Reduced Nitric Oxide Bioavailability Research Articles

Abstract Mo103: Aberrant Trans- and De- Nitrosylation Underpins Nitrosative Stress in Cardiometabolic HFpEF

Background: Heart failure with preserved ejection fraction (HFpEF) is among the leading causes of cardiovascular mortality and morbidity. Recent reports suggest excessive myocardial protein S-nitrosylation (-SNO), a hallmark of nitrosative stress, contributes to HFpEF pathophysiology. However, the role of transnitrosylases, enzymes that induce protein-SNO, or denitrosylases, enzymes that eliminate protein-SNO, have not been investigated in the context of HFpEF. Research Questions and Goals: We sought to investigate the regulation of protein nitrosylation dynamics in a rat model of cardiometabolic HFpEF. Methods: Echocardiographic assessment, invasive hemodynamic measurements, and exercise testing were conducted in WKY or ZSF1 obese (Ob) rats to evaluate HFpEF severity. Nitric oxide (NO) bioavailability and protein-SNO levels were determined in the heart tissue. Endothelial-dependent vascular reactivity was assayed to further evaluate NO signaling. In parallel, single-cell (sc) RNA sequencing was performed to determine the transcriptional changes in trans- and denitrosylases, as well as NO synthases. Results: We observed progressive deterioration in LV diastolic function (significantly elevated E/e’ and LV end-diastolic pressure) and exercise performance in ZSF1 Ob when compared to WKY controls. Notably, ZSF1 Ob rats exhibited an age-dependent elevation in protein-SNO levels, despite significant reduction in NO bioavailability and signaling. Targeted sc transcriptomic analysis revealed significantly elevated expression of transnitrosylases such as hemoglobin subunit α and β in cardiomyocytes, endothelial cells, and cardiac fibroblasts from ZSF1 Ob hearts. In contrast, significantly reduced expression of denitrosylases such as thioredoxin 2 was found in the ZSF1 Ob cardiomyocytes. No change was observed in expression of NO synthases (nNOS, iNOS, eNOS). Conclusion: Herein, we demonstrate a profound disconnect between insufficient NO bioavailability and nitrosative stress in ZSF1 Ob rat model of HFpEF. Our data suggests the pathological accumulation of nitroso - proteins, may be attributed in part to the derangement of transnitrosylases and denitrosylases expression. Restoration of physiological protein nitrosylation dynamics may represent a novel therapeutic approach for HFpEF, and warrants further investigation.

Can the dietary inorganic nitrate supplementation mitigate the vascular dysfunction in elderly with Alzheimer’s disease?

Aging and Alzheimer disease (AD) are both characterized by progressive reduction in nitric oxide bioavailability and endothelial function impairment. Promising studies on young healthy individuals underly the positive effect of dietary nitrate supplementation on endothelial function. However, the pharmacokinetics of the plasma nitrate and nitrite (PN) and the accompanying endothelial responsiveness following the oral nitrate ingestion in healthy elderly (HE) and patients with AD is not clear. Our hypothesis was that due to the reduction of nitric oxide bioavailability during aging and AD, an equal dose of oral nitrate (ON) would ameliorate the endothelial responsiveness more in the HE and even more in patients with AD in comparison to healthy young subjects (HY). Therefore, with the aim of investigating the pharmacokinetics of PN and the time-course endothelial responsiveness in response to a single dose ON, 10 HY (25±4 YRS), 10 HE (72±8 YRS) and 13 AD (73±7 YRS) were recruited. PN levels and endothelial responsiveness (single-passive leg movement (ΔPLM)) were measured in the recruited individuals prior to (T0) and hourly for 4 hours (T1, T2, T3, and T4) after: a) a single dose of ON; and b) a single dose of placebo. As expected at T0 both PN and ΔPLM were significantly reduced in the HE and even more reduced in the AD in comparison to the HY (all p<0.001). No changes in PN nor ΔPLM were detected in any group following placebo intake. PN increased significantly in all three groups at T1 (p<0.001) and constantly increased up to T4, but no differences were detected between time points T1 to T4 in any group. ΔPLM increased significantly in all three groups at T2, reaching highest values at T4 (p<0.001). However, AD exhibited significantly lower ΔPLM values at any time point compared to HY (p<0.0001) and HE (p<0.001). Significant correlation between PN and ΔPLM was found (p<0.001, R2=0.1654). Contrary to our hypothesis, the results of this study suggest that the PN pharmacokinetics following a single dose of ON is similar in HY, HE and AD. Moreover, the expected time-course amelioration of the endothelial responsiveness was reduced in the HE and even more reduced in the AD, suggesting that other factors, rather than the nitric oxide bioavailability, are playing a role in the age- and AD-related vascular dysfunction. Ricerca di Base 2017 - RBVR17XRSA - Effectiveness of exercise and nitrate supplementation on progression of dementia in patients with Alzheimer’s disease. 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.

Loss of nitric oxide bioavailability promotes arterial stiffening in a tissue transglutaminase-dependent manner

Although arterial stiffening undeniably contributes to cardiovascular disease, the cellular and molecular mechanisms leading to increased arterial stiffness are not well understood. Increased arterial stiffness in disease states such as obesity and type 2 diabetes typically coexists with indices of endothelial dysfunction including diminished nitric oxide (NO) bioavailability. In fact, it is commonly assumed that endothelial dysfunction precedes, and causally contributes to, arterial stiffening; yet the molecular underpinnings for this causal relationship remain elusive. We and others previously showed that activation of tissue transglutaminase (TG2), a crosslinking enzyme that also works as a G-protein and cellular scaffold, promotes cytoskeletal actin polymerization and stiffening of vascular smooth muscle cells (VSMC), thus contributing to whole-artery stiffening. It is also reported that NO is an endogenous inhibitor of TG2. Accordingly, herein, we tested the hypothesis that increased exposure of VSMC to NO suppresses TG2 activity and actin polymerization. Conversely, we hypothesized that loss of NO bioavailability promotes arterial stiffening, and that this effect is mediated by TG2 activity and downstream activation of LIM kinase (LIMK), an enzyme that modulates fibrillar (F)-actin severing via cofilin phosphorylation. All differences reported herein are significant at P<0.05. In support of our hypotheses, we show that treatment of cultured human aortic VSMC with the NO donor S-nitrosoglutathione (200μM for 2 hours) reduces TG2 activity (N-epsilon gamma glutamyl Lysine presence) and F-actin formation (phalloidin staining) induced by TG2 activation (dithiothreitol, 200μM). Furthermore, we show that exposure of mouse isolated mesenteric arteries to the NO synthase inhibitor L-NAME (300μM for 18 hours) causes an increase in arterial stiffness ( i.e., increased incremental modulus of elasticity) as determined using pressure myography and videomicroscopy. Of note, L-NAME-induced arterial stiffening is prevented when arteries are co-treated with a TG2 inhibitor (cystamine, 1mM) or LIMK inhibitor (LIMKi, 1μM). Taken together, these findings support the notion that NO is a key downregulator of TG2 activity and demonstrate that reduced NO bioavailability promotes arterial stiffening in a VSMC TG2-LIMK-F-actin dependent manner. As such, this work provides insight into plausible molecular mechanisms by which endothelial dysfunction leads to arterial stiffening. R01HL153264 (to LAML and JP), and MU Research Excellence Program (REP) postdoctoral fellowship (to FIRP). 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.

AT1 receptors modulate ethanol-induced loss of anticontractile effect of perivascular adipose tissue

Ethanol consumption activates renin-angiotensin-aldosterone system (RAAS), which plays a major role in the pro-contractile and hypertensive effects linked to ethanol. We hypothesized that ethanol consumption induces loss of the anticontractile effect of perivascular adipose tissue (PVAT)through RAAS-mediated mechanisms. We examined the contribution of angiotensin II type 1 receptors (AT1R) to ethanol-induced PVAT dysfunction. With this purpose, male Wistar Hannover rats were treated with ethanol 20 % (in volume ratio) and/or losartan (antagonist of AT1R; 10 mg/kg/day, gavage) for 9 weeks. Losartan prevented the increase in blood pressure and the loss of the anticontractile effect of PVAT induced by ethanol consumption. PVAT dysfunction occurred after 3 and 9 weeks of treatment with ethanol in an endothelium-dependent manner. Blockade of AT1R prevented ethanol-induced reduction of adiponectin levels in PVAT from ethanol-treated rats. Functional assays revealed that ethanol impaired the anticontractile effect of PVAT-derived angiotensin (1–7) and endothelial nitric oxide (NO). In conclusion, AT1R are implicated in ethanol-induced loss of the anticontractile effect of PVAT. In PVAT, AT1R activation decreases the production of adiponectin, a PVAT-derived factor that promotes vasorelaxation in an endothelium-dependent manner. In the endothelium, AT1R favors the production of superoxide (O2•-) leading to a reduction in NO bioavailability. These responses impair the vasodilator action induced by PVAT-derived angiotensin (1–7), which occurs via Mas receptors located in endothelial cells. Ethanol-induced PVAT dysfunction favors vascular hypercontractility, a response that could contribute to the hypertensive state associated with ethanol consumption.

Aging in Normotensive and Spontaneously Hypertensive Rats: Focus on Erythrocyte Properties.

Erythrocyte deformability, crucial for oxygen delivery to tissues, plays an important role in the etiology of various diseases. As the factor maintaining the erythrocyte deformability, nitric oxide (NO) has been identified. Reduced NO bioavailability also plays a role in the pathogenesis of hypertension. Our aim was to determine whether aging and hypertension affect erythrocyte deformability and NO production by erythrocytes in experimental animals divided into six groups according to age (7, 20 and 52 weeks), labeled WKY-7, WKY-20 and WKY-52 for normotensive Wistar-Kyoto (WKY) rats, and SHR-7, SHR-20 and SHR-52 for spontaneously hypertensive rats (SHR). The filtration method for the determination of erythrocyte deformability and the fluorescent probe DAF-2 DA for NO production were applied. Deformability and NO production by erythrocytes increased at a younger age, while a decrease in both parameters was observed at an older age. Strain-related differences in deformability were observed at 7 and 52 weeks of age. SHR-7 had reduced deformability and SHR-52 had increased deformability compared with age-matched WKY. Changes in NO production under hypertensive conditions are an unlikely primary factor affecting erythrocyte deformability, whereas age-related changes in deformability are at least partially associated with changes in NO production. However, an interpretation of data obtained in erythrocyte parameters observed in SHRs of human hypertension requires precaution.

Sildenafil attenuates oxidative stress and endothelial dysfunction in lead-induced hypertension.

Lead (Pb) reduces NO bioavailability, impairs the antioxidant system, and increases the generation of reactive oxygen species (ROS). Pb-induced oxidative stress may be responsible for the associated endothelial dysfunction. Sildenafil has shown nitric oxide (NO)-independent action, including antioxidant effects. Therefore, we examined the effects of sildenafil on oxidative stress, reductions of NO and endothelial dysfunction in Pb-induced hypertension. Wistar rats were distributed into three groups: Pb, Pb + sildenafil and Sham. Blood pressure and endothelium-dependent vascular function were recorded. We also examined biochemical determinants of lipid peroxidation and antioxidant function. ROS levels, NO metabolites and NO levels in human umbilical vein endothelial cells (HUVECs) were also evaluated. Sildenafil prevents impairment of endothelium-dependent NO-mediated vasodilation and attenuates Pb-induced hypertension, reduces ROS formation, enhances superoxide dismutase (SOD) activity and antioxidant capacity in plasma and increases NO metabolites in plasma and HUVECs culture supernatants, while no changes were found on measurement of NO released from HUVECs incubated with plasma of the Pb and Pb + sildenafil groups compared with the sham group. In conclusion, sildenafil protects against ROS-mediated inactivation of NO, thus preventing endothelial dysfunction and attenuating Pb-induced hypertension, possibly through antioxidant effects.

Elderberry extract improves molecular markers of endothelial dysfunction linked to atherosclerosis.

Endothelial dysfunction (ED), secondary to diminished nitric oxide (NO) production and oxidative stress, is an early subclinical marker of atherosclerosis. Reduced NO bioavailability enhances the adhesion of monocytes to endothelial cells and promotes atherosclerosis. Elderberry extract (EB) is known to contain high levels of anthocyanins which could exert vascular protective effects. Specifically, we investigated the functional capacity of EB on various markers of ED. Human umbilical vein endothelial cells (HUVEC) were pretreated with EB 50 μg/mL and stimulated with TNF-α 10 ng/mL. Cell viability, apoptosis, oxidative stress; eNOS, Akt, Nrf2, NOX-4, and NF-κB at the protein level were measured. A co-culture model was used to determine whether EB could prevent the adhesion of monocytes (THP-1) to HUVECs. Moreover, the expression of adhesion molecules and pro-inflammatory cytokines were also measured. It wasdemonstrated that EB prevented TNF-α induced apoptosis and reactive oxygen species production in HUVECs. Additionally, EB upregulated Akt and eNOS activity, and Nrf2 expression in response to TNF-α, whereas it decreased NOX-4 expression and NF-κB activity. EB prevented the adhesion of monocytes to HUVECs, as well as reduced IL-6 and MCP-1 levels, which was associated with inhibition of VCAM-1 expression. Our results demonstrate that EB upregulates key cellular markers of endothelial function and ameliorates markers of ED. EB could be used as a potential nutritional aid for preventing atherosclerosis progression.

Sevoflurane and isoflurane anesthesia induce redox imbalance, but only sevoflurane impairs vascular contraction.

Volatile anesthetics may cause vascular dysfunction; however, underlying effects are unclear. The aim of the present study was to investigate whether sevoflurane and isoflurane affect vascular function, nitric oxide (NO) bioavailability, and biomarkers of oxidative stress and inflammation. Wistar rats were divided into three experimental groups: Not anesthetized (control group) or submitted to anesthesia with isoflurane (Iso group) or sevoflurane (Sevo group). Hemodynamic parameters were monitored during anesthesia, and blood gas values and biochemical determinants were analyzed. Isometric contractions were recorded in aortic rings. Vasoconstriction induced by potassium chloride (KCl) and phenylephrine (Phe) were measured. No differences in hemodynamic parameters and blood gasses variables were observed. Impaired KCl and Phe-induced contractions were observed in endothelium-intact aorta of Sevo compared to Iso and Control groups. Redox imbalance was found in Sevo and Iso groups. Reduced NO bioavailability and increased activity of matrix metalloproteinase 2 (MMP-2) were observed in Sevo, but not in the Iso group. While reduced IL-10 and IL-1β were observed in Sevo, increases in IL-1β in the Iso group were found. Sevoflurane, but not isoflurane, anesthesia impairs vasocontraction, and reduced NO and cytokines and increased MMP-2 activity may be involved in vascular dysfunction after sevoflurane anesthesia.

Nitric Oxide Precursors and Dimethylarginines as Risk Markers for Accelerated Measured GFR Decline in the General Population

Nitric oxide (NO) deficiency is associated with endothelial dysfunction, hypertension, atherosclerosis, and chronic kidney disease (CKD). Reduced NO bioavailability is hypothesized to play a vital role in kidney function impairment and CKD. We investigated the association of serum levels of endogenous inhibitors of NO, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), and precursors of NO, arginine, citrulline, and ornithine, with a decline in glomerular filtration rate (GFR) and new-onset CKD. In a prospective cohort study of 1407 healthy, middle-aged participants of Northern European origin in the Renal Iohexol Clearance Survey (RENIS), GFR was measured repeatedly with iohexol clearance during a median follow-up time of 11 years. GFR decline rates were analyzed using a linear mixed model, new-onset CKD (GFR< 60 ml/min per 1.73 m2) was analyzed with interval-censored Cox regression, and accelerated GFR decline (the 10% with the steepest GFR decline) was analyzed with logistic regression. Higher SDMA was associated with slower annual GFR decline. Higher levels of citrulline and ornithine were associated with accelerated GFR decline (odds ratio [OR], 1.43; 95% confidence interval [CI] 1.16-1.76 per SD higher citrulline and OR 1.23; 95% CI 1.01 to 1.49 per SD higher ornithine). Higher citrulline was associated with new-onset CKD, with a hazard ratio of 1.33 (95% CI 1.07-1.66) per SD higher citrulline. Associations between NO precursors and the outcomes suggest that NO metabolism plays a significant role in the pathogenesis of age-related GFR decline and the development of CKD in middle-aged people.

Biological Assessment of the NO-Dependent Endothelial Function.

Nitric oxide (NO) is implicated in numerous physiological processes, including vascular homeostasis. Reduced NO bioavailability is a hallmark of endothelial dysfunction, a prequel to many cardiovascular diseases. Biomarkers of an early NO-dependent endothelial dysfunction obtained from routine venous blood sampling would be of great interest but are currently lacking. The direct measurement of circulating NO remains a challenge due by its high reactivity and short half-life. The current techniques measure stable products from the NO signaling pathway or metabolic end products of NO that do not accurately represent its bioavailability and, therefore, endothelial function per se. In this review, we will concentrate on an original technique of low temperature electron paramagnetic resonance spectroscopy capable to directly measure the 5-α-coordinated heme nitrosyl-hemoglobin in the T (tense) state (5-α-nitrosyl-hemoglobin or HbNO) obtained from fresh venous human erythrocytes. In humans, HbNO reflects the bioavailability of NO formed in the vasculature from vascular endothelial NOS or exogenous NO donors with minor contribution from erythrocyte NOS. The HbNO signal is directly correlated with the vascular endothelial function and inversely correlated with vascular oxidative stress. Pilot studies support the validity of HbNO measurements both for the detection of endothelial dysfunction in asymptomatic subjects and for the monitoring of such dysfunction in patients with known cardiovascular disease. The impact of therapies or the severity of diseases such as COVID-19 infection involving the endothelium could also be monitored and their incumbent risk of complications better predicted through serial measurements of HbNO.

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&amp;lt;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&amp;lt;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

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.

Inspiratory muscle strength training for lowering blood pressure and improving endothelial function in postmenopausal women: comparison with "standard of care" aerobic exercise.

Background: High blood pressure (BP), particularly systolic BP (SBP), is the major modifiable risk factor for cardiovascular diseases and related disorders of aging. SBP increases markedly with aging in women such that the prevalence of above-normal SBP (i.e., ≥120 mmHg) in postmenopausal women exceeds rates in age-matched men. This increase in SBP is associated with vascular endothelial dysfunction, mediated by excessive reactive oxygen species-induced oxidative stress and consequent reductions in nitric oxide bioavailability. Moderate-intensity aerobic exercise is a recommended lifestyle strategy for reducing SBP. However, adherence to aerobic exercise guidelines among postmenopausal women is low (<30%) and aerobic exercise does not consistently enhance endothelial function in estrogen-deficient postmenopausal women. High-resistance inspiratory muscle strength training (IMST) is a time-efficient, adherable lifestyle intervention that involves inhaling against resistance through a handheld device (30 breaths/day). Here, we present the protocol for a randomized controlled trial investigating the efficacy of 3 months of high-resistance IMST compared to guideline-based, “standard-of-care” aerobic exercise training for decreasing SBP and improving endothelial function in estrogen-deficient postmenopausal women with above-normal SBP (120–159 mmHg) at baseline (ClinicalTrials.gov Identifier: NCT05000515). Methods: A randomized, single-blind, parallel-group design clinical trial will be conducted in 72 (36/group) estrogen-deficient postmenopausal women with above-normal SBP. Participants will complete baseline testing and then be randomized to either 3 months of high-resistance IMST (30 breaths/day, 6 days/week, 75% maximal inspiratory pressure) or moderate-intensity aerobic exercise training (brisk walking 25 min/day, 6 days/week, 40–60% heart rate reserve). Outcome measures will be assessed after 3 months of either intervention. Following end-intervention testing, participants will abstain from their assigned intervention for 6 weeks, after which BP and endothelial function will be assessed to evaluate the potential persistent effects of the intervention on the primary and secondary outcomes. Discussion: This study is designed to compare the effectiveness of time-efficient, high-resistance IMST to guideline-based aerobic exercise training for lowering SBP and improving endothelial function, and interrogating potential mechanisms of action, in estrogen-deficient postmenopausal women. Clinical Trial Registration: ClinicalTrials.gov, Identifier: NCT05000515.

Red blood cell eNOS is cardioprotective in acute myocardial infarction

Red blood cells (RBCs) were shown to transport and release nitric oxide (NO) bioactivity and carry an endothelial NO synthase (eNOS). However, the pathophysiological significance of RBC eNOS for cardioprotection in vivo is unknown. Here we aimed to analyze the role of RBC eNOS in the regulation of coronary blood flow, cardiac performance, and acute myocardial infarction (AMI) in vivo. To specifically distinguish the role of RBC eNOS from the endothelial cell (EC) eNOS, we generated RBC- and EC-specific knock-out (KO) and knock-in (KI) mice by Cre-induced inactivation or reactivation of eNOS. We found that RBC eNOS KO mice had fully preserved coronary dilatory responses and LV function. Instead, EC eNOS KO mice had a decreased coronary flow response in isolated perfused hearts and an increased LV developed pressure in response to elevated arterial pressure, while stroke volume was preserved. Interestingly, RBC eNOS KO showed a significantly increased infarct size and aggravated LV dysfunction with decreased stroke volume and cardiac output. This is consistent with reduced NO bioavailability and oxygen delivery capacity in RBC eNOS KOs. Crucially, RBC eNOS KI mice had decreased infarct size and preserved LV function after AMI. In contrast, EC eNOS KO and EC eNOS KI had no differences in infarct size or LV dysfunction after AMI, as compared to the controls. These data demonstrate that EC eNOS controls coronary vasodilator function, but does not directly affect infarct size, while RBC eNOS limits infarct size in AMI. Therefore, RBC eNOS signaling may represent a novel target for interventions in ischemia/reperfusion after myocardial infarction.

Flavored and Nicotine-Containing E-Cigarettes Induce Impaired Angiogenesis and Diabetic Wound Healing via Increased Endothelial Oxidative Stress and Reduced NO Bioavailability.

The prevalent use of electronic cigarettes (e-cigarettes) has increased exponentially in recent years, especially in youth who are attracted to flavored e-cigarettes. Indeed, e-cigarette or vaping product use-associated lung injury (EVALI) cases started to emerge in the United States in August 2019, resulting in 2807 hospitalized cases and 68 deaths as of 18 February 2020. In the present study, we investigated, for the first time, whether flavored and nicotine containing e-cigarettes induce endothelial dysfunction to result in impaired angiogenesis and wound healing particularly under diabetic condition. Nicotine containing e-cigarettes with various contents of nicotine (0, 1.2%, 2.4%), and flavored e-cigarettes of classic tobacco, mint, menthol, and vanilla or fruit from BLU (nicotine 2.4%) or JUUL (nicotine 3%), were used to treat endothelial cells in vitro and streptozotocin-induced diabetic mice in vivo. Endothelial cell superoxide production, determined by dihydroethidium (DHE) fluorescent imaging and electron spin resonance (ESR), was markedly increased by exposure to e-cigarette extract (e-CSE) in a nicotine-content dependent manner, while nitric oxide (NO) bioavailability detected by DAF-FM fluorescent imaging was substantially decreased. All of the different flavored e-cigarettes examined also showed significant effects in increasing superoxide production while diminishing NO bioavailability. Endothelial cell apoptosis evaluated by caspase 3 activity was markedly increased by exposure to e-CSE prepared from flavored and nicotine containing e-cigarettes. Endothelial monolayer wound assays revealed that nicotine-containing and flavored e-cigarettes induced impaired angiogenic wound repair of endothelial cell monolayers. Furthermore, vascular endothelial growth factor (VEGF) stimulated wound healing in diabetic mice was impaired by exposure to e-CSEs prepared from nicotine-containing and flavored e-cigarettes. Taken together, our data demonstrate for the first time that flavored and nicotine-containing e-cigarettes induce endothelial dysfunction through excessive ROS production, resulting in decreased NO bioavailability, increased endothelial cell apoptosis, and impairment in angiogenesis and wound healing, especially under diabetic condition. These responses of endothelial dysfunction likely underlie harmful effects of e-cigarettes in endothelial-rich organs, such as heart and lungs. These data also indicate that rigorous regulation on e-cigarette use should be enforced in diabetic and/or surgical patients to avoid severe consequences from impaired angiogenesis/wound healing.

Dietary nitrate supplementation attenuates the mechanoflex in older women with early menopause: A pilot study

Women who reached early menopause (EM, 40‐44 yrs) have a greater risk for cardiovascular disease than women with normal menopause (NM, 50‐54 yrs), which may be attributed to a longer duration of estrogen loss and a greater reduction in nitric oxide bioavailability in both the peripherals and center. Previous studies have shown that dietary nitrate supplementation improves cardiovascular function in older women, but how autonomic neural control changes in daily living, such as during exercise, has been involved is unclear. We examined the hypothesis that 2wks continuous use of dietary nitrate supplements would attenuate the exercise pressor reflex in older women with EM.Six EM (65±4 [SD] yrs) and 8 NM (64±2 yrs) women participated in this randomized, placebo‐controlled, double‐blind, cross‐over pilot study. All subjects underwent 2 wks of beetroot juice (BRJ; 140 ml containing ~800 mg nitrate per day) or placebo (140 ml nitrate‐depleted BRJ, daily) intervention with ≥14 days of wash‐out. Before and after each intervention period, subjects performed SHG using the dominant hand at 40% of maximum voluntary contraction (MVC) until fatigue, followed by 2 min of PECA with an upper arm cuff inflated to 250 mmHg. Heart rate (HR; ECG), blood pressure (BP; finger photoplethysmography), muscle sympathetic nerve activity (MSNA; microneurography), and respiration (capnography) were continuously measured. In addition, 24‐h ambulatory BP standard deviation (SD) was monitored before and after each intervention (an indication of BP variability).There were no differences in baseline HR, BP and MSNA, MVC, and the time to fatigue between the EM and NM groups before and after intervention (all p&gt;0.05). At the first 30 s of SHG, the EM group showed significant decreases in MSNA burst frequency (DBF; ‐4.5±6.5 in EM vs. 2.5±6.2 bursts/min in NM, p=0.065) and burst incidence (DBI; ‐10.6±12.6 vs. 2.6±8.4 bursts/100 heartbeats, p=0.037) after BRJ intervention compared with the NM group (Figure1A and B). However, there were no differences between groups at fatiguing exercise and during PECA after the intervention. The interventional changes in 24‐h diastolic BP SD had a positive correlation with ΔMSNA BF (r=0.55, p=0.053) and BI (r=0.58, p=0.036) at the first 30 s of SHG in all subjects and EM individuals were in the left‐lower area (Figure2 A and B).These results suggest that dietary nitrate supplementation may attenuate and mechanoreflex, but not the metaboreflex, in older women with EM during static hand grip, which has a relation with BP variability during ambulation. Certainly, these preliminary observations need to be verified in more older women in future studies.

How do we kNOw the individual contribution of eNOS and nNOS for cerebral blood flow regulation?

How do we kNOw the individual contribution of eNOS and nNOS for cerebral blood flow regulation?

Placental Ischemia Says “NO” to Proper NOS-Mediated Control of Vascular Tone and Blood Pressure in Preeclampsia

In this review, we first provide a brief overview of the nitric oxide synthase (NOS) isoforms and biochemistry. This is followed by describing what is known about NOS-mediated blood pressure control during normal pregnancy. Circulating nitric oxide (NO) bioavailability has been assessed by measuring its metabolites, nitrite (NO2) and/or nitrate (NO3), and shown to rise throughout normal pregnancy in humans and rats and decline postpartum. In contrast, placental malperfusion/ischemia leads to systemic reductions in NO bioavailability leading to maternal endothelial and vascular dysfunction with subsequent development of hypertension in PE. We end this article by describing emergent risk factors for placental malperfusion and ischemic disease and discussing strategies to target the NOS system therapeutically to increase NO bioavailability in preeclamptic patients. Throughout this discussion, we highlight the critical importance that experimental animal studies have played in our current understanding of NOS biology in normal pregnancy and their use in finding novel ways to preserve this signaling pathway to prevent the development, treat symptoms, or reduce the severity of PE.

Chronic mercury exposure induces oxidative stress in female rats by endothelial nitric oxide synthase uncoupling and cyclooxygenase-2 activation, without affecting oestrogen receptor function.

Mercury has been shown to be a significant health risk factor and is positively associated with cardiovascular diseases. Evidence reveals that men are more likely to develop cardiovascular diseases than women during reproductive age. However, the effects of mercury in females remain poorly investigated, despite the finding that female hormones demonstrate a cardioprotective role. In the present study, we evaluated whether chronic mercury chloride exposure could alter blood pressure and vascular function of the female rat aorta. Ten-week-old female Wistar rats were divided into two groups: control (vehicle) and mercury treated (first dose of 4.6 μg/kg, subsequent daily doses of 0.07 μg/kg), im. Mercury treatment did not modify systolic blood pressure (SBP) but increased vascular reactivity due to the reduction of nitric oxide bioavailability associated with the increase in reactive oxygen species from endothelial nitric oxide synthase (eNOS) uncoupling. Furthermore, increased participation of the cyclooxygenase-2 pathway occurred through an imbalance in thromboxane 2 and prostacyclin 2. However, the oestrogen signalling pathway was not altered in either group. These results demonstrated that chronic exposure to mercury in females induced endothelial dysfunction and, consequently, increased aortic vascular reactivity, causing vascular damage to the female rat aorta and representing a risk of cardiovascular diseases.

Arginase-1 Released into CSF After Aneurysmal Subarachnoid Hemorrhage Decreases Arginine/Ornithine Ratio: a Novel Prognostic Biomarker

We hypothesized that the enzyme arginase-1 is released into the cerebrospinal fluid (CSF) during red blood cell lysis and contributes to dysregulated metabolism of the nitric oxide (NO) precursor L-arginine during aneurysmal subarachnoid hemorrhage (SAH). This prospective case–control study included 43 patients with aneurysmal SAH and ventricular drainage for clinical reasons. Longitudinal CSF samples (99) were obtained in the course of SAH. Patients were dichotomized regarding the occurrence of cerebral vasospasm syndrome (CVS) (N = 19). Arginase-1 and the amino acids L-arginine and L-ornithine were quantified in CSF. Outcome assessments included delayed cerebral ischemia (DCI) and functional status after 3 months using the modified Rankin Scale (mRS). Arginase-1 was released into the CSF of SAH patients whereas this enzyme was undetectable in controls. Compared to patients without CVS, arginase-1 levels were higher in CVS patients until day 14 after clinical event. The well-known surrogate parameter for arginase acitivity, the L-arginine to L-ornithine ratio (Arg/Orn), correlated with CSF arginase-1 levels. Arg/Orn was reduced in patients with CVS from disease onset (days 1–3, p = 0.0009) until day 14. Logistic regression analysis of early Arg/Orn was predictive for CVS (p = 0.008) and DCI (p = 0.035), independent of age, Hunt and Hess grade, and intraventricular blood. Arg/Orn < 2.71 at disease onset predicted CVS with a sensitivity of 86.7% and specificity of 72.2%. Arg/Orn ≥ 2.71 predicted excellent functional outcome. We propose a novel mechanism contributing to NO deprivation during SAH: arginase-1 is released from erythrocytes into the CSF, leading to L-arginine consumption and reduced NO bioavailability. Furthermore, Arg/Orn is a robust predictor for occurrence of CVS, DCI, and functional outcome 3 months after aneurysmal SAH. Our data provide a novel prognostic biomarker and may contribute to the development of novel therapeutic strategies in SAH. Clinical Trial Registration-URL: http://www.drks.de. Unique identifier: DRKS00015293, date of registration: 13.09.2018.