Nitric Oxide Bioavailability Research Articles

A protective erythropoietin evolutionary landscape, NLRP3 inflammasome regulation, and multisystem inflammatory syndrome in children.

The low incidence of pediatric severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and the associated multisystem inflammatory syndrome (MIS-C) lack a unifying pathophysiological explanation, impeding effective prevention and therapy. Activation of the NACHT, LRR, and PYD domains-containing protein(NLRP) 3 inflammasome in SARS-CoV-2 with perturbed regulation in MIS-C, has been reported. We posit that, early age physiological states and genetic determinants, such as certain polymorphisms of renin-angiotensin aldosterone system (RAAS) molecules, promote a controlled RAAS hyperactive state, and form an evolutionary landscape involving an age-dependent erythropoietin (EPO) elevation, mediating ancestral innate immune defenses that, through appropriate NLRP3 regulation, mitigate tissue injury and pathogen invasion. SARS-CoV-2-induced downregulation of angiotensin-converting enzyme (ACE)2 expression in endothelial cells (EC), impairment of endothelial nitric oxide (NO) synthase (eNOS) activity and downstream NO bioavailability, may promote a hyperactive RAAS with elevated angiotensin II and aldosterone that, can trigger, and accelerate NLRP3 inflammasome activation, while EPO-eNOS/NO abrogate it. Young age and a protective EPO evolutionary landscape may successfully inhibit SARS-CoV-2 and contain NLRP3 inflammasome activation. By contrast, increasing age and falling EPO levels, in genetically susceptible children with adverse genetic variants and co-morbidities, may lead to unopposed RAAS hyperactivity, NLRP3 inflammasome dysregulation, severe endotheliitis with pyroptotic cytokine storm, and development of autoantibodies, as already described in MIS-C. Our haplotype estimates, predicted from allele frequencies inpopulation databases, are in concordance with MIS-C incidence reports in Europeans but indicate lower risks for Asians and African Americans. Targeted Mendelian approaches dissecting the influence of relevant genetic variants are needed.

Towards a better understanding of arterial calcification disease progression in CKD: investigation of early pathological alterations.

Cardiovascular disease remains the leading cause of death in chronic kidney disease (CKD) patients, especially in those undergoing dialysis and kidney transplant surgery. CKD patients are at high risk of developing arterial media calcifications (AMC) and arterial stiffness. We hypothesized that investigation of disease progression at an early stage could provide novel insights in understanding AMC etiology. An adenine diet was administered to male Wistar rats to induce AMC. Rats were sacrificed after 2, 4 and 8 weeks. AMC was measured by assessment of aortic calcium and visualized using histology. Arterial stiffness was measured in vivo by ultrasound and ex vivo by applying cyclic stretch of physiological magnitude on isolated arterial segments, allowing us to generate the corresponding pressure-diameter loops. Further, ex vivo arterial reactivity was assessed in organ baths at 2 and 4 weeks to investigate early alterations in biomechanics/cellular functionality. CKD rats showed a time-dependent increase in aortic calcium which was confirmed on histology. Accordingly, ex vivo arterial stiffness progressively worsened. Pressure-diameter loops showed a gradual loss of arterial compliance in CKD rats. Additionally, viscoelastic properties of isolated arterial segments were altered in CKD rats. Furthermore, after 2 and 4 weeks of adenine treatment, a progressive loss in basal, nitric oxide (NO) levels was observed, which was linked to an increased vessel tonus and translates into an increasing viscous modulus. Our observations indicate that AMC-related vascular alterations develop early after CKD induction prior to media calcifications being present. Preventive action, related to restoration of NO bioavailability, might combat AMC development.

Pharmacokinetics of L-Citrulline in Neonates at Risk of Developing Bronchopulmonary Dysplasia-Associated Pulmonary Hypertension.

Options to treat pulmonary hypertension (PH) in neonates with bronchopulmonary dysplasia (BPD) are few and largely ineffective. Improving the bioavailability of nitric oxide (NO) might be an efficacious treatment for BPD-PH. When administered orally, the NO-L-arginine precursor, L-citrulline, increases NO production in children and adults, however, pharmacokinetic (PK) studies of oral L-citrulline have not been performed in infants and children. This study characterized the PK of enterally administered L-citrulline in neonates at risk of developing BPD-PH to devise a model-informed dosing strategy. Ten premature neonates (≤28 weeks gestation) were administered a single dose of 150 mg/kg (powder form solubilized in sterile water) oral L-citrulline at 32 ± 1 weeks postmenstrual age. Due to the need to limit blood draws, time windows were used to maximize the sampling over the dosing interval by assigning neonates to one of two groups (ii) samples collected pre-dose and at 1- and 2.5-h post-dose, and (ii) pre-dose and 0.25- and 3-h post-dose. The L-arginine concentrations (µmol/L) and the L-citrulline (µmol/L) plasma concentration-time data were evaluated using non-compartmental analysis (Phoenix WinNonlin version 8.1). Optimal dosage strategies were derived using a simulation-based methodology. Simulated doses of 51.5 mg or 37.5 mg/kg given four times a day produced steady-state concentrations close to a target of 50 µmol/L. The volume of distribution (V/F) and clearance (CL/F) were 302.89ml and 774.96ml/h, respectively, with the drug exhibiting a half-life of 16 minutes. The AUC from the time of dosing to the time of last concentration was 1473.3h*μmol/L, with Cmax and Tmax of 799μmol/L and 1.55h, respectively. This is the first PK study in neonates presenting data that can be used to inform dosing strategies in future randomized controlled trials evaluating enteral L-citrulline as a potential treatment to reduce PH associated with BPD in premature neonates. Clinical trials.gov Identifier: NCT03542812.

Novel Possible Biomarkers for the Cardiovascular Disease Prognosis

Background:Cardiovascular disease (CVD), especially heart failure (HF) as its common final pathway, is the leading cause of morbidity and mortality worldwide. Furthermore, oxidative and inflammatory processes represent fundamental underlying mechanisms for the development and progression of HF. Of interest, in recent years the development of markers with diagnostic and prognostic value for this pathology and other related CVD has been revalued.
 Objectives:This study was done to quantify and evaluate inflammatory markers, such as ultrasensitive C-reactive protein (uCRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and heat shock protein 70 (Hsp70) in the serum of patients with HF and to compare them with healthy individuals, also correlate the values obtained from oxidative stress markers and nitric oxide (NO) bioavailability previously investigated in these patients with the coexistence or not of secondary pulmonary hypertension (SPH) associated with HF.
 Methods: The determination of all parameters was achieved with standardized, reproducible, accurate, and affordable biochemical methods.
 Results: The values obtained for uCRP, IL-6, and TNF-α were following the pattern of oxidative markers previously found in these patients. These findings indicate the coexistence of oxidative stress and inflammation during HF. Of particular interest, such markers are more exacerbated when were associated with SPH, increasing its value as possible biomarkers in this pathology. However, the found levels of Hsp70 were controversial.
 Conclusion: The pattern of oxidative-inflammatory markers suggests their value as possible biomarkers in this cardiovascular disease. Nevertheless, additional studies are needed to assess in greater detail the importance of the relationship between serum Hsp70 expression and SPH-associated or non-SPH morbidity in HF.

Markers of Endothelial Dysfunction Are Attenuated by Resveratrol in Preeclampsia.

Preeclampsia (PE) is characterized by great endothelial dysfunction, decreased nitric oxide (NO) bioavailability, and higher levels of arginase activity. In the present study, we investigated the potential modulatory effects of trans-resveratrol (RSV) on arginase and endothelial dysfunction biomarkers in endothelial cells exposed to plasma from patients with PE and healthy pregnant (HP) women, and umbilical arteries from patients with PE. Human umbilical vein endothelial cells (HUVECs) were incubated with pooled plasma from 10 HP or 10 PE pregnant women and RSV; umbilical arteries from patients with PE were incubated with RSV; intracellular NO and total reactive oxygen species (ROS) levels were assessed using a probe that interacted with these radicals; total arginase activity was evaluated measuring the urea produced; total antioxidant capacity was measured using the ferric reduction ability power (FRAP) assay; and endothelial dysfunction biomarkers were assessed using qPCR in endothelial cells and umbilical arteries. RSV increased NO levels and decreased total arginase activity in endothelial cells incubated with plasma from patients with PE. In addition, RSV increased total antioxidant capacity and downregulated endothelial dysfunction biomarkers, such as intercellular adhesion molecule-1 (ICAM-1), von Willebrand factor (vWF), and Caspase-3, (CASP-3), in endothelial cells and umbilical arteries from PE patients. RSV treatment positively modulated the L-arginine-NO pathway, decreased arginase activity, and increased antioxidant capacity, in addition to downregulating endothelial dysfunction biomarkers.

Epigallocatechin gallate (EGCG) alleviates vascular dysfunction in angiotensin II-infused hypertensive mice by modulating oxidative stress and eNOS

Epigallocatechin gallate (EGCG) has been shown to have antihypertensive activity. However, the role of epigallocatechin gallate (EGCG) in improving vascular function via modulation of endothelial nitric oxide synthase (eNOS) in hypertensive subjects is not well researched. Angiotensin II-infused hypertensive mice (8–10 weeks old) received EGCG (50 mg/kg/day) for 14 days via oral gavage. The arterial systolic blood pressure (SBP) was measured using the tail-cuff method every three days. At the end of the treatment, the vascular reactivity of the isolated aortae was studied using wire myographs. The level of nitric oxide (NO), cyclic guanosine monophosphate (cGMP) and tetrahydrobiopterine (BH4) were determined using assay kits while the presence of proteins (NOS, p-eNOS and NOx-2) were determined using by Western blotting. In vivo treatment with EGCG for 14 days significantly attenuated the increase in SBP, alleviated the vascular dysfunction, increased the vascular cGMP and BH4 level as well as the expression of p-eNOS and decreased elevated ROS level and NOx-2 protein in angiotensin II-infused hypertensive mice. Collectively, treatment with EGCG in hypertensive mice exerts a blood pressure lowering effect which is partly attributed to the improvement in the vascular function due to its ability to reduce vascular oxidative stress in the aortic tissue leading to a decrease in eNOS uncoupling thus increasing NO bioavailability.

Endothelial dysfunction in COVID-19: an overview of evidence, biomarkers, mechanisms and potential therapies.

The fight against coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is still raging. However, the pathophysiology of acute and post-acute manifestations of COVID-19 (long COVID-19) is understudied. Endothelial cells are sentinels lining the innermost layer of blood vessel that gatekeep micro- and macro-vascular health by sensing pathogen/danger signals and secreting vasoactive molecules. SARS-CoV-2 infection primarily affects the pulmonary system, but accumulating evidence suggests that it also affects the pan-vasculature in the extrapulmonary systems by directly (via virus infection) or indirectly (via cytokine storm), causing endothelial dysfunction (endotheliitis, endothelialitis and endotheliopathy) and multi-organ injury. Mounting evidence suggests that SARS-CoV-2 infection leads to multiple instances of endothelial dysfunction, including reduced nitric oxide (NO) bioavailability, oxidative stress, endothelial injury, glycocalyx/barrier disruption, hyperpermeability, inflammation/leukocyte adhesion, senescence, endothelial-to-mesenchymal transition (EndoMT), hypercoagulability, thrombosis and many others. Thus, COVID-19 is deemed as a (micro)vascular and endothelial disease. Of translational relevance, several candidate drugs which are endothelial protective have been shown to improve clinical manifestations of COVID-19 patients. The purpose of this review is to provide a latest summary of biomarkers associated with endothelial cell activation in COVID-19 and offer mechanistic insights into the molecular basis of endothelial activation/dysfunction in macro- and micro-vasculature of COVID-19 patients. We envisage further development of cellular models and suitable animal models mimicking endothelial dysfunction aspect of COVID-19 being able to accelerate the discovery of new drugs targeting endothelial dysfunction in pan-vasculature from COVID-19 patients.

Involvement of shedding induced by ADAM17 on the nitric oxide pathway in hypertension.

A Disintegrin and Metalloprotease 17 (ADAM17), also called tumor necrosis factor-ɑ (TNF-ɑ) convertase (TACE), is a well-known protease involved in the sheddase of growth factors, chemokines and cytokines. ADAM17 is also enrolled in hypertension, especially by shedding of angiotensin converting enzyme type 2 (ACE2) leading to impairment of angiotensin 1–7 [Ang-(1–7)] production and injury in vasodilation, induction of renal damage and cardiac hypertrophy. Activation of Mas receptor (MasR) by binding of Ang-(1–7) induces an increase in the nitric oxide (NO) gaseous molecule, which is an essential factor of vascular homeostasis and blood pressure control. On the other hand, TNF-ɑ has demonstrated to stimulate a decrease in nitric oxide bioavailability, triggering a disrupt in endothelium-dependent vasorelaxation. In spite of the previous studies, little knowledge is available about the involvement of the metalloprotease 17 and the NO pathways. Here we will provide an overview of the role of ADAM17 and Its mechanisms implicated with the NO formation.

Initiation of 3,3-dimethyl-1-butanol at midlife prevents endothelial dysfunction and attenuates in vivo aortic stiffening with ageing in mice.

Vascular dysfunction: develops progressively with ageing; increases the risk of cardiovascular diseases (CVD); and is characterized by endothelial dysfunction and arterial stiffening, which are primarily mediated by superoxide-driven oxidative stress and consequently reduced nitric oxide (NO) bioavailability and arterial structural changes. Interventions initiated before vascular dysfunction manifests may have more promise for reducing CVD risk than interventions targeting established dysfunction. Gut microbiome-derived trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk, and can be suppressed by 3,3-dimethyl-1-butanol (DMB). We investigated whether DMB supplementation could prevent age-related vascular dysfunction in C57BL/6N mice when initiated prior to development of dysfunction. Mice received drinking water with 1% DMB or normal drinking water (control) from midlife (18 months) until being studied at 21, 24 or 27 months of age, and were compared to young adult (5 month) mice. Endothelial function [carotid artery endothelium-dependent dilatation (EDD) to acetylcholine; pressure myography] progressively declined with age in control mice, which was fully prevented by DMB via higher NO-mediated EDD and lower superoxide-related suppression of EDD (normalization of EDD with the superoxide dismutase mimetic TEMPOL). In vivo aortic stiffness (pulse wave velocity) increased progressively with age in controls, but DMB attenuated stiffening by ∼ 70%, probably due to preservation of endothelial function, as DMB did not affect aortic intrinsic mechanical (structural) stiffness (stress-strain testing) nor adventitial abundance of the arterial structural protein collagen. Our findings indicate that long-term DMB supplementation prevents/attenuates age-related vascular dysfunction, and therefore has potential for translation to humans for reducing CV risk with ageing. KEY POINTS: Vascular dysfunction, characterized by endothelial dysfunction and arterial stiffening, develops progressively with ageing and increases the risk of cardiovascular diseases (CVD). Interventions aimed at preventing the development of CV risk factors have more potential for preventing CVD relative to those aimed at reversing established dysfunction. The gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk and can be suppressed by supplementation with 3,3-dimethyl-1-butanol (DMB). In mice, DMB prevented the development of endothelial dysfunction and delayed and attenuated in vivo arterial stiffening with ageing when supplementation was initiated in midlife, prior to the development of dysfunction. DMB supplementation or other TMAO-suppressing interventions have potential for translation to humans for reducing CV risk with ageing.

Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Aromatic Aldehydes as Drug Candidates for the Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is caused by a single-point mutation, and the ensuing deoxygenation-induced polymerization of sickle hemoglobin (HbS), and reduction in bioavailability of vascular nitric oxide (NO), contribute to the pathogenesis of the disease. In a proof-of-concept study, we successfully incorporated nitrate ester groups onto two previously studied potent antisickling aromatic aldehydes, TD7 and VZHE039, to form TD7-NO and VZHE039-NO hybrids, respectively. These compounds are stable in buffer but demonstrated the expected release of NO in whole blood in vitro and in mice. The more promising VZHE039-NO retained the functional and antisickling activities of the parent VZHE039 molecule. Moreover, VZHE039-NO, unlike VZHE039, significantly attenuated RBC adhesion to laminin, suggesting this compound has potential in vivo RBC anti-adhesion properties relevant to vaso-occlusive events. Crystallographic studies show that, as with VZHE039, VZHE039-NO also binds to liganded Hb to make similar protein interactions. The knowledge gained during these investigations provides a unique opportunity to generate a superior candidate drug in SCD with enhanced benefits.

New Genetic Biomarkers of the Overlap Syndrome Tension-Type Headache and Arterial Hypertension.

Background: Nitric oxide (NO) is an important autocrine and paracrine signaling molecule that plays a crucial role in cardiovascular physiology and pathology regulation. NO is an important molecule involved in regulation of cerebral and extra cerebral cranial blood flow and arterial diameters. Reduced bioavailability of NO in the endothelium is an important precursor for impaired vasodilation and arterial hypertension (AH). Furthermore, NO is involved in nociceptive processing. A NO-induced biphasic response with immediate and a delayed headache is typical for chronic tension-type headaches (TTH) in humans. The aim was to study the association of allelic variants and genotypes of the single nucleotide variant (SNV) rs3782218 of the NOS1 gene with the TTH and AH overlap syndrome development in middle age adults. Materials and Methods: We observed 91 Caucasian participants who resided in Krasnoyarsk city: group 1 (TTH and AH overlap syndrome)—30 patients; group 2 (AH without headache)—30 patients; group 3 (control)—31 healthy volunteers. The diagnosis of AH was based on criteria of the European Society of Cardiology and the European Society of Hypertension (2018) и criteria of the Russian Society of Cardiology (2020). Diagnosis of TTH was based on criteria of the International Classification of Headache Disorders (2018). Real-time polymerase chain reaction was used for the determination of allelic variants and genotypes of the SNV rs3782218 of the NOS1 gene in all groups of participants. Results: The frequency of the minor allele T of rs3782218 was statistically significantly higher by 16.7 times in group 1 (TTH and AH) compared to group 3 (control): 26.7% versus 1.6%, respectively (p-value = 0.000065) and 3.2 times higher in group 1 (TTH and AH) compared to group 2 (AH without headache): 26.7% versus 8.3%, respectively (p-value = 0.008). The frequency of the heterozygous (CT) genotype was statistically significantly higher in group 1 (TTH and AH) compared to group 3 (control): 40.0% versus 3.2% (p-value = 0.000454) and in group 1 (TTH and AH) compared to group 2 (AH without headache): 40.0% versus 16.7% (p-value = 0.045). The minor allele T was statistically significantly associated with a high risk of developing the TTH and AH overlap syndrome compared with the controls (odds ratio (OR) = 22.2 (95% confidential interval (CI): 2.8–173.5)) and compared with AH without headache (OR = 4.0 (95% CI: 1.4–11.8)). Although the frequency of the minor allele T was 5.2 times higher in group 2 (AH without headache) compared with group 3 (control), there were not statistically significantly differences (p-value = 0.086). Conclusion: Thus, the minor allele T of rs3782218 of the NOS1 gene is an important genetic biomarker for a high risk of developing the TTH and AH overlap syndrome in hypertensive patients.

Induction of periodontitis and oral microbiome dysbiosis in a mouse model with heart failure with preserved ejection fraction results in worsening hypertension and diastolic dysfunction

Abstract Introduction Heart Failure with Preserved Ejection Fraction (HFpEF) is characterized by increased inflammation and disruption of the nitric oxide (NO) pathway. Oral microbiota has an essential role in the generation of a large portion of the NO bioavailability pool in the human body. Periodontitis (PD) is a common inflammatory condition that contributes to the development of chronic low-grade inflammation, oral microbiota dysbiosis, and dysregulation of the NO hemostasis. Purpose In this study, we sought to investigate the direct effect of PD induction on HFpEF manifestation in a mouse model. Methods HFpEF was induced in 11-week-old C57/black male mice by high-fat diet and inhibition of NO synthase using L-NAME (Nω-nitro-l-arginine-methyl-ester) (a “2-hit-model”) for 10 weeks. PD was induced by oral infection with P. gingivalis. Diastolic function of the left ventricle (LV) was assessed by high-frequency echocardiography. Blood pressure (BP) was measured using CODA non-invasive tail-cuff system. Results Induction of PD resulted in a significantly impaired diastolic function demonstrated by more pronounced decrease in e' and increase in E/e' ratio compared to HFpEF without PD or control mice (E/e': 39.7±2.6 vs. 30.1±1.9 vs. 24.3±2.1 for PD-HFpEF [n=15], HFpEF [n=15], and controls [n=10], respectively, p<0.001). While LV ejection fraction (EF) was similar, global longitudinal strain (GLS) was decreased in the HFpEF group and further decreased in the PD-HFpEF group compared to controls (p<0.001). BP was elevated in the HFpEF mice and PD induction resulted in a more remarkable increase in BP (systolic BP: 124.7±3.3 vs. 112.7±3.8 vs. 94.8±2.2 mmHg, respectively, p<0.001). Conclusions Induction of PD in a mouse model with HFpEF results in a more pronounced BP elevation and diastolic dysfunction compared to HFpEF without PD. Extensive molecular experiments are ongoing to explore the mechanisms responsible for the increased HFpEF severity in the setting of PD. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Israel Science Foundation

Electronic hookah vaping reduces plasma concentrations of nitrate/nitrite and increases systemic oxidative stress

Abstract Background Electronic (e-) hookah–a new category of vaping devices–has increased in use among young adults in recent years. E-hookahs utilize the placement of e-bowls on traditional water pipes, allowing the flavored aerosol to pass through a water-filled base before being inhaled. Though advertised as a safe tobacco alternative, e-hookahs deliver flavored nicotine by creating an aerosol of fine particles and other free radicals that may increase cardiovascular disease risk. While acute e-hookah vaping impairs endothelial function, the underlying mechanism is not completely understood. Purpose To determine the mechanistic involvement of nitric oxide (NO) and oxidative stress underlying the acute e-hookah induced endothelial dysfunction. Methods Healthy young adult chronic hookah smokers (n=7; 26±1 years of age, 24.4±0.8 kg-m2; mean ±SE) were randomized to vape two 30-minute sessions of flavored e-hookah and sham vaping, separated by a 7-day washout period. Flow-mediated dilation (FMD) of the brachial artery was performed with the use of ultrasound. Circulating plasma nitrite/nitrate (NOx), as an index of nitric oxide (NO) concentration, and lipid peroxidation marker 8-isoprostane concentrations, as well as smoking exposure biomarkers (plasma nicotine and exhaled carbon monoxide) were measured before and after the vaping sessions. Results As compared to unchanged parameters with sham vaping (p=ns), e-hookah vaping induced an acute reduction in FMD by −27±3% (pre- vs. post-vaping, p<0.001), suggesting impairment in endothelial function. These vascular changes were accompanied by significant reductions in circulating plasma levels of NOx and increases in 8-iso prostaglandin F2a levels (−41±11% and +28±11%, respectively; p<0.001). Plasma nicotine concentrations increased 4-fold more after vaping e-hookah, whereas exhaled CO levels did not change (p=ns). Conclusions Our data demonstrate that e-hookah vaping transiently increases systemic oxidative stress and decreases NOx levels, the latter implies reduced NO bioavailability and is in line with the observed impairment in endothelial function, expressed by acute FMD reductions. Thus, in contrast to the unsubstantiated beliefs that e-hookah vaping is benign, in overtly healthy young adults, e-hookah vaping causes acute vascular functional impairment, likely mediated by oxidative stress. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of California Tobacco-Related Disease Research Program, University of California

Endothelial dysfunction due to decreased nitric oxide bioavailability in dysglycaemic subjects and first-degree relatives of type 2 diabetic patients

Abstract Background/Introduction Oxidative stress plays an important role in the pathogenesis of type 2 diabetes mellitus. Purpose We aimed to investigate oxidative stress and nitrites/nitrates concentrations at baseline and during postprandial hyperglycaemia in 40 first-degree relatives (FDR) of type 2 diabetic patients with normal oral glucose test (OGTT), 40 subjects with abnormal OGTT (dysglycaemic) and 20 subjects with normal OGTT without parental history of diabetes (normoglycaemic). Methods Perfused boundary region (PBR) of the sublingual microvessels with a diameter 5–25μm (Microscan, Glycocheck), a marker of endothelial glycocalyx integrity, coronary flow reserve (CFR) in the distal left anterior descending coronary artery, left ventricular global longitudinal strain (GLS), malondialdehyde (MDA), protein carbonyls (PC) and nitrites/nitrates plasma levels were assessed at 0 and 120 min of a 2-hour OGTT. Insulin sensitivity was evaluated using Matsuda and insulin sensitivity index (ISI). Results In all subjects, there were no significant changes in MDA and PC at 120 min after OGTT (p>0.05). Compared with normoglycaemic subjects, in FDR and dysglycaemic subjects, nitrites/nitrates levels were significantly decreased at 120 min (−24% versus −30% versus −3%, respectively) with a parallel increase in PBR (+7% versus +10% versus −3%) and a reduction in CFR (−11% versus −14% versus −6%) and GLS (−4% versus −4% versus +0.1%) (p<0.05 for all comparisons). The percent reduction of nitrites/nitrates levels was associated with abnormal values of Matsuda and ISI, reversely related with the percent increase of PBR (r=−0.60, p=0.001) and positively related with the percent decrease of CFR (r=0.39, p=0.038) and GLS (r=0.48, p=0.008). Conclusion Insulin resistance determines acute endothelial responses during OGTT leading to reduced nitric oxide production with resultant coronary and myocardial dysfunction in FDR and dysglycaemic subjects. Funding Acknowledgement Type of funding sources: None.

Enhancing NO pathway improves diastolic function in a hamster model of heart failure with preserved ejection fraction associated with nonalcoholic steatohepatitis

Abstract Background and aims Heart failure with preserved ejection fraction (HFpEF) is a complex clinical phenotype frequently associated to the presence of multiple non-cardiac comorbidities including nonalcoholic steatohepatitis (NASH) commonly seen in metabolic syndrome and obesity. In this context, the enhanced production of ROS due to systemic inflammatory state participates to the myocardial structural and functional alterations by limiting nitric oxide bioavailability. We thus evaluated the effect of the phosphodiesterase type 5 (PDE5) inhibitor, vardenafil, on cardiac function and remodeling in a NASH hamster model. Method Male Golden Syrian hamsters were fed for 20 weeks with a free choice diet consisting in free access to control chow diet with normal drinking water or a high fat/high cholesterol diet with 10% fructose enriched drinking water. At 15 weeks hamsters were randomized on E/A ratio and ejection fraction to receive either vardenafil (10mg/kg/day orally for 5 weeks) or vehicle before cardiac and metabolic parameters evaluation. Results After 20 weeks of free choice diet, hamsters presented a large increase in hepatic lipid contents and developed NASH. Echocardiography showed that systolic function was preserved with ejection fraction in normal range whatever the diet. In contrast, E/A ratio was significantly increased in free choice fed hamsters (2,1±0,1 vs 1,4±0,04) and E'/A' ratio inverted when compared to control (0,8±0,02 vs 1,3±0,04). This was associated with a decreased isovolumic relaxation time (IVRT) and an increased E/E' ratio signing relaxation impairment and higher filling pressure, respectively. Moreover, pro-BNP was largely increased in free choice fed hamsters when compared to controls. While vardenafil had no impact on NASH, diastolic function was largely improved as evidenced by the significant decrease in E/A and E/E' ratios along with the increase in E'/A' ratio and IVRT. This effect was corroborated by the normalization of the pro-BNP plasma levels induced by the 5 week-treatment with vardenafil. Conclusion The present data demonstrate that free choice fed hamsters developed NASH and showed the ultimate stage of diastolic dysfunction characterized by a restrictive profile combining compliance and relaxation impairments as well as higher filling pressure. While no effect on NASH was observed, enhancing NO pathway with vardenafil treatment significantly improves diastolic function in this hamster model of HFpEF. Funding Acknowledgement Type of funding sources: None.

HIGH-INTENSITY STATINS COMBINED WITH EICOSAPENTAENOIC ACID (EPA) IMPROVES ENDOTHELIAL FUNCTION DURING EXPOSURE TO OXIDIZED LDL

BackgroundDuring atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL).Methods and ResultsHuman umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01).ConclusionIn combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials. BackgroundDuring atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL). During atherosclerosis, endothelial cell (EC) dysfunction results in reduced nitric oxide (NO) bioavailability and increased cytotoxic peroxynitrite (ONOO−). This loss of NO bioavailability results in abnormal vasodilation and inflammatory changes. Eicosapentaenoic acid (EPA) administered as icosapent ethyl (IPE) reduced cardiovascular (CV) events in high-risk patients treated with statins (REDUCE-IT). We tested the effects of high-intensity statins and EPA in ECs exposed to oxidized LDL (oxLDL). Methods and ResultsHuman umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01). Human umbilical vein ECs (HUVECs) were pretreated with 20 mg/dL oxLDL for 20 min, then treated with atorvastatin (active metabolite, ATM) and rosuvastatin (rosuva) at 1.0 μM ± EPA (10μM) for 1 hr. Cells were stimulated with calcium and assayed for the NO/ONOO− release ratio using nanosensors. ECs exposed to oxLDL showed a 60% reduction in NO release compared with vehicle (386±29 to 156±18 nM, p< 0.001) concomitant with a pronounced increase in ONOO− release (205±31 to 283±16 nM, p< 0.001), resulting in a >70% decrease in the NO/ONOO− release ratio (p < 0.001). ECs treated with ATM had an improved NO/ONOO− release ratio (53%) that increased in combination with EPA by 216% (p < 0.01). Similar results were observed for EPA in combination with rosuvastatin. When either statin was combined with EPA, there was also decreased ONOO− release compared to statin alone (p < 0.01). ConclusionIn combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials. In combination with high intensity statins, EPA enhanced NO bioavailability in dysfunctional human ECs. The ability of EPA to reverse vascular EC dysfunction may lead to reduced ischemic events in statin-treated patients, as evidenced in outcome trials.

Vascular Diseases in PNH

PNH is a condition in which uncontrolled complement activity leads to systemic complications, principally through intravascular hemolysis and platelet activation. It arises through a somatic mutation of the phosphatidylinositol glycan A (PIG-A) gene in bone marrow stem cells, 1,2 resulting in disruption to glycosylphosphatidylinositol (GPI) biosynthesis, 3. Among the deficient proteins are the complement regulatory proteinsCD55 andCD59, resulting in increased complement sensitivity of PNH cells, intravascular hemolysis, promotion of inflammatory mediators, and systemic hemoglobin release4 . Patients with PNH can present with multisystemic clinical manifestations due to intravascular hemolysis, thrombosis and bone marrow failure5 . Symptoms are therefore often non-specific, ranging from loss of vision (due to retinal thrombosis), headache and nausea/vomiting (due to cerebral thrombosis), pulmonary hypertension (due to pulmonary embolism), anaemia, through to pain and swelling in the lower extremities (due to deep vein thrombosis), renal failure and other symptoms affecting different systems6 . Thromboembolism is the most common cause of mortality in patients with PNH and accounts for approximately 40% to 67% of deaths of which the cause is known. Further, 29% to 44% of patients with PNH have been reported to have at least 1 thromboembolic event during the course of their disease, although the reason(s) a thrombotic event may suddenly occur remains an enigma7,8,9. Platelet activation, complementmediated hemolysis, impaired nitric oxide (NO) bioavailability, impairment of the fibrinolytic system, and inflammatory mediators are all proposed mechanisms and thought to be responsible for the increased thrombotic risk in patients with PNH. Multiple factors are likely to contribute to any one thrombotic event in patients with PNH. 10 Therapeutic strategies include terminal complement blockade and bone marrow transplantation. Eculizumab, a monoclonal antibody complement inhibitor, is highly effective and the only licensed therapy for PNH.11 The therapeutic anti-C5 antibody eculizumab (Soliris, Alexion) has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in a majority of PNH patients.12,13 Yet, persistent C3 activation occurring during eculizumab treatment may lead to progressive deposition of C3 fragments on affected erythrocytes and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematologic benefit of anti-C5 treatment.14,15 Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current complement-targeted treatment.16,17

Low-grade endotoxemia and NOX2 in patients with coronary microvascular angina.

Endothelial dysfunction and oxidative stress were hypothesized to be involved in the pathogenesis of coronary microvascular angina (MVA). NADPH oxidase-2 (NOX2) activation could provoke increased oxidative stress and endothelial dysfunction, but data on MVA have not been provided yet. This study aimed to evaluate the interaction among NOX2 activation, serum lipopolysaccharide (LPS) levels, as well as oxidative stress production as potential causes of endothelial dysfunction in MVA patients. In this study, we wanted to compare serum levels of soluble NOX2-dp (sNOX2-dp), H2O2 production, hydrogen peroxide breakdown activity (HBA), nitric oxide (NO) bioavailability, endothelin 1 (ET-1), serum zonulin (as intestinal permeability assay), and LPS in 80 consecutive subjects, including 40 MVA patients and 40 controls (CT), matched for age and sex. Compared with CT, MVA patients had significantly higher values of sNOX2-dp, H2O2, ET-1, LPS, and zonulin. Conversely HBA and NO bioavailability were significantly lower in MVA patients. Simple linear regression analysis showed that sNOX2 was associated with serum LPS, serum zonulin, H2O2, and ET-1. Furthermore, an inverse correlation between sNOX2, HBA, and nitric oxide bioavailability was observed. Multiple linear regression analysis showed that LPS and zonulin emerged as the only independent predictive variables associated with sNOX2. This study provides the first report attesting that patients with MVA have high LPS levels, NOX2 activation, and an imbalance between pro-oxidant and antioxidant systems, in favor of the oxidizing molecules that could be potentially implicated in the endothelial dysfunction and vasoconstriction of this disease.

T Lymphocyte-Derived Exosomes Transport MEK1/2 and ERK1/2 and Induce NOX4-Dependent Oxidative Stress in Cardiac Microvascular Endothelial Cells.

Background Activation of endothelial cells by inflammatory mediators secreted by CD4+ T lymphocytes plays a key role in the inflammatory response. Exosomes represent a specific class of signaling cues transporting a mixture of proteins, nucleic acids, and other biomolecules. So far, the impact of exosomes shed by T lymphocytes on cardiac endothelial cells remained unknown. Methods and Results Supernatants of CD4+ T cells activated with anti-CD3/CD28 beads were used to isolate exosomes by differential centrifugation. Activation of CD4+ T cells enhanced exosome production, and these exosomes (CD4-exosomes) induced oxidative stress in cardiac microvascular endothelial cells (cMVECs) without affecting their adhesive properties. Furthermore, CD4-exosome treatment aggravated the generation of mitochondrial reactive oxygen species (ROS), reduced nitric oxide (NO) levels, and enhanced the proliferation of cMVECs. These effects were reversed by adding the antioxidant apocynin. On the molecular level, CD4-exosomes increased NOX2, NOX4, ERK1/2, and MEK1/2 in cMVECs, and ERK1/2 and MEK1/2 proteins were found in CD4-exosomes. Inhibition of either MEK/ERK with U0126 or ERK with FR180204 successfully protected cMVECs from increased ROS levels and reduced NO bioavailability. Treatment with NOX1/4 inhibitor GKT136901 effectively blocked excessive ROS and superoxide production, reversed impaired NO levels, and reversed enhanced cMVEC proliferation triggered by CD4-exosomes. The siRNA-mediated silencing of Nox4 in cMVECs confirmed the key role of NOX4 in CD4-exosome-induced oxidative stress. To address the properties of exosomes under inflammatory conditions, we used the mouse model of CD4+ T cell-dependent experimental autoimmune myocarditis. In contrast to exosomes obtained from control hearts, exosomes obtained from inflamed hearts upregulated NOX2, NOX4, ERK1/2, MEK1/2, increased ROS and superoxide levels, and reduced NO bioavailability in treated cMVECs, and these changes were reversed by apocynin. Conclusion Our results point to exosomes as a novel class of bioactive factors secreted by CD4+ T cells in immune response and represent potential important triggers of NOX4-dependent endothelial dysfunction. Neutralization of the prooxidative aspect of CD4-exosomes could open perspectives for the development of new therapeutic strategies in inflammatory cardiovascular diseases.

Role of BH4 deficiency as a mediator of oxidative stress-related endothelial dysfunction in menopausal women.

Endothelial function (brachial artery flow-mediated dilation [FMD]) is reduced in estrogen-deficient postmenopausal women, mediated, in part, by reduced nitric oxide (NO) bioavailability, secondary to tetrahydrobiopterin (BH4) deficiency and oxidative stress. FMD is increased, but not fully restored, in postmenopausal women after acute intravenous vitamin C (VITC; superoxide scavenger) or oral BH4 supplementation. In vitro studies demonstrate that coadministration of VITC with BH4 prevents endothelial nitric oxide synthase (eNOS) uncoupling and reductions in NO by peroxynitrite. To investigate mechanisms of endothelial dysfunction in women, we assessed the separate and combined effects of VITC and BH4 to determine whether coadministration of VITC + BH4 improves FMD in healthy postmenopausal women (n = 19, 58 ± 5 yr) to premenopausal (n = 14, 36 ± 9 yr) levels, with exploratory testing in perimenopausal women (n = 8, 51 ± 3 yr). FMD was measured during acute intravenous infusions of saline (control) and VITC (∼2-3 g) ∼3 h after a single dose of oral BH4 (KUVAN, 10 mg/kg body wt) or placebo (randomized crossover, separated by ∼1 mo). Under the placebo condition, FMD was reduced in postmenopausal compared with premenopausal women during the saline infusion (5.6 ± 0.7 vs. 11.6 ± 0.9%, P < 0.001) and increased in postmenopausal women during VITC (+3.5 [1.4, 5.6]%, P = 0.001) and acute BH4 (+1.8 [0.37, 3.2]%, P = 0.01) alone. Coadministration of VITC + BH4 increased FMD in postmenopausal women (+3.0 [1.7, 4.3]%, P < 0.001), but FMD remained reduced compared with premenopausal women (P = 0.02). Exploratory analyses revealed that VITC + BH4 did not restore FMD in perimenopausal women to premenopausal levels (P = 0.045). Coadministration of VITC + BH4 does not restore FMD in menopausal women, suggesting that additional mechanisms may be involved.NEW & NOTEWORTHY Endothelial function is reduced across the menopausal stages related to increased oxidative stress associated with estrogen deficiency. In vitro studies demonstrate that coadministration of VITC with BH4 prevents endothelial nitric oxide synthase (eNOS) uncoupling and reductions in NO by peroxynitrite; however, this remains untested in humans. We demonstrate that the coadministration of BH4 + VITC does not restore endothelial function in perimenopausal and postmenopausal women to the level of premenopausal women, suggesting that other mechanisms contribute.