Preservation Of Endothelial Function Research Articles

Relation Between Endothelial Dysfunction and Exercise Training-Mediated Adaptation in Cardiovascular Risk Factors, Cardiorespiratory Fitness, and Vascular Health in Humans: A secondary analysis

Abstract Purpose A priori cardiovascular (CV) health status may impact reductions in risk factors and CV mortality and morbidity following exercise training, although this is not fully understood. Therefore, the purpose of the study was to examine if endothelial function (assessed via flow mediated dilation; FMD%), predicts the magnitude of change in CV risk factors or fitness following exercise training. Methods We pooled data from 338 individuals who underwent supervised exercise training (8–26 weeks). Using recent sex- and age-specific reference values for flow-mediated dilation (FMD%), we categorised participants as having preserved endothelial function (P-EF) (> 50th percentile of reference value, 56 females, 67 males, 46 ± 17 years) or reduced endothelial function (R-EF) (< 50th percentile of reference value, 67 females, 148 males, 48 ± 17 years). The effects of exercise training on cardiovascular risk factors (BMI, cholesterol, glucose and triglycerides), cardiorespiratory fitness (VO2peak) and vascular function (FMD%) were examined using a two-way mixed design general linear model. Results Exercise training significantly improved physical fitness (P < 0.001), with no difference in the magnitude of improvement between P-EF and R-EF. Modest but significant reductions were present in BMI, blood pressure and total cholesterol (all P < 0.005), with no difference between P-EF and R-EF groups in the magnitude of changes in these variables with training. Exercise training did not significantly alter glucose, triglycerides, high density lipoprotein (HDL) and low-density lipoprotein (LDL) (all P > 0.05). Conclusion Individuals with reduced and preserved a priori endothelial function status can obtain benefits from exercise in terms of risk factor modification and fitness change. Therefore, exercise has the potential to be beneficial in all clinical groups.

Endothelial function, arterial stiffness and heart rate variability of patients with cardiovascular diseases hospitalized due to COVID-19

BackgroundThe novel coronavirus disease (COVID-19) may cause vascular (e.g., endothelial dysfunction, and arterial stiffness), cardiac, autonomic (e.g., heart rate variability [HRV]), and systemic inflammatory response via direct viral attack, hypoxia-induced injury, or immunological dysregulation, especially in those patients with pre-existing cardiovascular diseases (CVD). However, to date, no study has shown prevalence of endothelial dysfunction, arterial stiffness and heart rate variability assessed by bedside peripheral arterial tonometry in patients with previous CVD hospitalized in the acute phase of COVID-19. ObjectiveThis study aimed to assess the prevalence of endothelial dysfunction, arterial stiffness, and altered HRV in patients with CVD hospitalized due to COVID-19. MethodsThis cross-sectional study was conducted from July 2020 to February 2021. Included male and female adult patients aged 40 to 60 years with previous CVD and diagnosed with COVID-19. Anthropometric data, comorbidities, and blood tests were analyzed. Endothelial function, arterial stiffness, and HRV were assessed using peripheral arterial tonometry (PAT), and the statistical significance was set at 5%. ResultsFourteen (51.8%) patients presented endothelial dysfunction (reactive hyperemia index = 1.2 ± 0.3) and enhancement in the high-frequency component of HRV (p < 0.05). There was a high prevalence of endothelial dysfunction, especially in patients with chronic heart failure (10 (71.4%)). Patients with preserved endothelial function showed a high augmentation index normalized to a heart rate of 75 bpm (p < 0.01), suggesting arterial stiffness. ConclusionPatients with CVD hospitalized due to COVID-19 presented endothelial dysfunction assessed using PAT, which could be used as a biomarker for arterial stiffness and altered HRV. The possibility of detecting vascular and autonomic changes during phase II of COVID-19 may help to prevent possible long-term complications.

PS-BPC02-2: CLINICAL IMPACTS OF ENDOTHELIUM-DEPENDENT FLOW-MEDIATED VASODILATION ASSESSMENT ON PRIMARY ALDOSTERONISM

Objective: Primary aldosteronism (PA) is defined as autonomous aldosterone production from the adrenal glands and represents the most common and treatable cause of hypertension. Also, it is well known that the incidence of cardiovascular morbidity is higher among patients with PA than among those with essential hypertension (EH). Histologically, PA is divided into two major subtypes, aldosterone-producing adenoma (APA) and bilateral idiopathic hyperplasia (IHA). However, the nature of vascular function in PA patients remains to be determined. The aim of this study was to determine the vascular function and investigate the implications of vascular function assessments in the patients with PA. Design and Method: All patients were diagnosed with PA according to the guidelines of the Japan Endocrine Society. Flow-mediated dilation (FMD), as an index of endothelial function, and cardio-ankle vascular index (CAVI), as an index of arterial stiffness, were retrospectively compared between 42 patients with APA, 37 patients with IHA, and 42 patients with EH. These values were also compared with background factors, KCNJ5 mutation and clinical outcome in terms of blood pressure reduction after adrenalectomy in the APA group. Results: No differences were found among the three groups (APA, IHA and EH) with regard to age, sex, duration of hypertension, body mass index, blood pressure, renal function and hemoglobin A1c. FMD was significantly lower in the APA group (4.8 ± 2.1%) and IHA group (4.1 ± 1.9%) than in the EH group (5.7 ± 2.1%). CAVI did not differ significantly among APA, IHA and EH groups (7.9 ± 1.3, 7.8 ± 1.0, 7.8 ± 1.1, respectively). Although no significant correlations were seen between FMD and background factors (i.e. age, body mass index, renal function, glucose and lipid metabolic parameters, and hormonal data) in the IHA group, FMD correlated negatively with BMI and plasma aldosterone concentration in the APA group (rs = -0.313, rs = -0.342, respectively). No significant difference in FMD (4.8 ± 1.4%, 4.5 ± 2.1%, respectively) or CAVI (8.1 ± 1.8, 8.0 ± 1.1, respectively) was seen between the KCNJ5-mutant group and the wild-type group. A cut-off FMD of 4.3% was selected for complete clinical success after adrenalectomy, offering 78% sensitivity and 80% specificity. Conclusions: Patients with PA displayed impaired endothelial function. Complete clinical success after adrenalectomy was associated with preserved endothelial function. This study provides a better understanding of FMD assessment in patients with PA.

Diosmin and its glycocalyx restorative and anti-inflammatory effects on injured blood vessels.

The endothelium, a crucial homeostatic organ, regulates vascular permeability and tone. Under physiological conditions, endothelial stimulation induces vasodilator endothelial nitric oxide (eNO) release and prevents adhesion molecule accessibility and leukocyte adhesion and migration into vessel walls. Endothelium dysfunction is a principal event in cardiovascular disorders, including atherosclerosis. Minimal attention is given to an important endothelial cell structure, the endothelial glycocalyx (GCX), a negatively charged heterogeneous polysaccharide that serves as a protective covering for endothelial cells and enables endothelial cells to transduce mechanical stimuli into various biological and chemical activities. Endothelial GCX shedding thus plays a role in endothelial dysfunction, for example by increasing vascular permeability and decreasing vessel tone. Consequently, there is increasing interest in developing therapies that focus on GCX repair to limit downstream endothelium dysfunction and prevent further downstream cardiovascular events. Here, we present diosmin (3',5,7-trihydroxy-4'-methoxyflavone-7-rhamnoglucoside), a flavone glycoside of diosmetin, which downregulates adhesive molecule expression, decreases inflammation and capillary permeability, and upregulates eNO expression. Due to these pleiotropic effects of diosmin on the vasculature, a possible unidentified mechanism of action is through GCX restoration. We hypothesize that diosmin positively affects GCX integrity along with GCX-related endothelial functions. Our hypothesis was tested in a partial ligation left carotid artery (LCA) mouse model, where the right carotid artery was the control for each mouse. Diosmin (50 mg/kg) was administered daily for 7 days, 72h after ligation. Within the ligated mice LCAs, diosmin treatment elevated the activated eNO synthase level, inhibited inflammatory cell uptake, decreased vessel wall thickness, increased vessel diameter, and increased GCX coverage of the vessel wall. ELISA showed a decrease in hyaluronan concentration in plasma samples of diosmin-treated mice, signifying reduced GCX shedding. In summary, diosmin supported endothelial GCX integrity, to which we attribute diosmin's preservation of endothelial function as indicated by attenuated expression of inflammatory factors and restored vascular tone.

Spectral Analysis of Resting Near‐Infrared Spectroscopy Shows Preserved Endothelial Function after Aerobic Training Exercise in Chronic Kidney Disease

This study aims to evaluate the effectiveness of aerobic exercise training on microcirculatory function among individuals with chronic kidney disease (CKD). Microcirculatory dysfunction is a prevalent complication in CKD that progresses as CKD evolves. Endothelial dysfunction is an early manifestation of microvascular disease and contributes to increased cardiovascular‐related mortality in CKD. Evidence indicates that endothelial dysfunction might be linked to deficits in blood perfusion and oxygen delivery. Aerobic exercise has been shown to improve cardiorespiratory function and blood pressure control for adults with CKD. Previous work has validated correspondence of specific frequency intervals in resting microvascular blood flow fluctuations with physiological events. Specifically, the spectrum amplitude from three frequency intervals (0.0095 – 0.02 Hz, 0.02 – 0.06 Hz, 0.06 – 0.15 Hz) have been attributed to microvascular regulation by endothelial, neurogenic, and myogenic origins, respectively. We hypothesized that 12 weeks of aerobic exercise would improve endothelial function in CKD.Ten CKD participants (Table 1) were randomized into treatment and control groups that performed progressive aerobic cycling exercise (N = 5) for 20‐45 min up to 80% of heart rate reserve and non‐aerobic, stretching exercise (N = 5). Each group performed activities 3 days/week for 12 weeks. Resting microvascular blood flow was measured in the forearm for 5 minutes using total hemoglobin (tHb) signal from near‐infrared spectroscopy. Reactive hyperemic index (RHI) was measured using peripheral artery tonometry at the upper arm, providing an accepted, single‐value index of endothelial function. Both measures were obtained at the beginning (baseline) and end of the 12‐week intervention (EOS), respectively. The resting tHb signal was analyzed using wavelet transform to obtain time‐frequency information on microcirculation regulated by endothelial, neurogenic, and myogenic functions. Differences at baseline and EOS were assessed using pairwise Student’s t‐test (statistical significance: p &lt; 0.05). The Pearson correlation coefficient between RHI and individual mean spectral amplitude was also obtained.Endothelial spectral amplitudes significantly decreased (p = 0.007) in the stretching group over the 12‐week intervention, suggesting decreased endothelial function (Fig.1). There was no significant change in the endothelial spectral amplitude (p = 0.16) in the cycling group. Changes in individual endothelial spectral amplitude and RHI were moderately correlated (r = 0.57 and p = 0.02).In conclusion, spectral analysis of resting microvascular blood flow in CKD suggests preserved endothelial function over 12 weeks of progressive aerobic cycling exercise, compared with a decline in those who performed a non‐aerobic stretching exercise. Ongoing work focuses on adapting and applying this wavelet methodology to MRI‐derived signals that provide temporo‐spatially resolved information on tissue oxygenation and perfusion. Resting microcirculatory properties yielded from this work may be useful for monitoring disease progression and guiding targeted therapeutic strategies to improve microvascular function in CKD.

Targeting the ASMase/S1P pathway protects from sortilin-evoked vascular damage in hypertension.

Sortilin has been positively correlated with vascular disorders in humans. No study has yet evaluated the possible direct effect of sortilin on vascular function. We used pharmacological and genetic approaches coupled with study of murine and human samples to unravel the mechanisms recruited by sortilin in the vascular system. Sortilin induced endothelial dysfunction of mesenteric arteries through NADPH oxidase 2 (NOX2) isoform activation, dysfunction that was prevented by knockdown of acid sphingomyelinase (ASMase) or sphingosine kinase 1. In vivo, recombinant sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of sphingosine-1-phosphate receptor 3 (S1P3) and gp91phox/NOX2 resulted in preservation of endothelial function and blood pressure homeostasis after 14 days of systemic sortilin administration. Translating these research findings into the clinical setting, we detected elevated sortilin levels in hypertensive patients with endothelial dysfunction. Furthermore, in a population-based cohort of 270 subjects, we showed increased plasma ASMase activity and increased plasma levels of sortilin, S1P, and soluble NOX2-derived peptide (sNOX2-dp) in hypertensive subjects, and the increase was more pronounced in hypertensive subjects with uncontrolled blood pressure. Our studies reveal what we believe is a previously unrecognized role of sortilin in the impairment of vascular function and in blood pressure homeostasis and suggest the potential of sortilin and its mediators as biomarkers for the prediction of vascular dysfunction and high blood pressure.

Differential effects of obesity on visceral versus subcutaneous adipose arteries: role of shear-activated Kir2.1 and alterations to the glycocalyx.

Obesity imposes well-established deficits to endothelial function. We recently showed that obesity-induced endothelial dysfunction was mediated by disruption of the glycocalyx and a loss of Kir channel flow sensitivity. However, obesity-induced endothelial dysfunction is not observed in all vascular beds: visceral adipose arteries (VAAs), but not subcutaneous adipose arteries (SAAs), exhibit endothelial dysfunction. To determine whether differences in SAA versus VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx, mice were fed a normal rodent diet, or a high-fat Western diet to induce obesity. Flow-induced vasodilation (FIV) was measured ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant-negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAAs of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAAs, but had no effect on VAAs, from obese mice and humans. Obesity abolished Kir shear sensitivity in VAA endothelial cells and significantly altered the VAA glycocalyx. In contrast, Kir shear sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were less pronounced. We reveal distinct differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA versus VAA endothelial function with obesity.NEW & NOTEWORTHY We identified a role for endothelial Kir2.1 in the differences observed in VAA versus SAA endothelial function with obesity. The endothelial glycocalyx, a regulator of Kir activation by shear, is unequally perturbed in VAAs as compared with SAAs, which we propose results in a near complete loss of VAA endothelial Kir shear sensitivity and endothelial dysfunction. We propose that these differences underly the preserved endothelial function of SAA in obese mice and humans.

Overexpression of dimethylarginine dimethylaminohydrolase 1 protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling.

Cardiovascular complications are the leading cause of death, and elevated levels of asymmetric dimethyarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are implicated in their pathophysiology. We investigated the role of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme hydrolyzing ADMA, in prevention of cardiovascular remodeling during hypertension. We hypothesized that the animals overexpressing DDAH1 will be protected from angiotensin II (ANG II)-induced end organ damage. Angiotensin II (ANG II) was infused in two doses: 0.75 and 1.5 mg/kg/day in DDAH1 transgenic mice (DDAH1 TG) and wild-type (WT) littermates for 2 or 4 wk. Echocardiography was performed in the first and fourth weeks of the infusion, systolic blood pressure (SBP) was measured weekly, and cardiac hypertrophy and vascular remodeling was assessed by histology. Increase in SBP after 1 wk of ANG II infusion was not different between the groups, whereas TG mice had lower SBP at later time points. TG mice were protected from cardiovascular remodeling after 2 wk of ANG II infusion in the high dose and after 4 wk in the moderate dose. TG mice had higher left ventricular lumen-to-wall ratio, lower cardiomyocyte cross-sectional area, and less interstitial fibrosis compared with WT controls. In aorta, TG mice had less adventitial fibrosis, lower medial thickness with preserved elastin content, lower counts of inflammatory cells, lower levels of active matrix metalloproteinase-2, and showed better endothelium-dependent relaxation. We demonstrated that overexpression of DDAH1 protects from ANG II-induced cardiovascular remodeling and progression of hypertension by preserving endothelial function and limiting inflammation.NEW & NOTEWORTHY We showed that overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects from angiotensin II-induced cardiovascular damage, progression of hypertension, and adverse vascular remodeling in vivo. This protective effect is associated with decreased levels of asymmetric dimethylarginine, preservation of endothelial function, inhibition of cardiovascular inflammation, and lower activity of matrix metalloproteinase-2. Our findings are highly clinically relevant, because they suggest that upregulation of DDAH1 might be a promising therapeutic approach against angiotensin II-induced end organ damage.

Mitogen and Stress-Activated Kinases 1 and 2 Mediate Endothelial Dysfunction.

Inflammation promotes endothelial dysfunction, but the underlying mechanisms remain poorly defined in vivo. Using translational vascular function testing in myocardial infarction patients, a situation where inflammation is prevalent, and knock-out (KO) mouse models we demonstrate a role for mitogen-activated-protein-kinases (MAPKs) in endothelial dysfunction. Myocardial infarction significantly lowers mitogen and stress kinase 1/2 (MSK1/2) expression in peripheral blood mononuclear cells and diminished endothelial function. To further understand the role of MSK1/2 in vascular function we developed in vivo animal models to assess vascular responses to vasoactive drugs using laser Doppler imaging. Genetic deficiency of MSK1/2 in mice increased plasma levels of pro-inflammatory cytokines and promoted endothelial dysfunction, through attenuated production of nitric oxide (NO), which were further exacerbated by cholesterol feeding. MSK1/2 are activated by toll-like receptors through MyD88. MyD88 KO mice showed preserved endothelial function and reduced plasma cytokine expression, despite significant hypercholesterolemia. MSK1/2 kinases interact with MAPK-activated proteins 2/3 (MAPKAP2/3), which limit cytokine synthesis. Cholesterol-fed MAPKAP2/3 KO mice showed reduced plasma cytokine expression and preservation of endothelial function. MSK1/2 plays a significant role in the development of endothelial dysfunction and may provide a novel target for intervention to reduce vascular inflammation. Activation of MSK1/2 could reduce pro-inflammatory responses and preserve endothelial vasodilator function before development of significant vascular disease.

Clinical impacts of endothelium-dependent flow-mediated vasodilation assessment on primary aldosteronism

ObjectivePrimary aldosteronism (PA) is divided into two major subtypes, aldosterone-producing adenoma (APA) and bilateral idiopathic hyperplasia (IHA) and is associated with a higher risk of cardiovascular events. However, the nature of vascular function in PA patients remains to be determined. The aim of this study was to determine the vascular function and investigate the implications of vascular function assessments in the patients.MethodsFlow-mediated dilation (FMD), as an index of endothelial function, and cardio-ankle vascular index (CAVI), as an index of arterial stiffness, were retrospectively compared between 42 patients with APA, 37 patients with IHA, and 42 patients with essential hypertension (EH). These values were also compared with background factors, KCNJ5 mutation and clinical outcome in terms of blood pressure reduction after adrenalectomy in the APA group.ResultsFMD was significantly lower in the APA group (4.8 ± 2.1%) and IHA group (4.1 ± 1.9%) than in the EH group (5.7 ± 2.1%). CAVI did not differ significantly among groups. Although no significant correlations were seen between FMD and background factors in the IHA group, FMD correlated negatively with BMI and plasma aldosterone concentration in the APA group (rs = −0.313, rs = −0.342, respectively). KCNJ5 mutational status was not associated with FMD value. High FMD was associated with blood pressure normalization after adrenalectomy in the APA group.ConclusionsPatients with PA displayed impaired endothelial function. Complete clinical success after adrenalectomy was associated with preserved endothelial function. This study provides a better understanding of FMD assessment in patients with PA.

Effects of Pyridoxamine Treatment on Large Artery Stiffness, Endothelial Function, and Cognitive Impairment in Old Mice

Large artery stiffness increases with advancing age and is associated with cerebrovascular dysfunction and cognitive impairment. One mechanism for age-related increases in large artery stiffness is collagen cross-linking caused by advanced glycation end-products (AGEs). However, the effect of inhibiting AGEs formation on age-related large artery stiffening, cerebrovascular dysfunction, and cognitive impairment is still unclear. Pyridoxamine is a form of vitamin B6 that prevents the formation of AGEs. Therefore, the purpose of this study was to investigate the effects of pyridoxamine on large artery stiffness, cerebral artery function, and cognitive function in old mice. Old male C57BL6 mice, 18-24 months of age, consumed either pyridoxamine (2 g/L) or vehicle control in drinking water for 6-months (n = 31 per group). For reference, the old mice were compared to a group of young male C57BL6 mice (5-7 months, n = 28). We measured large artery stiffness by aortic pulse wave velocity (PWV) at pre-treatment and at 6 months post-intervention. At 6 months post-intervention, we assessed cognitive function by nest building and the Morris Water Maze test, wheretime spent in the target quadrant during a probe trial was recorded. We also measured endothelial function in isolated and pressurized middle cerebral arteries (MCAs). From pre- to post- intervention, aortic PWV in pyridoxamine-treated mice decreased 16.8% (p=0.02), while aortic PWV in old control mice remained the same (p=0.43). MCA endothelium-dependent dilation, measured by the maximal dilation to acetylcholine (ACh), was 13.2% greater in old pyridoxamine-treated when compared with old control mice (p=0.01) and not different from young control mice (p=0.90). All groups had similar MCA endothelium-independent dilation, measured by the maximal dilation to sodium nitroprusside (p>0.05). Incubation withnitric oxide synthase inhibitor L-NAME reduced the maximal dilation to ACh in the MCA for all groups (p<0.001) to values that were similar across all groups (p>0.05), suggesting nitric oxide bioavailability as a mechanism for preserved endothelial function with pyridoxamine treatment. In the presence of NADPH oxidase inhibitor apocynin, maximal dilation to ACh was reduced in MCAs from old pyridoxamine-treated and young control mice (p<0.001), but not from old control mice (p=0.22), suggesting that pyridoxamine preserved the contribution of NADPH oxidase to the vasodilatory response. Compared with the young control mice, nest building scores were 57.5% lower in old control (p=0.005) and 37.5% lower in old pyridoxamine treated (p=0.08) mice. For the Morris Water Maze test, young control mice spent more time in the target zone compared to old control and old pyridoxamine-treated mice (p<0.05), and there was no difference between old groups (p=0.78). In conclusion, pyridoxamine treatment in old mice reduces large artery stiffness and preserves cerebral artery endothelial function by maintaining the contributions of nitric oxide and NADPH oxidase. However, pyridoxamine treatment in old mice appears to have minimal effects on cognitive function.

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

Beta3-adrenergic stimulation restores endothelial mitochondrial dynamics and prevents pulmonary arterial hypertension

Abstract Background Endothelial dysfunction is one of the most important hallmarks of pulmonary arterial hypertension (PAH). This leads to anomalous production of vasoactive mediators that are responsible for a higher vascular tone and a subsequent increase in pulmonary artery pressure (PAP), and to an increased vascular permeability that favors perivascular inflammation and remodeling, thus worsening the disease. Therefore, preservation of the endothelial barrier could become a relevant therapeutic strategy. Purpose In previous studies, others and we have suggested the pharmacological activation of the β3-adrenergic receptor (AR) as a potential therapeutic strategy for pulmonary hypertension (PH) due to left heart disease. However, its potential use in other forms of PH remain unclear. The aim of the present study was to elucidate whether the β3-AR agonist mirabegron could preserve pulmonary endothelium function and be a potential new therapy in PAH. Methods For this purpose, we have evaluated the effect of mirabegron (2 and 10 mg/kg·day) in different animal models, including the monocrotaline and the hypoxia-induced PAH models in rats and mice, respectively. Additionally, we have used a transgenic mouse model with endothelial overexpression of human β3-AR in a knockout background, and performed in vitro experiments with human pulmonary artery endothelial cells (HPAECs) for mechanistic experiments. Results Our results show a dose dependent effect of mirabegron in reducing mean PAP and Right Ventricular Systolic Pressure in both mice and rats. In addition, the use of transgenic mice has allowed us to determine that pulmonary endothelial cells are key mediators of the beneficial role of β3-AR pathway in ameliorating PAH. Mechanistically, we have shown in vitro that activation of β3-AR with mirabegron protects HPAECs from hypoxia-induced ROS production and mitochondrial fragmentation by restoring mitochondrial fission/fusion dynamics. Conclusions This protective effect of mirabegron would lead to endothelium integrity and preserved pulmonary endothelial function, which are necessary for a correct vasodilation, avoiding increased permeability and remodeling. Altogether, the current study demonstrates a beneficial effect of the β3-AR agonist mirabegron that could open new therapeutic avenues in PAH. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Programa de Atracciόn de Talento, Comunidad de Madrid

Maintenance of endothelial function following acute resistance exercise in females is associated with a tempered blood pressure response.

Acute resistance exercise-induced hypertensive stimulus impairs endothelial function in males. Because prior work indicates that female subjects have a tempered blood pressure response to acute resistance exercise, we hypothesized that, compared with males, females would better maintain endothelial function following resistance exercise and that this preservation would be associated with a lower hypertensive stimulus. Twenty-nine young healthy males (n = 16) and females (n = 13) participated in this study. All subjects performed a session of resistance exercise (leg extension) at the same relative workload [65% of one repetition maximum (1RM)]. The blood pressure response to exercise was characterized, and brachial artery flow-mediated dilation (FMD) was measured before and after. Males performed a second experimental trial in which the blood pressure response to resistance exercise was matched to that of females. This was accomplished by reducing the workload (~25% of 1RM). When resistance exercise was performed at the same relative workload, the systolic blood pressure was greater in males compared with females. Congruently, resistance exercise caused a significant impairment in FMD in males but not in females (P < 0.001). When the exercise workload was reduced in males, they no longer displayed an impairment in FMD following resistance exercise and, as such, the sex difference was abolished (P > 0.05). The present study reveals that acute resistance exercise transiently impairs endothelial function in young, healthy males but not in age-matched females. Notably, the maintenance of endothelial function in females is associated with a lower blood pressure response during resistance exercise.NEW & NOTEWORTHY The present data demonstrate for the first time that acute resistance exercise impairs endothelial function in young, healthy male but not female subjects. In addition, we show that the preservation of endothelial function in females is associated with a mitigated blood pressure response during resistance exercise. Accordingly, this work portrays a sexual dimorphism in the barostress response, and ensuing vascular effects, to resistance exercise.

Elevated GTP Cyclohydrolase I Pathway in Endothelial Progenitor Cells of Overweight Premenopausal Women.

Background/Aims. Sexual differences exist in endothelial progenitor cells (EPCs), and various cardiovascular risk factors are associated with the preservation of endothelial function in premenopausal women. However, it is unclear whether differences in endothelial function and circulating EPCs exist between overweight premenopausal women and age-matched men. Methods. We compared EPC counting and functions in normal-weight and overweight premenopausal women and men, evaluated endothelial function in each group, and detected the expression of the guanosine triphosphate cyclohydrolase I (GTPCH I) pathway. Results. The number of EPCs was lower in the male group than in the female group, regardless of normal-weight or overweight status, and there was no significant difference between the different weight groups among females or males. Endothelial function and EPC migration and proliferation were preserved in overweight premenopausal women compared with overweight men as were nitric oxide (NO) levels in plasma and secreted by EPCs. Endothelial function, the circulating EPC population, and NO levels were not different between normal-weight and overweight premenopausal women. Flow-mediated dilatation was significantly correlated with EPC function, plasma NO levels, and EPC-secreted NO. Conclusions. This investigation provides the first evidence for sex-based differences in EPC activity and endothelial function in overweight middle-aged individuals; these differences are associated with alterations in NO production and may partly occur through downregulation of the GTPCH I pathway. The present results provide new insights into the mechanism underlying the preserved endothelial function in overweight premenopausal women and may uncover a potential therapeutic target for endothelial repair in overweight population.

The effects of pulmonary rehabilitation on endothelial function and arterial stiffness in patients with chronic obstructive pulmonary disease

Cardiovascular disease is a major cause of mortality in chronic obstructive pulmonary disease (COPD) and endothelial dysfunction may enhance the mortality risk. Exercise training has shown to be beneficial for improvement of endothelial function in patients with cardiovascular disease, but this remains unclear in COPD. Thus, this study aimed to assess the effect of exercise-based pulmonary rehabilitation (PR) on endothelium function, arterial stiffness and plasma nitrite levels in patients with COPD. Patients with COPD engaged a 48-session PR program. Reactive hyperaemia index (RHI), augmentation index (AIx), and heart rate (HR) assed by peripheral arterial tonometry (PAT), plasma nitrite levels, systemic blood pressure, functional capacity (six-minute walk test) and the BODE index were assessed at baseline and after 24 and 48 sessions of PR. Plasma nitrite levels were also assessed before and after the first session of PR. Twenty-one subjects were included and completed 24 PR sessions, and 16 subjects completed 48 sessions. It was observed that a poorer AIx adjusted for HR in frequent COPD exacerbators (4.67 ± 16.5 vs. 20.9 ± 12.9; p = .02). PR improved functional capacity (380 ± 107 m vs. 442 ± 115 m; p < .001) and the BODE index (6 [2.8] vs. 4 [3]; p = .001), but did not change HR, systemic arterial pressure, RHI, AIx, and plasma nitrite levels during the follow-up. Plasma nitrite levels reduced after the first session of PR (0.074 [0.079] μM vs. 0.061 [0.04] μM; p = .027). The acute change in plasma nitrite levels correlated with RHI in patients with preserved endothelial function (r = 0.71; p = .01). Although exercise-based PR improved functional capacity and the BODE index, it did not change endothelial function and arterial stiffness in patients with COPD.

Abstract P120: Mir-338-3p is Down-regulated in Subcutaneous Small Arteries of Hypertensive Patients With Chronic Kidney Disease

Background: Hypertension (HTN) and chronic kidney disease (CKD) are among the most prevalent global health conditions that cause millions of deaths per year. They are associated with vascular damage characterized by vascular remodeling, stiffening and endothelial dysfunction. miRNAs (miRs) are a class of small non-coding RNA that regulate gene expression by binding to their target messenger RNAs (mRNAs), thereby leading to mRNA degradation or translational repression. Their implication in vascular injury remains unclear. We aimed to identify differentially expressed (DE) miRs in small arteries of HTN and CKD human subjects to gather insight into pathophysiological molecular mechanisms in these conditions. Methods and Results: Normotensive, HTN [systolic blood pressure (BP) &gt;135 mmHg or diastolic BP of 85-115 mmHg with BpTRU] and CKD subjects (eGFR&lt;60mL/min/m 2 ) (n=15-16) were studied. Small arteries were isolated from subcutaneous gluteal biopsies and RNA extracted for small and total RNA sequencing using Illumina HiSeq-2500. EdgeR identified DE miRs ( P &lt;0.05) uniquely associated with HTN (3 up and 6 down) or CKD (42 up and 39 down) or with both groups (2 down). Reverse transcription-quantitative PCR (RT-qPCR) was used to confirm miRNA differential expression. Correlation between RNA sequencing and RT-qPCR data was demonstrated for 3 miRs from 14 tested: mir-146a-5p (r=0.56, P &lt;10 -4 ), miR-338-3p (r=0.91, P &lt;10 -16 ) and mir-374a-3p (r=0.57, P &lt;10 -4 ). The best correlated DE miRNA, miR-338-3p was down-regulated by 76% ( P &lt;10 -4 ) and uniquely associated with CKD. TargetScan predicted that the up-regulated mRNA encoding glutathione peroxidase 3 ( GPX3 , 1.57 fold, P &lt;0.001) to be a miR-338-3p target. Both miR-338-3p and GPX3 were found to be highly expressed in human aortic endothelial cells (HAECs) by RT-qPCR. Target prediction was validated by showing using RT-qPCR that GPX3 expression was up-regulated 1.73±0.28 fold in HAECs transfected with anti-miR-338-3p ( P &lt;0.05). Conclusion: miR-338-3p down-regulation was found in small arteries, uniquely associated with CKD. GPX3 , which may be important for preservation of endothelial function, is a miR-338-3p potential target. miR-338-3p and associated target may play a role in vascular remodeling in CKD.

Chronic treatment with paeonol improves endothelial function in mice through inhibition of endoplasmic reticulum stress-mediated oxidative stress.

Endoplasmic reticulum (ER) stress leads to endothelial dysfunction which is commonly associated in the pathogenesis of several cardiovascular diseases. We explored the vascular protective effects of chronic treatment with paeonol (2'-hydroxy-4'-methoxyacetophenone), the major compound from the root bark of Paeonia suffruticosa on ER stress-induced endothelial dysfunction in mice. Male C57BL/6J mice were injected intraperitoneally with ER stress inducer, tunicamycin (1 mg/kg/week) for 2 weeks to induce ER stress. The animals were co-administered with or without paeonol (20 mg/kg/oral gavage), reactive oxygen species (ROS) scavenger, tempol (20 mg/kg/day) or ER stress inhibitor, tauroursodeoxycholic acid (TUDCA, 150 mg/kg/day) respectively. Blood pressure and body weight were monitored weekly and at the end of treatment, the aorta was isolated for isometric force measurement. Protein associated with ER stress (GRP78, ATF6 and p-eIF2α) and oxidative stress (NOX2 and nitrotyrosine) were evaluated using Western blotting. Nitric oxide (NO) bioavailability were determined using total nitrate/nitrite assay and western blotting (phosphorylation of eNOS protein). ROS production was assessed by en face dihydroethidium staining and lucigenin-enhanced chemiluminescence assay, respectively. Our results revealed that mice treated with tunicamycin showed an increased blood pressure, reduction in body weight and impairment of endothelium-dependent relaxations (EDRs) of aorta, which were ameliorated by co-treatment with either paeonol, TUDCA and tempol. Furthermore, paeonol reduced the ROS level in the mouse aorta and improved NO bioavailability in tunicamycin treated mice. These beneficial effects of paeonol observed were comparable to those produced by TUDCA and tempol, suggesting that the actions of paeonol may involve inhibition of ER stress-mediated oxidative stress pathway. Taken together, the present results suggest that chronic treatment with paeonol preserved endothelial function and normalized blood pressure in mice induced by tunicamycin in vivo through the inhibition of ER stress-associated ROS.

Cytosolic Active Caspase 3 Promotes Endothelial Barrier Integrity after Thrombin Stimulation

BackgroundWe have previously shown that MK2 deficiency leads to cytoplasmic sequestration of active caspase 3, prevention of apoptosis and is associated with a preserved endothelial barrier function in response to endotoxin challenge. While caspase 3 is widely described as the executioner of apoptosis, it has numerous cytosolic substrates that are not related to execution of apoptosis. Therefore, we hypothesized that cytosolic caspase 3 promotes endothelial barrier function.MethodsHuman lung micro‐vascular endothelial cells (HMVECs) were exposed to a non‐apoptotic, thrombin, stimulus and endothelial barrier function was assessed using electric cell‐substrate impedance sensing (ECIS). After exposures, a subset of HMVECs were evaluated for actin cytoskeletal rearrangement using phalloidin staining. In addition, HMVEC cell lysates were harvested for protein analyses. Caspase 3 knockdown or inhibition was achieved using RNA interference or the pharmacological inhibitor q‐VD‐OPH, respectively.ResultsExposure of HMVECs to thrombin demonstrated a dose dependent transient decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery on ECIS. Thrombin activation of HMVECs led to cytosolic caspase 3 activation, confirmed by fluorescent microscopy. Inhibition of caspases with q‐VD‐OPH in HMVECs led to a similar drop in thrombin‐induced endothelial barrier function but notably there was no subsequent barrier recovery. The lack of barrier recovery was not a result of endothelial cell death but rather a failure of de‐remodeling of thrombin‐induced actin stress fiber formation. Caspase 3 knockdown in HMVECs also resulted in lack of de‐remodeling after thrombin‐induced actin stress fiber formation.ConclusionTaken together, our results suggest cytosolic active caspase 3 promotes endothelial barrier integrity.

Improvement in erectile function in a rat model of high cholesterol diet-induced atherosclerosis by atorvastatin in a manner that is independent of its lipid-lowering property.

The purpose of the present study is to explore the effects of a lipid-lowering drug atorvastatin, a three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in the treatment of erectile dysfunction (ED) in a rat model of atherosclerosis (AS) and the possible mechanisms underneath. A high-cholesterol diet was administrated to Sprague-Dawley rats in an attempt to induce an ASED model, which was later confirmed by abdominal aorta histopathology and erectile function evaluation. ASED rats were further assigned to non-treatment group, atorvastatin low-dose treatment group (5mgkg-1 day-1 ), high-dose group (10mgkg-1 day-1 ) and sildenafil (1.5mgkg-1 day-1 ) treatment group. Lipid profile, erectile function, oxidative stress biochemical markers, endothelial nitric oxide synthase (eNOS) and extracellular superoxide dismutase (SODEX ) mRNA expression were evaluated after 8-week treatment duration. Erectile function was impaired in AS rat model, which was preserved in atorvastatin and sildenafil intervention groups. The oxidative stress biochemical markers were attenuated, while eNOS and SODEX mRNA expression were restored in atorvastatin and sildenafil groups, which were found to be involved in ED pathogenesis. However, the lipid profile remained unaltered in the treatment group, and it was elevated in ASED rats. This kind of lipid-lowering agent, or atorvastatin, has the utilisation potential in ASED treatment, even before lipid profiles altered. This effect on erectile function preservation of atorvastatin was attributed to its preservation of endothelial function, possibly through amelioration of oxidative stress and improvement in eNOS expression.