leading to significantly higher mortality. Salt-sensitive hypertension in humans, as well as in various experimental models of hypertension (e.g., Ang II-induced, Dahl-salt sensitive [DSS] rats, deoxycorticosterone acetate [DOCA]-salt hypertensive rats), is characterized by significantly elevated levels of plasma endothelin-1 (ET-1), oxidative stress, and kidney dysfunction. ET-1 is often considered a final common pathway in orchestrating cardiovascular dysfunction through a range of proinflammatory mediators such as Ang II, cytokines, hypoxia, thrombin, reactive oxygen species (ROS), oxidized LDL, and vasopressin, all of which stimulate ET-1 production. In this capacity, we report the discovery of a gut microbiota-derived metabolite, indole-3-acetic acid (IAA), that simultaneously blocks ET-1 receptors (ETAR and ETBR) and the ET-biosynthetic enzyme ECE-1. Our data demonstrate that IAA inhibits ET-1-induced constriction of resistance mesenteric arteries, ROS production in human aortic smooth muscle cells, activation of cultured endothelial cells, adhesion molecule expression, and leukocyte adhesion. IAA inhibited purified human ECE-1 and suppressed Ang-II-induced and hypoxia-elicited ET-1 production from endothelial cells in vitro. Administration of a once-daily, extended-release formulation of IAA inhibits blood pressure elevation and kidney dysfunction in both DSS and Ang II-induced hypertension. IAA treatment preserved endothelial nitric oxide (NO) production and maintained smooth muscle sensitivity to a NO donor, sodium nitroprusside. Furthermore, IAA inhibited the expression of ROS-associated proteins such as Nox2 and Nox4, reduced oxidative damages, and decreased the production of ET-1 in vivo. Overall, IAA is the first compound that, through novel dual targeting of both the angiotensin and endothelin systems, demonstrates profound antihypertensive action in both salt-sensitive and angiotensin II-induced hypertension. Thus, IAA has the potential to effectively treat hypertension that does not respond to conventional therapies. American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nitrate-rich beetroot juice supplementation in midlife and older adults with renal dysfunction increases vascular endothelial function and changes the circulating milieu to improve endothelial cell nitric oxide production and oxidative stress

BACKGROUND: Midlife and older (ML/O) adults with mild-to-moderate renal dysfunction (MRD) are at increased risk for cardiovascular disease (CVD). A key antecedent to CVD in ML/O adults with MRD is vascular endothelial dysfunction, mediated by declines in nitric oxide (NO) bioavailability due to excess reactive oxygen species (ROS)-related oxidative stress. We have shown that targeting the nitrate-nitrite-NO pathway with sodium nitrite in heathy ML/O adults improves endothelial function and changes the circulating milieu to lower endothelial cell (EC) ROS bioactivity. However, if nitrate-rich beetroot juice (N-BRJ) is effective for improving endothelial function in ML/O men and women with MRD is unknown. HYPOTHESES: We tested the hypotheses that chronic N-BRJ in ML/O men and women with MRD would: 1) improve endothelial function and 2) induce changes to the circulating milieu to increase EC NO production and lower EC ROS bioactivity. We also assessed possible sex differences in the effcacy of N-BRJ for improving endothelial function. METHODS: Twenty-six ML/O men and women with MRD (estimated glomerular filtration rate [eGFR; CKD-EPI]: 40-89 mL/min/1.73m2) underwent three months of supplementation with N-BRJ (70 mL with 6.45 mmol nitrate/day) (n=13, 7 women; age: 65±2 years; eGFR: 69.2±3.3 mL/min/1.73m2) or nitrate-depleted BRJ (placebo, PBO) (n=13, 7 women; age: 68±2 years; eGFR: 79.8±3.1 mL/min/1.73m2) in a randomized, placebo-controlled, parallel-group design clinical trial. Endothelial function was assessed by brachial artery flow-mediated dilation (FMDBA). Smooth muscle sensitivity to NO was assessed by brachial artery dilation to sublingual nitroglycerin. Human aortic ECs (HAECs) in culture were exposed to 10% subject serum (n=10[6-7 women]/group) collected before and after N-BRJ and PBO supplementation. Acetylcholine-stimulated NO production (DAR-4M-AM) and whole-cell ROS bioactivity (CellROX) were assessed in HAECs using immunofluorescence. Results: N-BRJ supplementation increased FMDBA by 28% (pre: 4.6±0.7%, post: 5.9±0.8%; p=0.03). FMDBA did not change with PBO (pre: 4.6±0.9%, post: 4.7±0.8%; p>0.05). Traditional CVD risk factors (i.e., blood pressure, cholesterol) and smooth muscle sensitivity to NO were unchanged in both groups (p>0.05). Preliminary assessments suggest greater improvements in endothelial function with N-BRJ in women (Δ=post-pre intervention; ΔFMDBA=1.6±0.4) compared to men (ΔFMDBA=0.9±1.0). Compared to pre-intervention, NO production was (trending) 7% higher (p=0.05) and ROS bioactivity was 25% lower (p=0.02) in HAECs exposed to serum collected post N-BRJ. Serum from PBO had no effects (p>0.05). CONCLUSION: N-BRJ supplementation in ML/O adults with MRD holds promise for improving endothelial function, in part by inducing changes to the circulating milieu that increase NO production and decrease oxidative stress in ECs. The effects on endothelial function may be greater in women compared to men. Targeting the nitrate-nitrite-NO pathway with N-BRJ may be an effective strategy for improving endothelial function in ML/O adults with renal dysfunction to possibly reduce CVD risk. K01DK115524 (MJR); R01AG013038 (DRS); F32HL167552 (KOM); NIH/NCATS CTSA UL1TR002535. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of endothelial cell-selective adhesion molecule in the regulation of pulmonary vascular resistance

Endothelial cell-selective adhesion molecule (ESAM) is a member of the endothelial cell junction adhesion molecules family, which plays an important role in the maintenance of endothelial barrier function in the pulmonary circulation. The role of ESAM in affecting pulmonary vascular resistance remains unknown. To examine the role of ESAM in pulmonary vascular resistance, whole lungs were isolated from 16-18 weeks old ESAM knock out (KO) mice and wild type (WT) littermates, ventilated and perfused. We found that pulmonary vascular resistance is significantly increased in the lung of ESAM KO mice compared to WT mice. Bradykinin-induced, endothelium-dependent reduction in the pulmonary vascular resistance was impaired in ESAM KO mice, whereas the direct NO donor, sodium nitroprusside-induced endothelium independent responses remained similar in WT and ESAM KO mice. U46619 mediated increases in pulmonary vascular resistance was significantly higher in ESAM KO mice. Inhibition of the NO synthesis with the L-NAME (0.2 mM for 10 minutes) augmented the U46619-mediated increase in pulmonary vascular resistance in WT mice, but it did not affect the U46619 response in the ESAM KO mice. The mRNA expression of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor, was increased in the lungs of ESAM KO mice, whereas expression of collagen isoforms (type I and type III) and fibronectin were similar in WT and ESAM KO mice. Collectively, we propose that ESAM plays an important role in the maintenance of pulmonary vascular resistance, likely via preserving endothelial nitric oxide production in pulmonary arteries. AHA predoctoral award ID 917057. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypothermic Oxygenated Perfusion Improves Vascular and Contractile Function by Preserving Endothelial Nitric Oxide Production in Cardiac Grafts Obtained With Donation After Circulatory Death.

Cardiac donation after circulatory death is a promising option to increase graft availability. Graft preservation with 30 minutes of hypothermic oxygenated perfusion (HOPE) before normothermic machine perfusion may improve cardiac recovery as compared with cold static storage, the current clinical standard. We investigated the role of preserved nitric oxide synthase activity during HOPE on its beneficial effects. Using a rat model of donation after circulatory death, hearts underwent insitu ischemia (21 minutes), were explanted for a cold storage period (30 minutes), and then reperfused under normothermic conditions (60 minutes) with left ventricular loading. Three cold storage conditions were compared: cold static storage, HOPE, and HOPE with Nω-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor). To evaluate potential confounding effects of high coronary flow during early reperfusion in HOPE hearts, bradykinin was administered to normalize coronary flow to HOPE levels in 2 additional groups (cold static storage and HOPE with Nω-nitro-L-arginine methyl ester). Cardiac recovery was significantly improved in HOPE versus cold static storage hearts, as determined by cardiac output, left ventricular work, contraction and relaxation rates, and coronary flow (P<0.05). Furthermore, HOPE attenuated postreperfusion calcium overload. Strikingly, the addition of Nω-nitro-L-arginine methyl ester during HOPE largely abolished its beneficial effects, even when early reperfusion coronary flow was normalized to HOPE levels. HOPE provides superior preservation of ventricular and vascular function compared with the current clinical standard. Importantly, HOPE's beneficial effects require preservation of nitric oxide synthase activity during the cold storage. Therefore, the application of HOPE before normothermic machine perfusion is a promising approach to optimize graft recovery in donation after circulatory death cardiac grafts.

Pathophysiological basis of hepatopulmonary syndrome

Circulatory changes with increased blood flow and vasodilatation/vasoconstriction imbalance are an integral consequence of liver cirrhosis and portal hypertension and can affect the pulmonary circulation with the development of vascular disorders, with hepatopulmonary syndrome (HPS) being the most common. HPS is a serious pulmonary complication of progressive liver disease, resulting in a poor clinical prognosis. Vascular tone decrease, monocytic infiltration of pulmonary vessels, formation of intrapulmonary arteriovenous shunts, dysfunction of alveolar type II cells, destruction of the endothelial glycocalyx are important in the pathogenesis of HPS. Abnormalities of pulmonary capillaries lead to hypoxemia caused by a violation of the ventilation/perfusion ratio, diffusion disorders, and the development of arteriovenous anastomoses. Infiltration of the pulmonary vessels by monocytes is one of the key factors of HPS. This migration is facilitated by the intestinal microbiota translocation into the portal bloodstream with increased expression of proinflammatory cytokines (tumor necrosis factor α, interleu­kins 1, 6), leading to the activation of monocytes. Monocytes located in the pulmonary circulation promote the vasodilation through the activation of inducible nitric oxide (NO) synthase and thus NO production. This is also associated with endothelial dysfunction due to a decreased hepatic secretion of bone morphogenetic protein 9 and increased endothelin 1, endothelial overexpression of endothelin B receptors, and increased endothelial NO production. Proangiogenic factors such as vascular endothelial growth factor, platelet-derived growth factor, and placental growth factor play an important role in the proliferation of pulmonary capillaries. Circulation of tumor necrosis factor α, bile acids and monocyte infiltration in the pulmonary circulation lead to increased apoptosis of alveolar type II cells and decreased surfactant synthesis. Chronic inflammation in HPS disrupts the continuity of the endothelial glycocalyx layer. This article provides an overview of the current knowledge on the pathogenesis of HPS, summarizes many features of the disease based on the literature research in MEDLINE database on the PubMed platform.

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

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

Oral curcumin phytosome supplementation improves anthropometric measures of adiposity and enhances endothelial function in rats on a high-fat-diet regimen

Objectives: Curcumin has a protective role in endothelial function and nitric oxide (NO) production in animal models of different diseases; however, the role of curcumin on aortic reactivity in rats placed on a high-fat diet (HFD) remains unclear. This study aims to determine whether oral curcumin phytosome supplementation can reduce adiposity and enhance endothelial function. Materials and Methods: Rats were assigned to one of three groups: normal diet (ND), HFD for 20 weeks, and HFD supplemented with curcumin phytosome (HFD + Curcumin). Anthropometric measures were recorded weekly for the three groups, until the end of the feeding regimen. After 20 weeks of feeding on HFD, myographic investigations were conducted on thoracic aortic rings dissected from HFD and HFD + Curcumin rats. The response to high potassium chloride (KCl), incremental doses of phenylephrine (Phe) before and after L-NAME treatment, acetylcholine (ACh), or sodium nitroprusside (SNP), was evaluated. ACh-induced relaxation was also assessed in HFD + Curcumin rats, after preincubation with chromium III-mesoporhyrin. Results: HFD rats exhibited increased adiposity measures, some of which were negatively correlated with vasorelaxation response to ACh. HFD + Curcumin rats had reduced anthropometric measures, compared to HFD rats. Aortic rings from HFD and HFD + Curcumin rats exhibited comparable contractile responses to KCl and Phe. The difference in contractile response to Phe before and after L-NAME incubation was greater for HFD + Curcumin rats. ACh induced greater vasorelaxant responses in HFD + Curcumin rats. There was no group difference in the relaxant response to SNP. In HD + Curcumin rats, chromium III mesoporphyrin significantly reduced ACh-induced relaxations. Conclusion: Oral curcumin phytosome supplementation could reduce adiposity in rats placed on an HFD and may have enhanced basal and stimulated NO release from the endothelium, and heme oxygenase-1 may partly mediate this curcumin protective role. This study provides evidence that this curcumin formulation, taken as a daily supplement, may be effective in providing some protection against adiposity-associated adverse cardiovascular disorders.

Asymmetric and symmetric dimethylarginine in high altitude pulmonary hypertension (HAPH) and high altitude pulmonary edema (HAPE)

Introduction: High altitude exposure may lead to high altitude pulmonary hypertension (HAPH) and high altitude pulmonary edema (HAPE). The pathophysiologic processes of both entities have been linked to decreased nitric oxide (NO) availability.Methods: We studied the effect of acute high altitude exposure on the plasma concentrations of asymmetric (ADMA) and symmetric dimethylarginine (SDMA), L-arginine, L-ornithine, and L-citrulline in two independent studies. We further investigated whether these biomarkers involved in NO metabolism were related to HAPH and HAPE, respectively. Fifty (study A) and thirteen (study B) non-acclimatized lowlanders were exposed to 4,559 m for 44 and 67 h, respectively. In contrast to study A, the participants in study B were characterized by a history of at least one episode of HAPE. Arterial blood gases and biomarker concentrations in venous plasma were assessed at low altitude (baseline) and repeatedly at high altitude. HAPE was diagnosed by chest radiography, and HAPH by measuring right ventricular to atrial pressure gradient (RVPG) with transthoracic echocardiography. AMS was evaluated with the Lake Louise Score (LLS) and the AMS-C score.Results: In both studies SDMA concentration significantly increased at high altitude. ADMA baseline concentrations were higher in individuals with HAPE susceptibility (study B) compared to those without (study A). However, upon high altitude exposure ADMA only increased in individuals without HAPE susceptibility, while there was no further increase in those with HAPE susceptibility. We observed an acute and transient decrease of L-ornithine and a more delayed but prolonged reduction of L-citrulline during high altitude exposure. In both studies SDMA positively correlated and L-ornithine negatively correlated with RVPG. ADMA was significantly associated with the occurrence of HAPE (study B). ADMA and SDMA were inversely correlated with alveolar PO2, while L-ornithine was inversely correlated with blood oxygenation and haemoglobin levels, respectively.Discussion: In non-acclimatized individuals ADMA and SDMA, two biomarkers decreasing endothelial NO production, increased after acute exposure to 4,559 m. The observed biomarker changes suggest that both NO synthesis and arginase pathways are involved in the pathophysiology of HAPH and HAPE.

Effects of circulating endothelial microvesicles isolated from adults with obesity on endothelial cell inflammation, apoptosis, and nitric oxide production.

Circulating endothelial cell-derived microvesicles (EMVs) have been shown to be elevated with obesity and associated with endothelial dysfunction; however, their direct effect on endothelial cells is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with obesity on endothelial cell inflammation, apoptosis, and nitric oxide (NO) production. EMVs (CD144+ microvesicles) were identified, enumerated, and isolated from plasma by flow cytometry from 24 sedentary adults: 12 normal-weight adults [8 M/4 F; age: 55 ± 6 yr; body mass index (BMI): 24.3 ± 0.7 kg/m2; EMV: 144 ± 53 EMVs/µL] and 12 adults with obesity (6 M/6 F; 59 ± 7 yr; BMI: 31.0 ± 1.1 kg/m2; EMV: 245 ± 89 EMVs/µL). Human umbilical vein endothelial cells were cultured and treated with EMVs from either normal-weight adults or adults with obesity. EMVs from obese adults induced significantly higher release of interleukin (IL)-6 (108.2 ± 7.7 vs. 90.9 ± 10.0 pg/mL) and IL-8 (75.4 ± 9.8 vs. 59.5 ± 11.5 pg/mL) from endothelial cells vs. EMVs from normal-weight adults, concordant with greater intracellular expression of phosphorylated NF-κB p65 (Ser536; active NF-κB) [145.0 ± 34.1 vs. 114.5 ± 30.4 arbitrary units (AU)]. Expression of phosphorylated p38-MAPK (15.4 ± 5.7 vs. 9.2 ± 2.5 AU) and active caspase-3 (168.2 ± 65.5 vs. 107.8 ± 40.5 AU), markers of cell apoptosis, was higher in cells treated with obesity-related EMVs. Phosphorylated endothelial nitric oxide synthase (eNOS) (Ser1177) expression (23.5 ± 7.2 vs. 34.7 ± 9.7 AU) and NO production (6.9 ± 1.4 vs. 8.7 ± 0.7 µmol/L) were significantly lower in the cells treated with EMVs from obese adults. These data indicate that circulating EMVs from adults with obesity promote a proinflammatory, proapoptotic, and NO-compromised endothelial phenotype. Circulating EMVs are a potential mediator of obesity-related endothelial dysfunction.NEW & NOTEWORTHY In the present study, we determined the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from adults with obesity on endothelial cell inflammation, apoptosis, and nitric oxide (NO) production in vitro. Circulating EMVs harvested from adults with obesity promoted a proinflammatory, proapoptotic, and NO-compromised endothelial phenotype. Elevated circulating EMVs in adults with obesity, independent of other cardiometabolic risk factors, are a potential novel systemic mediator of obesity-related endothelial dysfunction and vascular risk.

Dapagliflozin-Induced Myocardial Flow Reserve Improvement is not Associated with HDL Ability to Stimulate Endothelial Nitric Oxide Production.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors have shown controversial results in modulating plasma lipids in clinical trials. Most studies found slight increases in high-density lipoprotein (HDL) cholesterol but few have provided evidence on HDL functionality with disappointing results. However, there is broad agreement that these drugs provide cardiovascular protection through several mechanisms. Our group demonstrated that dapagliflozin improves myocardial flow reserve (MFR) in patients with type 2 diabetes (T2D) with coronary artery disease (CAD). The underlying mechanisms are still unknown, although in vitro studies have suggested the involvement of nitric oxide (NO). To investigate changes in HDL-mediated modulation of NO production with dapagliflozin and whether there is an association with MFR. Sixteen patients with CAD-T2D were enrolled and randomized 1:1 to dapagliflozin or placebo for 4weeks. Blood samples were collected before and after treatment for each group. The ability of HDL to stimulate NO production in endothelial cells was tested in vitro by incubating human umbilical vein endothelial cells (HUVEC) with apoB-depleted (apoB-D) serum of these patients. The production of NO was assessed by fluorescent assay, and results were expressed as fold versus untreated cells. Change in HDL-mediated NO production remained similar in dapagliflozin and placebo group, even after adjustment for confounders. There were no significant correlations between HDL-mediated NO production and MFR either at baseline or after treatment. No changes were found in HDL cholesterol in either group, while low-density lipoprotein cholesterol (LDL cholesterol) significantly decreased compared to baseline only in treatment group (p = 0.043). In patients with T2D-CAD, beneficial effects of dapagliflozin on coronary microcirculation seem to be unrelated to HDL functions. However, HDL capacity to stimulate NO production is not impaired at baseline; thus, the effect of drug treatments would be negligible. To conclude, we can assume that HDL-independent molecular pathways are involved in the improvement of MFR in this population. EudraCT No. 2016-003614-27; ClinicalTrials.gov Identifier: NCT03313752.

Update of Potential Biomarkers in Risk Prediction and Monitoring of Atherosclerosis in Systemic Lupus Erythematosus to Prevent Cardiovascular Disease.

Systemic lupus erythematosus is a chronic connective tissue disease associated with an increased risk of premature atherosclerosis. It is estimated that approximately 10% of SLE patients develop significant atherosclerosis each year, which is responsible for premature cardiovascular disease that is largely asymptomatic. This review summarizes the most recent reports from the past few years on biomarkers of atherosclerosis in SLE, mainly focusing on immune markers. Persistent chronic inflammation of the vascular wall is an important cause of cardiovascular disease (CVD) events related to endothelial dysfunction, cell proliferation, impaired production and function of nitric oxide and microangiopathic changes. Studies on pathogenic immune mediators involved in atherosclerosis will be crucial research avenues for preventing CVD.

The pathophysiology of diabetic foot: a narrative review.

An aging population and changes in dietary habits have increased the incidence of diabetes, resulting in complications such as diabetic foot ulcers (DFUs). DFUs can lead to serious disabilities, substantial reductions in patient quality of life, and high financial costs for society. By understanding the etiology and pathophysiology of DFUs, their occurrence can be prevented and managed more effectively. The pathophysiology of DFUs involves metabolic dysfunction, diabetic immunopathy, diabetic neuropathy, and angiopathy. The processes by which hyperglycemia causes peripheral nerve damage are related to adenosine triphosphate deficiency, the polyol pathway, oxidative stress, protein kinase C activity, and proinflammatory processes. In the context of hyperglycemia, the suppression of endothelial nitric oxide production leads to microcirculation atherosclerosis, heightened inflammation, and abnormal intimal growth. Diabetic neuropathy involves sensory, motor, and autonomic neuropathies. The interaction between these neuropathies forms a callus that leads to subcutaneous hemorrhage and skin ulcers. Hyperglycemia causes peripheral vascular changes that result in endothelial cell dysfunction and decreased vasodilator secretion, leading to ischemia. The interplay among these four preceding pathophysiological factors fosters the development and progression of infections in individuals with diabetes. Charcot neuroarthropathy is a chronic and progressive degenerative arthropathy characterized by heightened blood flow, increased calcium dissolution, and repeated minor trauma to insensate joints. Directly and comprehensively addressing the pathogenesis of DFUs could pave the way for the development of innovative treatment approaches with the potential to avoid the most serious complications, including major amputations.

Evaluation of the antioxidant activity and nitric oxide production effect of formulated crispy vegetables from thermal processing of Amaranthus viridis and Sauropus androgynous

Background: Amaranthus viridis and Sauropus androgynous are common edible vegetables consumed by the natives of Asia. This research aimed to carry out a comparative evaluation on the antioxidant activity and nitric oxide production effect of crispy vegetables formula of A. viridis and S. androgynous made by thermal processing.Methods: A. viridis and S. androgynous fresh vegetable leaves were dried, processed and formulated into crispy vegetables: A. viridis crispy vegetables, S. androgynous crispy vegetables and 50:50% (w/w) mixture of A. viridis and S. androgynous crispy vegetables. Total phenolic and flavonoids contents of the three crispy vegetables were evaluated using Folin Ciocalteu colorimetric and aluminum chloride assay. Results: S. androgynous crispy vegetable had higher phenolic (36.55 ± 0.01 mg GAE/g) and flavonoid (81.17±0.00 mg QE/g) contents than A. viridis crispy vegetables (16.55±0.00 mg GAE/g and 40.58±0.01 QE/g respectively) and 50:50% mixed crispy vegetable formula of A. viridis and S. androgynous (26.01 ± 0.00 mg GAE/g; 59.38±0.00 QE/g respectively). S. androgynous crispy vegetable sheet also demonstrated a better antioxidant activity against the radical effect of DPPH (15.43±0.07%), ABTS (34.04±0.05%) and FRAP (1.73±0.01) than A. viridis (13.45±0.11%, 26.35±0.17 &amp;; 1.56±0.01 respectively) and was comparable with mixed formula (14.23±0.43%; 32.31±0.12%, 1.81±0.03 respectively). A significant dose-dependent cytotoxic effect was induced by the three crispy vegetables with IC50 of 8.33±0.69, 7.84±0.35, 7.86±0.33 respectively on HUVECs. The results also showed that mixed crispy vegetables (50:50%) promoted endothelial nitric oxide production. Conclusion:The study concludes that the antioxidant activity of the crispy vegetables reflects the total phenolic and total flavonoid content as their functional properties. Our findings also suggest that thermal processing of foods could have significant effect on the antioxidant activity of functional foods; however, A. viridis and S. androgynous prepared in crispy form may pose no potential threat to the body like the fresh vegetables.Keywords:Amaranthus viridis, S. androgynous, crispy vegetables, antioxidant, nitric oxide, hypertension, Thailand

Association between endothelial nitric oxide synthase (eNOS) gene variants and nitric oxide production in preeclampsia: a case–control study in Ghana

Evidence suggests that a major cause of PE is endothelial dysfunction emanating from the reduced bioavailability of Nitric oxide (NO). Variants of endothelial nitric oxide synthase (eNOS) gene may lead to decreased NO levels. We explored the association between eNOS gene variants and nitric oxide levels among preeclamptic women in the Ghanaian population. This case–control study included 75 preeclamptic women and 75 healthy normotensive pregnant women attending antenatal care at the Nkawie-Toase Government Hospital, Ghana. A well-structured questionnaire was used to collect socio-demographic, obstetric and clinical data. Blood was obtained for DNA extraction; the gene variants were determined using PCR and RFLP. Preeclamptic women had significantly lower NO concentration compared to the normotensives (p < 0.0001) and was significantly different between VNTR variants (p < 0.0001). A significant difference in VNTR intron 4 distribution was also observed between the preeclamptic and normotensive women with 4c4c” (12.0%) and “4a4c” (1.3%) genotypes found predominantly in preeclamptic women (p < 0.0001). There was significantly higher distribution of “TC” genotype in preeclamptic women (44.0%) compared to normotensives (22.7%) (p = 0.019). However, possessing “4a4b” (cOR: 0.17, 95% CI 0.04–0.64) and “4b4b” (cOR: 0.09, 95% CI 0.02–0.38) significantly decreased the likelihood of experiencing preeclampsia by 83% and 91% respectively. Nitric oxide is reduced in preeclamptic women. NO levels in preeclampsia are altered by VNTR intron 4 variants but not T786C variants. Possessing VNTR intron 4 “4b” allele decreases the risk of PE while the “4c” allele increases the risk of PE. There is the need for eNOS variant screening and nitric oxide estimation among pregnant women for early prediction of women at risk of preeclampsia.

Progranulin Maintains Blood Pressure and Vascular Tone Dependent on EphrinA2 and Sortilin1 Receptors and Endothelial Nitric Oxide Synthase Activation.

Background The mechanisms determining vascular tone are still not completely understood, even though it is a significant factor in blood pressure management. Many circulating proteins have a significant impact on controlling vascular tone. Progranulin displays anti-inflammatory effects and has been extensively studied in neurodegenerative illnesses. We investigated whether progranulin sustains the vascular tone that helps regulate blood pressure. Methods and Results We used male and female C57BL6/J wild type (progranulin+/+) and B6(Cg)-Grntm1.1Aidi/J (progranulin-/-) to understand the impact of progranulin on vascular contractility and blood pressure. We found that progranulin-/- mice display elevated blood pressure followed by hypercontractility to noradrenaline in mesenteric arteries, which is restored by supplementing the mice with recombinant progranulin. In ex vivo experiments, recombinant progranulin attenuated the vascular contractility to noradrenaline in male and female progranulin+/+ arteries, which was blunted by blocking EphrinA2 or Sortilin1. To understand the mechanisms whereby progranulin evokes anticontractile effects, we inhibited endothelial factors. N(gamma)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) prevented the progranulin effects, whereas indomethacin (cyclooxygenase inhibitor) affected only the contractility in arteries incubated with vehicle, indicating that progranulin increases nitric oxide and decreases contractile prostanoids. Finally, recombinant progranulin induced endothelial nitric oxide synthase phosphorylation and nitric oxide production in isolated mesenteric endothelial cells. Conclusions Circulating progranulin regulates vascular tone and blood pressure via EphrinA2 and Sortilin1 receptors and endothelial nitric oxide synthase activation. Collectively, our data suggest that deficiency in progranulin is a cardiovascular risk factor and that progranulin might be a new therapeutic avenue to treat high blood pressure.

Human pulmonary microvascular endothelial cell DDAH1-mediated nitric oxide production promotes pulmonary smooth muscle cell apoptosis in co-culture.

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in preterm infants, and pulmonary hypertension (PH) develops in 25%-40% of patients with BPD, increasing morbidity and mortality. BPD-PH is characterized by vasoconstriction and vascular remodeling. Nitric oxide (NO) is a pulmonary vasodilator and apoptotic mediator made in the pulmonary endothelium by NO synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous eNOS inhibitor, primarily metabolized by dimethylarginine dimethylaminohydrolase-1 (DDAH1). Our hypothesis is that DDAH1 knockdown in human pulmonary microvascular endothelial cells (hPMVEC) will result in lower NO production, decreased apoptosis, and greater proliferation of human pulmonary arterial smooth muscle cells (hPASMC), whereas DDAH1 overexpression will have the opposite effect. hPMVECs were transfected with small interfering RNA targeting DDAH1 (siDDAH1)/scramble or adenoviral vector containing DDAH1 (AdDDAH1)/AdGFP for 24 h and co-cultured for 24 h with hPASMC. Analyses included Western blot for cleaved and total caspase-3, caspase-8, caspase-9, β-actin; trypan blue exclusion for viable cell numbers; terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL); and BrdU incorporation. Small interfering RNA targeting DDAH1 (siDDAH1) transfected into hPMVEC resulted in lower media nitrites, cleaved caspase-3 and caspase-8 protein expression, and TUNEL staining; and greater viable cell numbers and BrdU incorporation in co-cultured hPASMC. Adenoviral-mediated transfection of the DDAH1 gene (AdDDAH1) into hPMVEC resulted in greater cleaved caspase-3 and caspase-8 protein expression and lower viable cell numbers in co-cultured hPASMC. Partial recovery of hPASMC viable cell numbers after AdDDAH1-hPMVEC transfection was observed when media were treated with hemoglobin to sequester NO. In conclusion, hPMVEC-DDAH1-mediated NO production positively regulates hPASMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.NEW & NOTEWORTHY BPD-PH is characterized by vascular remodeling. NO is an apoptotic mediator made in the pulmonary endothelium by eNOS. ADMA is an endogenous eNOS inhibitor metabolized by DDAH1. EC-DDAH1 overexpression resulted in greater cleaved caspase-3 and caspase-8 protein expression and lower viable cell numbers in co-cultured SMC. After NO sequestration, SMC viable cell numbers partially recovered despite EC-DDAH1 overexpression. EC-DDAH1-mediated NO production positively regulates SMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.

Extremely Low-Frequency Electromagnetic Stimulation (ELF-EMS) Improves Neurological Outcome and Reduces Microglial Reactivity in a Rodent Model of Global Transient Stroke.

Extremely low-frequency electromagnetic stimulation (ELF-EMS) was demonstrated to be significantly beneficial in rodent models of permanent stroke. The mechanism involved enhanced cerebrovascular perfusion and endothelial cell nitric oxide production. However, the possible effect on the neuroinflammatory response and its efficacy in reperfusion stroke models remains unclear. To evaluate ELF-EMS effectiveness and possible immunomodulatory response, we studied neurological outcome, behavior, neuronal survival, and glial reactivity in a rodent model of global transient stroke treated with 13.5 mT/60 Hz. Next, we studied microglial cells migration and, in organotypic hippocampal brain slices, we assessed neuronal survival and microglia reactivity. ELF-EMS improved the neurological score and behavior in the ischemia-reperfusion model. It also improved neuronal survival and decreased glia reactivity in the hippocampus, with microglia showing the first signs of treatment effect. In vitro ELF-EMS decreased (Lipopolysaccharide) LPS and ATP-induced microglia migration in both scratch and transwell assay. Additionally, in hippocampal brain slices, reduced microglial reactivity, improved neuronal survival, and modulation of inflammation-related markers was observed. Our study is the first to show that an EMF treatment has a direct impact on microglial migration. Furthermore, ELF-EMS has beneficial effects in an ischemia/reperfusion model, which indicates that this treatment has clinical potential as a new treatment against ischemic stroke.