Endothelial Nitric Oxide Synthase Inhibitor Research Articles

The effect of P2X1 receptor on vascular responses in the diabetic rat model

ABSTRACT
 Objective: Although it is known that there are changes in the vascular purinergic system in diabetes, it is unknown whether P2X1-mediated vascular responses are affected. In this study, we aimed to investigate the vascular responses mediated by P2X1 receptor activation in streptozotocin-induced diabetes model. 
 Method: Animals were divided into two groups as diabetes and control. Diabetes was induced by 65 mg/kg single dose of streptozotocin. After 12 weeks, second branches of the mesenteric artery were isolated and placed into the wire myograph to evaluate the vascular responses to ATP and P2X1 receptor agonist. Vascular responses were also examined in the presence of endothelial nitric oxide synthase, cyclooxygenase or K+ channel inhibitors, to determine the possible mechanism/s of relaxation responses. 
 Results: In diabetes group relaxation responses to ATP and P2X1 receptor agonist were lower compared to control group. Vascular relaxation responses to P2X1 receptor agonist were significantly decreased in both groups in the presence of endothelial nitric oxide synthase inhibitor. Cyclooxygenase inhibitor and K+ channels inhibitors significantly blocked vascular relaxation responses in diabetes group but not in control animals. 
 Conclusion: The results of this study revealed that vascular P2X1 receptor-mediated relaxation responses are decreased in diabetes in diabetes and the pathways mediating these responses were changed.

Taxifolin as a Major Bioactive Compound in the Vasorelaxant Effect of Different Pigmented Rice Bran Extracts.

Cardiovascular disease is one of the leading causes of morbidity and mortality in recent years. The intake of polyphenol rich diets has been associated with improved cardiovascular function and reduced cardiovascular risks. Oryza sativa L. is one of the most common cereals worldwide. Rice bran, a byproduct of the rice milling process, contains many bioactive ingredients, including polyphenols, polysaccharides, proteins, and micronutrients. It is also consumed as a healthy diet in the form of rice bran oil and powder in many Asian countries like Japan, South Korea, and India for its several health benefits as a natural antioxidant. Thus, this study evaluated the vasorelaxant effect of ethanolic extracts of brown, green, red, and black rice bran and investigated its underlying vasorelaxant mechanism. Among the four rice bran extracts (RBEs) examined, the red rice bran extract (RRBE) had a strong endothelium-dependent vasorelaxant effect, which was markedly prevented by N-ω-nitro-L-arginine [endothelial nitric oxide synthase (eNOS) inhibitor], wortmannin [phosphoinositide-3 kinase (PI3K) inhibitor], and 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (inhibitor of guanylate cyclase). Likewise, RRBE induced the phosphorylation of eNOS and Src in cultured endothelial cells, thereby stimulating NO formation. Altogether, these findings propose that RRBE induces endothelium-dependent relaxation, involving at least in part, NO-mediated signaling through the PI3K/eNOS pathway. Further, LC-PDA analysis conducted on the four RBEs also revealed that RRBE highly contained taxifolin, which is an active flavanonol that induces endothelium-dependent vasorelaxation, compared to other RBEs. Subsequently, the underlying mechanism of taxifolin was assessed through vascular reactivity studies with pharmacological inhibitors similar to that of RRBE. These findings deciphered a distinct difference in vasorelaxant effects between RRBE and the other RBEs. We also observed that RRBE induced a potent endothelium-dependent NO-mediated relaxation in coronary artery rings, which involved the Src/PI3K pathway that activates eNOS. Additionally, taxifolin exhibited, at least in part, similar vasoprotective effects of RRBE. Therefore, we propose that RRBE may serve as natural sources of functional phytochemicals that improve cardiovascular diseases associated with disturbed NO production and endothelial dysfunction.

3-Hydroxyphenylacetic Acid: A Blood Pressure-Reducing Flavonoid Metabolite.

Regular intake of polyphenol-rich food has been associated with a wide variety of beneficial health effects, including the prevention of cardiovascular diseases. However, the parent flavonoids have mostly low bioavailability and, hence, their metabolites have been hypothesized to be bioactive. One of these metabolites, 3-hydroxyphenylacetic acid (3-HPAA), formed by the gut microbiota, was previously reported to exert vasorelaxant effects ex vivo. The aim of this study was to shed more light on this effect in vivo, and to elucidate the mechanism of action. 3-HPAA gave rise to a dose-dependent decrease in arterial blood pressure when administered i.v. both as a bolus and infusion to spontaneously hypertensive rats. In contrast, no significant changes in heart rate were observed. In ex vivo experiments, where porcine hearts from a slaughterhouse were used to decrease the need for laboratory animals, 3-HPAA relaxed precontracted porcine coronary artery segments via a mechanism partially dependent on endothelium integrity. This relaxation was significantly impaired after endothelial nitric oxide synthase inhibition. In contrast, the blockade of SKCa or IKCa channels, or muscarinic receptors, did not affect 3-HPAA relaxation. Similarly, no effects of 3-HPAA on cyclooxygenase nor L-type calcium channels were observed. Thus, 3-HPAA decreases blood pressure in vivo via vessel relaxation, and this mechanism might be based on the release of nitric oxide by the endothelial layer.

Role of H2S in Regulation of Vascular Tone in Metabolic Disorders.

We studied the effect of the H2S donor (NaHS, 1-500 μM) on the contractile responses of isolated aortic smooth muscle segments from rats with metabolic syndrome induced by high-fat, high-carbohydrate diet. It was found that the vasorelaxing effect of NaHS (5-100 μM) decreased in under conditions of MS. The endothelial NO synthase inhibitor L-NAME (100 μM) suppressed the effect of NaHS, while cystathionine-gamma-lyase inhibitor PAG (100 μM) decreased the vasodilating effects of acetylcholine (0.1-100 μM). Application of endogenous NO precursor L-arginine (1 mM) potentiated in the effects of H2S donor NaHS. Thus, the contractile activity of vascular smooth muscles in metabolic syndrome is determined by not only the effect of H2S, but also the influence of NO.

THE VASCULAR ENDOTHELIUM FUNCTION INDICATORS IN ORAL LIQUID OF IQOS SMOKING ADOLESCENTS

Heated tobacco products (HTP) were introduced to the world market as an alternative to conventional tobacco cigarettes. Active advertising, readily available to purchase and the public's perception that HTP are a safer alternative to conventional tobacco cigarettes lead to increasing their consumption by young age group in Ukraine. Despite the claims by the tobacco corporations about reduction of adverse effects the HTP on the activity of cardiovascular system, the published studies of potential vascular endothelial consequences were not found. Therefore, the aim of our study is to assess the HTP effect on vascular endothelial cells by determination of the nitrate, nitrite, S-nitrosothiols, and endothelin-1 levels in saliva of smoking adolescents. The object and research methods. Thirty adolescents aged 14-17 years old were divided to three groups. Group I was introduced by ten healthy non-smoking adolescents (control group). Group II consists of ten smoking adolescents have smoked the IQOS for two years. Group III is formed by ten smoking adolescents who have smoked the IQOS for three years. The salivary nitrate and nitrite levels were measured using the Griess assay such as an indicator of NO production. The salivary S-nitrosothiols were measured spectrophotometrically. The salivary endothelin-1 level was measured with ELISA kit. Statistical analysis of the data was carried out using the STATISTICA 7.0 program. The data were presented as Median values (Me), lower and upper quartile, i.e. Me [Lq; Uq]. Statistical significance was accepted if p < 0.05. Results. The salivary nitrate concentrations in groups II (Me = 14.11 [13.98; 14.52] μmol/l) and III (Me = 12.06 [11.44; 12.31] μmol/l) were significantly decreased compared to group I (Me = 15.89 [14.98; 16.89] μmol/l, KW test: H = 23.59, p=0.000. The values of nitrite in groups II and III were the following: Me = 2.32 [1.47; 2.48] μmol/l and 1.37 [1.25; 1.44] μmol/l, respectively. The indices were lower in 1.41 and 2.36 times than in group I: Me = 3. 24 [3.05; 3.78] μmol/l, KW test: Н = 22.92, p = 0.000. The levels of salivary S-nitrosothiols in group II are: Me = 1.02 [0.84; 1.27] μmol/l and group III Me = 0.36 [0.25; 0.62] μmol/l were reduced in 1.75 and 4.97 times compared to group I: Me = 1.79 [1.67; 1.97] μmol/l, KW test: Н = 24.46, р = 0.000. We assume that the reduction of salivary nitrate and nitrite concentrations is associated with a low level of S-nitrosothiols which serve as deposited and released nitric oxide (NO). Probably, the decrease of nitrates and nitrites is associated with the inhibition of endothelial NO synthase. The salivary endothelin-1 level was significantly higher in group II: Ме=0.58 [0.53; 0.73] pmol/ml and in group III: Ме = 0.82 [0.79; 0.93] pmol/ml than in group I: Ме = 0.17 [0.14; 0.26] pmol/ml, KW test: Н = 23.94, р = 0.000. It should be noted, the significant differences between data of nitrate, nitrite, S-nitrosothiol and endothelin-1 content in groups II and III were found (Mann-Whitney U test, nitrates - Z = 3.62, p = 0.000; nitrites - Z = 2.91 , p = 0.003; S-nitrosothiols - Z = 3.74, p = 0.00; endothelin-1 - Z = 3.36, p = 0.000). These results showed interrelation between adverse IQOS effect on vascular endothelium of adolescents duration of IQOS smoke. Conclusions. The adverse effect of heated tobacco product (IQOS) on the biochemical indicators of the vascular endothelium function of adolescents was found in our experimental study. The salivary nitrate, nitrite and S-nitrosothiols levels were significantly decreased in IQOS smoking adolescents. The salivary endothelin-1 content was significantly elevated in IQOS smoking adolescents. The revealed changes indicate the endothelial dysfunction in young smokers. The development of pathological processes in the vascular endothelium depends on the smoking duration.

Concentration of Zearalenone, Alpha-Zearalenol and Beta-Zearalenol in the Myocardium and the Results of Isometric Analyses of the Coronary Artery in Prepubertal Gilts.

The carry-over of zearalenone (ZEN) to the myocardium and its effects on coronary vascular reactivity in vivo have not been addressed in the literature to date. Therefore, the objective of this study was to verify the hypothesis that low ZEN doses (MABEL, NOAEL and LOAEL) administered per os to prepubertal gilts for 21 days affect the accumulation of ZEN, α-ZEL and β-ZEL in the myocardium and the reactivity of the porcine coronary arteries to vasoconstrictors: acetylcholine, potassium chloride and vasodilator sodium nitroprusside. The contractile response to acetylcholine in the presence of a cyclooxygenase (COX) inhibitor, indomethacin and / or an endothelial nitric oxide synthase (e-NOS) inhibitor, L-NAME was also studied. The results of this study indicate that the carry-over of ZEN and its metabolites to the myocardium is a highly individualized process that occurs even at very low mycotoxin concentrations. The concentrations of the accumulated ZEN metabolites are inversely proportional to each other due to biotransformation processes. The levels of vasoconstrictors, acetylcholine and potassium chloride, were examined in the left anterior descending branch of the porcine coronary artery after oral administration of ZEN. The LOAEL dose clearly decreased vasoconstriction in response to both potassium chloride and acetylcholine (P < 0.05 for all values) and increased vasodilation in the presence of sodium nitroprusside (P = 0.021). The NOAEL dose significantly increased vasoconstriction caused by acetylcholine (P < 0.04), whereas the MABEL dose did not cause significant changes in the vascular response. Unlike higher doses of ZEN, 5 μg/kg had no negative influence on the vascular system.

Violet LED induces vasodilation in rat aortic rings by soluble guanylate cyclase-dependent mechanism and increases SOD activity.

We found several studies that have used the aortic rings as an experimental model, mainly for the testing of new drugs or new therapies that try to reverse or prevent endothelial dysfunction or characterize its mechanism of action in a biological system, creating the knowledge necessary to obtain the treatment of those several diseases, where many of these treatments involve photobiomodulation therapies. We also found numerous wavelengths represented by different colors of LASER or LED in which frequently, the mechanism of action in biological systems is unknown. This study has as main objective to investigate the effects of the Violet LED Light (405 nm) by using isolated aortic rings, looking for nitric oxide (NO) release, and evaluating if Violet LED Light can modulate the superoxide dismutase (SOD) activity. We performed a vascular reactivity study in isolated aortic rings from normotensive rats with a single LED application. Besides it, the rings were pre-incubated with soluble guanylate cyclase (sGC) inhibitor or endothelial NO synthase inhibitor and subsequently underwent the application of the Violet LED. The cell viability and nitric oxide release in cell culture of human umbilical codon vein cells (HUVEC) were analyzed. In the vascular reactivity experiment, we observed a peak of vasodilation when applying light to the aortic rings. The soluble guanylate cyclase inhibitor abolished the relaxation induced by the Violet LED Light. However, the NO synthase inhibitor did not modify the Violet LED effect. In an isolated system, we verified that the Violet LED Light can increase SOD activity. Our results suggest that Violet LED Light induces vasodilation by a mechanism dependent on sGC activation, and not by NOS activation, and part of this effect could be due to the increase of SOD activity.

Cardiovascular and Respiratory Toxicity of Protamine Sulfate in Zebrafish and Rodent Models.

Protamine sulfate (PS) is the only available option to reverse the anticoagulant activity of unfractionated heparin (UFH), however it can cause cardiovascular and respiratory complications. We explored the toxicity of PS and its complexes with UFH in zebrafish, rats, and mice. The involvement of nitric oxide (NO) in the above effects was investigated. Concentration–dependent lethality, morphological defects, and decrease in heart rate (HR) were observed in zebrafish larvae. PS affected HR, blood pressure, respiratory rate, peak exhaled CO2, and blood oxygen saturation in rats. We observed hypotension, increase of HR, perfusion of paw vessels, and enhanced respiratory disturbances with increases doses of PS. We found no effects of PS on human hERG channels or signs of heart damage in mice. The hypotension in rats and bradycardia in zebrafish were partially attenuated by the inhibitor of endothelial NO synthase. The disturbances in cardiovascular and respiratory parameters were reduced or delayed when PS was administered together with UFH. The cardiorespiratory toxicity of PS seems to be charge–dependent and involves enhanced release of NO. PS administered at appropriate doses and ratios with UFH should not cause permanent damage of heart tissue, although careful monitoring of cardiorespiratory parameters is necessary.

Pterostilbene and its nicotinate derivative ameliorated vascular endothelial senescence and elicited endothelium-dependent relaxations via activation of sirtuin 1.

Vascular endothelial cell senescence is a leading cause of age-associated diseases and cardiovascular diseases. Interventions and therapies targeting endothelial cell senescence and dysfunction would have important clinical implications. This study evaluated the effect of 10 resveratrol analogues, including pterostilbene (Pts) and its derivatives, against endothelial senescence and dysfunction. All the tested compounds at the concentrations from 10-9 M to 10-6 M did not show cytotoxicity in endothelial cells by MTT assay. Among the 10 resveratrol analogues, Pts and Pts nicotinate attenuated the expression of senescence-associated β-galactosidase, downregulated p21 and p53, and increased the production of nitric oxide (NO) in both angiotensin II - and hydrogen peroxide - induced endothelial senescence models. In addition, Pts and Pts nicotinate elicited endothelium-dependent relaxations, which were attenuated in the presence of endothelial NO synthase (eNOS) inhibitor L-NAME or sirtuin 1 (SIRT1) inhibitor sirtinol. Pts and Pts nicotinate did not alter SIRT1 expression but enhanced its activity. Both Pts and Pts nicotinate have high binding activities with SIRT1, according to surface plasmon resonance results and the molecular docking analysis. Inhibition of SIRT1 by sirtinol reversed the anti-senescent effects of Pts and Pts nicotinate. Moreover, Pts and Pts nicotinate shared similar ADME (absorption, distribution, metabolism, excretion) profiles and physiochemical properties. This study suggests that the Pts and Pts nicotinate ameliorate vascular endothelial senescence and elicit endothelium-dependent relaxations via activation of SIRT1. These two compounds may be potential drugs for the treatment of cardiovascular diseases related to endothelial senescence and dysfunction.

Asymmetric Dimethylarginine: a Key Player in the Pathophysiology of Endothelial Dysfunction, Vascular Inflammation and Atherosclerosis in Rheumatoid Arthritis?

Patients with rheumatoid arthritis (RA), a chronic and disabling autoimmune condition that is characterized by articular and extra-articular manifestations and a pro-inflammatory and pro-oxidant state, suffer from premature atherosclerosis and excessive cardiovascular disease burden. A key step in the pathogenesis of atherosclerosis is impaired synthesis of the endogenous messenger nitric oxide (NO) by endothelial cells which, in turn, alters local homeostatic mechanisms and favors vascular damage and plaque deposition. While the exact mechanisms of endothelial dysfunction in RA remain to be established, there is good evidence that RA patients have relatively high circulating concentrations of the methylated arginine asymmetric dimethylarginine (ADMA), a potent endogenous inhibitor of endothelial NO synthase (eNOS). This review discusses the biological and pathophysiological role of ADMA, the interplay between ADMA, inflammation and oxidative stress, and the available evidence on the adverse impact of ADMA on endothelial function and atherosclerosis and potential ADMA-lowering therapies in RA patients.

Effect of NOS Inhibitors and Anticoagulants on Nitric Oxide Production in a Tissue-factor Induced Rat DIC Model.

We examined the mechanism of nitric oxide (NO) production in a tissue-factor (TF)-induced disseminated intravascular coagulation (DIC) model in rats, using inducible nitric oxide synthase (iNOS) inhibitor (L-NIL), endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME), Factor Xa inhibitor (DX-9065a), and thrombin inhibitor argatroban. Experimental DIC was induced by sustained infusion of 3.75 U/kg TF for 4 h via the tail vein. We then investigated the effect of these four agents on TF-induced DIC. Administration of L-NIL or L-NAME during induction of TF-induced DIC did not affect hemostatic markers, whereas elevated plasma levels of NO metabolites (NOX) were significantly suppressed by co-administration of L-NAME. A significant increase in eNOS-mRNA expression was observed in the TF-induced DIC model. Argatroban almost completely suppressed eNOS-mRNA expression. eNOS plays an important role in the NO production in the TF-induced DIC, and thrombin is a key stimulant of eNOS-mRNA expression in this model.

Alpha-Adrenergic Agonists Stimulate Fluid Secretion in Lacrimal Gland Ducts.

PurposeThe role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms.MethodsFluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca2+ level [Ca2+i] were investigated with microfluorometry.ResultsBoth norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [].ConclusionsOur results prove the direct role of α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of β-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.

Aging differentially impacts vasodilation and angiogenesis in arteries from the white and brown adipose tissues.

Aging adipose tissues (ATs) manifest reduced vascularity and increased hypoxia and inflammation that contribute to local and systemic metabolic dysfunction. However, the mechanisms that underlie these age-related changes are incompletely understood. In this study, we sought to examine insulin-stimulated vasodilation and angiogenesis in the arterial vasculature from three major AT depots, perigonadal white (pgWAT), subcutaneous white (scWAT) and brown (BAT) from young and old mice. Here, we demonstrate that in young mice, insulin-stimulated vasodilation is lower in feed arteries from pgWAT compared to scWAT (p<0.05), but no differences were found between feed arteries in other AT depots (p>0.05). Insulin-stimulated vasodilation was lower in old compared to young feed arteries from all three AT depots (p<0.05 for all). In the presence of endothelial nitric oxide synthase inhibitor, L-NAME, insulin-stimulated vasodilation was decreased in young (p<0.05), but was unaffected in old (p>0.05) from all AT depots. We also observed no age-related differences in endothelium-independent dilation, as assessed by sodium nitroprusside (p>0.05). We next investigated angiogenic capacity of the vasculature in these AT depots. In young mice, BAT vasculature demonstrated the highest angiogenic potential, followed by pgWAT and scWAT. We found that aging decreased angiogenic sprout formation in pgWAT and BAT (both p<0.05), but increased angiogenic potential in scWAT (p<0.05), indicating dissimilar impact of aging on angiogenesis in different AT depots. Collectively, these data suggest that aging leads to a consistent impairment in insulin-stimulated vasodilation and reduction in NO bioavailability in all three AT, although aging differentially impacts angiogenic capacity across different AT depots.

Vascular toxicity associated with anti-angiogenic drugs.

Over the past two decades, the treatment of cancer has been revolutionised by the highly successful introduction of novel molecular targeted therapies and immunotherapies, including small-molecule kinase inhibitors and monoclonal antibodies that target angiogenesis by inhibiting vascular endothelial growth factor (VEGF) signaling pathways. Despite their anti-angiogenic and anti-cancer benefits, the use of VEGF inhibitors (VEGFi) and other tyrosine kinase inhibitors (TKIs) has been hampered by potent vascular toxicities especially hypertension and thromboembolism. Molecular processes underlying VEGFi-induced vascular toxicities still remain unclear but inhibition of endothelial NO synthase (eNOS), reduced nitric oxide (NO) production, oxidative stress, activation of the endothelin system, and rarefaction have been implicated. However, the pathophysiological mechanisms still remain elusive and there is an urgent need to better understand exactly how anti-angiogenic drugs cause hypertension and other cardiovascular diseases (CVDs). This is especially important because VEGFi are increasingly being used in combination with other anti-cancer dugs, such as immunotherapies (immune checkpoint inhibitors (ICIs)), other TKIs, drugs that inhibit epigenetic processes (histone deacetylase (HDAC) inhibitor) and poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors, which may themselves induce cardiovascular injury. Here, we discuss vascular toxicities associated with TKIs, especially VEGFi, and provide an up-to-date overview on molecular mechanisms underlying VEGFi-induced vascular toxicity and cardiovascular sequelae. We also review the vascular effects of VEGFi when used in combination with other modern anti-cancer drugs.

Ac2-26 Alleviates Brain Injury after Cardiac Arrest and Cardiopulmonary Resuscitation in Rats via the eNOS Pathway.

Background Brain injury is the leading cause of death following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Ac2-26 and endothelial nitric oxide synthase (eNOS) have been shown to reduce neuroinflammation. This study is aimed at determining the mechanism by which Ac2-26 protects against inflammation during brain injury following CA and CPR. Methods Sixty-four rats were randomized into sham, saline, Ac2-26, and Ac2-26+L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor) groups. Rats received Ac2-26, Ac2-26+L-NIO, or saline after CPR. Neurologic function was assessed at baseline, 24, and 72 hours after CPR. At 72 hours after resuscitation, serum and brain tissues were collected. Results Blood-brain barrier (BBB) permeability increased, and the number of surviving neurons and neurological function decreased in the saline group compared to the sham group. Anti-inflammatory and proinflammatory factors, neuron-specific enolase (NSE) levels, and the expression of eNOS, phosphorylated (p)-eNOS, inducible nitric oxide synthase (iNOS), and oxidative stress-related factors in the three CA groups significantly increased (P < 0.05). BBB permeability decreased, and the number of surviving neurons and neurological function increased in the Ac2-26 group compared to the saline group (P < 0.05). Ac2-26 increased anti-inflammatory and reduced proinflammatory markers, raised NSE levels, increased the expression of eNOS and p-eNOS, and reduced the expression of iNOS and oxidative stress-related factors compared to the saline group (P < 0.05). The effect of Ac2-26 on brain injury was reversed by L-NIO (P < 0.05). Conclusions Ac2-26 reduced brain injury after CPR by inhibiting oxidative stress and neuroinflammation and protecting the BBB. The therapeutic effect of Ac2-26 on brain injury was largely dependent on the eNOS pathway.

Relationship Between Plasma Osteopontin and Arginine Pathway Metabolites in Patients With Overt Coronary Artery Disease.

IntroductionOsteopontin (OPN) is involved in ectopic calcification. Its circulating form is upregulated in coronary artery disease (CAD) patients. Circulating OPN levels positively correlate with oxidative stress, one of the major triggers of endothelial dysfunction. Endothelial dysfunction is, in turn, associated with reduced nitric oxide (NO) bioavailability due to the impaired arginine pathway. The aim of this study was to better understand the correlations between OPN, oxidative stress markers, and the arginine pathway metabolites.Methods and ResultsELISA and mass spectrometry techniques have been used to evaluate circulating OPN and arginine pathway/oxidative stress metabolites, respectively, in twenty-five control subjects and thirty-three patients with overt atherosclerosis. OPN positively correlates with 2,3-dinor-8isoPGF2a levels (p = 0.02), ornithine (p = 0.01), ADMA (p = 0.001), SDMA (p = 0.03), and citrulline (p = 0.008) levels only in CAD patients. In addition, citrulline positively correlated with ADMA (p = 0.02) levels, possibly as result of other sources of citrulline biosynthetic pathways.ConclusionThe association between OPN and impaired arginine/NO pathway could play a role in the inhibition of endothelial NO synthase (eNOS) and/or in the arginase activation in the context of CAD patients. However, further studies are needed to verify the cause-effect relationship between OPN, oxidative stress, and arginine/NO pathway dysregulation.

Selective and sensitive fluorescence imaging reveals microenvironment-dependent behavior of NO modulators in the endothelial system

Nitric oxide (NO) is a second messenger playing crucial roles in the signaling of a variety of cellular functions. Due to its pathophysiological significance, various NO modulators have been developed to explore NO pathways and some have been used as therapies. These modulators are often used directly to observe pharmacological effects in cell lines, but their actual effect on intracellular NO level is seldom analyzed. Herein, facilitated by a selective and sensitive fluorescence probe, we observed that some NO modulators displayed unexpected behaviors with both NO scavenger carboxy-PTIO and endothelial nitric oxide synthase (eNOS) inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME) failing to decrease intracellular free NO level in EA. hy926 cells while NO donor diethylamine-NONOate (DEA·NONOate) and eNOS activator calcimycin (A23187) failing to increase free NO level in human umbilical vein endothelial cell line (HUV-EC-C), although the reagents were confirmed to work normally in the primary human umbilical vein endothelial cells (primary HUVECs) and RAW 264.7 macrophage cells. Further research suggested that these unusual behaviors might be attributed to the cellular microenvironments including both the NO synthase (NOS) level and the endogenous glutathione (GSH) level. Genetically manipulating eNOS level in both cells restores the expected response, while decreasing GSH level restores the ability of DEA·NONOate to increase NO level in HUV-EC-C. These results reveal that the cellular microenvironment has a profound impact on pharmacological effect. Our study suggests GSH as a reservoir for NO in live cells and highlights the value of chemical probes as valuable tools to reveal microenvironment-dependent pharmacological effects.

Assessment of endothelium-dependent vasodilation in the clinic: what endothelial factor are we studying?

Objective . To study the contribution of the main endothelial vasodilators using different functional tests. Design and methods . Blood flow in the microvessels of the forearm skin was assessed in 30 healthy volunteers (20-21 years) using high-frequency Doppler ultrasound. Three functional tests were performed: iontophoresis with acetylcholine, a test with reactive hyperemia and a temperature test with local heating of the forearm skin to 42 °C. To analyze the mechanisms of endothelium-dependent vasodilation we carried out iontophoresis of vasoactive substances: L-Nitro-Arginine Methyl Ester (L-NAME) — inhibitor of endothelial NO synthase (eNOS), tetraethylammonium (TEA) — inhibitor of endothelial hyperpolarizing factor (EDHF), diclofenac sodium — inhibitor of cyclooxygenase (COX). Results . During the test with acetylcholine was characterized by a rapid increase in the volume rate of blood flow for 2 minutes, from 4 minutes there was a slow decrease in the volume rate of blood flow. The blockade of nitric oxide and prostacyclin led to a slowdown in blood flow growth and the blockade of EDHF to accelerate the recovery of the reaction of microvessels to acetylcholine. In postocclusion test changes in blood flow occur only under blockade of nitric oxide, while the blockade of EDHF and prostacyclin showed no effect on reactive hyperemia. Similar results were obtained by local heating of the skin. Conclusions. Nitric oxide plays a leading role in all tests, including local heat test and flow-mediated dilation. Endothelial hyperpolarizing factor is significant only in the mechanism of acetylcholine-induced vasodilation.

The Nitric Oxide Contribution to Reflex Cutaneous Vasodilation During Intradermal Microdialysis is Similar When Quantified Either Within or Between Sites

Nitric oxide (NO) is an important component of the reflex cutaneous vasodilatory response during whole body heating (WBH). The NO contribution to reflex cutaneous vasodilation can be quantified via local intradermal microdialysis delivery of the endothelial NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME), either (1) within a single site by perfusing L‐NAME after a set rise in oral temperature (ΔTor) is reached, or (2) between two sites, one of which acts as a control while the other is perfused with L‐NAME throughout WBH. It is unclear, however, whether these two methods of quantifying the NO contribution to reflex cutaneous vasodilation yield similar results.PurposeTo compare the NO contribution to reflex cutaneous vasodilation when calculated either within a site or between two sites. We hypothesized that the NO contribution to reflex cutaneous vasodilation would be similar when quantified either within or between sites.MethodsRetrospective analysis of skin blood flow responses during whole‐body heat stress (water‐perfused suit) was performed for 11 young (22 ± 1 yr) and 48 middle‐aged/older (64 ± 5 yr) adults. Two intradermal microdialysis fibers placed in the ventral skin of the forearm were perfused with either lactated Ringer’s (control) or 20 mM L‐NAME during WBH. After ΔTor = 1°C was reached, mean body temperature was clamped and 20 mM L‐NAME was added to the control site until a post‐L‐NAME plateau was established. Red cell flux was measured continuously at each site (laser‐Doppler flowmetry) and cutaneous vascular conductance (CVC = flux/MAP) was expressed as a percentage of maximum (%CVCmax; 28mM sodium nitroprusside + 43°C). The NO contribution to reflex vasodilation was calculated in two ways for each subject: (1) as the difference in %CVCmax between the ΔTor = 1°C and post‐L‐NAME plateau within the control site, and (2) as the difference in %CVCmax between the control and L‐NAME sites at ΔTor = 1°C.ResultsWhen analyzed as a single group, there were no differences between methods of quantifying the NO contribution to reflex vasodilation (17.3 ± 1.6% vs. 21.7 ± 2.9%, p = 0.09). Similarly, there were no differences between methods when the young (26.1 ± 4.9% vs. 36.0 ± 8.5%, p = 0.20) and middle‐aged/older (15.2 ± 1.5% vs. 18.3 ± 2.9%, p = 0.26) groups were analyzed separately. Maximal CVC was similar between the young (1.85 ± 0.19) and middle‐aged/older (1.66 ± 0.14) groups (p = 0.53). There was a negative correlation between age and NO contribution (R2 = 0.42; p = 0.02) with no difference in slope (p = 0.46) or intercept (p = 0.18) between the two methods.ConclusionQuantification of the NO contribution to reflex cutaneous vasodilation yields similar results when calculated either within a site or between two sites. Importantly, the well‐documented age‐related reduction in the NO contribution to reflex vasodilation is evident regardless of the method used. These data suggest that either of these two methods are effective for quantifying the NO contribution to reflex cutaneous vasodilation, regardless of the magnitude of the overall vasodilatory response.Support or Funding InformationSupported by NIH T‐32 Grant #5T32AG049676‐03

Vascular Endothelial Dysfunction Induced by a Western‐Style Diet Can Be Transferred via Fecal Microbiota Transplant in Mice

Consumption of a Western‐style diet (WD; high fat and sugar, low fiber) is associated with vascular endothelial dysfunction, which increases risk of cardiovascular and metabolic diseases. Endothelial dysfunction is largely mediated by reduced nitric oxide (NO) bioavailability, caused by increased superoxide‐driven oxidative stress and inflammation. WD is also associated with adverse changes to the gut microbiome—a potential link between WD and vascular dysfunction and a promising target for therapeutic intervention.PURPOSETo determine if WD‐induced vascular dysfunction can be transferred solely via the gut microbiome using fecal microbiota transplants (FMT).METHODSYoung male C57BL/6 mice were fed either control (CON) or WD chow beginning at 3 mo of age. Starting at age 3–5 mo, mice were administered FMTs (feces collected pre‐treatment), via oral gavage, from either their own cohort (i.e., sham condition: CON+CON, N=5; WD+WD, N=12) or from the other cohort of mice (CON+WD, N=5; WD+CON, N=12) for 8–16 weeks. Endothelium‐dependent dilation (EDD; ex vivo carotid artery dilation to increasing doses of acetylcholine) and associated mechanisms were assessed following the intervention.RESULTSBody mass at sacrifice was higher in WD (35.9±0.3 g) vs. CON‐fed mice (CON: 33.4±1.0 g, p&lt;0.01) but was not affected by FMT (both p&gt;0.14). WD‐related impairments in peak EDD (CON+CON: 98.5±1% vs. WD‐WD: 83.3±1%, p&lt;0.01) were attenuated by FMT from CON‐fed mice (WD+CON: 90.6±0.4%; p=0.02 vs. WD+WD), whereas FMT from WD‐fed mice induced a similar impairment in peak EDD (CON+WD: 86.0±2%) to WD chow alone. These changes were NO‐mediated, as addition of the endothelial NO synthase inhibitor L‐NAME abolished differences in peak EDD (CON+CON: 26±3% vs. CON+WD: 26±3%, p=0.91; WD‐WD: 32±1%, vs. WD+CON: 26±2%, p=0.44). FMT did not alter smooth muscle sensitivity to NO, assessed as dilation with the NO donor sodium nitroprusside (both cohorts p&gt;0.17), or aortic superoxide production (electron paramagnetic resonance spectroscopy; both p&gt;0.34). As endothelial function can also be influenced by systemic inflammation, we cultured visceral white adipose tissue for 24 h and measured pro‐inflammatory cytokine production in culture media. In preliminary results (N=4–10/group), FMT from WD‐fed mice increased adipose tissue release of interleukin (IL)‐1β (CON+CON: 60±28 vs. CON+WD: 163±68 pg/ml per 1g of tissue), interferon (IFN)□ (CON+CON: 19±4 vs. CON+WD: 65±25 pg/ml/g), and tumor necrosis factor (TNF)□ (CON+CON: 548±80 vs. CON+WD: 693±27 pg/ml/g) to higher levels than WD‐fed mice (WD+WD: mice, IL‐1β: 89±8 pg/ml/g, IFN□: 43±6 pg/ml/g). However, FMT from CON‐fed mice did not consistently affect adipose inflammation in WD‐fed mice.CONCLUSIONSOur data indicate that WD‐induced vascular endothelial dysfunction: 1) can be transferred by FMT via alterations in NO bioavailability and possibly systemic inflammation; and 2) can be ameliorated by FMT from healthy mice. The gut microbiome may be a promising target for the treatment of WD‐induced vascular dysfunction and cardiovascular diseases.Support or Funding InformationR01 HL134887, F32 HL140875