Arterial Endothelium Research Articles

Ferrostatin-1 Reversed Chronic Intermittent Hypoxia-Induced Ferroptosis in Aortic Endothelial Cells via Reprogramming Mitochondrial Function.

Chronic intermittent hypoxia (CIH) related arterial endothelium injury is a common cause of cardiovascular system injury. However, the mechanism still needs to be clarified. In this study, we aimed to clarify the role and mechanism of ferrostatin-1 (Fer-1) in CIH-related rat arterial endothelial cells (ROAEC) ferroptosis. ROAEC was divided into control group, CIH group, and CIH+ Fer-1 group. Cell viability was detected by cell counting kit 8 kits (CCK8). The apoptotic rate, reactive oxygen species (ROS) levels, Fe2+ levels, and lipid ROS levels were detected by flow cytometry. Malondialdehyde (MDA) levels and nicotinamide adenine dinucleotide (NAD+)/NADH ratio were detected via Elisa kits. The mRNA and protein levels of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected by qRT-PCR and Western blot. Mitochondrial structure and function were observed by transmission electron microscope (TEM) and mitochondrial membrane potential (MMP). Central carbon metabolism was measured to compare metabolites among each group. After the CIH exposure, ROAEC cell viability decreased; The levels of cell apoptosis, ROS, Fe2+, MDA, and lip ROS increased; The levels of NAD+/NADP ratio decreased; The mRNA and protein levels of GPX4 and SLC7A11 decreased (all p<0.05). Co-cultured with Fer-1 reversed the levels of apoptosis rate, cell viability, ROS, Fe2+, MAD, lipid ROS, NAD+/NADH ratio and the mRNA and protein expression of GPX4 and SLC7A11 (all p<0.05). The TEM results showed that damaged mitochondrial membrane and the matrix spillover in the CIH group. The results of the JC-1 assay showed decreased MMP in the CIH group. Fer-1 treatment ameliorated the mitochondrial injury. The results of central carbon metabolism found that CIH altered the metabolites in the TCA cycle, which were reversed by Fer-1 treatment. CIH-induced ferroptosis in ROAEC, which were reversed by Fer-1 via reprogramming mitochondrial function.

O95: The role of the complement system in mediating ischaemic cholangiopathy in DCD liver transplantation

Abstract Background The discrepancy between available organs and the number of patients on the waiting list have led to an increasing number of donor after circulatory death (DCD) liver grafts being used. DCD liver grafts suffer from a higher risk of ischaemic cholangiopathy, the pathophysiology of which is poorly understood. We hypothesized that the complement system mediates damage to the arterial endothelium, thereby affecting the ability of cholangiocytes to regenerate. Methods Livers declined for clinical transplantation were perfused for up to 10 hours with anti-coagulated packed red cells at 37° with or without complement inhibition. Levels of circulating complement components, tissue binding of complement proteins, and organ damage markers were measured. Results 5 DBD and 5 DCD livers were perfused, and 5 further DCD livers being perfused with eculizumab. C3 production was significantly higher in the untreated DCD cohort compared to DBD livers at 10 hours (52.68 mg/ml vs 8.62 mg/ml, p&amp;lt;0.005). C5b-9 deposition on the arterial endothelium of the portal tracts was significantly higher in the untreated DCD cohort compared to the treated DCD livers (mean 0.9 R.U vs 0.4 R.U, p&amp;lt;0.05). There was a non-significant difference in bile production and composition between the treated DCD and untreated DCD cohort. Conclusions There is evidence that the complement system mediates the damage to the arterial endothelium within the portal tracts of the liver. This damage could be ameliorated by complement inhibition. Further work to correlate this findings with clinical outcomes will be required.

Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2O2-induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.

A Study of transcription factor MEF2A gene polymorphisms in patients with coronary artery disease.

MEF2A (myocyte enhancer factor-2A) is a transcription factor of the MEF2 family. It has been recognized as the cause of coronary artery disease in the absence of any other clinical characteristic. It is involved in vascular development and is most commonly found in the coronary artery endothelium. The goal of this case-control study was to see if there was a link between polymorphisms in the MEF2A gene and coronary artery disease. A case-control study was carried out to look into the possible significance of MEF2A polymorphisms as a risk factor for coronary artery disease. This research included 225 patients and 225 healthy controls. A biochemical examination was carried out to evaluate the risk factors for developing this condition. The polymorphisms of Mef2A (1250 C > T in exon 8 and 452 G > T, 481 A > G in exon 11) were found using the PCR-RFLP technique. All identified risk variables, such as hypercholesterolemia, diabetes mellitus, and hypertriglyceridemia, were shown to be statistically significant in the current study for coronary artery disease occurrence. The most polymorphisms were found in MEF2A 1250 C > T, MEF2A 452 G > T, and MEF2A 481 A > G. The genotyping results for MEF2A 1250, MEF2A 452, and MEF2A 481 were (X2 = 2.985; P = 0.235), (X2 = 4.371; P = 0.112), and (X2 = 4.025; P = 0.134), respectively. In conclusion, we identified a much higher incidence of MEF2A in people with coronary artery disease, and MEF2A may play a crucial role in cardiovascular pathophysiology. Patients and controls had considerably different genetics and frequency of alleles.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

FSH Is Responsible for Androgen Deprivation Therapy-Associated Atherosclerosis in Mice by Exaggerating Endothelial Inflammation and Monocyte Adhesion.

Androgen deprivation therapy (ADT) is the mainstay treatment for advanced prostate cancer. But ADTs with orchiectomy and gonadotropin-releasing hormone (GnRH) agonist are associated with increased risk of cardiovascular diseases, which appears less significant with GnRH antagonist. The difference of follicle-stimulating hormone (FSH) in ADT modalities is hypothesized to be responsible for ADT-associated cardiovascular diseases. We administered orchiectomy, GnRH agonist, or GnRH antagonist in male ApoE-/- mice fed with Western diet and manipulated FSH levels by testosterone and FSH supplementation or FSH antibody to investigate the role of FSH elevation on atherosclerosis. By combining lipidomics, in vitro study, and intraluminal FSHR (FSH receptor) inhibition, we delineated the effects of FSH on endothelium and monocytes and the underlying mechanisms. Orchiectomy and GnRH agonist, but not GnRH antagonist, induced long- or short-term FSH elevation and significantly accelerated atherogenesis. In orchiectomized and testosterone-supplemented mice, FSH exposure increased atherosclerosis. In GnRH agonist-treated mice, blocking of short FSH surge by anti-FSHβ antibody greatly alleviated endothelial inflammation and delayed atherogenesis. In GnRH antagonist-treated mice, FSH supplementation aggravated atherogenesis. Mechanistically, FSH, synergizing with TNF-α (tumor necrosis factor alpha), exacerbated endothelial inflammation by elevating VCAM-1 (vascular cell adhesion protein 1) expression through the cAMP/PKA (protein kinase A)/CREB (cAMP response element-binding protein)/c-Jun and PI3K (phosphatidylinositol 3 kinase)/AKT (protein kinase B)/GSK-3β (glycogen synthase kinase 3 beta)/GATA-6 (GATA-binding protein 6) pathways. In monocytes, FSH upregulated CD29 (cluster of differentiation 29) expression via the PI3K/AKT/GSK-3β/SP1 (specificity protein 1) pathway and promoted monocyte-endothelial adhesion both in vitro and in vivo. Importantly, FSHR knockdown by shRNA in endothelium of carotid arteries markedly reduced GnRH agonist-induced endothelial inflammation and atherosclerosis in mice. FSH is responsible for ADT-associated atherosclerosis by exaggerating endothelial inflammation and promoting monocyte-endothelial adhesion.

Diagnostic criteria of endothelial dysfunction, subclinical atherosclerosis and their relationship with NOS3 and HIF1A gene polymorphism in military personnel in extreme conditions of the arctic zone

The state of the arterial vascular endothelium against the background of subclinical atherosclerosis and its relationship with gene polymorphism in military personnel serving in the Arctic climatic zone, depending on the duration of service, was evaluated. In total, 251 male servicemen aged 28–40 years were examined. The group of servicemen of the Arctic climate zone included 99 people, who were divided into three groups according to the duration of military service in the Arctic: group 1 included 8 (8%) servicemen with up to 5 years of military service; group 2, 21 (21%) people with 5–10 years; group 3, 70 (71%) servicemen with 10 years. The control group included 152 military personnel aged 28–40 years from the temperate climate zone. All participants underwent a test with reactive hyperemia, elastography, measurement of the thickness of the intima–media complex on the brachial artery, ultrasound examination of the common carotid arteries, photoplethysmography, pulse oximetry, spirometry, anthropometry, biochemical blood analysis, and determination of gene polymorphism using polymerase chain reaction. The results revealed that endothelium-dependent vasodilation rats in groups 2 and 3 were 1. 7 and 2. 7 times less than that in group 1 (p 0. 05). In 11 (16%) military personnel of group 3, thickening of the intima–media complex was found in 50% of the adjacent areas of the common carotid arteries. In groups 2 and 3, the total blood cholesterol level exceeded the value by 10% and 14%, respectively, compared with group 1 (p 0. 05). The artery wall was rigid in carriers of the T/T genotypes of the HIF1A and NOS3 genes and was most elastic in the G/T genotypes of the NOS3 gene and C/C of the HIF1A gene. In general, military personnel serving in the Arctic climatic zone with a service duration of 10 years have endothelial dysfunction and peripheral vascular stiffness with subclinical atherosclerosis phenomena, particularly in military personnel with the T/T genotypes of HIF1A (rs11549465) and NOS3 (rs1799983). We believe that the leading pathogenetic link of endothelial dysfunction of arterial vessels is the resistance of myocytes to the effects of vasodilation factors expressed by the vascular endothelium, particularly nitric oxide. The longer the service experience of military personnel under conditions of polar hypoxia, the higher the probability of developing endothelial dysfunction.

Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation.

Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by a high mortality rate. Pulmonary arterial endothelium cells (PAECs) serve as a primary sensor of various environmental cues, such as shear stress and hypoxia, but PAEC dysfunction may trigger vascular remodelling during the onset of PH. This study aimed to illustrate the role of Sirtuin 7 (SIRT7) in endothelial dysfunction during PH and explore the potential therapeutic strategy for PH. SIRT7 levels were measured in human and murine experimental PH samples. Bioinformatic analysis, immunoprecipitation, and deacetylation assay were used to identify the association between SIRT7 and Krüpple-like factor 4 (KLF4), a key transcription factor essential for endothelial cell (EC) homeostasis. Sugen5416 + hypoxia (SuHx)-induced PH mouse models and cell cultures were used for the study of the therapeutic effect of SIRT7 for PH. SIRT7 level was significantly reduced in lung tissues and PAECs from PH patients and the SuHx-induced PH mouse model as compared with healthy controls. Pulmonary endothelium-specific depletion of Sirt7 increased right ventricular systolic pressure and exacerbated right ventricular hypertrophy in the SuHx-induced PH model. At the molecular level, we identified KLF4 as a downstream target of SIRT7, which deacetylated KLF4 at K228 and inhibited the ubiquitination-proteasome degradation. Thus, the SIRT7/KLF4 axis maintained PAEC homeostasis by regulating proliferation, migration, and tube formation. PAEC dysfunction was reversed by adeno-associated virus type 1 vector-mediated endothelial overexpression of Sirt7 or supplementation with nicotinamide adenine dinucleotide (NAD)+ intermediate nicotinamide riboside which activated Sirt7; both approaches successfully reversed PH phenotypes. The SIRT7/KLF4 axis ensures PAEC homeostasis, and pulmonary endothelium-specific SIRT7 targeting might constitute a PH therapeutic strategy.

Effects of Herbs on Blood Pressure, Diabetes Mellitus, and Hyperlipidemia

Overweight and obesity in youth is a worldwide public health problem. Overweight and obesity in childhood and adolescents have a substantial effect upon many systems, resulting in clinical conditions such as metabolic syndrome, early atherosclerosis, dyslipidemia, hypertension and type 2 diabetes (T2D). Obesity and the type of body fat distribution are still the core aspects of insulin resistance and seem to be the physiopathologic links common to metabolic syndrome, cardiovascular disease and T2D. In presence of high levels of blood LDL particles, free radicals (ROS), macrophages, cytokines, platelets, histamine, 5HT get involved in LDL oxidation, leading to formation of atherosclerotic plaques which get deposited in endothelium of coronary arteries causing coronary artery disease (CAD). Psyllium fibers and Ficus Carica (FC) are being considered to be useful in treating hyperlipidemia, hypertension, and hyperglycemia. This study was conducted to determine hypotensive, hypolipidemic, and hypoglycemic potential of Psyllium husk and FC. Study was conducted in National hospital, Lahore, Pakistan from January to April 2019. Seventy five hyperlipidemic, hypertensive and diabetic patients were enrolled selected from the hospital with their consent. They were divided in three groups ie 25 patients in each group. Group-1 was on placebo, group-2 was on 15 grams psyllium husk to be used in three divided doses. Group-3 was on 12 grams FC to be taken in three divided doses daily for two months. Their Lipid profile, FBS, and hypertension were determined before and after two months therapy by these two agents. Two analyze data we used SPSS version 10.01.001 2015 and t test was applied to determine changes in the tested parameters. In two months therapy by psyllium husk in 24 patients TG, TC, LDL-c was reduced 21.48, 10.09, 24.17 mg/dl respectively. HDL was increased 12.38 mg/dl in this group. FBS reduction was 28.66 mg/dl, systolic BP 6.30, and diastolic BP reduced in this group 7.52 mm of mg. We then concluded that FC and PH have good enough potential to normalize lipid profile, reduce BP, and FBS in patients suffering from metabolic syndrome.

Development of in vitro microfluidic models to study endothelial responses to pulsatility with different mechanical circulatory support devices.

Continuous-flow ventricular assist devices (CFVAD) and counterpulsation devices (CPD) are used to treat heart failure (HF). CFVAD can diminish pulsatility, but pulsatile modes have been implemented to increase vascular pulsatility. The effects of CFVAD in a pulsatile mode and CPD support on the function of endothelial cells (ECs) are yet to be investigated. In this study, two in vitro microfluidic models for culturing ECs are proposed to reproduce blood pressure (BP) and wall shear stress (WSS) on the arterial endothelium while using these medical devices. The layout and parameters of the two microfluidic systems were optimized based on the principle of hemodynamic similarity to efficiently simulate physiological conditions. Moreover, the unique design of the double-pump and double afterload systems could successfully reproduce the working mode of CPDs in an in vitro microfluidic system. The performance of the two systems was verified by numerical simulations and in vitro experiments. BP and WSS under HF, CFVAD in pulsatile modes, and CPD were reproduced accurately in the systems, and these induced signals improved the expression of Ca2+, NO, and reactive oxygen species in ECs, proving that CPD may be effective in normalizing endothelial function and replacing CFVAD to a certain extent to treat non-severe HF. This method offers an important tool for the study of cell mechanobiology and a key experimental basis for exploring the potential value of mechanical circulatory support devices in reducing adverse events and improving outcomes in the treatment of HF in the future.

FUNDC1/USP15/Drp1 ameliorated TNF-α-induced pulmonary artery endothelial cell proliferation by regulating mitochondrial dynamics.

Mitochondrial dysfunction in pulmonary artery endothelial cells (PAECs) is related to the pathogenesis of pulmonary hypertension (PH). The mitochondrial receptor protein FUN14 domain containing 1 (FUNDC1) was found to be involved in pulmonary artery smooth muscle cell proliferation in PH. However, its role in PAECs remains unclear. We investigated FUNDC1 expression in the pulmonary artery endothelium in both monocrotaline-induced animal models and TNF-α-stimulated cell models. Additionally, the effect of FUNDC1 on PAECs proliferation and its possible mechanism were also investigated. We observed decreased FUNDC1 protein levels in animals and in vitro in PAECs. FUNDC1 deficiency in PAECs upregulated the expression of the deubiquitination enzyme ubiquitin-specific peptidase 15 (USP15), enhanced dynamin-related protein1 (Drp1)-mediated mitochondrial division, and increased mitochondrial ROS levels via the deubiquitination of Drp1. Additionally, FUNDC1 deficiency increased aerobic glycolysis, the production of ATP and lactic acid, and glucose uptake. FUNDC1 overexpression inhibited PAECs proliferation. Moreover, FUNDC1 overexpression in combination with a mitochondrial division or aerobic glycolysis inhibitor enhanced its inhibitory effect on cell proliferation. Our study findings suggest that FUNDC1 deficiency induced by inflammation can promote PAECs proliferation by regulating mitochondrial dynamics and cell energy metabolism via the USP15/Drp1 pathway.

Functional state of the endothelium in men after mine-explosive injury during the war in Ukraine

Mine-explosive trauma (MET) is the most common trauma type and one of the main disability causes of the able-bodied contingent. Among the mechanisms of the cardiovascular and cerebrovascular disorders development after MET, endothelial dysfunction (ED) plays an important role. The study aims - to assess the functional state of the microvessels and large arteries endothelium in men after a mine-explosive injury. Materials and methods: the functional state of the skin microvessels endothelium was determined using a two-channel laser Doppler flowmeter BLF-21D "Transonic Systems Inc" (USA) during the test with the creation of reactive hyperemia. The function of the large arteries endothelium was determined using the D.S. Celermajer test, with post-occlusion reactive hyperemia according to the generally accepted method on the Nemio XG device, SSA-580A (Toshiba) with a high-frequency linear sensor with a central frequency of 7 MHz. We examined men with mild MET (average age 37.7 ± 1.5 years). The control group was represented by 43 practically healthy men without MET (average age 41.8 ± 2.1 years). Results: dysfunction of the microvessels endothelium was found in most persons with MET (69.1%), compared to men without MET (41.9%). Dysfunction of the large vessels endothelium was found in 40.4% of examined persons with MET. Persons with MET have a combination of large and small vessel endothelial dysfunction in 23.4% of cases, at the same time persons without MET have a higher proportion of isolated microvessel endothelial dysfunction (34.9%). Conclusions: men with MET often have arterial endothelial dysfunction with microvascular endothelial dysfunction together, whereas men without MET have predominantly isolated microvascular endothelial dysfunction. ________________________________________________________________________________________ Keywords: mine-explosive injury; functional state of the endothelium; microcirculation.

A Phase II Study to Evaluate the Safety and Efficacy of Defibrotide in Children with High Risk Kawasaki Disease (IND 127812)

Background: Kawasaki disease (KD) is the leading cause of acquired heart disease in children in the developed world. It is an acute, generalized vasculitis, particularly in the coronary arteries, that occurs predominantly in infants and young children (Newburger et al, 2016). The acute phase of KD produces diffuse vascular inflammation that is thought to be associated with activation of the IL-1 pathway. Endothelium, located in the innermost layer of the coronary arteries, serves as an interface between the vascular media and circulating inflammatory cells and inflammatory cytokines. The coronary artery endothelium is the first target of the inflammatory attack in the early stages of KD (Qiu et al, Frontiers Cardiovascular Medicine, 2022) (Liu et al, Molecular Cell Biochem, 2021). There are pathologic changes during an acute arteritis with neutrophilic infiltration leading to necrosis in all the vessel walls, although the usual target is the medium sized extra-parenchymal muscular arteries. Current treatment is directed at reducing inflammation with IVIG and aspirin. Usually, this leads to rapid clinical improvement. However, 10-20% of patients are IVIG resistant and require further methods of reducing inflammation (Newburger et al, 2004). These patients that are IVIG resistant, have recrudescent fevers and evidence of ongoing inflammation, with subsequent increased risk for aneurysm formation (Tremoulet et al, 2008). Furthermore, other particular patient groups have been identified as having a higher risk (HR) of coronary artery aneurysms. Boys have a higher incidence of developing KD, but even accounting for that, are at a higher risk for developing coronary artery aneurysms (Belay et al, 2006). Defibrotide is a polydisperse mixture of predominantly single-stranded oligonucleotides (90% single-stranded and 10% double-stranded) derived from porcine intestinal mucosa. Several in vivo studies indicate that defibrotide primarily targets endothelium, particularly in small vessels, and appears to reduce endothelial cell injury and stabilize endothelial function (Falanga et al, Leukemia, 2003) (Cairo et al, BJH, 2020). Defibrotide is currently approved for SOS/VOD, with renal or pulmonary dysfunction post HSCT and has been well studied in infants with SOS post HSCT (Corbacioglu et al, Lancet, 2012). We hypothesized that Defibrotide will be safe and well tolerated in HR KD patients who fail IVIG and may be effective in difficult to treat KD patients, by mitigating ongoing endothelial dysfunction. Primary Aims: To determine the safety and efficacy of defibrotide in infants with HR KD. Design/Methods: Patients with KD as defined by the American Heart Association, have HR features (Table 1), age birth - 11 years of age, PT/PTT WNL, platelet count ≥100K/mm 3, and without active bleeding, hypersensitivity to defibrotide or receiving systemic anti-coagulant therapy, were eligible. Defibrotide, generously supplied by Jazz Pharmaceuticals, was administered within 96 hours of the conclusion of IVIG at 6.25 mg/kg IV Q6H (total daily dose 25 mg/kg/day) and continued for 7 days or until the patient was discharged from hospital, whichever occurred first (IND 127812) (NCT04777422). Stopping rules included: ≥ 15% grade III-IV allergic reaction or anaphylaxis or hemorrhage. All patients were treated with current standard of care, including: IVIG, aspirin (max 5mg/kg/dose), antibiotics, and intravenous fluids. Complete blood counts, coagulation studies and echocardiogram were performed at baseline, day 10, day 21, and 6 weeks after defibrotide was first given for disease assessment. Results: We have preliminary enrolled 2 patients (mean age of 31 months, range 15 months to 4 years) with a gender ratio of M/F 1/1 (Table 2). The median dose of defibrotide per patient was 5.5 (range 1-11) and the average hospital admission length was 5.5 days (range 5-6 days). No patients demonstrated heart abnormalities on baseline echocardiogram. There were no serious adverse events (SAE) probably or directly related to defibrotide, as evaluated by investigators. One patient had a grade II epistaxis, but no patients had any SAEs. Conclusion: This preliminary data suggests defibrotide is safe in children with HR KD failing IVIG. Further patients will be required to determine the safety and preliminary efficacy of defibrotide in children with HR KD. Supported in part by a research grant from Jazz Pharmaceuticals.

Bacterial butyrate mediates the anti-atherosclerotic effect of silybin

Silybin (SIL) is a versatile bioactive compound used for improving liver damage and lipid disorders and is also thought to be beneficial for atherosclerosis (AS). The goal of this study was to investigate the efficacy of SIL in the treatment of AS in ApoE−/−mice fed a high-fat diet and explore the mechanism underlying treatment outcomes. We found that SIL significantly alleviated AS-related parameters, including the extent of aortic plaque formation, hyperlipidemia, and adhesion molecule secretion in the vascular endothelium. 16 S rRNA gene sequencing analysis, together with the application of antibiotics, showed that intestinal butyrate-producing bacteria mediated the ameliorative effect of SIL on AS. Further analysis revealed that SIL facilitated butyrate production by increasing the level of butyryl-CoA: acetate CoA-transferase (BUT). The increased expression of monocarboxylic acid transporter-1 (MCT1) induced by butyrate and MCT4 induced by SIL in the apical and basolateral membranes of colonocytes, respectively, resulted in enhanced absorption of intestinal butyrate into the circulation, leading to the alleviation of arterial endothelium dysfunction. Moreover, the SIL-mediated increase in intestinal butyrate levels restored gut integrity by upregulating the expression of tight junction proteins and promoting gut immunity, thus inhibiting the AS-induced inflammatory response. This is the first study to show that SIL can alleviate AS by modulating the production of bacterial butyrate and its subsequent absorption.

Obesogenic diet disrupts tissue-specific mitochondrial gene signatures in the artery and capillary endothelium.

Endothelial cells (ECs) adapt to the unique needs of their resident tissue and metabolic perturbations, such as obesity. We sought to understand how obesity affects EC metabolic phenotypes, specifically mitochondrial gene expression. We investigated the mesenteric and adipose endothelium because these vascular beds have distinct roles in lipid homeostasis. Initially, we performed bulk RNA sequencing on ECs from mouse adipose and mesenteric vasculatures after a normal chow (NC) diet or high-fat diet (HFD) and found higher mitochondrial gene expression in adipose ECs compared with mesenteric ECs in both NC and HFD mice. Next, we performed single-cell RNA sequencing and categorized ECs as arterial, capillary, venous, or lymphatic. We found mitochondrial genes to be enriched in adipose compared with mesentery under NC conditions in artery and capillary ECs. After HFD, these genes were decreased in adipose ECs, becoming like mesenteric ECs. Transcription factor analysis revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) had high specificity in NC adipose artery and capillary ECs. These findings were recapitulated in single-nuclei RNA-sequencing data from human visceral adipose. The sum of these findings suggests that mesenteric and adipose arterial ECs metabolize lipids differently, and the transcriptional phenotype of the vascular beds converges in obesity due to downregulation of PPAR-γ in adipose artery and capillary ECs.NEW & NOTEWORTHY Using bulk and single-cell RNA sequencing on endothelial cells from adipose and mesentery, we found that an obesogenic diet induces a reduction in adipose endothelial oxidative phosphorylation gene expression, resulting in a phenotypic convergence of mesenteric and adipose endothelial cells. Furthermore, we found evidence that PPAR-γ drives this phenotypic shift. Mining of human data sets segregated based on body mass index supported these findings. These data point to novel mechanisms by which obesity induces endothelial dysfunction.

Tsantan Sumtang, a traditional Tibetan medicine, protects pulmonary vascular endothelial function of hypoxia-induced pulmonary hypertension rats through AKT/eNOS signaling pathway

Ethnopharmacological relevanceTsantan Sumtang (TS), originated from the Four Tantras, is an empirical Tibetan medicine prescription, which has been widely used for treating cardiovascular diseases in the clinic in Qinghai Province of China. Our previous studies found that TS alleviated hypoxia-induced pulmonary hypertension (HPH) in rats. However, the effect and bioactive fractions of TS on hypoxia-injured pulmonary vascular endothelium are unknown. Aim of the studyTo investigate the effect, bioactive fractions and pharmacological mechanism of TS on hypoxia-injured pulmonary vascular endothelium in vivo and in vitro. Materials and methodsIn vivo studies, HPH animal model was established, and TS was administrated for four weeks. Then, hemodynamic indexes, ex vivo pulmonary artery perfusion experiment, morphological characteristics, nitric oxide (NO) production, and the protein expression of protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) and AMP-activated protein kinase (AMPK)/eNOS signaling were determined. In vitro studies, 1% O2-induced pulmonary artery endothelial cells (PAECs) injury model was applied for screening bioactive fractions of TS by cell proliferation assay and NO production measurement. The associated proteins of AKT/eNOS signaling were further measured to elucidate underlying mechanism of bioactive fraction of TS via using phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. Ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) was used to reveal the chemical profile of bioactive fraction of TS. ResultsTS showed protective effect on the integrity of distal pulmonary arterial endothelium in HPH rats. Tsantan Sumtang dilated pulmonary arterial rings in HPH rats. TS enhanced NO bioavailability in lung tissue via regulating AKT/eNOS signaling. Furthermore, in the cellular level, cell viability as well as NO content of hypoxia-injured PAECs were elevated by fraction 17 of water extract of TS (WTS), through activating the AKT/eNOS signaling. Ellagic acid could be one of compositions in fraction 17 of WTS to produce NO in hypoxia-injured PAECs. ConclusionTS restored pulmonary arterial endothelial function in HPH rats. The bioactive fraction 17 was screened, which protected hypoxia-injured PAECs via upregulating AKT/eNOS signaling.

EPCO-48. THE SINGLE-CELL AND SPATIAL TRANSCRIPTOMIC ARCHITECTURE OF MENINGEAL SOLITARY FIBROUS TUMORS PHENOCOPIES CEREBRAL VASCULAR DEVELOPMENT AND HOMEOSTASIS

Abstract INTRODUCTION Meningeal solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms that are associated with local recurrence and distant metastasis. The cell states and spatial transcriptomic architecture of SFTs are unknown. METHODS Single-cell RNA sequencing or spatial transcriptomic sequencing were performed on 40,022 cells or 23,682 spatial transcriptomes from 12 meningeal SFT samples. Datasets were corrected for batch effects using Harmony and integrated using uniform maniform approximation and projection (UMAP). Clusters were defined using automated cell type classification, cell signature gene sets, cell cycle analysis, and differentially expressed marker genes. Trajectory analyses were performed using RNA velocity and pseudotime. Cell-cell communication analysis was performed using CellChat. RESULTS were validated using immunofluorescence and immunohistochemistry and generalized using comparisons to 30,934 single-cell transcriptomes from 6 meningioma samples, 139,134 perinatal human brain vascular single-cell transcriptomes, 84,138 adult human brain vascular single-cell transcriptomes, and DNA methylation profiles from 8 SFT and 221 meningioma samples. RESULTS Unsupervised hierarchical clustering of DNA methylation profiles revealed molecular distinction of meningeal SFTs from meningiomas. Deconvolution of vascular single-cell types showed SFT cell clusters resembling perinatal or adult fibroblasts, perinatal mitotic endothelia, and adult venous or arterial endothelia. UMAP and trajectory analyses showed SFTs were comprised of 8 interchangeable tumor cell states that were associated with cell adhesion (VCAM1, NCAM2), cell stress (EGR1), cell signaling (NOTCH3), extracellular matrix remodeling (ECM) (ADAMTS6, PLCG2), or protein synthesis pathways (RPL27A, RPL37). Cell-cell communication analyses identified single-cell and spatial interactions between VCAM1- or NCAM2-expressing SFT cells and endothelia or immature neurons in the tumor microenvironment, respectively. Single-cell deconvolution demonstrated evolution of ECM and protein synthesis SFT cells in paired primary/recurrent samples, and evolution of ECM SFT cells and macrophages in paired primary/metastatic samples. CONCLUSIONS The single-cell and spatial transcriptomic architecture of meningeal SFT reveals dynamic cell states that phenocopy cerebral vascular development and homeostasis.

Endothelial Hemoglobin Alpha Mediates Iron Regulation of Endothelial Nitric Oxide

Iron deficiency is associated with heart failure, chronic kidney disease, and chronic inflammation. All of these pathologies are associated with endothelial dysfunction and impaired nitric oxide (NO) signaling. Previous clinical studies have found iron deficiency anemia (IDA) increases endothelial NO signaling and attributed this change to the reduction in circulating hemoglobin. However, while free hemoglobin is a potent scavenger of NO, it has been shown in ex vivo preparations that endothelial NO signaling is unaffected by the presence of erythrocytes suggesting changes in circulating hemoglobin do not alter NO signaling. For these reasons we focused on endothelial iron regulation. Using single cell RNA sequencing data of isolated mesenteric and adipose endothelial cells generated by our lab, we have observed arterial endothelial cells have higher transferrin receptor expression compared to capillary, veinous, and lymphatic endothelial cells. This suggests that the arterial endothelium, which controls blood flow and is especially susceptible to endothelial dysfunction, may be particularly sensitive to changes in iron status. Additionally, our lab has previously shown the alpha chain of hemoglobin (Hbα) is expressed in the endothelium of small arteries and scavenges NO. We therefore hypothesized endothelial Hbα, rather than blood hemoglobin is responsible for the increased NO signaling in IDA. We first tested this hypothesis in a mouse model of IDA in which we were able to replete vascular iron stores. C57BL/6 mice were fed an iron deficient (2-6 ppm Fe) or nutrient matched control diet (48 ppm Fe) beginning at weaning. IDA mice underwent phlebotomies (10% blood volume) at 9 and 11 weeks old to aid in the progression of anemia. To restore vascular iron stores, a subset of IDA mice received a single i.p. injection of iron dextran (FeDex; 20 mg/kg) at 12 weeks. One week later complete blood counts were assessed, tissues were collected, and blood flow studies were performed. As expected, IDA mice had low blood hemoglobin (Con: 15.54 ± 0.43 vs. IDA: 6.04 ± 0.35 g/dL; p &amp;lt; 0.0001). FeDex did not rescue the anemia (8.55 ± 0.74 g/dL; p &amp;lt; 0.0001 vs. Con), but did rescue vascular iron stores as measured by ferritin light chain protein. To investigate NO signaling, we used laser speckle contrast imaging to measure changes in in vivo blood flow in response to the NO synthase inhibitor L-NAME. The magnitude of the response to L-NAME is an indicator of NO signaling. IDA mice exhibited an exaggerated response to L-NAME indicating increased NO signaling. Rescuing vascular iron stores with FeDex restored the L-NAME response back to control levels suggesting vascular iron stores, rather than the anemia itself increased NO signaling. In order to investigate the role of endothelial Hbα, we repeated these studies in endothelial specific Hbα knockout mice (Hba1 fl/fl-Cdh5-CreER T2+) developed by us. Lack of endothelial Hbα did not prevent the increased response to L-NAME in IDA mice, but did prevent the rescue by FeDex. These data suggest a model in which endothelial Hbα participates in regulating NO in response to iron, but not the increased NO in IDA.

Advancements in the Regulation of Different-Intensity Exercise Interventions on Arterial Endothelial Function.

Normal-functioning endothelium is crucial to maintaining vascular homeostasis and inhibiting the development and progression of cardiovascular diseases such as atherosclerosis. Exercise training has been proven effective in regulating arterial endothelial function, and the effect of this regulation is closely related to exercise intensity and the status of arterial endothelial function. With this review, we investigated the effects of the exercise of different intensity on the function of arterial endothelium and the underlying molecular biological mechanisms. Existing studies indicate that low-intensity exercise improves arterial endothelial function in individuals who manifest endothelial dysfunction relative to those with normal endothelial function. Most moderate-intensity exercise promotes endothelial function in individuals with both normal and impaired arterial endothelial function. Continuous high-intensity exercise can lead to impaired endothelial function, and high-intensity interval exercise can enhance both normal and impaired endothelial function. In addition, it was demonstrated that the production of vasomotor factors, oxidative stress, and inflammatory response is involved in the regulation of arterial endothelial function under different-intensity exercise interventions. We posit that this synthesis will then provide a theoretical basis for choosing the appropriate exercise intensity and optimize the prescription of clinical exercise for persons with normal and impaired endothelium.

Japanese Quince Fruit Juice Exerts a Cardioprotective Effect in a Model of Diet-Induced Metabolic Syndrome in Rats

Summary The current study aimed to evaluate the influence of Japanese quince (Chaenomeles japonica) fruit juice (JQFJ) on the myocardium and on the coronary arteries of rats with diet-induced metabolic syndrome (MS). Male Wistar rats (n=50) were divided into 5 groups: Control, MS, MS+JQFJ2.5, MS+JQFJ5 and MS+JQFJ10. MS was induced with a high-fat high-fructose diet for 10 weeks. During that period, all animals were daily orally treated with distilled water (Control and MS groups) or with JQFJ at doses of 2.5 ml/kg, 5 ml/kg and 10 ml/kg (the other three groups, respectively). At the end of the experiment, the myocardium and coronary arteries were examined histopathologically. In group MS, necrotic endothelial cells and exposed basal membrane were observed. JQFJ at 2.5 ml/kg reduced the impairment but activated endothelial cells were still found. JQFJ at 5 ml/kg and 10 ml/kg prevented coronary artery endothelium damage, preserving the normal morphology. The myocardium in MS group presented with cardiomyocyte degeneration and increased distance between the cells. In group MS+JQFJ2.5, the degeneration was decreased. In groups MS+JQFJ5 and MS+JQFJ10, the histology of the myocardium resembled that of the control group. In the current histopathological evaluation, JQFJ prevented the MS-induced impairment of myocardium and coronary arteries.