Endothelium-dependent Vasodilation Research Articles

Veno-Arterial Extracorporeal Membrane Oxygenation (ECMO) Impairs Bradykinin-Induced Relaxation in Neonatal Porcine Coronary Arteries.

Extracorporeal membrane oxygenation (ECMO) is a lifesaving support for respiratory and cardiovascular failure. However, ECMO induces a systemic inflammatory response syndrome that can lead to various complications, including endothelial dysfunction in the cerebral circulation. We aimed to investigate whether ECMO-associated endothelial dysfunction also affected coronary circulation. Ten-day-old piglets were randomized to undergo either 8 h of veno-arterial ECMO (n = 5) or no treatment (Control, n = 5). Hearts were harvested and coronary arteries were dissected and mounted as 3 mm rings in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U46619, concentration–response curves to the endothelium-dependent vasodilator bradykinin (BK) and the nitric oxide (NO) donor (endothelium-independent vasodilator) sodium nitroprusside (SNP) were performed. Relaxation to BK was studied in the absence or presence of the NO synthase inhibitor Nω-nitro-L-arginine methyl ester HCl (L-NAME). U46619-induced contraction and SNP-induced relaxation were similar in control and ECMO coronary arteries. However, BK-induced relaxation was significantly impaired in the ECMO group (30.4 ± 2.2% vs. 59.2 ± 2.1%; p < 0.0001). When L-NAME was present, no differences in BK-mediated relaxation were observed between the control and ECMO groups. Taken together, our data suggest that ECMO exposure impairs endothelium-derived NO-mediated coronary relaxation. However, there is a NO-independent component in BK-induced relaxation that remains unaffected by ECMO. In addition, the smooth muscle cell response to exogenous NO is not altered by ECMO exposure.

Arterial stiffness, endothelial dysfunction and impaired fibrinolysis are pathogenic mechanisms contributing to cardiovascular risk in ANCA-associated vasculitis

Cardiovascular disease is a complication of systemic inflammatory diseases including anti-neutrophil cytoplasm antibody-associated vasculitis (AAV). The mechanisms of cardiovascular morbidity in AAV are poorly understood, and risk-reduction strategies are lacking. Therefore, in a series of double-blind, randomized case-control forearm plethysmography and crossover systemic interventional studies, we examined arterial stiffness and endothelial function in patients with AAV in long-term disease remission and in matched healthy volunteers (32 each group). The primary outcome for the case-control study was the difference in endothelium-dependent vasodilation between health and AAV, and for the crossover study was the difference in pulse wave velocity (PWV) between treatment with placebo and selective endothelin-A receptor antagonism. Parallel invitro studies of circulating monocytes and platelets explored mechanisms. Compared to healthy volunteers, patients with AAV had 30% reduced endothelium-dependent vasodilation and 50% reduced acute release of endothelial active tissue plasminogen activator (tPA), both significant in the case-control study. Patients with AAV had significantly increased arterial stiffness (PWV: 7.3 versus 6.4 m/s). Plasma endothelin-1 was two-fold higher in AAV and independently predicted PWV and tPA release. Compared to placebo, both selective endothelin-A and dual endothelin-A/B receptor blockade reduced PWV and increased tPA release in AAV in the crossover study. Mechanistically, patients with AAV had increased platelet activation, more platelet-monocyte aggregates, and altered monocyte endothelin receptor function, reflecting reduced endothelin-1 clearance. Patients with AAV in long-term remission have elevated cardiovascular risk and endothelin-1 contributes to this. Thus, our data support a role for endothelin-blockers to reduce cardiovascular risk by reducing arterial stiffness and increasing circulating tPA activity.

Glycyrrhizin ameliorates vascular endothelial cell senescence by inhibiting HMGB1 in HFD/STZ-induced diabetic rats and human umbilical vein endothelial cells

The senescence and dysfunction of vascular endothelial cells are important features of diabetic vascular disease. High mobility group box-1(HMGB1) may be involved in vascular injury in response to high glucose. Glycyrrhizin (GL) is an HMGB1 inhibitor that significantly reduces HMGB1. However, the relationship between HMGB1 and vascular ageing in diabetes is not clear, the protective mechanism of GL against vascular injury in type 2 diabetes mellitus (T2DM) is unclear too. This study aims to examine the role of HMGB1 in vascular endothelial cell senescence and the protective effects of GL on vascular aging in high fat diet/streptozotocin (HFD/STZ) induce type 2 diabetic rats.After induction of diabetes, GL (150 mg/kg/d) was treated by gavage for 4 weeks. Results showed that compared with the Control group, the serum level of HMGB1 was increased in rats with type 2 diabetes, while the expression of HMGB1 mRNA and protein in the thoracic aorta was upregulated, with a decrease in endothelium-dependent vasodilation function and an increase in aging degree in the thoracic aorta. However, the above indicators were significantly improved after GL treatment. In HUVECs, we found that treated with HMGB1 (50, 100 and 200 ng/ml) for 48 h induced cells senescence and GL (50, 100 mg/L) significantly inhibited high-glucose-induced endothelial cell senescence, meanwhile GL (50, 100 mg/L) significantly inhibited the high-glucose-induced HMGB1 release and upregulated p53 expression. In conclusion, GL as an HMGB1 inhibitor, attenuates endothelium-dependent relaxation impairment and vascular ageing in an animal model of diabetes and high-glucose-induced endothelial cell senescence.

SOLUBLE GUANYLYL CYCLASE ACTIVATION RESCUES HYPEROXIA-INDUCED DYSFUNCTION OF VASCULAR RELAXATION.

Introduction: Perioperative alterations in perfusion lead to ischemia and reperfusion injury, and supplemental oxygen is administered during surgery to limit hypoxic injury but can lead to hyperoxia. We hypothesized that hyperoxia impairs endothelium-dependent and endothelium-independent vasodilation but not the vasodilatory response to heme-independent soluble guanylyl cyclase activation. Methods: We measured the effect of oxygen on vascular reactivity in mouse aortas. Mice were ventilated with 21% (normoxia), 60% (moderate hyperoxia), or 100% (severe hyperoxia) oxygen during 30 minutes of renal ischemia and 30 minutes of reperfusion. After sacrifice, the thoracic aorta was isolated, and segments mounted on a wire myograph. We measured endothelium-dependent and endothelium-independent vasodilation with escalating concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, and we measured the response to heme-independent soluble guanylyl cyclase activation with cinaciguat. Vasodilator responses to each agonist were quantified as the maximal theoretical response ( Emax ) and the effective concentration to elicit 50% relaxation (EC 50 ) using a sigmoid model and nonlinear mixed-effects regression. Aortic superoxide was measured with dihydroethidium probe and high-performance liquid chromatography quantification of the specific superoxide product 2-hydroxyethidium. Results: Hyperoxia impaired endothelium-dependent (ACh) and endothelium-independent (SNP) vasodilation compared with normoxia and had no effect on cinaciguat-induced vasodilation. The median ACh Emax was 76.4% (95% confidence interval = 69.6 to 83.3) in the normoxia group, 53.5% (46.7 to 60.3) in the moderate hyperoxia group, and 53.1% (46.3 to 60.0) in the severe hyperoxia group ( P &lt; 0.001, effect across groups), while the ACh EC 50 was not different among groups. The SNP Emax was 133.1% (122.9 to 143.3) in normoxia, 128.3% (118.1 to 138.6) in moderate hyperoxia, and 114.8% (104.6 to 125.0) in severe hyperoxia ( P &lt; 0.001, effect across groups), and the SNP EC 50 was 0.38 log M greater in moderate hyperoxia than in normoxia (95% confidence interval = 0.18 to 0.58, P &lt; 0.001). Cinaciguat Emax and EC 50 were not different among oxygen treatment groups (median range Emax = 78.0% to 79.4% and EC 50 = -18.0 to -18.2 log M across oxygen groups). Aorta 2-hydroxyethidium was 1419 pmol/mg of protein (25th-75th percentile = 1178-1513) in normoxia, 1993 (1831-2473) in moderate hyperoxia, and 2078 (1936-2922) in severe hyperoxia ( P = 0.008, effect across groups). Conclusions: Hyperoxia, compared with normoxia, impaired endothelium-dependent and endothelium-independent vasodilation but not the response to heme-independent soluble guanylyl cyclase activation, and hyperoxia increased vascular superoxide production. Results from this study could have important implications for patients receiving high concentrations of oxygen and at risk for ischemia reperfusion-mediated organ injury.

Comparison of the Ameliorating Effects of Valsartan and Amlodipine on Vascular Endothelial Dysfunction and Oxidative Stress in Elderly Patients with Type H Hypertension.

Objective To explore whether long-term administration of valsartan or amlodipine can improve vascular endothelial function and reduce the production of reactive oxygen species in patients with H-type hypertension, so as to provide a reference for clinical treatment. Methods A total of 82 elderly patients with type H hypertension who were admitted to our hospital from March to August 2017 were selected as the research subjects. The study included a 4-week continuous irrigation period followed by a 24-week randomized treatment period. Forty patients in the valsartan group and 42 in the amlodipine group were treated with 5 mg amlodipine or 80 mg valsartan for 24 weeks. Clinical efficacy, 24 h mean DBP, SBP, and 24HSBP, DBP coefficient of variation, cardiac fatty acid-binding protein (H-FABP), vascular pseudohemophilia factor (VWF), nitric oxide (NO), endothelium-dependent vasodilation function (FMD), nonendothelium-dependent vasodilation function (NMD), malondialdehyde (MDA), GSH-Px, and SOD levels were observed. Results The total effective rate was 80.0% (32/40) in the valsartan group and 85.71% (36/42) in the amlodipine group, and the difference was not statistically significant (P > 0.05). After 24 weeks of treatment, the 24 h mean SBP, SDP, 24HSBP, and DBP variation coefficients of the two groups were significantly decreased, and there was no statistical significance between the two groups (P > 0.05). After treatment, the values of H-FABP, VWF, NO, and MDA in both groups decreased compared with before treatment, while the values of FMD%, NMD%, SOD, and GSH-Px increased compared with before treatment (P > 0.05), and the levels of H-FABP, VWF, and NO in the valsartan group were lower than those in the amlodipine group. Meanwhile, FMD%, NMD%, SOD, and GSH-Px levels were higher than those in the amlodipine group (P > 0.05). Conclusion Valsartan and amlodipine reduce blood pressure, improve vascular endothelial function, and inhibit oxidative stress in elderly patients with H-type hypertension on average. However, valsartan has a better effect on vascular endothelial dysfunction and oxidative stress in elderly patients with H-type hypertension.

Abstract P3138: The Regulatory Role Of Sirtuin 6 In Coronary Microvascular Dysfunction

Introduction: Coronary microvascular dysfunction (CMD) is characterized by impaired endothelial-dependent vasodilation. These impairments are seen in diabetic cardiomyopathy, ischemia with no obstructive coronary artery disease, and heart failure with preserved ejection fraction (HFpEF), but detailed mechanisms have yet to be elucidated. Sirtuin 6 (Sirt6), an important member of the Sirtuin family, has been implicated in obesity, insulin resistance, type 2 diabetes mellitus (T2DM), and cardiovascular diseases. Sirt6 protects EC from premature senescence, oxidative stress, and atherosclerosis by sustaining high eNOS levels and preserving cell replication. Sirt6 was reported to regulate endothelial dilation in atherosclerosis in Sirt6 heterozygous mice, but how Sirt6 regulates coronary microvascular function remains to be determined. Methods: Sirt6 knockout and wild-type (WT) mice were fed a diet high in fat and sugar (HFHS) for six months, and blood lipid and glucose were measured. Coronary arteries were isolated, and endothelial-dependent vasodilation (EDD) was assessed using myography (DMT). Echocardiography and treadmill exercise exertion tests were performed to evaluate cardiac function. Myocardial blood flow (MBF) was measured by doppler. Cardiac fibrosis was detected using trichrome staining, and molecular pathways were elucidated via gene and protein analysis. Results: Our preliminary data show that Sirt6 was downregulated in diabetes. The EDD of coronary arterioles was impaired, and the mediator of coronary vasodilation switched from NO to H 2 O 2 in the Sirt6 knockout mice. Compared to the WT mice, myocardial blood flow is decreased, ejection fraction (EF) was not changed, the running distance was reduced, and E/E’ ratio was increased in Sirt6 knockout mice. The Sirt6 targeted proteins were identified. Conclusions: Sirt6 regulated coronary microvascular function, and ablation of Sirt6 caused CMD and diastolic dysfunction. Further genetic profiling will elucidate the pathways and mechanisms converging with Sirt6 to regulate microvascular function.

PCSK9 Promotes Endothelial Dysfunction During Sepsis Via the TLR4/MyD88/NF-κB and NLRP3 Pathways.

Endothelial dysfunction often accompanies sepsis. We aimed to explore the role of PCSK9 in septic endothelial dysfunction. Sepsis was induced by lipopolysaccharide (LPS) treatment of human umbilical vein endothelial cells (HUVECs) in vitro and cecal ligation and puncture (CLP) surgery in mice in vivo. Evolocumab (EVC) and Pep 2-8, PCSK9 inhibitors, were subsequently used to determine the role of PCSK9 in sepsis-induced endothelial dysfunction in vitro and in vivo, respectively. In addition, the TLR4 agonist, Kdo2-Lipid A ammonium (KLA), was used to determine the related mechanism. Protein expression of eNOS, VE-cadherin, PCSK9, TLR4, MyD88, p-p65, p65, NLRP3, ASC, and caspase-1 p20 in mice aortas and HUVECs was measured by western blotting, while mRNA expression of TNFα, IL-1β, and IL-18 was determined by qRT-PCR. The level of inflammatory cytokines of mouse aortas was visualized by immunohistochemistry. Vasodilation of the aorta was detected by vascular reactivity experiments. The 96-h survival rate after CLP was assessed. Our findings showed that the expression of eNOS and VE-cadherin decreased, and PCSK9 expression increased, in septic HUVECs or mice. Inhibition of PCSK9 increased eNOS and VE-cadherin expression. Activation of the TLR4/MyD88/NF-κB and NLRP3 pathways may be responsible for PCSK9-induced endothelial dysfunction in sepsis. Vascular reactivity tests and survival studies showed that inhibition of PCSK9 could prevent the decrease in endothelium-dependent vasodilation function and improve the survival rates of septic mice. In summary, our results suggested that increased PCSK9 expression during sepsis activated the TLR4/MyD88/NF-κB and NLRP3 pathways to induce inflammation, which resulted in vascular endothelial dysfunction and decreased survival rates. Thus, inhibition of PCSK9 may be a potential clinical therapeutic target to improve vascular endothelial function in sepsis.

Mirabegron relaxes arteries from human visceral adipose tissue through antagonism of α1-adrenergic receptors

AimAs inadequate perfusion has emerged as a key determinant of adipose tissue dysfunction in obesity, interest has grown regarding possible pharmacological interventions to prevent this process. Mirabegron has proved to improve insulin sensitivity and glucose homeostasis in obese humans via stimulation of β3-adrenoceptors which also seem to mediate endothelium-dependent vasodilation in disparate human vascular beds. We characterized, therefore, the vasomotor function of mirabegron in human adipose tissue arteries and the underlying mechanisms. MethodsSmall arteries (116–734 μm) isolated from visceral adipose tissue were studied ex vivo in a wire myograph. After vessels had been contracted, changes in vascular tone in response to mirabegron were determined under different conditions. ResultsMirabegron did not elicit vasorelaxation in vessels contracted with U46619 or high-K+ (both P > 0.05). Notably, mirabegron markedly blunted the contractile effect of the α1-adrenergic receptor agonist phenylephrine (P < 0.001) either in presence or absence of the vascular endothelium. The anti-contractile action of mirabegron on phenylephrine-induced vasoconstriction was not influenced by the presence of the selective β3-adrenoceptor blocker L-748,337 (P < 0.05); lack of involvement of β3-adrenoceptors was further supported by absent vascular staining for them at immunohistochemistry. ConclusionsMirabegron induces endothelium-independent vasorelaxation in arteries from visceral adipose tissue, likely through antagonism of α1-adrenoceptors.

Abstract P3023: The Regulatory Role Of Mir-21 In Heart Failure With Preserved Ejection Fraction (hfpef)

Introduction: Coronary microvascular dysfunction (CMD) is characterized by impaired endothelial-dependent vasodilation. These impairments are seen in diabetic cardiomyopathy, ischemia with no obstructive coronary artery disease, and heart failure with preserved ejection fraction (HFpEF), but detailed mechanisms have yet to be elucidated. Moreover, how microRNA (miR-21) regulates CMD in HFpEF is not entirely understood. Methods: miR-21 knockout and wild-type (WT) mice were fed a diet high in fat and sugar (HFHS) for 6 months and blood lipid and glucose were measured. Echocardiography and treadmill exercise exertion tests were performed to assess cardiac function. Myocardial blood flow (MBF) under stress was measured by contrast echocardiography or doppler after the treatment with different dosages of norepinephrine and mean arterial blood pressure was measured simultaneously by femoral pressure catheter. Cardiac fibrosis was detected using trichrome staining and molecular pathways were elucidated via gene and protein analysis. Results: Our preliminary data show decreased MBF during stress in WT mice fed a HFHS diet. While ejection fraction was not changed, cardiac index, stroke volume, and running distance were decreased. Furthermore, E/E’ ratio was increased in WT mice fed HFHS diet compared to the WT mice fed a chow diet, suggesting heart failure and impaired diastolic function with normal systolic function. Moreover, perivascular fibrosis was increased in the WT mice fed a HFHS diet compared to WT mice fed a chow diet. However, the ablation of miR-21 reversed all these changes in the mice fed a HFHS diet. Conclusions: miR-21 regulates CMD and ameliorated HFpEF. Further investigation will elucidate the pathways and mechanisms converging with miR-21 to regulate HFpEF.

HIV and Schistosoma Co-Exposure Leads to Exacerbated Pulmonary Endothelial Remodeling and Dysfunction Associated with Altered Cytokine Landscape.

HIV and Schistosoma infections have been individually associated with pulmonary vascular disease. Co-infection with these pathogens is very common in tropical areas, with an estimate of six million people co-infected worldwide. However, the effects of HIV and Schistosoma co-exposure on the pulmonary vasculature and its impact on the development of pulmonary vascular disease are largely unknown. Here, we have approached these questions by using a non-infectious animal model based on lung embolization of Schistosoma mansoni eggs in HIV-1 transgenic (HIV) mice. Schistosome-exposed HIV mice but not wild-type (Wt) counterparts showed augmented pulmonary arterial pressure associated with markedly suppressed endothelial-dependent vasodilation, increased endothelial remodeling and vessel obliterations, formation of plexiform-like lesions and a higher degree of perivascular fibrosis. In contrast, medial wall muscularization was similarly increased in both types of mice. Moreover, HIV mice displayed an impaired immune response to parasite eggs in the lung, as suggested by decreased pulmonary leukocyte infiltration, small-sized granulomas, and augmented residual egg burden. Notably, vascular changes in co-exposed mice were associated with increased expression of proinflammatory and profibrotic cytokines, including IFN-γ and IL-17A in CD4+ and γδ T cells and IL-13 in myeloid cells. Collectively, our study shows for the first time that combined pulmonary persistence of HIV proteins and Schistosoma eggs, as it may occur in co-infected people, alters the cytokine landscape and targets the vascular endothelium for aggravated pulmonary vascular pathology. Furthermore, it provides an experimental model for the understanding of pulmonary vascular disease associated with HIV and Schistosoma co-morbidity.

The state of platelet and plasma hemostasis as a predictor of coronary blood flow in patients with acute myocardial infarction.

Aim To study the relationship of the platelet function and plasma homeostasis with the blood flow in the infarct-related artery (IRA) and with the course of acute myocardial infarction (AMI).Material and methods This study included 93 patients with AMI (75 patients with ST-elevation AMI and 18 patients without ST segment elevation). 63 patients had TIMI 0-1 blood flow in the IRA and 30 patients had TIMI 2-3. Rotational thromboelastometry, impedance aggregometry, the endothelium-dependent vasodilation (EDVD) test, and the thrombodynamics test were performed for all patients. The primary clinical endpoint included the totality of in-hospital complications of AMI, and the secondary endpoint included the totality of out-of-hospital complications of AMI. Major bleedings (BARC 3-5) and minor bleedings (BARC 1-2) were evaluated separately.Results Patients with IRA TIMI 0-1 flow were characterized by a shorter blood clotting time (BCT), larger thrombus size and density, more intense platelet aggregation induced by arachidonic acid and ADP, and lower values of the EDVD test. It was found that the parameters of platelet aggregation induced by arachidonic acid (AUC Asa) in combination with BCT allowed assessment of the severity of IRA blood flow disorder (sensitivity 76 %, specificity 71 %) in patients with AMI, regardless of the presence of ST segment elevation on the ECG. In addition, the incidence of the primary endpoint was greater in patients with IRA TIMI 0-1 flow (41.3% and 16.7%, respectively; p=0.015). In patients with TIMI 2-3 flow in the long-term period of the disease, the incidence of minor bleedings was significantly higher (8.5% and 30.4 %, respectively; p=0.045).Conclusion Compared to patients with preserved blood flow, patients with AMI and IRA TIMI 0-1 flow are characterized by endothelial dysfunction and more intense processes of thrombogenesis and platelet aggregation. It has been shown for the first time that the combination of two simple criteria for assessing hemostasis (AUC Asa; BCT) allows assessment of the degree of IRA blood flow disorder in patients with AMI.

Mitochondrial Ca2+ Uptake Drives Endothelial Injury By Radiation Therapy.

Background:Radiation therapy strongly increases the risk of atherosclerotic vascular disease, such as carotid stenosis. Radiation induces DNA damage, in particular in mitochondria, but the upstream and downstream signaling events are poorly understood. The objective of this study was to define such mechanisms.Methods:Endothelial-specific MCU (mitochondrial Ca2+ uniporter) knockout and C57Bl6/J mice with or without a preinfusion of a mitoTEMPO (mitochondrial reactive oxygen species [ROS] scavenger) were exposed to a single dose of cranial irradiation. 24, and 240 hours postirradiation, vascular reactivity, endothelial function, and mitochondrial integrity were assessed ex vivo and in vitro.Results:In cultured human endothelial cells, irradiation with 4 Gy increased cytosolic Ca2+ transients and the mitochondrial Ca2+ concentration ([Ca2+]mt) and activated MCU. These outcomes correlated with increases in mitochondrial ROS (mtROS), loss of NO production, and sustained damage to mitochondrial but not nuclear DNA. Moreover, irradiation impaired activity of the ETC (electron transport chain) and the transcription of ETC subunits encoded by mitochondrial DNA (mtDNA). Knockdown or pharmacological inhibition of MCU blocked irradiation-induced mtROS production, mtDNA damage, loss of NO production, and impairment of ETC activity. Similarly, the pretreatment with mitoTEMPO, a scavenger of mtROS, reduced irradiation-induced Ca2+ entry, and preserved both the integrity of the mtDNA and the production of NO, suggesting a feed-forward loop involving [Ca2+]m and mtROS. Enhancement of DNA repair in mitochondria, but not in the nucleus, was sufficient to block prolonged mtROS elevations and maintain NO production. Consistent with the findings from cultured cells, in C57BL/6J mice, head and neck irradiation decreased endothelium-dependent vasodilation, and mtDNA integrity in the carotid artery after irradiation. These effects were prevented by endothelial knockout of MCU or infusion with mitoTEMPO.Conclusions:Irradiation-induced damage to mtDNA is driven by MCU-dependent Ca2+ influx and the generation of mtROS. Such damage leads to reduced transcription of mitochondrial genes and activity of the ETC, promoting sustained mtROS production that induces endothelial dysfunction. Our findings suggest that targeting MCU and mtROS might be sufficient to mitigate irradiation-induced vascular disease.

Hyperoxaluria Induces Endothelial Dysfunction in Preglomerular Arteries: Involvement of Oxidative Stress.

Urolithiasis is a worldwide problem and a risk factor for kidney injury. Oxidative stress-associated renal endothelial dysfunction secondary to urolithiasis could be a key pathogenic factor, similar to obesity and diabetes-related nephropathy. The aim of the present study was to characterize urolithiasis-related endothelial dysfunction in a hyperoxaluria rat model of renal lithiasis. Experimental approach: Endothelial dysfunction was assessed in preglomerular arteries isolated from control rats and in which 0.75% ethylene glycol was administered in drinking water. Renal interlobar arteries were mounted in microvascular myographs for functional studies; superoxide generation was measured by chemiluminescence and mRNA and protein expression by RT-PCR and immunofluorescence, respectively. Selective inhibitors were used to study the influence of the different ROS sources, xanthine oxidase, COX-2, Nox1, Nox2 and Nox4. Inflammatory vascular response was also studied by measuring the RNAm expression of NF-κB, MCP-1 and TNFα by RT-PCR. Results: Endothelium-dependent vasodilator responses were impaired in the preglomerular arteries of the hyperoxaluric group along with higher superoxide generation in the renal cortex and vascular inflammation developed by MCP-1 and promoted by NF-κB. The xanthine oxidase inhibitor allopurinol restored the endothelial relaxations and returned superoxide generation to basal values. Nox1 and Nox2 mRNA were up-regulated in arteries from the hyperoxaluric group, and Nox1 and Nox2 selective inhibitors also restored the impaired vasodilator responses and normalized NADPH oxidase-dependent higher superoxide values of renal cortex from the hyperoxaluric group. Conclusions: The current data support that hyperoxaluria induces oxidative stress-mediated endothelial dysfunction and inflammatory response in renal preglomerular arteries which is promoted by the xanthine oxidase, Nox1 and Nox2 pathways.

Antihypertensive Activity in High Salt-Induced Hypertensive Rats and LC-MS/MS-Based Phytochemical Profiling of Melia azedarach L. (Meliaceae) Leaves.

Melia azedarach L. leaves have been traditionally used but not scientifically evaluated for antihypertensive activity. The focus of the present work was to carry out the detailed phytochemical profiling and antihypertensive potential of methanolic extract and subsequent fractions of this plant. The tandem mass spectrometry-based phytochemical profiling of M. azedarach extract (Ma.Cr) and fractions was determined in negative ionization mode while molecular networking was executed using the Global Natural Product Social (GNPS) molecular networking platform. This study resulted in the identification of 29 compounds including flavonoid O-glycosides, simple flavonoids, triterpenoidal saponins, and cardenolides as the major constituents. Ma.Cr at the concentration of 300 mg/kg resulted in a fall in blood pressure (BP), i.e., 81.44 ± 2.1 mmHg in high salt-induced hypertensive rats in vivo, in comparison to normotensive group, i.e., 65.36 ± 1.8 mmHg at the same dose. A decrease in blood pressure was observed in anaesthetized normotensive and hypertensive rats treated with extract and various fractions of M. azedarach. A reasonable activity was observed for all fractions except the aqueous fraction. The highest efficacy was shown by the ethyl acetate fraction, i.e., 77.06 ± 3.77 mmHg in normotensive and 88.96 ± 1.3 mmHg in hypertensive anaesthetized rats. Ma.Cr and fractions showed comparatively better efficacy towards hypertensive rats as compared to rats with normal blood pressure. Blood pressure-lowering effects did not change upon prior incubation with atropine. In vitro testing of Ma.Cr and polarity-based fractions resulted in L-NAME sensitive, endothelium-dependent vasodilator effects on aortic tissues. Pretreatment of aorta preparations with Ma.Cr and its fractions also blocked K+-induced precontractions indicating Ca2+ channel blocking activity comparable to verapamil. The extract and polarity-based fractions did not reveal a vasoconstrictor response in spontaneously beating isolated rat aorta. Ma.Cr and fractions when used in atrial preparations resulted in negative inotropic and chronotropic effects. These effects in atrial preparations did not change in the presence of atropine. These effects of extract and fractions explained the antihypertensive potential of M. azedarach and thus provided a scientific basis for its ethnopharmacological use in the treatment of hypertension. Among the constituents observed, flavonoids and flavonoid O-glycosides were previously reported for antihypertensive potential.

MTOR contributes to endothelium-dependent vasorelaxation by promoting eNOS expression and preventing eNOS uncoupling

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

Maternal perfluorooctane sulfonic acid exposure during rat pregnancy causes hypersensitivity to angiotensin II and attenuation of endothelium-dependent vasodilation in the uterine arteries †.

Epidemiological studies show a strong association between environmental exposure to perfluorooctane sulfonic acid (PFOS) and preeclampsia and fetal growth restriction; however, the underlying mechanisms are unclear. We tested the hypothesis that gestational PFOS exposure leads to pregnancy complications via alterations in uterine vascular endothelium-independent angiotensin II-related mechanisms and endothelium-derived factors such as nitric oxide. Pregnant Sprague-Dawley rats were exposed to PFOS 0.005, 0.05, 0.5, 5, 10, and 50μg/mL through drinking water from gestational day 4 to 20, and dams with PFOS 50μg/mL were used to assess mechanisms. PFOS exposure dose dependently increased maternal blood pressure but decreased fetal weights. Uterine artery blood flow was lower and resistance index was higher in the PFOS dams. In PFOS dams, uterine artery contractile responses to angiotensin II were significantly greater, whereas contractile responses to K+ depolarization and phenylephrine were unaffected. Plasma angiotensin II levels were not significantly different between control and PFOS dams; however, PFOS exposure significantly increased Angiotensin II type 1 receptor (AGTR1) and decreased AGTR2 protein levels in uterine arteries. Endothelium-dependent relaxation response to acetylcholine was significantly reduced with decreased endothelial nitric oxide synthase expression in the uterine arteries of PFOS dams. Left ventricular hypertrophy and fibrosis were observed, along with increased ejection fraction and fractional shortening in PFOS dams. These results suggest that elevated maternal PFOS levels decrease uterine blood flow and increase vascular resistance via heightened angiotensin II-mediated vasoconstriction and impaired endothelium-dependent vasodilation, which provides a molecular mechanism linking elevated maternal PFOS levels with gestational hypertension and fetal growth restriction.

Roles of Reactive Oxygen Species in Vascular Complications of Diabetes: Therapeutic Properties of Medicinal Plants and Food

The rising prevalence of chronic metabolic disorders, such as obesity and type 2 diabetes, most notably associated with cardiovascular diseases, has emerged as a major global health concern. Reactive oxygen species (ROS) play physiological functions by maintaining normal cellular redox signaling. By contrast, a disturbed balance occurring between ROS production and detoxification of reactive intermediates results in excessive oxidative stress. Oxidative stress is a critical mediator of endothelial dysfunction in obesity and diabetes. Under a hyperglycemic condition, the antioxidant enzymes are downregulated, resulting in an increased generation of ROS. Increases in ROS lead to impairment of endothelium-dependent vasodilatations by reducing NO bioavailability. Chronic treatments with antioxidants were reported to prevent the development of endothelial dysfunction in diabetic patients and animals; however, the beneficial effects of antioxidant treatment in combating vascular complications in diabetes remain controversial as antioxidants do not always reverse endothelial dysfunction in clinical settings. In this review, we summarize the latest progress in research focused on the role of ROS in vascular complications of diabetes and the antioxidant properties of bioactive compounds from medicinal plants and food in animal experiments and clinical studies to provide insights for the development of therapeutic strategies.

Endothelial cyclin I reduces vulnerability to angiotensin II-induced vascular remodeling and abdominal aortic aneurysm risk.

Retinoblastoma protein (Rb) supports vasoprotective E2F Transcription Factor 1 (E2f1)/Dihydrofolate Reductase (Dhfr) pathway activity in endothelial cells. Cyclin I (Ccni) promotes Cyclin-Dependent Kinase-5 (Cdk5)-mediated Rb phosphorylation. Therefore, we hypothesized that endothelial Ccni may regulate cardiovascular homeostasis, vessel remodeling, and abdominal aortic aneurysm (AAA) formation. Aortic CCNI mRNA expression was analyzed in the Gene Expression Omnibus (GEO) GSE57691 cohort consisting of AAA patients (n=39) and healthy controls (n=10). We employed wild-type (WT) mice and endothelial Ccni knockout (Ccnifl/flTie2-Cre) mice to conduct in vivo and ex vivo experimentation using an Angiotensin (Ang) II hypertension model and a CaCl2 AAA model. Mice were assessed for Rb/E2f1/Dhfr signaling, biopterin (i.e., biopterin [B], dihydrobiopterin [BH2], and tetrahydrobiopterin [BH4]) production, cardiovascular homeostasis, vessel remodeling, and AAA formation. Aortic CCNI mRNA expression was downregulated in AAA patients. Both Ang II- and CaCl2-induced WT mice showed aortic Ccni upregulation coupled with vasculoprotective upregulation of Rb/E2f1/Dhfr signaling and biopterins. Endothelial Ccni knockout downregulated medial Rb/E2f1/Dhfr signaling and biopterins in Ang II-induced hypertensive mice, which exacerbated eNos uncoupling and H2O2 production. Endothelial Ccni knockout impaired in vivo hemodynamic responses and endothelium-dependent vasodilatation in ex vivo mesenteric arteries in response to Ang II. Endothelial Ccni knockout exacerbated mesenteric artery remodeling and AAA risk in response to Ang II and CaCl2. Endothelial Ccni acts as a critical negative regulator of eNos uncoupling-mediated ROS generation and thereby reduces vulnerability to hypertension-induced vascular remodeling and AAA development in mice.

Spinosin ameliorates insulin resistance by suppressing reactive oxygen species-associated inflammation.

Objective(s):Spinosin is the predominant C-glycoside flavonoid derived from the seeds of Zizyphus jujuba var. Spinosa (Rhamnaceae). The present study aimed to investigate the effects of spinosin on insulin resistance (IR) in vascular endothelium.Materials and Methods:The anti-IR effect of spinosin was evaluated in a high-fat diet (HFD) treated mice model. The effects of spinosin pretreatment on reactive oxygen species (ROS)-associated inflammation in Human umbilical vein endothelial cells (HUVEC) were evaluated by western blot analysis and reverse transcription-polymerase chain reaction. The effect of spinosin on insulin-mediated endothelium-dependent vasodilation of rat aortae was further evaluated. Results:Spinosin at 20 mg/kg alleviates increased mice’s body weight, fasting serum glucose, oral glucose tolerance, serum insulin, insulin resistance index, and serum lipid of HFD-treated mice. Spinosin at 20 μM suppressed ROS overproduction, and inhibited ROS-related HUVEC inflammation by inhibiting mRNA expression of tumor necrosis factor-α and interleukin-6. In addition, spinosin at 0.1 μM showed a vasodilation effect of isoprenaline-pretreated rat aortae and increased insulin-mediated NO production in endothelial cells. These effects were shown to be related to the spinosin regulating serine/tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) facilitated/phosphoinositide 3-kinase (PI3K) signaling. Conclusion:Spinosin may ameliorate IR and ROS-associated inflammation, and increase endothelial NO production by mediating IRS-1/PI3K/endothelial nitric oxide synthase (eNOS) pathway.

Endothelial cell membrane cholesterol content regulates the contribution of TRPV4 channels in ACh-induced vasodilation in rat gracilis arteries.

Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48 h of chronic hypoxia (CH). CH couples endothelial transient receptor potential subfamily V member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium-dependent hyperpolarization (EDH) pathway does not present in control animals. TRVPV4 channel activity has been shown to be regulated by membrane cholesterol. Hence, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelium-dependent vasodilation. Male Sprague-Dawley rats were exposed to ambient atmospheric (atm.) pressure or 48-h of hypoxia (0.5atm). Vasodilation to acetylcholine (ACh) was determined using pressure myography in gracilis arteries. EC membrane cholesterol was depleted using methyl-β-cyclodextrin (MβCD) and supplemented with MβCD-cholesterol. Inhibiting TRPV4 did not affect ACh-induced vasodilation in normoxic controls. However, TRPV4 inhibition reduced resting diameter in control arteries suggesting basal activity. TRPV4 contributes to ACh-induced vasodilation in these arteries when EC membrane cholesterol is depleted. Inhibiting TRPV4 attenuated ACh-induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion in arteries from CH animals abolished the contribution of TRPV4 to ACh-induced vasodilation. Endothelial cell membrane cholesterol impedes the contribution of TRPV4 channels in EDH-mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.