Endothelial Nitric Oxide Formation Research Articles

Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. To uncover these mechanisms, force development was measured exvivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused exvivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

Carissa edulis Vahl (Apocynaceae) extract, a medicinal plant of Benin pharmacopoeia, induces potent endothelium-dependent relaxation of coronary artery rings involving nitric oxide

BackgroundHypertension is a major cardiovascular risk factor that affects most countries including those of Africa. Although Carissa edulis Vahl, Diodia scandens Sw. and Cleome gynandra L. are traditionally used in Benin as antihypertensive treatments with some efficacy mentioned by the local population, their biological activity on the cardiovascular system remains poorly studied. AimThe study investigated the vasoreactivity of the plants and assessed the underlying mechanisms using isolated arteries. Study designAqueous-ethanolic extracts of aerial parts of C. edulis, D. scandens and C. gynandra were prepared by maceration before being subjected to multi-step liquid-liquid fractionation with solvents of increasing polarity. The vasoreactivity of the extracts and fractions were assessed on isolated porcine coronary artery and rat aorta using organ chambers, the role of nitric oxide (NO) using NG-nitro-L-arginine (NO synthase inhibitor), prostanoids using indomethacin (cyclooxygenases inhibitor) and endothelium-dependent hyperpolarization using TRAM-34 plus UCL 1684 (inhibitors of calcium-dependent K+ channels), and the vascular uptake of polyphenols using Neu reagent. ResultsThe aqueous-ethanolic crude extract of C. edulis (CECE) induced potent relaxations that were exclusively endothelium-dependent and more pronounced than those to D. scandens and C. gynandra. The n-butanolic fraction of C. edulis (CEBF) was more active than the cyclohexane, dichloromethane, and ethyl acetate fractions. The relaxation induced by CECE and CEBF were inhibited by NG-nitro-L-arginine and affected neither by TRAM-34 plus UCL 1684 nor by indomethacin. CEBF induced sustained endothelium-dependent relaxations for at least 60 min, and inhibited, in a concentration-dependent manner, contractions to KCl, CaCl2, U46619 and serotonin in rings with endothelium. Analysis of CEBF by LCHRMS indicated the presence of polyphenols, terpenes, and alkaloids. Exposure of coronary artery and aorta rings to CEBF caused the accumulation of polyphenols predominantly in the endothelium. ConclusionC. edulis leaf extract induced pronounced endothelium-dependent relaxations and inhibited contractile responses by stimulating the endothelial formation of NO. LCHRMS analysis of the most active fraction, the butanolic fraction, revealed the presence of numerous compounds including polyphenols, terpenes, and alkaloids. The polyphenols of CEBF accumulated preferentially in the endothelium of the arterial wall. Thus, these observations support the folkloric use of C. edulis in hypertension.

ASSOCIATIONS BETWEEN PLASMA METABOLOMICS AND BLOOD PRESSURE ACROSS ETHNICITIES: THE HELIUS STUDY

Objective: Blood pressure (BP) is regulated by plasma metabolites from different neurohumoral and cardiometabolic systems. Since there are well-established differences in hypertension pathogenesis and treatment response between ethnicities, we hypothesized that plasma metabolites may be differently associated with BP across ethnic groups. Design and method: From the HELIUS study, 369 subjects (mean age 52 ± 11 years, 51%F) of African and non-African descent were included. Office systolic and diastolic BP levels were recorded. Plasma metabolites were measured with untargeted LC-MS from fasting plasma samples. Associations between metabolite profiles and BP were assessed with machine learning prediction models. Associations between the resulting best predictors and BP were assessed with linear regression models while adjusting for age, sex, renal function and diabetes, and interactions with ethnicity were tested. Results: Plasma metabolite profiles explained 14.1% of systolic BP variance and 10.6% of diastolic BP variance. Top predictors for both systolic and diastolic BP included N-formylmethionine, several acylcarnitines and polyunsaturated fatty acids such as hexadecadienoate. These metabolites were significantly associated with higher systolic BP with estimates ranging from 3.0–4.5 mmHg per 1 SD increase in the adjusted models. Associations with hexadecadienoate, dihomolinoleate and catecholamine metabolites, including vanillactate had significant interactions with ethnicity and were only significant in subjects of non-African descent. The top predictor N-formylmethionine was the most consistent predictor across ethnicities and sex. This metabolite was associated with higher levels of nitric oxide (NO)-related metabolites, including dimethylarginine (r = 0.42, p < 0.001) and citrulline (r = 0.24, p < 0.0001). Conclusions: Plasma metabolome composition explained a large proportion of BP variance, but best predicting metabolites were differently associated with BP across ethnicities. N-formylmethionine was the most consistent predictor for BP across ethnicities and sex. This metabolite was associated with metabolites in the NO-pathway. We hypothesize that formylmethionine affects BP through the inhibition of endothelial NO formation.

Understanding Myeloperoxidase-Induced Damage to HDL Structure and Function in the Vessel Wall: Implications for HDL-Based Therapies.

Atherosclerosis is a disease of increased oxidative stress characterized by protein and lipid modifications in the vessel wall. One important oxidative pathway involves reactive intermediates generated by myeloperoxidase (MPO), an enzyme present mainly in neutrophils and monocytes. Tandem MS analysis identified MPO as a component of lesion derived high-density lipoprotein (HDL), showing that the two interact in the arterial wall. MPO modifies apolipoprotein A1 (apoA-I), paraoxonase 1 and certain HDL-associated phospholipids in human atheroma. HDL isolated from atherosclerotic plaques depicts extensive MPO mediated posttranslational modifications, including oxidation of tryptophan, tyrosine and methionine residues, and carbamylation of lysine residues. In addition, HDL associated plasmalogens are targeted by MPO, generating 2-chlorohexadecanal, a pro-inflammatory and endothelial barrier disrupting lipid that suppresses endothelial nitric oxide formation. Lesion derived HDL is predominantly lipid-depleted and cross-linked and exhibits a nearly 90% reduction in lecithin-cholesterol acyltransferase activity and cholesterol efflux capacity. Here we provide a current update of the pathophysiological consequences of MPO-induced changes in the structure and function of HDL and discuss possible therapeutic implications and options. Preclinical studies with a fully functional apoA-I variant with pronounced resistance to oxidative inactivation by MPO-generated oxidants are currently ongoing. Understanding the relationships between pathophysiological processes that affect the molecular composition and function of HDL and associated diseases is central to the future use of HDL in diagnostics, therapy, and ultimately disease management.

Nanoencapsulation of the omega-3 EPA:DHA 6:1 formulation enhances and sustains NO-mediated endothelium-dependent relaxations in coronary artery rings and NO formation in endothelial cells

• Omega-3fatty acids stimulate the endothelial formation of vasoprotective nitric oxide. • EPA:DHA 6:1 nanoparticles caused greater sustained endothelium-dependent relaxation. • EPA:DHA 6:1 nanoparticles caused sustained formation of NO in endothelial cells. • EPA:DHA 6:1 nanoparticles-treated endothelial cells had greater anti-platelet effect. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) formulation with a ratio of 6:1 is a potent stimulator of the endothelial formation of nitric oxide (NO). The aim of the study was to investigate whether nanoencapsulation of EPA:DHA 6:1 followed by coating with gum increases its biological activity. Vascular reactivity was assessed using porcine coronary artery rings, the formation of NO in cultured endothelial cells (ECs) using DAF-FM and indirectly by platelet aggregation studies. Coated EPA:DHA 6:1 nanoparticles induced sustained relaxations of coronary artery rings that were greater in rings with than in those without endothelium, and more pronounced than with the native form. Treatment of ECs with coated EPA:DHA 6:1 nanoparticles caused greater and more sustained formation of NO and enhanced their anti-aggregatory effects. Thus, nanoencapsulation of EPA:DHA 6:1 is an attractive strategy to enhance the beneficial effect at the vascular endothelium.

Anthocyanin-rich blackcurrant intake by old rats improves blood pressure, vascular oxidative stress and endothelial dysfunction associated with SGLT1- and 2-mediated vascular uptake of anthocyanin

Aging-related endothelial dysfunction and vascular oxidative stress affect early arterial sites at risk. Anthocyanins uptake via sodium-glucose co-transporter 1 (SGLT1) are potent inducers of endothelial formation of nitric oxide (NO). This study examined if anthocyanin-rich blackcurrant (ARB) improves the endothelial function in old rats. Male Wistar rats (22-month old) received ARB (60 and 120 mg/kg/d) orally for 2 weeks. Systolic blood pressure (SBP) was assessed by tail-cuff sphygmomanometry, vascular reactivity using organ chambers, protein expression by immunofluorescence, oxidative stress using dihydroethidium, and anthocyanin uptake by Neu reagent. Old rats showed increased SBP, abolished endothelium-dependent hyperpolarization-mediated relaxation and increased contractile response to phenylephrine in the mesenteric artery, which were improved by ARB. Old aorta showed increased oxidative stress and expression levels of eNOS, which were improved by ARB. SGLT1 immunofluorescence predominantly in the endothelium was more pronounced in the aortic arch than the aorta and higher in old than young rats, whereas the SGLT2 signal was low. The ARB treatment induced a dose-dependent accumulation of anthocyanins in the aorta and aortic arch. An ARB purified extract promoted ex vivo greater anthocyanins uptake mostly in the endothelium in the aortic arch than aorta, and in old compared to young rats. The anthocyanins uptake was inhibited to a greater extent by a dual SGLT1/2 inhibitor than by a selective SGLT2 inhibitor in the aorta of young and old rats. Both SGLT inhibitors reduced also ex vivo the age-related vascular oxidative stress. The upregulation of SGLT1, and the greater SGLT1 and SGLT2-mediated uptake of anthocyanins predominantly in the endothelium at arterial sites at risk in old rats suggest that anthocyanins appear as interesting natural products to protect the endothelial function with increasing age.

Coronavirus 2019 Disease (COVID-19), Systemic Inflammation, and Cardiovascular Disease.

Acute respiratory failure associated with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread worldwide and presents critical challenges for the public health and medical communities The World Health Organization (WHO) has declared SARS-CoV-2 a public health emergency of international concern, with a global estimate of over 7 million human infections and more than 400000 deaths worldwide as of June 12, 2020 A 2020 report by the China Medical Treatment Expert Group for Coronavirus disease 2010 (COVID-19) (2) showed that the clinical spectrum of the viral infection is dominated by fever (up to 88 7% of patients during hospitalization) and cough (67 8% of patients) followed by symptoms such as headache, fatigue, or shortness of breath

Intake of omega-3 formulation EPA:DHA 6:1 by old rats for 2 weeks improved endothelium-dependent relaxations and normalized the expression level of ACE/AT1R/NADPH oxidase and the formation of ROS in the mesenteric artery

Omega-3 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect the cardiovascular system, in part, by stimulating the endothelial formation of nitric oxide (NO). EPA:DHA 6:1 has been identified as a potent omega 3 PUFA formulation to induce endothelium-dependent vasorelaxation and activation of endothelial NO synthase (eNOS). This study examined whether intake of EPA:DHA 6:1 (500 mg/kg/day) for 2 weeks improves an established endothelial dysfunction in old rats (20 months old), and, if so, the underlying mechanism was subsequently determined.In the main mesenteric artery rings, an endothelial dysfunction characterized by a blunted NO component, an abolished endothelium-dependent hyperpolarization component, and increased endothelium-dependent contractile responses (EDCFs) are observed in old rats compared to young rats. Age-related endothelial dysfunction was associated with increased vascular formation of reactive oxygen species (ROS) and expression of eNOS, components of the local angiotensin system, senescence markers, and cyclooxygenase-2 (COX-2), and the downregulation of COX-1. The EPA:DHA 6:1 treatment improved the NO-mediated relaxation, reduced the EDCF-dependent contractile response and the vascular formation of ROS, and normalized the expression level of all target proteins in the old arterial wall.Thus, the present findings indicate that a 2-week intake of EPA:DHA 6:1 by old rats restored endothelium-dependent NO-mediated relaxations, most likely, by preventing the upregulation of the local angiotensin system and the subsequent formation of ROS.

P6278Ageing is associated with increased endothelial sodium-glucose cotransporter 1 expression at arterial sites at risk promoting enhanced anthocyanin accumulation and improved vascular oxidative stress

Abstract Introduction Ageing is characterized by endothelial dysfunction and vascular oxidative stress affecting initially arterial sites at risk. Anthocyanin-rich products are potent stimulators of the endothelial formation of nitric oxide. Sodium-glucose co-transporter 1 (SGLT1) expression has been shown to be increased by oxidative stress and mediate anthocyanin uptake in endothelial cells. Purpose The study determined whether ageing is associated with an upregulation of SGLT1 in arterio-susceptible (aortic arch) and resistant (aorta) sites, and evaluated the vascular SGLT1-mediated anthocyanin uptake. In addition, the impact of a 2-week ingestion of an anthocyanin-rich blackcurrant concentrate (ARBC) by old rats on vascular anthocyanin uptake and oxidative stress, and systolic blood pressure (SBP) was assessed. Methods Male Wistar rats (22-month old) were either untreated or treated with ARBC (60 and 120 mg/kg/day) in the drinking water for 2 weeks. SGLT1 expression was assessed by immunofluorescence, anthocyanin accumulation by Neu A reagent using a purified extract (BCE) prepared from ARBC, oxidative stress by dihydroethidium using confocal microscopy, and SBP by tail-cuff sphingomanometry. Results SGLT1 immunofluorescence was observed predominantly in the endothelium and was higher in the aortic arch than the aorta in old rats whereas only low levels were observed in young rats (12-week old). Exposure of vascular sections to BCE resulted in anthocyanin uptake exclusively in the endothelium, which was higher in the aortic arch than the aorta, and more pronounced in old than young rats. Anthocyanin uptake induced by BCE in the aorta was markedly reduced by LX4211 (a SGLT1/2 inhibitor) both in old and young rats. A high level of oxidative stress was observed throughout the aortic wall of old compared to young rats, which was inhibited by LX4211. Ingestion of ARBC by old rats resulted in a dose-dependent accumulation of anthocyanins throughout the aorta wall and the aortic arch. The tissue accumulation of anthocyanins was associated with a reduced level of oxidative stress. Ageing was associated with increased SBP by about 8 mmHg, which was reduced by ARBC 60 and 120 mg/kg/day treatment by about 5 and 7 mmHg, respectively. Conclusion The present findings indicate that ageing is associated with an upregulation of SGLT1 predominantly in the endothelium and that this effect is more pronounced at the aortic arch than the aorta. The increased endothelial expression level of SGLT1 promoted a greater accumulation of anthocyanins sensitive to LX4211. In addition, a 2-week ingestion of ARBC by old rats resulted in the accumulation of anthocyanins throughout the arterial wall of the aortic arch and aorta, and resulted in a reduced level of oxidative stress and systolic blood pressure. Thus, SGLT1 may be an attractive target to restore vascular protection at arterial sites at risk by promoting endothelial and vascular uptake of anthocyanins.

Shear stress-induced endothelial adrenomedullin signaling regulates vascular tone and blood pressure.

Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress-induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress-induced release of adrenomedullin, which in turn activated its Gs-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This Gs/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gαs showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress-induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent Gs-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.

EPA:DHA 6:1 is a superior omega-3 PUFAs formulation attenuating platelets-induced contractile responses in porcine coronary and human internal mammary artery by targeting the serotonin pathway via an increased endothelial formation of nitric oxide

At arterial sites of endothelial denudation and dysfunction, activated platelets contribute to vascular injury through the release of potent contracting factors such as serotonin (5-HT). This study evaluated whether omega-3 polyunsaturated fatty acids (PUFAs), known to protect the vascular system, are able to prevent platelets-induced contractile responses in isolated arteries and, if so, to investigate the underlying mechanism and the importance of the omega-3 PUFAs formulation.Porcine coronary arteries (PCA), human internal mammary arteries (IMA) and washed human platelets were prepared and vascular reactivity was studied in organ chambers.In PCA rings, aggregating platelets caused concentration-dependent contractions that were significantly inhibited by the 5-HT2A receptor antagonist ketanserin, and by EPA:DHA 6:1 but not EPA:DHA 1:1 at 0.4% v/v. EPA:DHA 6:1 also prevented the 5-HT-induced contractions but affected only slightly those to the thromboxane A2 analogue U46619. The inhibitory effect of EPA:DHA 6:1 on platelets-induced contractions was not observed in rings without endothelium, and prevented by an eNOS inhibitor but not by inhibitors of endothelium-dependent hyperpolarization. In IMA rings, EPA:DHA 6:1 but not EPA:DHA 1:1 at 0.4% v/v significantly prevented the 5-HT-induced contraction, and induced greater endothelium-dependent relaxations than bradykinin and acetylcholine sensitive to an eNOS inhibitor.EPA:DHA 6:1 strongly inhibits platelets- and 5-HT-induced contractions in PCA rings and those to 5-HT in IMA rings most likely through an increased endothelial formation of NO. These findings suggest that the omega-3 PUFAs EPA:DHA 6:1 formulation may be of interest to prevent platelets-induced vascular injury at arterial sites of endothelial dysfunction.

O35 An anthocyanin-rich blackcurrant extract induced NO-mediated relaxation in coronary artery rings and eNOS phosphorylation in cultured endothelial cells: Role of sodium-glucose cotransporters 1 and 2

Introduction The beneficial cardiovascular effect of polyphenol-rich food and beverages has been attributed, at least in part, to an improved vascular function through an increased endothelial formation of nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH), two major vasoprotective mechanisms. Black currant is a rich source of anthocyanins, and, in particular, of glucoside- and rutinoside-conjugated delphinidin and cyanidin. This study evaluated the role of sodium-glucose cotransporters (SGLT) 1 and 2 in the blackcurrant extract (BCE)-induced NO-mediated endothelium-dependent relaxation and activation of endothelial NO synthase (eNOS). Methods The reactivity of porcine coronary artery rings was assessed in organ chambers, and the expression and phosphorylation level of proteins in cultured porcine coronary artery endothelial cells by Western blot analysis. Results BCE (310.0 ± 0.1 mg gallic acid equivalent/g) was prepared from a blackcurrant juice containing 2.7 g gallic acid equivalent/L of polyphenols using a Sephadex LH-20 column. BCE caused potent concentration-dependent relaxations of coronary artery rings with endothelium, which were significantly reduced by the dual SGLT1 and SGLT2 inhibitor LX4211, and also to some extent by the SGLT2 inhibitor canagliflozin but not dapagliflozin. In contrast, LX4211 did not affect relaxations to bradykinin, sodium nitroprusside and epigallocatechin gallate. BCE induced the phosphorylation of eNOS at the activator site Ser1177 in cultured endothelial cells, which was significantly prevented by LX4211, dapagliflozin and canagliflozin. Conclusion These observations indicate that BCE is a potent activator of eNOS and inducer of NO-mediated vasorelaxation possibly subsequent to the entry of conjugated anthocyanins via SGLT1/2 into endothelial cells.

Role of GPER in estrogen-dependent nitric oxide formation and vasodilation

Estrogens are potent regulators of vasomotor tone, yet underlying receptor- and ligand-specific signaling pathways remain poorly characterized. The primary physiological estrogen 17β-estradiol (E2), a non-selective agonist of classical nuclear estrogen receptors (ERα and ERβ) as well as the G protein-coupled estrogen receptor (GPER), stimulates formation of the vasodilator nitric oxide (NO) in endothelial cells. Here, we studied the contribution of GPER signaling in E2-dependent activation of endothelial NO formation and subsequent vasodilation. Employing E2 and the GPER-selective agonist G-1, we investigated eNOS phosphorylation and NO formation in human endothelial cells, and endothelium-dependent vasodilation in the aortae of wild-type and Gper-deficient mice. Both E2 and G-1 induced phosphorylation of eNOS at the activation site Ser1177 to similar extents. Endothelial NO production to E2 was comparable to that of G-1, and was substantially reduced after pharmacological inhibition of GPER. Similarly, the clinically used ER-targeting drugs 4OH-tamoxifen, raloxifene, and ICI182,780 (faslodex, fulvestrant™) induced NO formation in part via GPER. We identified c-Src, EGFR, PI3K and ERK signaling pathways to be involved in GPER-dependent NO formation. In line with activation of NO formation in cells, E2 and G-1 induced equally potent vasodilation in the aorta of wild-type mice. Gper deletion completely abrogated the vasodilator response to G-1, while reducing the response to E2 by ∼50%. These findings indicate that a substantial portion of E2-induced endothelium-dependent vasodilation and NO formation is mediated by GPER. Thus, selective targeting of vascular GPER may be a suitable approach to activate the endothelial NO pathway, possibly leading to reduced vasomotor tone and inhibition of atherosclerotic vascular disease.

Does Aldosterone Play a Significant Role for Regulation of Vascular Tone?

Besides the well-known renal effects of aldosterone, the hormone is now known to have direct vascular effects. Clinical observations underline substantial adverse effects of aldosterone on cardiovascular function. The source of systemic circulating aldosterone is the adrenal gland zona glomerulosa cells through stimulus-secretion coupling involving depolarization, opening of L- and T-type calcium channels and aldosterone synthase activation. Local formation and release in peripheral tissues such as perivascular fat is recognized. Where does aldosterone affect the vasculature? Mineralocorticoid receptors (MRs) are present in endothelial and vascular smooth muscle cells, and MR-independent pathways are also involved. The vascular effects of aldosterone are complex, both concentration and temporal and spatial aspects are relevant. The acute response includes vasodilation through endothelial nitric oxide formation and vasoconstrictor effects through endothelial-contracting cyclooxygenase-derived factors and a changed calcium handling. The response to aldosterone can change within the same blood vessels depending on the exposure time and status of the endothelium. Chronic responses involve changed levels of reactive oxygen radicals, endothelial Na-influx and smooth muscle calcium channel expression. Furthermore, perivascular cells for example mast cells have also been suggested to participate in the chronic response. Moreover, the vascular effect of aldosterone depends on the status of the endothelium which is likely the cause of the very different responses to aldosterone and MR treatment observed in human studies going from increased to decreased flow depending on whether the patient had prior cardiovascular disease with endothelial dysfunction or not. A preponderance of constrictor versus dilator responses to aldosterone could therefore be involved in the detrimental vascular actions of the hormone in the setting of endothelial dysfunction and contribute to explain the beneficial action of MR blockers on blood pressure and target organ injury.

Cardiovascular effects induced by northeastern Brazilian red wine: Role of nitric oxide and redox sensitive pathways

Red wines have been shown to protect the vascular system by increasing the endothelial formation of nitric oxide (NO). This study investigated the vasoactive properties of an alcohol-free Cabernet-Sauvignon northeastern Brazilian red wine – RIOSOL (RSCS) extract. The polyphenolic-rich RSCS contained 4.2 mg GAE/L of polyphenols, and its major compounds included quercetin, myricetin and kaempferol. In normotensive conscious rats, RSCS produced hypotension and tachycardia, which were attenuated by the endothelial NO synthase inhibitor (eNOS), L-NAME. In addition, after 2 weeks of oral treatment, RSCS reduced arterial pressure in L-NAME-treated hypertensive rats. RSCS caused NO-mediated relaxations in phenylephrine contracted mesenteric artery rings, and induced the formation of NO and superoxide anions and a redox-sensitive phosphorylation of Akt and eNOS in cultured endothelial cells. In conclusion, the findings indicate that an alcohol-free lyophilized RSCS induced hypotension and endothelium-dependent vasorelaxation as a consequence of the activation of the Akt-eNOS-NO pathway in a redox-sensitive manner.

P2Y₂ and Gq/G₁₁ control blood pressure by mediating endothelial mechanotransduction.

Elevated blood pressure is a key risk factor for developing cardiovascular diseases. Blood pressure is largely determined by vasodilatory mediators, such as nitric oxide (NO), that are released from the endothelium in response to fluid shear stress exerted by the flowing blood. Previous work has identified several mechanotransduction signaling processes that are involved in fluid shear stress-induced endothelial effects, but how fluid shear stress initiates the response is poorly understood. Here, we evaluated human and bovine endothelial cells and found that the purinergic receptor P2Y2 and the G proteins Gq/G11 mediate fluid shear stress-induced endothelial responses, including [Ca2+]i transients, activation of the endothelial NO synthase (eNOS), phosphorylation of PECAM-1 and VEGFR-2, as well as activation of SRC and AKT. In response to fluid shear stress, endothelial cells released ATP, which activates the purinergic P2Y2 receptor. Mice with induced endothelium-specific P2Y2 or Gq/G11 deficiency lacked flow-induced vasodilation and developed hypertension that was accompanied by reduced eNOS activation. Together, our data identify P2Y2 and Gq/G11 as a critical endothelial mechanosignaling pathway that is upstream of previously described mechanotransduction processes and demonstrate that P2Y2 and Gq/G11 are required for basal endothelial NO formation, vascular tone, and blood pressure.

Hydrogen sulfide inhibits endothelial nitric oxide formation and receptor ligand-mediated Ca(2+) release in endothelial and smooth muscle cells.

In the vascular system, ATP-sensitive K(+)-channels are a target for H2S. Recent evidence suggests that H2S may also modulate Na(+)- and Ca(2+)-permeable channels and intracellular Ca(2+) stores, but the influence of H2S on endothelial Ca(2+) dynamics and Ca(2+)-dependent activation of endothelial nitric oxide synthase (eNOS) is unclear. In this study, we investigated the effects of H2S on Ca(2+) signaling in endothelial and smooth muscle cells with special emphasis given to the role of H2S in modulating endothelial NO formation. Experiments were performed with endothelial cells from porcine aorta, the human endothelial cell line HMEC-1, and smooth muscle cells from rat aorta and trachea. Mobilization of intracellular Ca(2+) and Ca(2+) entry was monitored with Fura-2. Activity of eNOS was determined as conversion of incorporated l-[(3)H]arginine into l-[(3)H]citrulline. Incubation of endothelial cells with the H2S donors sodium hydrogen sulfide (NaHS) and GYY4137 blocked activation of eNOS by the receptor agonist ATP but not by the Ca(2+) ionophore A23187. Data revealed that H2S inhibited ATP-induced release of Ca(2+) from intracellular stores indicating that H2S attenuates eNOS activity by blocking capacitative Ca(2+) entry. A similar inhibitory effect of H2S on ATP-induced Ca(2+) release and Ca(2+) entry was also observed in human microvascular endothelial cells and smooth muscle cells. H2S antagonized Ca(2+) mobilization by receptor agonists and store-operated Ca(2+) entry thereby limiting eNOS activation and NO formation. The effect of H2S on Ca(2+) stores was not restricted to endothelial cells but was also observed in vascular and tracheal smooth muscle cells.

Effect of the oestrogen receptor antagonist fulvestrant on the cirrhotic rat lung.

It has been postulated that cirrhosis-related lung vasodilatation and the subsequent hepatopulmonary syndrome are partly explained by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In this study, we assessed whether the oestrogen receptor antagonist fulvestrant (F) improves cirrhosis-related lung abnormalities. Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham, and sham+F. Histological, immunohistochemical, and Western blot analyses were performed on lung samples. In the lung, the endothelial NO synthase and the nitrotyrosine protein expressions were increased in CBDL as compared to sham rats. Both parameters were significantly reduced by fulvestrant in the CBDL rats. Surprisingly, the level of pVASP (an indirect marker of NO formation and action) was decreased in CBDL rats, and fulvestrant had no effect on this parameter. The level of the vascular endothelial growth factor, the diameter of small lung vessels, and the number of macrophages were increased in CBDL lungs in comparison with sham lungs, and these parameters were unaffected by fulvestrant treatment. In conclusion, fulvestrant may not be relevant to improve lung abnormalities in cirrhosis because NO may not be biologically active and because key events contributing to the lung abnormalities are not affected by fulvestrant.

Abstract 174: Remote Ischemic Preconditioning Improves Digital Vascular Endothelial Function and Enhances Circulating Nitric Oxide Formation in Healthy Volunteers

Background: Remote ischemic preconditioning (RIPC) induced by brief episodes of ischemia of the limb protects against organ damage by ischemia-reperfusion. It has been reported that RIPC improves endothelial function of the forearm in normal subjects and patients with coronary artery disease. However, the digital vascular endothelial response to RIPC has not been determined in healthy volunteers. Methods: This study was performed to examine effect of RIPC on fingertip digital vascular endothelial function by using endothelial pulse amplitude tonometry (EndoPAT) device and alteration in circulating nitric oxide(NO) level in peripheral blood. Ten healthy adult subjects (5 men, 5 women, mean age 25.2 years) had 3 cycles of 5 min ischemia alternating with 5 min reperfusion of the forearm. Another 10 healthy volunteers (6 men, 4 women, mean age 26.2 years) were used as the control group of sham and the cuff was inflated for 3 times at 10 mmHg for 5 minutes with 5-minute intervals. Fingertip digital vascular endothelial function of the ipsilateral arm was measured prior to first ischemia, 3h and 6h after the last episode of ischemia. Blood samples were taken from the contralateral arm prior to first ischemia, 3h and 6h after the last episode of ischemia. Results: Fingertip digital vascular endothelial function was significantly increase in 3h and 6h after the last RIPC intervention compared with the baseline condition(p<0.05). In parallel the RIPC stimulus increased circulating NO formation(p<0.05). There is a close association between finger vascular endothelial function and NO formation(p<0.05). Conclusion: The present findings demonstrate for the first time that transient RIPC contributes to improvement of digital vascular endothelial function and increase in circulating NO production in healthy volunteers. Our data provide a novel evidence to support that RIPC might gain even wider application not only in disease but also in health.