Impaired Endothelial Function Research Articles

SGLT2 inhibition attenuates arterial dysfunction and decreases vascular F-actin content and expression of proteins associated with oxidative stress in aged mice

Aging of the vasculature is characterized by endothelial dysfunction and arterial stiffening, two key events in the pathogenesis of cardiovascular disease (CVD). Treatment with sodium glucose transporter 2 (SGLT2) inhibitors is now known to decrease cardiovascular morbidity and mortality in type 2 diabetes. However, whether SGLT2 inhibition attenuates vascular aging is unknown. We first confirmed in a cohort of adult subjects that aging is associated with impaired endothelial function and increased arterial stiffness and that these two variables are inversely correlated. Next, we investigated whether SGLT2 inhibition with empagliflozin (Empa) ameliorates endothelial dysfunction and reduces arterial stiffness in aged mice with confirmed vascular dysfunction. Specifically, we assessed mesenteric artery endothelial function and stiffness (via flow-mediated dilation and pressure myography mechanical responses, respectively) and aortic stiffness (in vivo via pulse wave velocity and ex vivo via atomic force microscopy) in Empa-treated (14 mg/kg/day for 6 weeks) and control 80-week-old C57BL/6 J male mice. We report that Empa-treated mice exhibited improved mesenteric endothelial function compared with control, in parallel with reduced mesenteric artery and aortic stiffness. Additionally, Empa-treated mice had greater vascular endothelial nitric oxide synthase activation, lower phosphorylated cofilin, and filamentous actin content, with downregulation of pathways involved in production of reactive oxygen species. Our findings demonstrate that Empa improves endothelial function and reduces arterial stiffness in a preclinical model of aging, making SGLT2 inhibition a potential therapeutic alternative to reduce the progression of CVD in older individuals.Graphical abstract

Alterations in aortic vasorelaxation in rats with epilepsy induced by the electrical amygdala kindling model

Cardiovascular alterations are frequently related to epilepsy in both clinical and experimental models, and have been hypothesized as a potential contributor to sudden unexpected death in epilepsy (SUDEP). Further, the frequency of generalized tonic-clonic seizures (GTCS) is a primary risk factor for SUDEP. Therefore, we aimed to evaluate the vascular response of rats subjected to the electrical amygdala kindling model of epilepsy. Male Wistar rats were randomly distributed into the following groups: without seizures (sham, n = 8), 5 GTCS (5 S, n = 5), and 10 GTCS (10 S, n = 6). One day after the last seizure, the rats were euthanized, and the thoracic aorta rings with (E+) and without (E-) endothelium were used to evaluate vascular reactivity ex vivo using the organ bath system. The maximum response to acetylcholine-induced vasorelaxation in the E+ aortic ring was lower in the 5 S group than in the sham and 10 S groups. A reduced concentration of sodium nitroprusside was required to induce vasorelaxation in the E- aortic rings. These results suggest an impairment in endothelial function and alterations in the nitric oxide (NO) pathway. In conclusion, epilepsy altered the vasorelaxation of the aortic rings and the number of seizures influenced these alterations; therefore, an analysis of endothelial function in patients with a high risk of SUDEP may be beneficial.

IDF21-0074 Role of methylglyoxal modified low density lipoprotein in type 2 diabetes mellitus and its role in periodontal disease

Background: Glycation, a physiological and pathological process mainly affects proteins, nucleic acids and lipids. Methylglyoxal (MGO) along with free radicals is known to contribute to the pathogenesis of several diseases, in disorders particularly having enhanced production of acetone and amino-acetone such as diabetes mellitus (DM). The accumulation of MGO induces β-cells dysfunction, insulin resistance as well as impairment of endothelial functions in DM patients. Also, the increased level of MGO intensifies ROS generation and AGEs formation which further accelerates the co-morbidities associated with type 2 diabetes mellitus (T2DM). There is an increased susceptibility and severity of periodontal diseases in case of patients with diabetes and vice-versa. Aim: The likely role of native low density lipoprotein (LDL) and glycated LDL in type 2 diabetes mellitus (T2DM) and periodontitis (PD) as well as in T2DM+PD patients has been evaluated. Method: Sera from T2DM+PD T2DM and PD patients (n=80), were screened for the presence of antibodies against MGO-LDL and native LDL. Besides, the conformational changes in LDL isolated from T2DM+PD, T2DM and PD patients (possibly due to glycoxidaton) were also studied by UV absorption, fluorescence and CD spectroscopy. ThT, Comet assay, SEM and TEM were also done. Cellular toxicity of the isolated LDL from both T2DM+PD, T2DM and PD patients was demonstrated by comet assay. Biochemical parameters like increase in carbonyl content, TBARS, TNF α and IL1β indicate LDL oxidation and increase in pro-inflammatory cytokines. Results: MGO-LDL (in comparison to parallel control) proved to be a potent antigen showing very high binding with T2DM+PD, T2DM and PD sera as observed in direct binding ELISA. The stronger binding of T2DM+PD, T2DM and PD auto-antibodies to MGO-LDL suggests the importance of this modification in the onset and pathogenesis of these diseases. Inhibition ELISA results showed very high percent inhibition of antibodies from T2DM+PD, T2DM and PD patients by MGO-LDL in comparison to native LDL pointing towards the possible role of glycoxidation of LDL in these patients. The modified LDL might have further generated high levels of circulating auto-antibodies in the sera of these patients. Further, to ascertain the specificity of auto-antibodies from T2DM+PD, T2DM and PD patients, competitive inhibition ELISA studies were done on IgG isolated from the sera of these patients. The samples of isolated IgG from T2DM+PD (T2DM+PD-IgG), T2DM (T2DM-IgG) and PD (PD-IgG) showed very strong recognition of MGO-LDL in comparison to the native counterpart. The specificity and affinity of T2DM+PD, T2DM and PD auto-antibodies towards MGO-LDL was further confirmed by gel retardation assay. The gel mobility results confirmed specificity of MGO-LDL towards auto-antibodies from T2DM and PD patients. Furthermore, high recognition of experimentally induced anti-MGO-LDL antibodies by T2DM+PD-LDL, T2DM-LDL and PD-LDL compared to NHS-LDL confirmed that the observed structural damage in T2DM+PD-LDL, T2DM-LDL and PD-LDL is due to glycoxidation. Discussion: The neo-epitopes on LDL created by its exposure to MGO appear to be responsible for induction and elevated levels of these antibodies, and thus MGO-LDL may be considered as potential antigenic candidate for auto-immune response in diabetes and periodontitis patients with a potential for biomarker development.

324 The role of CCN3 in lung endothelial identity and function

OBJECTIVES/GOALS: Idiopathic Pulmonary Fibrosis (IPF) is a fatal disease of lung scarring. Aberrant vascular remodeling is a contributor to IPF progression. We have identified CCN3 as an endothelial gene that is upregulated in resolving but not in persistent lung fibrosis in mice. Here we tested the role of CCN3 in lung microvascular endothelial function. METHODS/STUDY POPULATION: RNAi loss of function experiments were used to evaluate the role of CCN3 in lung endothelial biology. Human lung microvascular endothelial cells (HLMECs) were transfected with human CCN3 siRNA and analyzed via qPCR to assess expression of endothelial transcripts, via wound healing assay for assessment of migratory function, and via 2D tube formation assay for assessment of angiogenic function. To ascertain the effect of HLMECs on Normal Human Lung Fibroblasts (NHLFs), conditioned media (CM) from endothelial cells with control and CCN3 siRNA was applied to TGFβ primed fibroblasts and qPCR was used to measure expression levels of pro-fibrotic transcripts. Recombinant human CCN3 protein was subsequently used to confirm the gain of function role of CCN3 in lung endothelial biology using a subset of these assays. RESULTS/ANTICIPATED RESULTS: CCN3 is a secreted matricellular protein thought to be involved in angiogenesis, cell adhesion, cell migration, and inflammatory responses in endothelial cells. In other organs, CCN3 suppresses expression of fellow matricellular protein, CCN2 (CTGF); importantly, CCN2 is a known pro-fibrotic mediator of aberrant tissue remodeling in the fibrotic lung. Upon CCN3 knockdown in HLMECs, we observed reduced transcripts for inflammatory and pro-fibrotic genes, along with impaired endothelial function in wound healing and angiogenesis assays. CM from CCN3 knockdown endothelial cells enhanced the pro-fibrotic effects of TGFβ in NHLFs. Addition of recombinant CCN3 to HLMECs generated, conditioned media that reduced fibroblast pro-fibrotic activation. DISCUSSION/SIGNIFICANCE: We have shown that matricellular protein- CCN3 plays a fundamental role in endothelial identity and function and could be a promising therapeutic target in IPF. A future goal is to restore levels of genes such as CCN3 in the aged vasculature in the setting of lung fibrosis to test their capacity to promote vascular repair and fibrosis resolution.

Endothelium function biomarkers and carotid intima-media thickness changes in relation to NOS3 (rs2070744) and GNB3 (rs5443) genes polymorphism in the essential arterial hypertension.

Objective. The aim of the present study was to clarify the endothelial function biomarkers and carotid "intima media" thickness (IMT) changes in relation to GNB3 (rs5443) and NOS3 (rs2070744) genes polymorphism in the essential arterial hypertension (EAH). Methods. One-hundred EAH patients (48 - control) participated in the case-control study. Soluble vascular cell adhesion molecule (sVCAM-1), total NO metabolites (NO2 -+NO3 -), transcriptional activity of NOS3 gene, endothelium-dependent flow-mediated dilation of the brachial artery (FMD BA), and carotid IMT were studied. GNB3 (rs5443) and NOS3 (rs2070744) genotyping was performed by TaqMan probes (CFX96™Real-Time PCR). Results. The connection of NOS3 (rs2070744) with decreased total NO metabolites (F=71.11; p<0.001), reduced NOS3 genes transcription activity (F=8.71; p<0.001) and increased sVCAM-1 (F=6.96; p=0.002), especially in the C-allele carriers (particularly in CC-genotype patients with lower NO - 16.46% and 40.88%; p<0.001), lowered the transcription activity of NOS3 gene - 46.03% 7 times (p<0.001), and become higher sVCAM-1 - 35.48% and 89.48% (p<0.001), respectively. ANOVA did not confirm the association of GNB3 (rs5443) gene with endothelial function and carotid IMT. Severe EAH was associated with increased carotid IMT - 50.0% (p<0.001) and 57.14% (p=0.007), wider carotid arteries - 17.36% (p=0.012) and 21.79% (p=0.004), and decreased NOS3 genes transcription activity - 34.54% (p=0.003). Atherosclerotic plaques were unilateral - 24.77% (χ2=5.35; p=0.021) or bilateral - 27.62% (χ2=5.79; p=0.016). IMT---gt---0.9 mm was followed by a higher BP (p<0.001), FMD BA 11.80% decrease with compensatory increase in carotid arteries diameters - 17.38% and 21.99% (p<0.001) and sVCAM-1 by 20.49% (p=0.005). Conclusion. NOS3 (rs2070744), but not GNB3 (rs5443), gene associated with the essential arterial hypertension severity relying upon the endothelial function impairment and NOS3 genes reduced transcription activity.

Donor Simvastatin Treatment is More Effective in Donors with Low Plasma Levels of Arginine

<h3>Purpose</h3> Brain death may lead to a severe impairment of endothelial function and affect graft function. Recent study shows that simvastatin treatment to the organ donor decreases plasma biomarker levels of myocardial injury and heart failure and reduces the number of acute rejection episodes with hemodynamic compromise early after heart transplantation. However, the mechanisms of donor simvastatin treatment remain unknown. Therefore, we analyzed the plasma metabolomics of brain-dead heart transplant donors with or without simvastatin treatment to examine the molecular background of the beneficial effects. <h3>Methods</h3> In a prospective, double-blinded randomized controlled trial, 84 multiorgan donors were randomly assigned to receive either 80 mg of simvastatin (n=42) via nasogastric tube or to receive no simvastatin (n=42). Plasma samples from 84 human brain-dead multi-organ donors were collected immediately before organ procurement. We profiled over 100 targeted metabolites using mass spectrometry. <h3>Results</h3> We found that arginine and tryptophan were weakly negatively correlated with simvastatin treatment. Univariate Cox regression analysis showed that low levels of arginine were significantly associated with acute rejection and graft-related mortality (Figure 1 A,B). Additionally, we saw that donor simvastatin treatment significantly reduced the number of acute rejections by 41% (p=0.012) in low arginine group within the first year (Figure 1C). There was no difference in donor demographics between the groups. However, there was a statistically significantly higher need for mechanical circulatory support for hearts from donors with low arginine levels. <h3>Conclusion</h3> This study shows that donor simvastatin treatment could be more efficient in brain-dead heart transplant donors with low arginine. Further analysis will be conducted by exploring the interplay between arginine and simvastatin in detail. The complete results will be presented at the ISHLT2022 congress.

Microvascular dysfunction and neurovascular uncoupling are exacerbated in peripheral artery disease, increasing the risk of cognitive decline in older adults.

Peripheral artery disease (PAD) is a vascular pathology with high prevalence among the aging population. PAD is associated with decreased cognitive performance, but the underlying mechanisms remain obscure. Normal brain function critically depends on an adequate adjustment of cerebral blood supply to match the needs of active brain regions via neurovascular coupling (NVC). NVC responses depend on healthy microvascular endothelial function. PAD is associated with significant endothelial dysfunction in peripheral arteries, but its effect on NVC responses has not been investigated. This study was designed to test the hypothesis that NVC and peripheral microvascular endothelial function are impaired in PAD. We enrolled 11 symptomatic patients with PAD and 11 age- and sex-matched controls. Participants were evaluated for cognitive performance using the Cambridge Neuropsychological Test Automated Battery and functional near-infrared spectroscopy to assess NVC responses during the cognitive n-back task. Peripheral microvascular endothelial function was evaluated using laser speckle contrast imaging. We found that cognitive performance was compromised in patients with PAD, evidenced by reduced visual memory, short-term memory, and sustained attention. We found that NVC responses and peripheral microvascular endothelial function were significantly impaired in patients with PAD. A positive correlation was observed between microvascular endothelial function, NVC responses, and cognitive performance in the study participants. Our findings support the concept that microvascular endothelial dysfunction and neurovascular uncoupling contribute to the genesis of cognitive impairment in older PAD patients with claudication. Longitudinal studies are warranted to test whether the targeted improvement of NVC responses can prevent or delay the onset of PAD-associated cognitive decline.NEW & NOTEWORTHY Peripheral artery disease (PAD) was associated with significantly decreased cognitive performance, impaired neurovascular coupling (NVC) responses in the prefrontal cortex (PFC), left and right dorsolateral prefrontal cortices (LDLPFC and RDLPFC), and impaired peripheral microvascular endothelial function. A positive correlation between microvascular endothelial function, NVC responses, and cognitive performance may suggest that PAD-related cognitive decrement is mechanistically linked, at least in part, to generalized microvascular endothelial dysfunction and subsequent impairment of NVC responses.

Impaired Endothelial Function in Convalescent Phase of COVID-19: A 3 Month Follow Up Observational Prospective Study.

Background: Endothelial dysfunction has a role in acute COVID-19, contributing to systemic inflammatory syndrome, acute respiratory distress syndrome, and vascular events. Evidence regarding COVID-19 middle- and long-term consequences on endothelium are still lacking. Our study aimed to evaluate if COVID-19 severity could significantly affect the endothelial function after three months from the acute phase. Methods: We assessed endothelial function in outpatients with previous COVID-19 three months after negative SARS-CoV-2 molecular test by measuring flow-mediated dilation (FMD) in patients categorized according to a four-variable COVID-19 severity scale (“home care”; “hospital, no oxygen”; “hospital, oxygen”; “hospital requiring high-flow nasal canula, non-invasive ventilation, invasive mechanical ventilation, or extracorporeal membrane oxygenation”). FMD difference among COVID-19 severity categories was assessed with analysis of variance; we further clarified the relationship between FMD and previous COVID-19 severity with multivariate logistic models. Results: Among 658 consecutive COVID-19 subjects, we observed a significant linear trend of FMD reduction with the increase of the COVID-19 category (p < 0.0001). The presence of endothelial dysfunction was more frequent among hospitalized patients (78.3%) with respect to home-care patients (21.7%; p < 0.0001). COVID-19 severity was associated with increased endothelial dysfunction risk (OR: 1.354; 95% CI: 1.06–1.71; p = 0.011) at multivariate binary logistic analysis. FMD showed a significant direct correlation with PaO2 (p = 0.004), P/F ratio (p = 0.004), FEV1 (p = 0.008), and 6MWT (p = 0.0001). Conclusions: Hospitalized COVID-19 subjects showed an impaired endothelial function three months after the acute phase that correlated with pulmonary function impairment. Further studies are needed to evaluate if these subjects are at higher risk of developing pulmonary disease or future cardiovascular events.

Modified methods of polygepatography and peripheral arterial tonometry in the assessment of peripheral circulation regulation

Objective. The purpose of the work was to assess the application of modified ways of polyhepatography (PHG) and peripheral arterial tonometry (PAT) in the evaluation of regulation of peripheral circulation (capillary blood flow).Design and methods. We included 150 people, divided into four groups. The first group (n = 40) includes patients with stage II hypertension, moderate and high risk of cardiovascular complications. The second group (n = 40) includes patients with stable forms of coronary heart disease in combination with hypertension. The third group (n = 40) includes patients with chronic liver diseases. The fourth group (n = 30) consisted of subjects without anamnestic and objective data of pathology. All subjects underwent a comprehensive clinical and laboratory examination, an assessment of intrahepatic hemodynamics by the PGG method, an assessment of the endothelial function by the PAT method. A modified method of PAT was used to evaluate the central reaction of the peripheral blood flow regulation system (endothelium-independent vasodilation).Results. Endothelial dysfunction was found in patients with cardiovascular pathology and in patients with chronic liver diseases. A modified method of PAT showed a multidirectional reaction of peripheral blood flow to the test with local ischemia. A number of features were identified in the study groups when assessing disorders of intrahepatic microcirculation. Patients of group I had multidirectional disorders of arteriovenous inflow (45% cases, confidence interval (CI) from 27 % to 63 %) and outflow (37,5 %, CI from 22 % to 56 %) in the liver, while in patients of group II and group III, disorders of arteriovenous inflow were more common, 85 % (CI from 70 % to 95%) and 90% (82 % to 94%), respectively. Rheographic signs of bile passage disorders were more common in groups II and III. A significant negative relationship was established between endothelial dysfunction and the severity of intrahepatic microcirculation disorders (r = –0,35, p &lt; 0,001).Conclusions. Modified methods of peripheral arterial tone and PHG enable assessment of local and central mechanisms of blood flow regulation at the microvascular level in patients with hypertension, coronary heart disease and chronic liver diseases. The relationship between impaired endothelial function and intrahepatic microcirculation allows us to consider the liver as a target organ in cardiovascular pathology.

Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

Jumonji domain–containing protein-3 (JMJD3), a histone H3 lysine 27 (H3K27) demethylase, promotes endothelial regeneration, but its function in neointimal hyperplasia (NIH) of arteriovenous fistulas (AVFs) has not been explored. In this study, we examined the contribution of endothelial JMJD3 to NIH of AVFs and the mechanisms underlying JMJD3 expression during kidney failure. We found that endothelial JMJD3 expression was negatively associated with NIH of AVFs in patients with kidney failure. JMJD3 expression in endothelial cells (ECs) was also downregulated in the vasculature of chronic kidney disease (CKD) mice. In addition, specific knockout of endothelial JMJD3 delayed EC regeneration, enhanced endothelial mesenchymal transition, impaired endothelial barrier function as determined by increased Evans blue staining and inflammatory cell infiltration, and accelerated neointima formation in AVFs created by venous end to arterial side anastomosis in CKD mice. Mechanistically, JMJD3 expression was downregulated via binding of transforming growth factor beta 1–mediated Hes family transcription factor Hes1 to its gene promoter. Knockdown of JMJD3 enhanced H3K27 methylation, thereby inhibiting transcriptional activity at promoters of EC markers and reducing migration and proliferation of ECs. Furthermore, knockdown of endothelial JMJD3 decreased endothelial nitric oxide synthase expression and nitric oxide production, leading to the proliferation of vascular smooth muscle cells. In conclusion, we demonstrate that decreased expression of endothelial JMJD3 impairs EC regeneration and function and accelerates neointima formation in AVFs. We propose increasing the expression of endothelial JMJD3 could represent a new strategy for preventing endothelial dysfunction, attenuating NIH, and improving AVF patency in patients with kidney disease.

The effect of an mRNA vaccine against COVID-19 on endothelial function and arterial stiffness

To fight the COVID-19 pandemic, messenger RNA (mRNA) vaccines were the first to be adopted by vaccination programs worldwide. We sought to investigate the short-term effect of mRNA vaccine administration on endothelial function and arterial stiffness. Thirty-two participants (mean age 37 ± 8 years, 20 men) who received the BNT162b2 mRNA COVID-19 vaccine were studied in three sessions in a sequence-randomized, sham-controlled, assessor-blinded, crossover design. The primary outcome was endothelial function (assessed by brachial artery flow-mediated dilatation (FMD)), and the secondary outcomes were aortic stiffness (evaluated with carotid-femoral pulse wave velocity (PWV)) and inflammation (measured by high-sensitivity C-reactive protein (hsCRP) in blood samples). The outcomes were assessed prior to and at 8 h and 24 h after the 1st dose of vaccine and at 8 h, 24 h, and 48 h after the 2nd dose. There was an increase in hsCRP that was apparent at 24 h after both the 1st dose (−0.60 [95% confidence intervals [CI]: −1.60 to −0.20], p = 0.013) and the 2nd dose (maximum median difference at 48 h −6.60 [95% CI: −9.80 to −3.40], p < 0.001) compared to placebo. The vaccine did not change PWV. FMD remained unchanged during the 1st dose but decreased significantly by 1.5% (95% CI: 0.1% to 2.9%, p = 0.037) at 24 h after the 2nd dose. FMD values returned to baseline at 48 h. Our study shows that the mRNA vaccine causes a prominent increase in inflammatory markers, especially after the 2nd dose, and a transient deterioration of endothelial function at 24 h that returns to baseline at 48 h. These results confirm the short-term cardiovascular safety of the vaccine.

Neurobehavioral and neurobiochemical effect of atomoxetine and N-acetylcysteine in streptozotocin diabetes induced endothelial dysfunction and related dementia

Metabolic conditions like diabetes, is a major risk factor for the development of dementia of vascular origin. This study investigates the efficacy of atomoxetine (ATX) and N-acetylcysteine (NAC) in streptozotocin (STZ) diabetes induced vascular endothelium dysfunction and related dementia. Single dose STZ (50 mg/kg i.p) was administered to Albino Wistar rats (male, 200–250 g) by dissolving in citrate buffer. Morris water maze (MWM) and attentional set shifting tests (ASST) were used to assess the spatial learning, memory, reversal learning, and executive functioning in animals. Body weight, serum glucose, serum nitrite/nitrate, vascular endothelial function, aortic superoxide anion, brains’ oxidative markers (thiobarbituric acid reactive species-TBARS, reduced glutathione-GSH, superoxide dismutase-SOD, and catalase-CAT), inflammatory markers (IL-6, IL-10, TNF-α, and myeloperoxidase-MPO), acetylcholinesterase activity-AChE and histopathological changes were also assessed. Atomoxetine - ATX (2 mg kg−1/ 4 mg kg−1) and N-acetylcysteine- NAC (250 mg kg−1/ 500 mg kg−1) were used alone as well as in combination, as the treatment drugs. Donepezil (0.5 mg kg-1) was used as a positive control. STZ administered rats showed increase in serum glucose levels and decrease in body weight. Rats administered with STZ also showed reduction in learning, memory, reversal learning, executive functioning, impairment in endothelial function, increase in brains’ oxidative stress, inflammation, AChE activity and histopathological changes. Administration of ATX and NAC in two different doses as well as in combination, significantly attenuated the STZ induced diabetes induced impairments in the behavioral, endothelial, biochemical parameters and histopathological changes. Co-treatment of ATX and NAC was better in comparison to the doses when given alone. Hence, STZ administration caused diabetes induced dementia of vascular origin which was attenuated by the administration of ATX and NAC. Therefore, these agents may be studied further for the assessment of their full potential in diabetes induced dementia of vascular origin conditions.

Pequi Fruit Extract Increases Antioxidant Enzymes and Reduces Oxidants in Human Coronary Artery Endothelial Cells.

Reactive oxygen species (ROS) imbalance results in endothelial cell function impairment. Natural phenolic antioxidant compounds have been investigated as therapeutic alternatives. The fruit bark of Brazilian-native pequi (Caryocar brasiliense, Camb.) is rich in polyphenols. The HPLC-MS (High-Performance Liquid Chromatography coupled with Mass Spectrometry) analyses identified gallic acid and catechin in six out of seven ethanolic extract samples prepared in our lab. In this study, we examined the effects of ethanolic pequi extract on ROS levels in human coronary artery endothelial cells (HCAEC) subjected to hypoxia or oxidative stress. We first confirmed the oxidant scavenging capacity of the extract. Then, HCAEC pre-incubated with 10 or 25 μg/mL of extract were subjected to hypoxia for 48 h or 100 μM H2O2 for six hours and compared to the normoxia group. Total and mitochondrial ROS levels and cell proliferation were measured. Pequi significantly reduced cytosolic HCAEC ROS levels in all conditions. Mitochondrial ROS were also reduced, except in hypoxia with 10 μg/mL of extract. HCAEC proliferation increased when treated with 25 μg/mL extract under hypoxia and after H2O2 addition. Additionally, pequi upregulated oxidative stress defense enzymes superoxide dismutase (SOD-)1, SOD-2, catalase, and glutathione peroxidase. Together, these findings demonstrate that pequi bark extract increases antioxidative enzyme levels, decreases ROS, and favors HACEC proliferation, pointing to a protective effect against oxidative stress.

Roux-en-Y Gastric Bypass Modulates AMPK, Autophagy and Inflammatory Response in Leukocytes of Obese Patients.

Obesity is characterized by low-grade chronic inflammation, metabolic overload, and impaired endothelial and cardiovascular function. Roux-en-Y gastric bypass (RYGB) results in amelioration of the pro-oxidant status of leukocytes and the metabolic profile. Nevertheless, little is known about the precise mechanism that drives systemic and metabolic improvements following bariatric surgery. In this cohort study, we investigated the effect of RYGB on molecular pathways involving energy homeostasis in leukocytes in 43 obese subjects one year after surgery. In addition to clinical and biochemical parameters, we determined protein expression of systemic proinflammatory cytokines by Luminex®, different markers of inflammation, endoplasmic reticulum (ER) stress, autophagy/mitophagy by western blot, and mitochondrial membrane potential by fluorescence imaging. Bariatric surgery induced an improvement in metabolic outcomes that was accompanied by a systemic drop in hsCRP, IL6, and IL1β levels, and a slowing down of intracellular inflammatory pathways in leukocytes (NF-κB and MCP-1), an increase in AMPK content, a reduction of ER stress (ATF6 and CHOP), augmented autophagy/mitophagy markers (Beclin 1, ATG5, LC3-I, LC3-II, NBR1, and PINK1), and a decrease of mitochondrial membrane potential. These findings shed light on the specific molecular mechanisms by which RYGB facilitates metabolic improvements, highlighting the relevance of pathways involving energy homeostasis as key mediators of these outcomes. In addition, since leukocytes are particularly exposed to physiological changes, they could be used in routine clinical practice as a good sensor of the whole body’s responses.

γ Peptide Nucleic Acid-Based miR-122 Inhibition Rescues Vascular Endothelial Dysfunction in Mice Fed a High-Fat Diet.

The blood levels of microRNA-122 (miR-122) is associated with the severity of cardiovascular disorders, and targeting it with efficient and safer miR inhibitors could be a promising approach. Here, we report the generation of a γ-peptide nucleic acid (γPNA)-based miR-122 inhibitor (γP-122-I) that rescues vascular endothelial dysfunction in mice fed a high-fat diet. We synthesized diethylene glycol-containing γP-122-I and found that its systemic administration counteracted high-fat diet (HFD)-feeding-associated increase in blood and aortic miR-122 levels, impaired endothelial function, and reduced glycemic control. A comprehensive safety analysis established that γP-122-I affects neither the complete blood count nor biochemical tests of liver and kidney functions during acute exposure. In addition, long-term exposure to γP-122-I did not change the overall adiposity, or histology of the kidney, liver, and heart. Thus, γP-122-I rescues endothelial dysfunction without any evidence of toxicity in vivo and demonstrates the suitability of γPNA technology in generating efficient and safer miR inhibitors.

Long term non-invasive endothelial function assessment in patients with paediatric inflammatory multisystem syndrome temporarily associated with Covid 19 (PIMS TS)

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Patients with PIMS-TS present with features of vasculitis (bright coronary arteries and diffuse coronary ectasia on transthoracic echocardiography) and prothrombotic features (e.g. elevated D Dimers) indicating involvement of the endothelial layer in the inflammatory process. Impairment in endothelial function may contribute to the acute but also to possible long-term consequences in patients with PIMS-TS. The aim of this pilot study is to assess non-invasively the endothelial (dys)function using reactive hyperemic peripheral arterial tonometry (RH-PAT) 6 months after the acute inflammatory phase. Methods Ten patients with previous diagnosis of PIMS-TS were compared to age-matched controls. The endothelial function was assessed using the EndoPAT device which provides the reactive hyperemic index (RHI) of endothelial function in a 15-min test. Cardiac function indices by means of LV fractional shortening (FS) was also assessed. Results There were no significant differences regarding age (11.2 ± 3.0 vs 13.6 ± 2.4, p = 0.063), height, weight and body surface area, (BSA: 1.49 ± 0.36 vs 1.52 ± 0.25, p = 0.856) in patients with previous diagnosis of PIMS-TS and controls respectively. The two groups also had similar LV systolic function assessed by FS (36.3 ± 9.1% vs 36.7 ± 7.1%, p = 0.922). The RHI in the PIMS TS group was similar to the control group (1.65 ± 0.43 vs 1.81 ± 0.60, p = 0.533 respectively). Conclusions. Patients with PIMS-TS who may present with features of vasculitis during the acute phase, do not show evidence of endothelial dysfunction during the long term follow-up, suggesting resolution. Further studies are required to accurately determine the endothelial (dys)function during the acute phase of the inflammatory syndrome and course.

Association of COVID-19 with impaired endothelial glycocalyx, vascular function and myocardial efficiency four months after infection

Funding AcknowledgementsType of funding sources: None.AimsSARS-CoV-2 infection may lead to endothelial and vascular dysfunction. Weinvestigated alterations of arterial stiffness, endothelial coronary and myocardialfunction markers four months after COVID-19 infection.MethodsIn a case-control prospective study, we included 100 patients four months after COVID-19 infection, 50 age- and sex-matched healthy individuals. We measured a) pulse wave velocity (PWV), b) flow-mediated dilation (FMD) of brachial artery, c) coronary Flow Reserve (CFR) by Doppler echocardiography d) left ventricular (LV) global longitudinal strain (GLS), e) left ventricular myocardial work index, constructive work, wasted work and work efficiency and e) von-Willenbrand factor and thrombomodulin as endothelial biomarkers.ResultsCOVID-19 patients had lower CFR and FMD values than controls (2.39 ± 0.39 vs 3.31 ± 0.59, p = 0.0122, 5.12 ± 2.95% vs 8.12 ± 2.23%, p = 0.006 respectively). Compared to controls, COVID-19 patients had higher PWV (PWVc-f 12.32 ± 2.44 vs 10.11 ± 1.85 m/sec, p = 0.033) and impaired LV GLS (-19.11 ± 2.14% vs -20.41 ± 1.61%, p = 0.001). Compared to controls, COVID-19 patients had higher myocardial work index, and wasted work (2067.7 ± 325.9 mmHg% vs 1929.4 ± 312.7 mmHg%, p = 0.026, 104.6 ± 58.9 mmHg% vs 75.1 ± 52.6 mmHg%, p = 0.008, respectively), while myocardial efficiency was lower (94.8 ± 2.5% vs 96.06 ± 2.3%, p = 0.008). and thrombomodulin were higher in COVID-19 patients than controls (3716.63 ± 188.36 vs 2590.02 ± 156.51pg/ml, p < 0.001). MDA was higher in COVID-19 patients than controls (10.55 ± 2.45 vs 1.01 ± 0.50 nmole/L, p = 0.001). Residual cardiovascular symptoms at 4 months were associated with oxidative stress markers. Myocardial work efficiency was related with PWV (F=-0.309, p = 0.016) and vWillenbrand (F=-0.541, p = 0.037). Myocardial wasted work was related with PWV (F = 0.255, p = 0.047) and vWillenbrand (F = 0.610, p = 0.016).ConclusionsSARS-CoV-2 may cause vascular dysfunction, followed by a waste of cardiac work, in order to compensate for increased arterial stiffness 4 months after infection.

Isolated diastolic hypertension is not associated with endothelial dysfunction.

Hypertension is associated with an impairment of endothelial function. However, it is unclear whether isolated diastolic hypertension is associated with endothelial dysfunction. The purpose of this study was to investigate the association of endothelial function with isolated diastolic hypertension diagnosed by using two major hypertension guidelines: systolic blood pressure (BP) < 130 mmHg and diastolic BP ≥ 80 mmHg according to the 2017 American College of Cardiology/American Heart Association (ACC/AHA) hypertension guideline and systolic BP < 140 mmHg and diastolic BP ≥ 90 mmHg according to the 2018 European Society of Cardiology/European Society of Hypertension (ESC/ESH) hypertension guideline. We measured the flow-mediated vasodilation (FMD) of the brachial artery in subjects without systolic hypertension who were not treated with antihypertensive drugs. Of 3727 subjects (2813 men; mean age: 41.3 ± 10.9 years), 749 (20.1%) had isolated diastolic hypertension according to the 2017 ACC/AHA definition. Multiple logistic regression analysis revealed that isolated diastolic hypertension was not associated with endothelial dysfunction, defined as FMD < 7.0% (OR, 1.15; 95% CI, 0.98-1.35; P = 0.09). Of 4747 subjects (3727 men; mean age: 45.1 ± 10.8 years), 314 subjects (6.6%) had isolated diastolic hypertension according to the ESC/ESH definition. Multiple logistic regression analysis revealed that isolated diastolic hypertension was not associated with endothelial dysfunction after adjusting for age and sex (OR, 1.04; 95% CI, 0.82-1.32; P = 0.76). Isolated diastolic hypertension was not associated with endothelial dysfunction in individuals who were not treated with antihypertensive drugs regardless of the major hypertension guideline definition used.

The Association of Flow-Mediated Dilatation and Blood Parameters in Primary Raynaud's Phenomenon.

Introduction Raynaud's phenomenon (RP) is a multifactorial disorder. If any underlying disease cannot be determined to be responsible for RP, then it is considered to be the primary RP (pRP). We aimed to investigate the differences between laboratory markers and impaired endothelial function in pRP. Materials and Methods Forty-two pRP patients and 30 healthy individuals were included as the study and control groups, respectively. The endothelial function was evaluated with flow-mediated dilatation (FMD) of the brachial artery. The blood samples were obtained from both groups, and white blood cell (WBC), hemoglobin, platelets, mean platelet volume (MPV), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), D-dimer, fibrinogen, albumin, fibrinogen-to-albumin ratio (FAR), neutrophil-to-lymphocyte ratio (NLR), D-dimer-to-albumin ratio (DDAR), and monocyte chemoattractant protein-1 (MCP-1) parameters were studied. The blood parameters and FMD values obtained were compared between groups. Results The groups were similar in regard to age, gender, and smoking history (p < 0.05). There was no difference between the two groups in regard to hemoglobin, platelet, MPV, creatinine, ALT, D-dimer, albumin, FAR, NLR, and DDAR levels (p < 0.05). AST levels were slightly higher in the pRP group (p=0.027). Markedly increased WBC, fibrinogen, MPV, and MCP-1 values were detected in the pRP group (p=0.001), as well as higher abnormal FMD responses (p=0.001). There was a direct correlation between abnormal FMD response and serum MCP-1 values in patients with pRP (R: 0.308, R2: 0.095, p: 0.044). Conclusion It seems to be that MCP-1 levels are higher in patients with pRP, and increased values of MCP-1 levels seem to be related to impaired endothelial functions.

Comparable Impairment of Vascular Endothelial Function by a Wide Range of Electronic Nicotine Delivery Devices.

Electronic nicotine delivery systems (ENDS; ie, vaping devices) such as e-cigarettes, heated tobacco products, and newer coil-less ultrasonic vaping devices are promoted as less harmful alternatives to combustible cigarettes. However, their cardiovascular effects are understudied. We investigated whether exposure to aerosol from a wide range of ENDS devices, including a new ultrasonic vaping device, impairs endothelial function. We measured arterial flow-mediated dilation (FMD) in rats (n = 8/group) exposed to single session of 10 cycles of pulsatile 5-second exposure over 5 minutes to aerosol from e-liquids with and without nicotine generated from a USONICIG ultrasonic vaping device, previous generation e-cigarettes, 5% nicotine JUUL pods (Virginia Tobacco, Mango, Menthol), and an IQOS heated tobacco product; with Marlboro Red cigarette smoke and clean air as controls. We evaluated nicotine absorption and serum nitric oxide levels after exposure, and effects of different nicotine acidifiers on platelet aggregation. Aerosol/smoke from all conditions except air significantly impaired FMD. Serum nicotine varied widely from highest in the IQOS group to lowest in USONICIG and previous generation e-cig groups. Nitric oxide levels were not affected by exposure. Exposure to JUUL and similarly acidified nicotine salt e-liquids did not affect platelet aggregation rate. Despite lack of heating coil, the USONICIG under airflow conditions heated e-liquid to ~77°C. A wide range of ENDS, including multiple types of e-cigarettes with and without nicotine, a heated tobacco product, and an ultrasonic vaping device devoid of heating coil, all impair FMD after a single vaping session comparably to combusted cigarettes. The need to understand the cardiovascular effects of various ENDS is of timely importance, as we have seen a dramatic increase in the use of these products in recent years, along with the growing assumption among its users that these devices are relatively benign. Our conclusion that a single exposure to aerosol from a wide range of ENDS impairs endothelial function comparably to cigarettes indicates that vaping can cause similar acute vascular functional impairment to smoking and is not a harmless activity.