Endothelial Dysfunction Response Research Articles

Abstract 18714: Myocardial Infarction With Non-Obstructive Coronary Artery Disease in a Young Woman - The Cardinal Role of Coronary Reactivity Testing in Unveiling the Underlying Etiology

43-year old female presented to the emergency department with substernal chest pain. She had similar symptoms years ago without obstructive CAD on coronary angiogram and was diagnosed with myocarditis. Presenting ECG revealed new anterolateral T wave inversions (Fig 1), and high-sensitivity troponins peaked at 322 ng/L consistent with NSTEMI. TTE was significant for global hypokinesis of the left ventricle with a reduced ejection fraction of 35 to 40%. Invasive coronary angiogram showed no obstructive CAD or spontaneous coronary artery dissection however there was reduced flow in the mid to distal LAD raising suspicion for endothelial and microvascular dysfunction. Contrast-enhanced cardiac MRI showed myocardial edema in the anterior and anteroseptal walls with elevated myocardial T1 and T2 values, and early enhancement post gadolinium administration showed an acute LAD territorial infarct (Fig 2). Findings were consistent with myocardial infarction with non-obstructive coronary artery disease (MINOCA). She was started on carvedilol for empiric treatment of coronary artery spasm and microvascular dysfunction, along with aspirin and atorvastatin. Losartan was added for heart failure management. Outpatient invasive coronary reactivity testing showed severe epicardial (70% vasoconstriction) and microvascular (80% decrease in coronary blood flow) endothelial dysfunction in response to acetylcholine (Fig 3 and Fig 4) as underlying etiology of recent MINOCA. CFR was normal. L-arginine (NO precursor) was added to the medical regimen. Patient has not had recurrence of chest pain or ACS at 1 year follow-up and LVEF normalized on repeat echocardiogram. This case shows the importance of cardiac MRI in confirming the diagnosis of MINOCA in patients with no obstructive CAD, and the crucial role of coronary reactivity testing in diagnosing and treating severe coronary endothelial dysfunction, which is aligned with the AHA/ACC MINOCA consensus document.

Postnatal Overfeeding during Lactation Induces Endothelial Dysfunction and Cardiac Insulin Resistance in Adult Rats.

Early overnutrition is associated with cardiometabolic alterations in adulthood, likely attributed to reduced insulin sensitivity due to its crucial role in the cardiovascular system. This study aimed to assess the long-term effects of early overnutrition on the development of cardiovascular insulin resistance. An experimental childhood obesity model was established using male Sprague Dawley rats. Rats were organized into litters of 12 pups/mother (L12-Controls) or 3 pups/mother (L3-Overfed) at birth. After weaning, animals from L12 and L3 were housed three per cage and provided ad libitum access to food for 6 months. L3 rats exhibited elevated body weight, along with increased visceral, subcutaneous, and perivascular fat accumulation. However, heart weight at sacrifice was reduced in L3 rats. Furthermore, L3 rats displayed elevated serum levels of glucose, leptin, adiponectin, total lipids, and triglycerides compared to control rats. In the myocardium, overfed rats showed decreased IL-10 mRNA levels and alterations in contractility and heart rate in response to insulin. Similarly, aortic tissue exhibited modified gene expression of TNFα, iNOS, and IL-6. Additionally, L3 aortas exhibited endothelial dysfunction in response to acetylcholine, although insulin-induced relaxation remained unchanged compared to controls. At the molecular level, L3 rats displayed reduced Akt phosphorylation in response to insulin, both in myocardial and aortic tissues, whereas MAPK phosphorylation was elevated solely in the myocardium. Overfeeding during lactation in rats induces endothelial dysfunction and cardiac insulin resistance in adulthood, potentially contributing to the cardiovascular alterations observed in this experimental model.

Disturbed flow increases endothelial inflammation and permeability via a Frizzled-4-β-catenin-dependent pathway.

Multidirectional or disturbed flow promotes endothelial dysfunction and is associated with early atherogenesis. Here we investigated the role of Wnt signalling in flow-mediated endothelial dysfunction. The expression of Frizzled-4 was higher in cultured human aortic endothelial cells (ECs) exposed to disturbed flow compared to that seen for undisturbed flow, obtained using an orbital shaker. Increased expression was also detected in regions of the porcine aortic arch exposed to disturbed flow. The increased Frizzled-4 expression in cultured ECs was abrogated following knockdown of R-spondin-3. Disturbed flow also increased the nuclear localisation and activation of β-catenin, an effect that was dependent on Frizzled-4 and R-spondin-3. Inhibition of β-catenin using the small-molecule inhibitor iCRT5 or knockdown of Frizzled-4 or R-spondin-3 resulted in reduced expression of pro-inflammatory genes in ECs exposed to disturbed flow, as did inhibition of WNT5A signalling. Inhibition of the canonical Wnt pathway had no effect. Inhibition of β-catenin also reduced endothelial paracellular permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling. These data suggest the presence of an atypical Frizzled-4-β-catenin pathway that promotes endothelial dysfunction in response to disturbed flow.

Sex Differences in Vascular Response to Mental Stress and Adverse Cardiovascular Events Among Patients With Ischemic Heart Disease.

Microvascular measures of vascular dysfunction during acute mental stress may be important determinants of major adverse cardiovascular events (MACE), especially among younger and middle-aged women survivors of an acute myocardial infarction. In the MIMS2 study (Myocardial Infarction and Mental Stress 2), individuals who had been hospitalized for a myocardial infarction in the past 8 months were prospectively followed for 5 years. MACE was defined as a composite index of cardiovascular death and first/recurring events for nonfatal myocardial infarction and hospitalizations for heart failure. Reactive hyperemia index and flow-mediated dilation were used to measure microvascular and endothelial function, respectively, before and 30 minutes after a public-speaking mental stress task. Survival models for recurrent events were used to examine the association between vascular response to stress (difference between poststress and resting values) and MACE. Reactive hyperemia index and flow-mediated dilation were standardized in analyses. Of 263 patients (the mean age was 51 years; range, 25-61), 48% were women, and 65% were Black. During a median follow-up of 4.3 years, 64 patients had 141 adverse cardiovascular events (first and repeated). Worse microvascular response to stress (for each SD decrease in the reactive hyperemia index) was associated with 50% greater risk of MACE (hazard ratio, 1.50 [95% CI, 1.05-2.13]; P=0.03) among women only (sex interaction: P=0.03). Worse transient endothelial dysfunction in response to stress (for each SD decrease in flow-mediated dilation) was associated with a 35% greater risk of MACE (hazard ratio, 1.35 [95% CI, 1.07-1.71]; P=0.01), and the association was similar in women and men. Peripheral microvascular dysfunction with mental stress was associated with adverse events among women but not men. In contrast, endothelial dysfunction was similarly related to MACE among both men and women. These results suggest a female-specific mechanism linking psychological stress to adverse outcomes.

Abstract P407: Sex Differences in Microvascular Response to Mental Stress and Adverse Cardiovascular Events Among Patients With Ischemic Heart Disease

Introduction: Microvascular dysfunction during acute mental stress may be an important determinant of major adverse cardiovascular outcomes (MACE) especially among middle-aged women survivors of an acute myocardial infarction (MI). Hypothesis: There are sex differences in the association between microvascular dysfunction and MACE. Methods: The Myocardial Infarction and Mental Stress Study 2 enrolled individuals who had been hospitalized for an MI in the past 8 months at the age of 60 years or younger, with an oversampling of women. Reactive hyperemia index (RHI) was used to measure digital microvascular function before and 30 minutes after a public-speaking mental stress task. This assessment was contrasted with macrovascular endothelial function at the brachial artery through flow-mediated dilation (FMD). Participants were followed for 5-years, and MACE was defined as a composite of cardiovascular death, first/recurring events for nonfatal MI, and hospitalizations for heart failure. Cox proportional hazards models were used to examine vascular response to stress (difference between post-stress and resting values) and MACE adjusting for demographics, clinical risk factors, medications, psychological factors, and mental stress ischemia. Lower RHI and FMD values are indicative of greater microvascular and endothelial dysfunction, respectively. Results: Of 263 participants, the mean age was 51 years and 48% were women. There were 64 patients with 141 adverse cardiovascular events (first and repeated). In the overall sample, there was no significant association between microvascular response to stress and MACE, but there was a significant interaction with sex (p = 0.03). Each standard deviation decrease in RHI response to stress was associated with 50% greater risk of MACE (HR: 1.50, 95% CI: 1.05, 2.13) among women only, while no association was observed in men (HR 0.93, 95% CI: 0.69, 1.26). Transient endothelial dysfunction in response to stress (for each standard deviation decrease in FMD) was associated with 35% greater risk of MACE (HR: 1.35; 95% CI: 1.07, 1.71), and was similar in women and men. Conclusions: Peripheral microvascular dysfunction with mental stress is associated with MACE among women but not among men. In contrast, brachial artery endothelial dysfunction is similarly related to MACE among both men and women. These results suggest that microvascular dysfunction is a female-specific mechanism linking psychological stress to adverse outcomes.

Abstract P184: Racial Differences In Mental Stress-induced Transient Endothelial Dysfunction And Its Association With Cardiovascular Outcomes

Background: An established link exists between acute mental stress and the development of transient endothelial dysfunction (TED) as assessed by brachial artery flow-mediated vasodilation (FMD). Black individuals have a high burden of cardiovascular (CV) risk factors and social disadvantages, which may result in adverse vascular responses to stress. We investigated differences in TED with mental stress in Black and non-Black individuals with coronary artery disease (CAD), and whether TED is associated with CV outcomes in both groups. Methods: We enrolled 812 patients with stable CAD between June 2011 and March 2016 and followed them through February 2020. Study participants were subjected to a public-speaking mental stress task and FMD (derived from % vasodilation with brachial artery hyperemia testing) was assessed by ultrasound before and 30 minutes after mental stress. TED was defined as a lower post-stress FMD than pre-stress FMD. We compared pre-stress FMD, post-stress FMD, and TED between Black and non-Black participants. In both groups, we examined the association of TED with an adjudicated composite end point of CV death and myocardial infarction (MI) after adjusting for demographic, clinical, and socioeconomic factors. Both first and recurring events were counted and multivariable models for repeated events were used for analysis. Results: The mean (SD) age was 59 (10) years, and 328 (40%) were Black. Pre-stress FMD was lower in Black than non-Black participants [3.7 (2.8) vs. 4.9 (3.8) p<0.001], and significantly declined with mental stress in both groups. TED occurred more often in Black (76%) than non-Black patients (67%), age- and sex-adjusted OR, 1.6, 95% CI, 1.4-1.7). Race related differences in TED persisted after adjustment for demographic factors, CAD risk factors and medications (Black vs. non-Black OR, 1.6, 95% CI, 1.5-1.7). After further adjusting for socioeconomic indicators (education, income, and employment status), TED remained elevated in Black patients (OR, 1.4 (95% CI, 1.3-1.6). Over a median (interquartile range) follow-up period of 75 (65-82) months, 142 (18%) patients had at least one event. Development of TED with mental stress was associated with an increased risk for the study outcome both in Black (age- and sex-adjusted HR, 3.0; 95% CI, 1.3-6.8) and non-Black participants (HR, 1.7, 95% CI, 0.96-3.0), p=0.26 for interaction. Adjustment for demographic, clinical and socioeconomic factors did not diminish these associations. Conclusions: Among CAD patients, Black individuals are more likely than non-Black individuals to develop endothelial dysfunction with mental stress, which in turn is associated with an increased risk of adverse cardiovascular events. Endothelial dysfunction in response to psychological stress may represent an important mechanism for the disproportionate risk of adverse CV outcomes in Black patients.

Transcription of DNA-Methyltransferases in Endothelial Cells Exposed to Mitomycin C

DNA-methyltransferases catalyze DNA methylation in the CpG sites, which play an important role in the maintenance of genome stability. The association between DNA methylation and genotoxic stress resulting in the action of various clastogens has been shown. Genotoxic stress is one of the triggers of endothelial dysfunction. In this study, the transcription of DNMT1, DNMT3A and DNMT3B genes in coronary (HCAEC) and internal thoracic (HITAEC) artery endothelial cells exposed to alkylating mutagen mitomycin C was studied using quantitative polymerase chain reaction. In HCAEC exposed to mitomycin C, DNMT1 transcription is 1.7-fold higher compared to the unexposed control. After elimination of the mutagen from the cultures followed by 24-hours of cultivation, a 2-fold increase of transcription of DNMT3B in HCAEC exposed to mitomycin C compared to the control was observed. At the same time, no changes in transcription of the studied DNA-methyltransferases were found in HITAEC exposed to the mutagen. Thus, increased transcription of DNA-methyltransferase may be a possible molecular mechanism underlying endothelial dysfunction in response to mutagenic load in an in vitro experiment.

Cold pressor testing and sympathetic nervous system contribution to ischemia with no obstructive coronary artery disease: Results from the Women's Ischemia Syndrome Evaluation-Coronary Vascular Dysfunction Project

Study objectiveCold pressor testing (CPT) is a known stimulus of the sympathetic nervous system (SNS). To better understand sympathetic contribution to coronary blood flow regulation in women with suspected ischemia and no obstructive coronary arteries (INOCA), we compared myocardial perfusion reserve during CPT stress cardiac magnetic resonance (CMR) imaging between women with suspected INOCA and reference subjects. DesignProspective cohort. SettingAcademic hospital. Participants107 women with suspected INOCA and 21-age-matched reference women. InterventionsCPT stress CMR was performed with measurement of myocardial perfusion reserve index (MPRI), adjusted for rate pressure product (MPRIRPP). Invasive coronary function testing in a subset of INOCA women (n = 42) evaluated for endothelial dysfunction in response to acetylcholine, including impaired coronary diameter response ≤0% and coronary blood flow response (ΔCBF) <50%. Main outcome measureMPRIRPP. ResultsCompared to reference women, the INOCA group demonstrated higher resting RPP (p = 0.005) and CPT MPRIRPP (1.09 ± 0.36 vs 0.83 ± 0.18, p = 0.002). Furthermore, INOCA women with impaired ΔCBF (n = 23) had higher CPT MPRIRPP (p = 0.044) compared to reference women despite lower left ventricular ejection fraction (64 ± 7% vs 69 ± 2%, p = 0.005) and higher mass-to-volume ratio (0.79 ± 0.15 vs 0.62 ± 0.09, p < 0.0001). These differences in CPT MPRIRPP did not persist after adjusting for age, body mass index, and history of hypertension. CPT MPRIRPP among INOCA women did not differ based on defined acetylcholine responses. ConclusionsMyocardial perfusion reserve to CPT stress is greater among women with INOCA compared to reference subjects. This CPT response was also noted in women with coronary endothelial dysfunction, suggesting a greater contribution of the SNS to coronary flow than endothelial dysfunction. Therapies to modulate sympathetic activity should be studied in this population.

CFTR limits F-actin formation and promotes morphological alignment with flow in human lung microvascular endothelial cells.

Micro‐ and macrovascular endothelial dysfunction in response to shear stress has been observed in cystic fibrosis (CF), and has been associated with inflammation and oxidative stress. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) regulates endothelial actin cytoskeleton dynamics and cellular alignment in response to flow. Human lung microvascular endothelial cells (HLMVEC) were cultured with either the CFTR inhibitor GlyH‐101 (20 µM) or CFTRinh‐172 (20 µM), tumor necrosis factor (TNF)‐α (10 ng/ml) or a vehicle control (0.1% dimethyl sulfoxide) during 24 and 48 h of exposure to shear stress (11.1 dynes/cm2) or under static control conditions. Cellular morphology and filamentous actin (F‐actin) were assessed using immunocytochemistry. [Nitrite] and endothelin‐1 ([ET‐1]) were determined in cell culture supernatant by ozone‐based chemiluminescence and ELISA, respectively. Treatment of HLMVECs with both CFTR inhibitors prevented alignment of HLMVEC in the direction of flow after 24 and 48 h of shear stress, compared to vehicle control (both p < 0.05). Treatment with TNF‐α significantly increased total F‐actin after 24 h versus control (p < 0.05), an effect that was independent of shear stress. GlyH‐101 significantly increased F‐actin after 24 h of shear stress versus control (p < 0.05), with a significant (p < 0.05) reduction in cortical F‐actin under both static and flow conditions. Shear stress decreased [ET‐1] after 24 h (p < 0.05) and increased [nitrite] after 48 h (p < 0.05), but neither [nitrite] nor [ET‐1] was affected by GlyH‐101 (p > 0.05). CFTR appears to limit cytosolic actin polymerization, while maintaining a cortical rim actin distribution that is important for maintaining barrier integrity and promoting alignment with flow, without effects on endothelial nitrite or ET‐1 production.

Changes in the concentration of endothelin-1 and the state of blood flow in the uterine arteries of women in the third trimester of gestation with exacerbation of cytomegalovirus infection in the second trimester of pregnancy

Aim. To assess the change in the concentration of endothelin-1 (ET-1) and the state of uteroplacental blood flow in women in the third trimester of gestation with exacerbation of cytomegalovirus infection (CMVI) in the second trimester of pregnancy.Materials and methods. A comprehensive examination of 120 women with pregnancies uncomplicated and complicated by exacerbation of CMVI in the second trimester of gestation was carried out. The first group consisted of 30 women with a seronegative CMVI normal pregnancy, the second group – 30 patients with a latent course of CMVI and the development of chronic compensated placental insufficiency (CCPI), the third – 30 women with exacerbation of CMVI and CCPI, and the fourth – 30 patients with exacerbation of CMVI and chronic subcompensated placental insufficiency (CSPI).Results. With the development of CCPI at 30-34 weeks of gestation in women of the second group, compared with the first, the concentration of ET-1 in blood serum increased to 1.16±0.09 fmol/L (in the control – 0.86±0.08 fmol/L, p&lt;0,05). There was an increase in the systolic-diastolic ratio (SDR) in the right uterine artery (RUA) by 1.40 times (p=0.000001) and in the left uterine artery (LUA) by 1.46 times (p=0.000001), which illustrated the important role of endothelial activation in increasing resistance in the uterine artery basin. In the third group, in comparison with the first and second ones, an increase in the concentration of ET-1 was recorded to 1.57±0.13 fmol/L (p&lt;0.001 and p&lt;0.05, respectively), reflecting a more pronounced activation of the endothelium. In women of the third group, in comparison with the first and second groups, there was an increase in SDR in the RUA by 1.43 times (p=0.000001) and 1.01 times (p=0.046), respectively. Comparison of blood flow in the LUA in women in the third group in comparison with that in the first and second groups showed an increase in SDR by 1.47 times (p=0.000001) and 1.01 times (p=0.0462), respectively. At the same time, a strong positive correlation was recorded between the SDR in the RUA and the level of ET-1 (r=076; p&lt;0.001), which is a marker of an endothelium-dependent increase in vascular resistance. In women of the fourth group, the level of ET-1 was 2.15±0.17 fmol/L, which is significantly higher compared with group 1 (5.08 times, p=0.000001), with the second group (4.08 times, p=0.000001) and in comparison with the third group (1.23 times, p=0.0029). SDR in RUA in the fourth group compared to the first one increased by 1.62 times (p=0.000001), and compared to the second – by 1.15 times (p=0.00002). At the same time, in patients of the fourth group in comparison with the third group, SDR in RUA increased by 1.13 times (p=0.024). In the fourth group, in contrast to the first one, there was an increase in SDR in LUA by 1.58 times (p=0.000001), and in comparison, with the second group, SDR in LUA was 1.07 times higher (p=0.00021). Patients of the fourth group, in comparison with the third one, had higher SDR indices in the LUA (1.06 times, p=0.0207). There was a stronger relationship between the SDR value in RUA and the concentration of ET-1 (r=0.86; p&lt;0.001) and a weak correlation between SDR in LUA and ET-1 (r=0.47; p&lt;0.01), which reflects the influence of endothelial dysfunction in response to an increase in the tone of the uterine arteries.Conclusion. The development of CSPI in the third trimester of gestation in women who underwent an exacerbation of CMVI in the second trimester of pregnancy, in contrast to CCPI with a similar infectious pathology in pregnant women, is characterized by a more pronounced increase in vascular resistance in the uterine artery basin against the background of endothelial dysfunction.

The vascular response to acute hyperglycaemia: What is the role of exercise capacity?

The vascular response to acute hyperglycaemia: What is the role of exercise capacity?

Nicotinamide nucleotide transhydrogenase (NNT) regulates mitochondrial ROS and endothelial dysfunction in response to angiotensin II

Endothelial dysfunction is a critical, initiating step in the development of hypertension (HTN) and mitochondrial reactive oxygen species (ROS) are important contributors to endothelial dysfunction. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the nicotinamide nucleotide transhydrogenase (Nnt) gene that are associated with endothelial dysfunction and increased risk for HTN. NNT is emerging as an important enzyme that regulates mitochondrial NADPH levels and mitochondrial redox balance by supporting the thiol dependent peroxidase systems in the mitochondria. We have previously shown that the absence of NNT in C57Bl/6J animals promotes a more severe hypertensive phenotype through reductions in •NO and endothelial dependent vessel dilation. However, the impact of NNT on human endothelial cell function remains unclear. We utilized NNT directed shRNA in human aortic endothelial cells to test the hypothesis that NNT critically regulates mitochondrial redox balance and endothelial function in response to angiotensin II (Ang II). We demonstrate that NNT expression and activity are elevated in response to the mitochondrial dysfunction and oxidative stress associated with Ang II treatment. Knockdown of NNT led to a significant elevation of mitochondrial ROS production and impaired glutathione peroxidase and glutathione reductase activities associated with a reduction in the NADPH/NADP+ ratio. Loss of NNT also promoted mitochondrial dysfunction, disruption of the mitochondrial membrane potential, and impaired ATP production in response to Ang II. Finally, we observed that, while the loss of NNT augmented eNOS phosphorylation at Ser1177, neither eNOS activity nor nitric oxide production were similarly increased. The results from these studies clearly demonstrate that NNT is critical for the maintenance of mitochondrial redox balance and mitochondrial function. Loss of NNT and disruption of redox balance leads to oxidative stress that compromises eNOS activity that could have a profound effect on the endothelium dependent regulation of vascular tone.

Vascular Expression of Toll‐Like Receptor 4 and Myeloid Differentiation Factor‐88 Mediate Endothelial Dysfunction in Response to Lipopolysaccharide Independent of Circulating Immune Cells

The goal of this study was to test the hypothesis that vascular expression of toll‐like receptor 4 (TLR4) and its primary downstream signaling pathway, myeloid‐differentiation factor‐88 (MyD88), mediate endothelial dysfunction in response to lipopolysaccharide (LPS). Carotid arteries from wild‐type (C57Bl/6), Tlr4‐Lps‐d (Tlr4 mutant mice), and Myd88‐deficient (Myd88−/−) mice were incubated with either vehicle (saline) or LPS (0.05–0.50 μg/ml) for 22 hrs. LPS, but not vehicle, treatment produced concentration‐dependent impairment (P&lt;0.05) of endothelium‐dependent relaxation to acetylcholine (ACh) in wild‐type mice. In contrast, in carotid arteries from Tlr4‐Lps‐d or Myd88−/− mice endothelial responses to ACh following LPS incubation were found to be similar (P&gt;0.05) to responses in wild‐type mice incubated with vehicle. LPS has no effect (P&gt;0.05) on vascular response to sodium nitroprusside in any of the six experimental groups. Both basal and NADPH‐stimulated superoxide levels were selectively increased (P&lt;0.05) in vessels from wild‐type, but not Tlr4‐Lps‐d or Myd88−/−, mice treated with LPS. RNA expression revealed that both TLR4 and MyD88 are indeed expressed in the vascular wall under basal conditions and that LPS induces differential expression of a number of inflammatory gene products, including Tlr4, Myd88, and Nfkb1. Pathway analysis revealed a linkage between TLR4 and MyD88 in the vessels wall with IL‐6 and downstream targets such as Nox2 and Nox4. These findings serve to link activation of TLR4 and downstream signaling through MyD88 and NFkB1 with increases in vascular superoxide and functional impairment of endothelial function in response to LPS. Taken together, these findings demonstrate that both Tlr4 and Myd88 expression and activity within the vascular wall contributes to LPS‐induced impairment of endothelial function. The fact that experiments were performed ex vivo, demonstrate that the effects of LPS on endothelial function occur directly within the vascular wall independently of circulating immune cell activation.Support or Funding InformationSupported in part by NIH/NIBIB R01‐EB2006.

Role of PP2A in Racial Differences in Endothelial Dysfunction

BackgroundAfrican Americans (AA) exhibit a higher prevalence of endothelial dysfunction and an exaggerated response to inflammatory cytokines compared to Caucasian Americans (CA). Endothelial dysfunction is characterized by a reduction in endothelial nitric oxide synthase (eNOS) activity and ultimately a lower nitric oxide production. Data obtained from bovine endothelial cells show that a reduction in eNOS activity in response to an inflammatory stimulus is mediated by protein phosphatase 2A (PP2A). Additionally, higher PP2A activity is associated with a decrease in the phosphorylation of PP2A at the Tyrosine 307 residue (p‐PP2AY307). Therefore, the purpose of this study was to investigate the role of PP2A in racial differences seen in endothelial dysfunction in response to an inflammatory stimulus.MethodsHuman umbilical vein endothelial cells (HUVECs) (n=6 cell lines; 3 CA &amp; 3 AA) were cultured under two conditions: control or tumor necrosis factor‐α (TNF‐α) (10 ng/mL, 4 hours). Western blotting was used to measure protein expression of p‐PP2AY307, PP2A, p‐eNOSS1177 and eNOS. A two‐way ANOVA was used to assess the effects of race and conditions.ResultsNo racial differences were found in the protein expression of PP2A (Fig. 1B) and eNOS (Fig. 2B) at baseline or with TNF‐α stimulation. However, AA had a lower p‐PP2AY307 expression (p=0.021; Fig. 1A) compared to CA HUVECs under both conditions. Additionally, AA had a lower p‐eNOSS1177 expression (p=0.008; Fig. 2A) compared to CA HUVECs under both condition.ConclusionLower p‐PP2AY307 and p‐eNOSS1177 levels in AA endothelial cells could be attributed to an increase in PP2A activity that can ultimately inhibit eNOS phosphorylation and decrease nitic oxide production. Therefore, PP2A activity can explain, in part, racial differences observed in endothelial dysfunction and consequently cardiovascular diseases.Support or Funding InformationRacial differences in p‐PP2AY307 and PP2A protein expression in HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐PP2AY307 and B) Densiometric quantification and representative Western blot of PP2A. (# p= .02 in AA compared to CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 1Racial differences in p‐eNOSS1177 and eNOS protein expression in HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐eNOSS1177 and B) Densiometric quantification and representative Western blot of eNOS. (# p= .008 in AA compared to CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 2AcknowledgementsFunded partially by AU PGOP Fellowship.

TIPE1 accelerates atherogenesis by inducing endothelial dysfunction in response to oxidative stress

Atherosclerosis is an inflammatory disease of the arterial wall, which involves endothelial cells and immune cells. Endothelial dysfunction has been considered an important step in the initiation of the disease. TIPE1 is a newly identified protein of the TIPE family, and plays a vital role in inflammation and tumorigenesis. However, its role in atherogenesis remains unclear. In this study, we demonstrated that TIPE1 promoted atherogenesis by inducing endothelial dysfunction. When human umbilical vein endothelial cells (HUVECs) were exposed to oxidative stress, the level of TIPE1 was significantly up-regulated, and the ROS generation markedly increased in TIPE1 over-expressing HUVECs. As a result, the growth of HUVECs was inhibited, and the apoptosis was enhanced. However, the cell contact ability between HUVECs and THP-1 cells were augmented due to the up-regulation of adhesion molecules such as E-selectin and ICAM-1 induced by TIPE1 overexpression. Importantly, ApoE−/− mice injected with TIPE1 recombinant lentivirus developed significantly severe atherosclerosis accompanied by hyperglycemia, hypercholesterolemia and increased white blood count. These findings indicated that excessive ROS induced by the overexpression of TIPE1 in endothelial cells accelerated the process of atherogenesis.

Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress

IntroductionThe etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic. AimTo focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells. MethodsBovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 μM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10–15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (H2O2) was measured using the Amplex Red assay and intracellular H2O2 using a fluorescent sensor. In addition, ROS superoxide (O2•-) was measured using a O2•- chemiluminescence enhancer. Both H2O2 and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO−) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [14C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C16-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells’ dysfunction. Main Outcome MeasuresSingle high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway. ResultsHere, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O2•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildenafil protected the endothelial cells from radiation therapy-induced apoptosis. Strengths and LimitationsThis is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O2•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED. ConclusionsThis study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED.Wortel RC, Mizrachi A, Li H, et al. Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress. J Sex Med 2019;16:1721–1733.

Proteomic study of endothelial dysfunction in response to homocysteinylated albumin

Hyperhomocysteinemia is reported to be involved in the progression of cardiovascular and neurological diseases. The levels of homocysteine in blood can increase due to deficiency of folic acid, vitamin B12, or B6. Homocyteine gets converted to its cyclic analog, homocysteine thiolactone, by methionyl-tRNA synthetase during the process of translation, which can further react with the free amino groups of proteins forming an N-homocysteinylated product. The homocysteine-modified proteins have altered structure and function and can be deleterious to cells. Accumulation of such modified proteins in plasma could be one of the possible mechanisms for the development of cardiovascular complications in patients with hyperhomocysteinemia. To investigate this, albumin was modified with homocysteine thiolactone, and its effect on endothelial function was studied in Human Umbilical Vein Endothelial Cells (HUVECs) by SWATH MS-based proteomics approach. Treatment of HUVECs with homocysteinylated albumin for 24 h negatively affected cell viability, and led to altered abundance of about 53 proteins, out of the total 1410 identified in proteomic analysis. Thus, homocysteinylated proteins can lead to dysfunction in vascular endothelial cells and predispose to cardiovascular diseases.

PPARγ Target Gene Retinol Binding Protein 7 (RBP7) Protects Against Endothelial Dysfunction Induced by Mitochondrial Uncoupling

PPARγ is a nuclear receptor transcription factor that promotes an anti‐oxidant pathway in the endothelium. We previously reported that retinol binding protein 7 (RBP7) is an endothelial‐specific PPARγ target and that it is required for the protective effects of PPARγ in the endothelium. Transgenic mice expressing a dominant‐negative mutation in PPARγ specifically in the endothelium (E‐V290M) and RBP7 knockout mice (RBP7ko) exhibited increased oxidative stress and endothelial dysfunction in response to high fat diet, a cardiovascular stressor. Here, we hypothesize that RBP7 plays a protective role against endothelial dysfunction induced by the pro‐oxidant effects of mitochondrial uncoupling. We evaluated vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) in carotid arteries from 4 and 8 month old RBP7ko mice, infused with either saline or a sub‐pressor dose of Angiotensin II (Ang II; 120ng/kg/min) for 2 weeks. As expected, Ang II infusion did not affect systolic blood pressure after 2 weeks either in wild type (WT) (4 months old: 116.2±4.2 vs 113.9±4.1 mmHg; 8 months old: 111.3±3.8 vs 109.5±2.9 mmHg) or RBP7ko (4 months old: 112.9±1.1 vs 111.8±5.2 mmHg; 8 months old: 112.2±4.2 vs 113.9±4.1 mmHg). Endothelium‐dependent relaxation to ACh was not affected by Ang II in either 4 month‐ or 8 month‐old RBP7ko (4 month old: % relaxation ‐ saline, 89.77±1.3 vs Ang II, 93.85±2.0; 8 months old: % relaxation ‐ saline, 80.02±6.8 vs Ang II, 74.65±10.6) or WT littermates (4 months old: % relaxation ‐ saline, 98.14±0.3 vs Ang II, 94.58±1.7; 8 months old: % relaxation ‐ saline, 81.43±4.7 vs Ang II, 78.64±10.4). Next, to examine if RBP7 plays a role in mitochondrial uncoupling, the vessels were pre‐incubated with the mitochondrial uncoupler Carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; 1uM for 30 minutes). After CCCP, the relaxation responses to ACh in 4 month old mice were not altered in any of the groups. Interestingly, in 8 month old mice, we observed a trend towards impaired endothelial function in the carotid artery from Ang II‐infused RBP7ko compared with genotype‐matched saline‐treated mice (% relaxation: 51.96±3.8 vs 81.82±5.3, p = 0.06) as well as Ang II‐treated WT mice (% relaxation: 51.96±3.8 vs 77.1±2.0 p=0.08). There were no changes in SNP‐mediated relaxation in any of the groups. We conclude that a subpressor dose of Ang II by itself was not able to impair vascular function in 4‐ or 8‐month old mice. However in 8‐month old mice, Ang II infusion increased the susceptibility to CCCP‐dependent endothelial impairment, and RBP7 was required to mediate vascular protection against mitochondrial uncoupling triggered by CCCP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Concentrations of the Selected Biomarkers of Endothelial Dysfunction in Response to Antiepileptic Drugs: A Literature Review.

Epilepsy is a disease arising from morphological and metabolic changes in the brain. Approximately 60% of patients with seizures can be controlled with 1 antiepileptic drug (AED), while in others, polytherapy is required. The AED treatment affects a number of biochemical processes in the body, including increasing the risk of cardiovascular diseases (CVDs). It is indicated that the duration of AED therapy with some AEDs significantly accelerates the process of atherosclerosis. Most of AEDs increase levels of homocysteine (HCys) as well as may affect concentrations of new, nonclassical risk factors for atherosclerosis, that is, asymmetric dimethylarginine (ADMA) and homoarginine (hArg). Because of the role of these parameters in the pathogenesis of CVD, knowledge of HCys, ADMA, and hArg concentrations in patients with epilepsia treated with AED, both pediatric and adult, appears to be of significant importance.

HIV Proteins and Endothelial Dysfunction: Implications in Cardiovascular Disease.

With the success of antiretroviral therapy (ART), a dramatic decrease in viral burden and opportunistic infections and an increase in life expectancy has been observed in human immunodeficiency virus (HIV) infected individuals. However, it is now clear that HIV- infected individuals have enhanced susceptibility to non-AIDS (Acquired immunodeficiency syndrome)-related complications such as cardiovascular disease (CVD). CVDs such as atherosclerosis have become a significant cause of morbidity and mortality in individuals with HIV infection. Though studies indicate that ART itself may increase the risk to develop CVD, recent studies suggest a more important role for HIV infection in contributing to CVD independently of the traditional risk factors. Endothelial dysfunction triggered by HIV infection has been identified as a critical link between infection, inflammation/immune activation, and atherosclerosis. Considering the inability of HIV to actively replicate in endothelial cells, endothelial dysfunction depends on both HIV-encoded proteins as well as inflammatory mediators released in the microenvironment by HIV-infected cells. Indeed, the HIV proteins, gp120 (envelope glycoprotein) and Tat (transactivator of transcription), are actively secreted into the endothelial cell micro-environment during HIV infection, while Nef can be actively transferred onto endothelial cells during HIV infection. These proteins can have significant direct effects on the endothelium. These include a range of responses that contribute to endothelial dysfunction, including enhanced adhesiveness, permeability, cell proliferation, apoptosis, oxidative stress as well as activation of cytokine secretion. This review summarizes the current understanding of the interactions of HIV, specifically its proteins with endothelial cells and its implications in cardiovascular disease. We analyze recent in vitro and in vivo studies examining endothelial dysfunction in response to HIV proteins. Furthermore, we discuss the multiple mechanisms by which these viral proteins damage the vascular endothelium in HIV patients. A better understanding of the molecular mechanisms of HIV protein associated endothelial dysfunction leading to cardiovascular disease is likely to be pivotal in devising new strategies to treat and prevent cardiovascular disease in HIV-infected patients.