Modulation Of Endothelial Function Research Articles

Gut Microbiome and Carotid Artery Intima-Media Thickness: A Narrative Review of the Current Scenario.

Up to the last update, the gut microbiome (GM) had been associated with a different physiologic host process, including those affecting cardiovascular health. The carotid intima-media thickness (IMT) is an indicator of atherosclerosis and cardiovascular risk. The GM influence on atherosclerosis progression has garnered growing attention in recent years but the consensus in subclinical atherosclerosis remains elusive. The aim of this narrative review is to investigate the connection between the GM and carotid IMT, encompassing mechanisms like the microbiome impact on metabolite production, and systemic inflammation, and its effects on endothelial function. The literature analysis revealed that the GM appears to exert an influence on carotid IMT development, likely through mechanisms involving metabolites' production, systemic inflammation, and endothelial function modulation. Additional research, however, is needed to finely elucidate the relationship between the GM and atherosclerosis. Specifically, more extensive studies are required to pinpoint individuals at the highest risk of developing atherosclerosis based on their GM composition. This will facilitate the enhancement and optimization of cardiovascular disease prevention strategies and enable the treatments' customization for each patient. Further investigations are required to refine patient outcomes in the context of probiotics and other interventions aimed at improving microbiome composition and function.

Modulation of Endothelial Function by TMAO, a Gut Microbiota-Derived Metabolite.

Endothelial function is essential in the maintenance of systemic homeostasis, whose modulation strictly depends on the proper activity of tissue-specific angiocrine factors on the physiopathological mechanisms acting at both single and multi-organ levels. Several angiocrine factors take part in the vascular function itself by modulating vascular tone, inflammatory response, and thrombotic state. Recent evidence has outlined a strong relationship between endothelial factors and gut microbiota-derived molecules. In particular, the direct involvement of trimethylamine N-oxide (TMAO) in the development of endothelial dysfunction and its derived pathological outcomes, such as atherosclerosis, has come to light. Indeed, the role of TMAO in the modulation of factors strictly related to the development of endothelial dysfunction, such as nitric oxide, adhesion molecules (ICAM-1, VCAM-1, and selectins), and IL-6, has been widely accepted. The aim of this review is to present the latest studies that describe a direct role of TMAO in the modulation of angiocrine factors primarily involved in the development of vascular pathologies.

Rheum rhaponticum and Rheum rhabarbarum Extracts as Modulators of Endothelial Cell Inflammatory Response.

Inflammation, endothelial dysfunction, and alterations in blood physiology are key factors contributing to atherosclerosis and other cardiovascular disorders. Hence, modulation of endothelial function and reducing its pro-inflammatory and pro-thrombotic activity is considered one of the most important cardioprotective strategies. This study aimed to evaluate the anti-inflammatory potential of rhubarb extracts isolated from petioles and underground organs of Rheum rhabarbarum L. (garden rhubarb) and R. rhaponticum L. (rhapontic rhubarb) as well as two stilbenoids, typically found in these plants, i.e., rhapontigenin (RHPG) and its glycoside, rhaponticin (RHPT). Analysis of the anti-inflammatory effects of the indicated rhubarb-derived substances involved different aspects of the endothelial cells' (HUVECs) response: release of the inflammatory mediators; cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX) expression as well as the recruitment of leukocytes to the activated HUVECs. The ability of the rhubarb-derived extracts to inhibit COX-2 and 5-LOX activities was examined as well. The study was supplemented with the in silico analysis of major components of the analyzed extracts' interactions with COX-2 and 5-LOX. The obtained results indicated that the examined plant extracts and stilbenes possess anti-inflammatory properties and influence the inflammatory response of endothelial cells. Biochemical and in silico tests revealed significant inhibition of COX-2, with special importance of rhaponticin, as a compound abundant in both plant species. In addition to the reduction in COX-2 gene expression and enzyme activity, a decrease in the cytokine level and leukocyte influx was observed. Biochemical tests and computational analyses indicate that some components of rhubarb extracts may act as COX-2 inhibitors, with marginal inhibitory effect on 5-LOX.

Paricalcitol Improves the Angiopoietin/Tie-2 and VEGF/VEGFR2 Signaling Pathways in Adriamycin-Induced Nephropathy.

Renal endothelial cell (EC) injury and microvascular dysfunction contribute to chronic kidney disease (CKD). In recent years, increasing evidence has suggested that EC undergoes an endothelial-to-mesenchymal transition (EndoMT), which might promote fibrosis. Adriamycin (ADR) induces glomerular endothelial dysfunction, which leads to progressive proteinuria in rodents. The activation of the vitamin D receptor (VDR) plays a crucial role in endothelial function modulation, cell differentiation, and suppression of the expression of fibrotic markers by regulating the production of nitric oxide (NO) by activating the endothelial NO synthase (eNOS) in the kidneys. This study aimed to evaluate the effect of paricalcitol treatment on renal endothelial toxicity in a model of CKD induced by ADR in rats and explore mechanisms involved in EC maintenance by eNOS/NO, angiopoietins (Angs)/endothelium cell-specific receptor tyrosine kinase (Tie-2, also known as TEK) and vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) axis. The results show that paricalcitol attenuated the renal damage ADR-induced with antiproteinuric effects, glomerular and tubular structure, and function protection. Furthermore, activation of the VDR promoted the maintenance of the function and structure of glomerular, cortical, and external medullary endothelial cells by regulating NO production. In addition, it suppressed the expression of the mesenchymal markers in renal tissue through attenuation of (transforming growth factor-beta) TGF-β1/Smad2/3-dependent and downregulated of Ang-2/Tie-2 axis. It regulated the VEGF/VEGFR2 pathway, which was ADR-deregulated. These effects were associated with lower AT1 expression and VDR recovery to renal tissue after paricalcitol treatment. Our results showed a protective role of paricalcitol in the renal microvasculature that could be used as a target for treating the beginning of CKD.

GPR68 Inhibition with a Novel Group of Ogremorphin Inhibitors Upregulate Endothelial Barrier Function and Protect Against Bacterial Pathogens or Acidosis‐induced Inflammation in Lung Endothelium

BackgroundEndothelial dysfunction characterized by an increase in endothelial permeability and inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID‐19. Thus, drugs targeting the preservation and restoration of endothelial function represent an attractive therapeutic target to treat endothelial dysfunction‐derived cardiopulmonary diseases. G protein‐coupled receptors (GPCRs), especially a sub‐family of proton‐sensing GPCRs including GPR4 and GPR68, have been recently suggested to play a role in the modulation of endothelial function. In this study, we investigated the barrier protective and anti‐inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM‐1 in cultured human lung endothelial cells as well as in mice.MethodsEndothelial permeability was measured by monitoring transendothelial electrical resistance (TER) across human pulmonary arterial endothelial cells (HPAECs). Protein and mRNA expression levels of endothelial inflammation markers were analyzed by western blot and quantitative real time PCR, respectively. Acidosis was induced by switching cells to acidic pH (6.5) medium and luciferase‐based Tango assay was performed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heat‐killed Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples.ResultsBoth OGM8345 (1‐5 µM) and OGM‐1 (0.3‐1.5 µM) induced a robust dose‐dependent increase in basal EC barrier function that was evident by an 150‐200% increase in TER values. Both inhibitors also markedly attenuated LPS‐ and HKSA‐induced endothelial hyperpermeability. LPS or HKSA‐induced upregulation of inflammatory cytokines/chemokines genes TNF‐α, ICAM‐1, VCAM‐1, IL‐6, IL‐8, IL‐1β, and CXCL5 was significantly attenuated by OGMs as demonstrated by RT‐PCR analysis. Consistently, both OGMs suppressed LPS‐ and HKSA‐induced protein expression of phospho‐NFkB, VCAM‐1 and ICAM‐1. In contrast, pharmacologic inhibition of GPR4 by NE 52‐QQ57 failed to alleviate LPS or HKSA‐induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression as well as GPR68 activity that was inhibited by OGMs. Both OGMs attenuated vascular leak and lung inflammation caused by intratracheal injection of LPS or HKSA in C57BL/6 mice as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of inflammatory cells and protein content in bronchoalveolar lavage samples.ConclusionAltogether, these results establish an essential role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68‐selective inhibitors in improving endothelial dysfunction‐derived lung injuries.

G-protein subunit α-14 is dysregulated in human placentas from systemic lupus erythematosus pregnancies

IntroductionSystemic lupus erythematosus (SLE) is associated with placental dysfunctions during pregnancy, which may cause multiple adverse fetal and maternal outcomes. G-protein subunits α-11 (GNA11) and -14 (GNA14) participate actively in angiogenesis via the modulation of endothelial function. This study aimed to determine whether GNA11 and GNA14 levels in placental tissues differed between SLE and normal term pregnancies.Material and methodsTwenty-four individuals, including 14 patients with SLE and 10 normal pregnant women, were included in this study. The expression levels of GNA11 and GNA14 in the placentas were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting analyses. The localization of GNA11 and GNA14 in placental tissues was evaluated using immunohistochemistry. The correlations between relative mRNA and protein expression levels of GNA11,14 and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores were also analyzed.ResultsRT-qPCR revealed that the mRNA levels of GNA14, but not GNA11, were significantly decreased by 50% (P = 0.01) in SLE vs. normal term placentas. Western blotting showed that the protein levels of GNA14, but not GNA11, were significantly increased by 3.52-fold (P = 0.02) in SLE vs. normal term placentas. Using immunohistochemistry, positive staining for GNA11 and GNA14 was present in trophoblasts and villous stromal cells of the placental tissues. The expression levels of GNA11 and GNA14 were not significantly correlated with the SLEDAI scores (r2=0.02~0.24, P = 0.08~0.75).ConclusionsThe dysregulation of GNA14 in the placentas indicates a regulatory role during human SLE pregnancies.

A Betalain-rich Dietary Supplement, But Not PETN, Increases Vasodilation and Nitric Oxide: A Comparative, Single-dose, Randomized, Placebo-controlled, Blinded, Crossover Pilot Study

Nutraceutical supplements have demonstrated promise as agents for improving athletic performance and for positively affecting cardiovascular health and vigor through modulation of endothelial function at the cellular level. High-nitrate products, such as red beet juices and powders, have been observed to improve athletic performance potentially through increased nitric oxide (NO) concentrations in the blood. Similarly, a patented low nitrate, low sugar betalain-rich supplement has also been reported to significantly improve athletic performance. To the best of our knowledge, no acute clinical studies have been conducted that have demonstrated the comparative efficacies of high-nitrate or betalain-rich, low nitrate materials on measures of endothelial function in real time. In this acute single-dose, double-blinded, randomized, placebo-controlled, crossover study, we examined the effects of the betalain-rich low nitrate dietary supplement, (BRS, 50mg), in comparison to pentaerythritol tetranitrate (PETN, 40mg), a pharmaceutical drug that is a potent source of organic nitrate, and a placebo, on various measures of endothelial function for up to 4-hours post-ingestion. More specifically, in order to gauge post-treatment changes in endothelial function we measured flow-mediated dilation (FMD), nitrite (NO2)/nitrate (NO3) content, circulating nitrosyl-hemoglobin (NOHb) concentration, and cellular metabolic activity (CMA) measured as generation of reactive oxygen species, a side reaction of oxidative-reductive cellular metabolism. Ten participants completed all arms of the study. Results suggest that within 2 hours, BRS, but not PETN or placebo, resulted in significantly elevated levels of NOHb (a measure of bioavailable NO●) (p = 0.017) and increased vasodilation as measured by FMD, (p = 0.025). As expected, due to its high nitrate content, NO2/NO3 levels were increased by PETN within 2-hours (p = 0.048), but not by BRS or placebo. Finally, under these experimental conditions, PETN and BRS produced no significant changes for mitochondrial, NADPH-oxidase dependent or cellular CMA. These data provide preliminary support for single-dose effectiveness of BRS, but not PETN, on levels of bioavailable NO● and FMD, both important measures of endothelial function. Additionally, these data suggest potentially different mechanisms of action related to low nitrate BRS and organic nitrate PETN.

Some Aspects of Role of Nitric Oxide in the Mechanisms of Hypertension (Experimental Study).

BackgroundModulation of endothelial function is a therapeutic option to reduce some of the significant complications of hypertension. However, the relationship between endothelial dysfunction reduced nitric oxide (NO) production, and the development of hypertension is not fully understood. To establish a potential pathogenetic link between impaired NO synthesis and hypertension, we investigated the results of competitive interaction of the substrate of NO synthase, L-arginine, and its analog, an non-selective inhibitor of NO synthase, N-nitro-methyl ether-L-arginine (L-NAME), in experimental rats.MethodsArterial hypertension was induced in male Wistar rats by intraperitoneal administration of L-NAME (Sigma-Aldrich) for 4 - 7 weeks. During the last 3 weeks, to a separate group of animals simultaneously with L-NAME, L-arginine (Sigma-Aldrich) was administered. In animals monitored for systolic and diastolic pressure, the level of NO in blood samples was determined spectrophotometrically using a Griess reagent.ResultsAdministration of L-NAME for 4 - 7 weeks induced an irreversible decrease of NO content in blood, a reversible increase of systolic pressure (SP) and diastolic pressure (DP), and an irreversible increase in pulse pressure (PP). In rats against the background of 7 weeks of intraperitoneal administration of L-NAME, during the last 3 weeks, they were injected with L-arginine, the SP and DP indices returned to their initial values, PP decreased and the NO content in arterial blood increased.ConclusionsThe results of the study indicate the presence of residual endothelial dysfunction (characterized by insufficient NO) after the correction of hypertension. Therefore, in developing the new therapeutic approaches for the treatment of hypertension, it is necessary to include drugs that, in addition to correcting blood pressure, will support normalization, and potentiation of endothelial function and endogenous NO synthesis.

Sortilin evokes endothelial dysfunction and arterial hypertension through the dysregulation of sphingolipid metabolism and oxidative stress

Abstract Background Sortilin, a member of vacuolar protein sorting domain family Vps10, has been positively correlated with vascular and metabolic disorders in humans. Previous study has shown that, in response to Fas receptor stimulation, sortilin together with acid sphingomyelinase (ASMase) promote the clustering of lipid rafts and subsequent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in coronary endothelial cells. However, whether sortilin plays a role in endothelial cells function is currently unknown. Purpose To assess whether sortilin per se was able to influence vascular function, thereby contributing to the pathogenesis of cardiovascular diseases. Methods Pressure myography was used to study vascular reactivity of mesenteric arteries. To investigate the involvement of acid sphingomyelinase (ASM), we performed gene silencing approach and fluorometric activity assay. NADPH oxidase lucigenin assay was used to evaluate oxidative stress in endothelial cells and resistance vessels. The effects of circulating sortilin on cardiovascular system was evaluated by systemic delivery of recombinant sortilin protein to wild-type (WT), sphingosine-1-phosphate receptor 3 (S1P3) and NADPH oxidase 2 (gp91phox/NOX2) deficient mice. Systolic arterial blood pressure (SBP) was noninvasively registered in conscious mice by tail-cuff blood monitoring. Finally, to explore the translational relevance of sortilin, we measured sortilin and NOX2 soluble derived peptide levels using ELISA and quantified sphingosine-1-phosphate (S1P) by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in plasma of hypertensive patients. Results Here we demonstrated that sortilin evoked endothelial dysfunction in mesenteric arteries due to increased NADPH oxidase-derived oxidative stress. Knockdown of ASM successfully prevented impairment of endothelial function. Using the inhibitor of sphingosine kinase type 1 (SphK1), sortilin failed to evoke endothelial impairment as well as NADPH oxidase activation. In endothelial cells, sortilin induced S1P-dependent activation of Rac1/NOX2 signaling axis, which was prevented by TY-52156, an antagonist of lysosphingolipid receptor S1P3. In vivo sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of S1P3 and gp91phox/NOX2 resulted in preservation of endothelial function and SBP unchanged levels after 14 days of systemic sortilin administration. Finally, to translate these research findings into a clinical setting, we found that hypertensive patients have higher plasma levels of sortilin, ASMase, S1P and soluble NOX2 derived peptide than normotensive subjects. Conclusions These results demonstrate the pathologic role of sortilin in the modulation of endothelial function and arterial blood pressure, suggesting that sortilin and its mediators might represent novel therapeutic targets in vascular diseases and hypertension. Funding Acknowledgement Type of funding source: None

A brief discussion of the benefit and mechanism of vitamin D supplementation on coronavirus disease 2019.

Vitamin D exerts extraskeletal functions, including immunomodulatory activity, protection against respiratory tract infections and pleiotropic effects on the cardiovascular system. Since the outbreak of the coronavirus disease-2019 (COVID-19) pandemic, several articles have suggested the potential involvement of vitamin D in reducing the risk and severity of the disease. Epidemiological and observational studies support the hypothesis of a protective role of vitamin D but most studies are retrospective or based on small samples. However, the pandemic progression and the increased knowledge on the pathogenesis of COVID-19 have challenged the first evidence, suggesting also potential negative consequences derived by adequate vitamin D status. A cautious interpretation of the significance of low vitamin D25OH levels is advisable. The balance between over-activation of innate immunity and the exhaustibility of the adaptive immune response still needs to be clarified. In addition, the modulation of endothelial function, the down-regulation of renin, angiotensin-converting-enzyme (ACE) and angiotensin genes and the up-regulation of ACE2 expression is still an area of research. Speculative hypotheses and observational data have suggested a protective role of vitamin D in COVID-19. However, many unanswered questions remain, aberrant detrimental effects of adequate vitamin D25OH levels cannot be excluded and whether its adequacy may prevent the infection or improve clinical outcomes needs to be assessed by adequately sized and designed population-based studies and intervention trials.

Mediterranean diet and endothelial function in patients with coronary heart disease: An analysis of the CORDIOPREV randomized controlled trial

BackgroundEndothelial dysfunction is a crucial step in atherosclerosis development, and its severity is determinant for the risk of cardiovascular recurrence. Diet may be an effective strategy to protect the endothelium, although there is no consensus about the best dietary model. The CORonary Diet Intervention with Olive oil and cardiovascular PREVention (CORDIOPREV) study is an ongoing prospective, randomized, single-blind, controlled trial in 1,002 coronary heart disease (CHD) patients, whose primary objective is to compare the effect of 2 healthy dietary patterns (low-fat versus Mediterranean diet) on the incidence of cardiovascular events. Here, we report the results of one secondary outcome of the CORDIOPREV study: to evaluate the effect of these diets on endothelial function, assessed by flow-mediated dilation (FMD) of the brachial artery.Methods and findingsFrom the total participants taking part in the CORDIOPREV study, 805 completed endothelial function study at baseline and were randomized to follow a Mediterranean diet (35% fat, 22% monounsaturated fatty acids [MUFAs], and <50% carbohydrates) or a low-fat diet (28% fat, 12% MUFAs, and >55% carbohydrates), with endothelial function measurement repeated after 1 year. As secondary objectives and to explore different underlying mechanisms in the modulation of endothelial function, we quantified endothelial microparticles (EMPs) and endothelial progenitor cells (EPCs) and evaluated, in 24 preselected patients, in vitro cellular processes related to endothelial damage (reactive oxygen species, apoptosis, and senescence) and endothelial repair (cell proliferation and angiogenesis), as well as other modulators (micro-RNAs [miRNAs] and proteins). Patients who followed the Mediterranean diet had higher FMD (3.83%; 95% confidence interval [CI]: 2.91–4.23) compared with those in the low-fat diet (1.16%; 95% CI: 0.80 to 1.98) with a difference between diets of 2.63% (95% CI: 1.89–3.40, p = 0.011), even in those patients with severe endothelial dysfunction. We observed higher EPC levels (group difference: 1.64%; 95% CI: 0.79–2.13, p = 0.028) and lower EMPs (group difference: −755 EMPs/μl; 95% CI: −1,010 to −567, p = 0.015) after the Mediterranean diet compared with the low-fat diet in all patients. We also observed lower intracellular reactive oxygen species (ROS) production (group difference: 11.1; 95% CI: 2.5 to 19.6, p = 0.010), cellular apoptosis (group difference: −20.2; 95% CI: −26.7 to −5.11, p = 0.013) and senescence (18.0; 95% CI: 3.57 to 25.1, p = 0.031), and higher cellular proliferation (group difference: 11.3; 95% CI: 4.51 to 13.5, p = 0.011) and angiogenesis (total master segments length, group difference: 549; 95% CI: 110 to 670, p = 0.022) after the Mediterranean diet than the low-fat diet. Each dietary intervention was associated with distinct changes in the epigenetic and proteomic factors that modulate biological process associated with endothelial dysfunction. The evaluation of endothelial function is a substudy of the CORDIOPREV study. As in any substudy, these results should be treated with caution, such as the potential for false positives because of the exploratory nature of the analyses.ConclusionsOur results suggest that the Mediterranean diet better modulates endothelial function compared with a low-fat diet and is associated with a better balance of vascular homeostasis in CHD patients, even in those with severe endothelial dysfunction.Clinical trial registrationURL, http://www.cordioprev.es/index.php/en. clinicaltrials.gov number NCT00924937.

Endothelial Function in Obese Patients Treated with Bariatric Surgery.

PurposeBariatric surgery (BS) is becoming an increasingly frequent treatment option especially in people with morbid obesity, demonstrating that it is able to reduce total mortality and cardiovascular (CV) risk. Despite endothelial dysfunction is an essential requisite contributing to atherosclerosis and predicting CV events, only some studies have investigated the effects of BS on endothelial function with controversial results. In this study, the effects of weight loss on endothelial function were investigated in obese patients after BS and compared with patients after medical nutrition treatment (MNT).Patients and MethodsSeventeen obese patients who underwent BS procedures (9 adjustable gastric bands, 3 gastric by-passes and 5 biliopancreatic diversions) were included in the study and compared with 18 obese individuals who underwent MNT. Endothelial function was investigated by flow-mediated dilation (FMD) of the brachial artery. Also, carotid intima-media thickness (c-IMT) was measured as a marker of subclinical atherosclerosis.ResultsAt the end of follow-up, the mean weight loss was 18.8% in the BS group and 7.0% in the MNT group. After treatment, FMD significantly decreased in the BS group (mean ± SD before: 9.0 ± 4.7; after: 6.1 ± 2.9%; P= 0.04); however, no significant change was observed in the MNT group (before: 9.4 ± 5.8; after: 8.3 ± 5.3; P= 0.41). The modification of endothelial function was negatively correlated with c-IMT change in the BS group (r= −0.63; P <0.007).ConclusionA significant endothelial dysfunction occurred following BS but not after MNT. Furthermore, the decline of endothelial function observed in the BS group might have contributed to atherosclerosis.

P638High intensity interval exercise training improves endothelial function in patients with heart failure with preserved ejection fraction

Abstract Background Cardiac rehabilitation has traditionally invoved low-to moderate intensity continuous exercise training (MICT). There is growing evidence that high intensity interval exercise training (HIIT) shows similar or greater efficacy compared with MICT. However, the relationship between the training modality and its effects on endothelial function still remains to be elucidated. Purpose To evaluate the impact of high intensity interval exercise training versus low-to moderate intensity continuous exercise training on circulating blood markers of endothelial function: the stable end product of nitric oxide (NOx), S–nitrosothiols (RSNO–reservoir for bioavailable nitric oxide), dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in patients (pts) with heart failure with preserved ejection fraction (HFpEF). Methods A total of 43 pts with HFpEF (mean age 57.5±6.8y) admitted at residential rehabilitation center were enrolled in the study. Patients were randomized to HIIT, (HIIT group, n=20) and MICT, (MICTgroup, n=23). At baseline and 3 weeks later, in all pts values of NOx, RSNO, ADMA and SDMA were determined and exercise test was performed. Results After 3 weeks NOx increased significantly in both groups: in HIITgroup (from 34.7±7.5 to 48.5±8.3 μmol/l, P&lt;0.0001), in MICTgroup (from 35.0±9.5 to 42.5±10.0 μmol/l, P&lt;0.05), and value of NOx after 3 weeks was higher in HIIT than in MICT group (P&lt;0.05). Value of ADMA as well of SDMA decreased in both groups after 3 weeks, and this decrease was significant in HIIT group: ADMA in HIIT group (from 0.305±0.055 to 0.250±0.077 μmol/l, P&lt;0.005) and in MICT group (from 0.325±0.069 to 0.280±0.140 μmol/l, ns); SDMA in HIIT group (from 0.285±0.050 to 0.240±0.060 μmol/l, P&lt;0,005) and in MICT group (from 0.270±0.080 to 0.247±0.063 μmol/l, ns). Value of RSNO significantly increased in both groups, with higher increase in HIIT group: in HIIT group (from 3.0±1.5 to 5.1±1.7 μmol/l, P&lt;0.001), in MICTgroup (from 3.2±1.7 to 4.5±2.3 μmol/l, P&lt;0.05). Compared to the baseline, at the end of the study, exercise capacity (METs) was significantly higher in both groups (P&lt;0.0001). Higher increase in METs in HIIT group during exercise training (by 39.8%), compared to increase in MICT group (by 27.2%), resulted in significantly higher exercise tolerance in HIIT than in MICT group at the end of the study (P&lt;0.02). In both groups, adverse events during or after exercise training, were not occurred. Conclusions In pts with HFpEF high intensity interval exercise training as well as low-to moderate intensity continuous exercise training induced favorable modification of endothelial function, expressed through increase of NOx and RSNO, and decrease of ADMA and SDMA. More significant increase of NOx and RSNO, and significant decrease of ADMA and SDMA gives an advantage to HIIT over MICT exercise training in patients with HFpEF.

171-OR: Metformin Prevents Microvascular Insulin Resistance and Preserves Endothelial Function in High-Fat Diet-Fed Rats

Endothelial dysfunction and vascular insulin resistance coexist and both contribute to the development of type 2 diabetes mellitus (T2DM) and increased cardiovascular risks. Insulin relaxes precapillary arterioles to increase insulin delivery to and action in muscle via recruiting muscle microvasculature and this action is impaired in the insulin resistant states. Metformin is the first line therapy for T2DM and has been shown to improve endothelial function beyond its hypoglycemic effects. Here we aim to examine whether metformin could restore vascular insulin responses in the setting of insulin resistance induced by high fat diet (HFD) feeding via the modulation of endothelial function. Adult, male Sprague-Dawley rats were fed on HFD with or without metformin treatment for 2 weeks. A chow fed group served as control. After dietary intervention, rats were fasted overnight and received a euglycemic-hyperinsulinemic clamp for 120 min. Insulin-mediated muscle microvascular recruitment was assessed before and during insulin infusion using contrast-enhanced ultrasound. Distal saphenous artery was isolated and used for determination of endothelial function and vascular insulin responses. Compared with controls, HFD feeding abolished insulin-induced muscle microvascular recruitment in muscle, decreased insulin-mediated whole body glucose disposal and blunted the vasodilatory response to insulin in small resistance vessel. Metformin treatment significantly enhanced insulin’s microvascular action, effectively rescued small arteriole endothelial function, but did not improve insulin-stimulated whole body glucose disposal in HFD rats. We conclude that metformin could reverse microvascular insulin resistance and attenuate endothelial dysfunction induced by HFD feeding. There findings may provide a novel mechanistic insight into metformin’s cardiovascular protective effect in patients with diabetes. Disclosure J. Liu: None. W. Chai: None. L. Gong: None. K.W. Aylor: None. Z. Liu: None. Funding American Diabetes Association (1-17-ICTS-059 to Z.L.); National Institutes of Health (R01HL094722, R01DK102359)

Modulation of endothelial function by pro-inflammatory cytokines involved in chronic inflammatory diseases. Focus on IL-17A, IL-20, IL-23 and IL-9

Chronic inflammatory diseases (CID) such as rheumatoid arthritis, systemic lupus erythematosus, spondyloarthritis are associated with increased cardiovascular risk secondary to the presence of endothelial dysfunction. In addition to the well-known cytokines (TNF-α, IL-1β and IL-6), emerging data identified new cytokines such as IL-17A, IL-20, IL-23 and IL-9 key-players of the pathogenesis of CID. To date, whether these cytokines might contribute to CID-associated endothelial dysfunction is not known. This study investigated the effect of IL-17A, IL-20, IL-23 and IL-9 on endothelium-dependent relaxation in response to acetylcholine (Ach) in aortic rings from male Lewis rats. Thoracic aortic rings are incubated for 1 or 24 h at 37 °C with 2 concentrations of each cytokine (IL-17A: 250 pg/mL and 10 ng/mL; IL-20: 500 pg/mL and 5 ng/mL; IL-23: 80 pg/mL and 10 ng/mL; and IL-9: 300 pg/mL and 10 ng/mL). Incubation with 10 ng/ml TNF-α was used as a positive control and with vehicle as negative control. At the end of the incubation period, concentration-response curves to Ach (10 −11 –10 −4 mol/L) were assessed. As expected, a 24 h- but not 1 h-incubation with TNF-α reduced Ach-induced relaxation. The same result was obtained with IL-17A (10 ng/mL). By contrast, IL-20 did not change Ach-induced relaxation whatever the concentration and the incubation time. Impairment in vascular relaxation was observed after exposure to IL-9 (10 ng/mL), both after 1 h- and more severely after 24 h-incubation. As regards IL-23, an effect was observed only after 1 h incubation and with high concentration. Our data demonstrated that IL-17A, IL-23 and IL-9 but not IL-20 induced endothelial dysfunction, with different kinetics profiles. IL-9 exhibited the most important effect thus revealing a new putative role of this pleiotropic cytokine in CID-associated cardiovascular risk. Further studies are needed to confirm these data on animal models of diseases.

Angiopoietin-2, Renal Deterioration, Major Adverse Cardiovascular Events and All-Cause Mortality in Patients with Diabetic Nephropathy

Background/Aims: Diabetic nephropathy is the leading cause of end-stage renal disease and accounts for 30∼40% of patients requiring maintenance dialysis, thereby increasing the burden on health insurance programs. Diabetic nephropathy is also the strongest predictor of cardiovascular morbidity and mortality. The aim of this study was to examine whether angiopoietin-2 (Angpt2), a modulator of endothelial function, affects the clinical outcomes of diabetic patients. Methods: This study enrolled 236 patients with diabetes mellitus with estimated glomerular filtration rate (eGFR) < 60ml/min/1.73m² from January 2006 to December 2011, who were followed until June 2017. Clinical outcomes included renal outcomes (commencing dialysis and rapid decline in renal function (eGFR decline > 3 ml/min per 1.73 m²/year)), major adverse cardiovascular events (MACEs), and all-cause mortality. Results: Over a mean follow-up period of 3.9±2.7 years, 135 (57.2%) patients commenced dialysis, 106 (44.9%) had rapid decline in renal function, and 50 (21.2%) had MACEs or died from all-causes. Log-formed Angpt2 was significantly associated with increased risks of commencing dialysis (HR: 3.91, 95% CI: 1.56-9.76), rapid renal function decline (OR: 6.81, 95% CI: 1.06-43.88), and MACEs or all-cause mortality (HR: 6.34, 95% CI: 1.18-33.97) in the adjusted analysis. Patients in the highest quartile had hazard ratios of 2.90 and 3.11 for commencing dialysis and rapid renal function decline, respectively, compared to those in the lowest quartile after adjustments. Similar significant dose-response results were found in composite outcomes of either MACEs or all-cause mortality. Conclusion: Angpt2 is an independent predictor of adverse clinical outcomes in diabetic patients. Further studies are needed to identify the pathogenic role of Angpt2 in renal deterioration and cardiovascular complications of diabetes mellitus.

Abstract 29: Alpha-1-antitrypsin Protects High Density Lipoprotein From Functional Inactivation by Elastase

High density lipoproteins (HDL) are complexes of lipid and protein with several known atheroprotective functions. These functions are driven by specific lipids and proteins contained on the HDL particle and include reverse cholesterol transport, suppression of inflammation, and modulation of endothelial function. These activities are most important within atherosclerotic plaque, a harsh environment where HDL interact with macrophage foam cells, activated neutrophils, and dysfunctional endothelial cells. Neutrophils and macrophages secrete proteases, such as elastase, which damage structural components and soluble proteins and propagate inflammatory signaling. It has been suggested that, in plaque, HDL become damaged and dysfunctional. We recently characterized a subspecies of HDL that carries the protein alpha-1-antitrypsin (A1AT), an abundant plasma serine protease inhibitor. In the current study, we tested the hypothesis that A1AT enriched HDL are protected from proteolytic damage and functional inactivation by elastase, the main protease inhibited by A1AT. Human HDL was isolated by ultracentrifugation and was enriched with A1AT by co-incubation and unbound A1AT was removed. Treatment of native HDL with elastase resulted in significant proteolytic degradation of both apoA-I and apoA-II, visualized by coomassie stained SDS-PAGE. Analysis of lipoprotein size by one dimensional native gel electrophoresis revealed that pre-beta HDL were completely degraded by elastase. Compared to native HDL, A1AT enriched HDL samples were protected from protein and pre-beta particle degradation by elastase. We next tested the effect of elastase treatment on HDL function. In native HDL, elastase had damaging effects on ABCA1 mediated cholesterol efflux (-32%; p&lt;0.0001) and the ability to esterify free cholesterol (-14%; p&lt;0.02). A1AT enriched HDL displayed no loss of functionality upon treatment with elastase. Both of these activities are required for HDL to perform what is thought to be its most important function, reverse cholesterol transport. In conclusion, the data presented indicate that HDL particles which contain A1AT may represent a functionally important species of HDL, which have an advantage in the protease-rich plaque environment.

Study of endothelial function response to exercise training in hypertensive individuals (SEFRET): study protocol for a randomized controlled trial.

BackgroundEndothelial dysfunction is a hallmark of diabetes mellitus and systemic arterial hypertension (SAH) and an early maker for atherosclerosis. Aerobic exercise training is known to enhance endothelial function, but little is understood about the effects of resistance or combined exercise training on endothelial function. The aim of this study is to investigate the effect of a 12-week aerobic (AT), resistance (RT), or combined (aerobic and resistance, CT) training program on endothelial function and assess associated effects on blood pressure in individuals with SAH.Methods/designEighty-one subjects with SAH aged 18 to 70 years will be selected and randomly assigned to three types of exercise training: AT, RT or CT. The study will involve the following procedures and tests: anamnesis, anthropometric assessment, echocardiography, blood pressure measurements through ambulatory blood pressure monitoring, flow-mediated dilation, ergospirometry, one repetition maximum test (1-RM), and blood collection (number of circulating endothelial progenitor cells, number of circulating endothelial microparticles, lipid profile, glucose, glycated hemoglobin, and creatinine). The AT intervention will consist of a 40-min exercise session with progressive intensities ranging from 50 to 75 % of heart rate reserve. The RT intervention will consist of a 40-minute session with four sets of six to 12 repetitions with a rest period of 60 to 90 seconds between each set and each type of exercise. Weight loads will be adjusted to 60 to 80 % of 1-RM for six types of exercise. The CT intervention will consist of a 20-min aerobic exercise session, followed by an additional 20-min resistance exercise session; each resistance exercise will have two sets less to match the total training volume.DiscussionThe study results are expected evidence of cardiovascular protective effects of different types of exercise training through the modulation of endothelial function in hypertensive individuals. Knowing the magnitude of improvement of endothelium-dependent vasodilation for the different types of exercise training can provide scientific evidence for the prescription of exercise programs for vascular protection targeting hypertensive individuals.Trial registrationThe Brazilian Clinical Trials Registry (http://www.ensaiosclinicos.gov.br/) under RBR-9ygmdn and dated 1 March 2015.

Abstract P198: Differential Regulation of Endothelial Nitric Oxide Synthase Phosphorylation by Protease-Activated Receptors in Adult Human Endothelial Cells

Protease-activated receptors (PARs) have been shown to regulate endothelial nitric oxide synthase (eNOS) through the activation of specific sites on the enzyme. It has been established that phosphorylation of eNOS-Ser-1177 leads to the production of the potent vasodilator nitric oxide (NO), and is associated with PAR-2 activation; while phosphorylation of eNOS-Thr-495 decreases NO production, and is coupled to PAR-1 activation. In this study, we demonstrate a differential regulation of the eNOS/NO pathway by the PARs using primary adult human coronary artery endothelial cells (HCAEC). Thrombin and the PAR-1 activating peptide, TFLLR, which are known to phosphorylate eNOS-Thr-495 in bovine and human umbilical vein endothelial cells, phosphorylated eNOS-Ser-1177 in HCAECs, and increased NO production. The PAR-1 responses were blocked using SCH-79797, a PAR-1 inhibitor, and L-NAME was used to inhibit NO production. A PAR-2 specific ligand, SLIGRL, which has been shown to phosphorylate eNOS-Ser-1177 in bovine and human umbilical vein endothelial cells, primarily regulated eNOS-Thr-495 phosphorylation and suppressed NO production in the HCAECs. PAR-3, known for its non-signaling potential, was activated by TFRGAP, a PAR-3 mimicking peptide, and only induced phosphorylation of eNOS-Thr-495 with no effect on NO production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was calcium-dependent using the calcium chelator, BAPTA, and eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632. These data suggest a vascular bed specific differential coupling of PARs to the signaling pathways that regulate eNOS and NO production that may be responsible for the modulation of endothelial function associated with cardiovascular disease.

Letter by Kain Regarding Article, “Vitamin D Promotes Vascular Regeneration”

I read with interest the article by Wong et al1 on the effects of vitamin D supplementation on vascular repair. Outdoor physical activity promotes health and treats chronic conditions, for example, psoriasis, possibly by increasing biosynthesis and biological activity of the active hormone 1,25-dihydroxycholecalciferol (1,25-dihydroxy-vitamin-D3 [1,25-VitD3]) on endothelial function and vascular repair. The beneficial effects of physical activity on endothelial function can be mediated in a number of ways, including synthesis of molecular mediators, changes in neurohormonal release, and oxidant/antioxidant balance. Physical activity can also elicit systemic molecular pathways connected to angiogenesis and chronic anti-inflammatory action with consequent modification of endothelial function.2 It is already known that vitamin …