eNOS Expression Research Articles

Hydrogen sulfide prevents the vascular dysfunction induced by severe traumatic brain injury in rats by reducing reactive oxygen species and modulating eNOS and H2S-synthesizing enzyme expression

AimTo assess the effects of subchronic administration with NaHS, an exogenous H2S donor, on TBI-induced hypertension and vascular impairments. Main methodsAnimals underweministration does not prevent the body weight loss but slightly imnt a lateral fluid percussion injury, and the hemodynamic variables were measured in vivo by plethysmograph method. The vascular function in vitro, the ROS levels by the DCFH-DA method and the expression of H2S-synthesizing enzymes and eNOS by Western blot were measured in isolated thoracic aortas at day 7 post-TBI. The effect of L-NAME on NaHS-induced effects in vascular function was evaluated. Brain water content was determined 7 days after trauma induction. Body weight was recorded throughout the experimental protocol, whereas the sensorimotor function was evaluated using the neuroscore test at days −1 (basal), 2, and 7 after the TBI induction. Key findingsTBI animals showed: 1) an increase in hemodynamic variables and ROS levels in aortas; 2) vascular dysfunction; 3) sensorimotor dysfunction; and 4) a decrease in body weight, the expression of H2S-synthesizing enzymes, and eNOS phosphorylation. Interestingly, NaHS subchronic administration (3.1 mg/kg; i.p.; every 24 h for six days) prevented the development of hypertension, vascular dysfunction, and oxidative stress. L-NAME abolished NaHS-induced effects.Furthermore, NaHS treatment restored H2S-synthesizing enzymes and eNOS phosphorylation with no effect on body weight, sensorimotor impairments, or brain water content. SignificanceTaken together, these results demonstrate that H2S prevents TBI-induced hypertension by restoring vascular function and modulating ROS levels, H2S-synthesizing enzymes expression, and eNOS phosphorylation.

Chrysanthemum coronarium L. Protects against Premature Senescence in Human Endothelial Cells.

The senescence of vascular endothelial cells (EC) leads to vascular dysfunction. However, the molecular mechanisms of EC senescence and its associated pathophysiological changes have not yet been clearly studied. This study sought to inspect the Chrysanthemum coronarium L. (CC) extract's mechanism in preventing premature senescence of EC. A senescent endothelial cell model was created in human umbilical vein endothelial cells (HUVECs) with 100 µmol/L H2O2 treatment for 24 h. The effect of CC on senescent HUVECs was elucidated by measuring the activity of β-galactosidase (SA-β-gal), which exhibits an aging-related phenotype. SA-β-gal activity increased to 13.2 ± 2.85% in H2O2-treated HUVECs, whereas this activity was attenuated in the CC group. Immunoblot analyses revealed that p21, p53, and PAI-1 levels increased in the senescent HUVECs; however, the levels decreased in the HUVECs treated with various concentrations of CC (10, 20, and 50 μg/mL). The CC extract reduced the production of reactive oxygen species and reversed the decrease in NO production. Additionally, pretreatment with an Nω-nitro-l-arginine methyl ester (eNOS inhibitor) and nicotinamide (sirtuin 1 inhibitor) inhibited the anti-senescent effect of CC extract in HUVECs. Taken together, this study validated the novel endothelial protective effect of CC extract and its prevention of senescence in HUVECs through the mechanism regulated by eNOS and SIRT1 expression.

Effects of exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells on full-thickness skin defect in diabetic mice

Effects of exosomes from hepatocyte growth factor-modified human adipose mesenchymal stem cells on full-thickness skin defect in diabetic mice

Spirulina platensis Ameliorates Oxidative Stress Associated with Antiretroviral Drugs in HepG2 Cells.

Lately, Spirulina platensis (SP), as an antioxidant, has exhibited high potency in the treatment of oxidative stress, diabetes, immune disorder, inflammatory stress, and bacterial and viral-related diseases. This study investigated the possible protective role of Spirulina platensis against ARV-induced oxidative stress in HepG2 cells. Human liver (HepG2) cells were treated with ARVs ((Lamivudine (3TC): 1.51 µg/mL, tenofovir disoproxil fumarate (TDF): 0.3 µg/mL and Emtricitabine (FTC): 1.8 µg/mL)) for 96 h and thereafter treated with 1.5 µg/mL Spirulina platensis for 24 h. After the treatments, the gene and protein expressions of the antioxidant response pathway were determined using a quantitative polymerase chain reaction (qPCR) and Western blots. The results show that Spirulina platensis decreased the gene expressions of Akt (p < 0.0001) and eNOS (↓p < 0.0001) while, on the contrary, it increased the transcript levels of NRF-2 (↑p = 0.0021), Keap1 (↑p = 0.0002), CAT (↑p < 0.0001), and NQO-1 (↑p = 0.1432) in the HepG2 cells. Furthermore, the results show that Spirulina platensis also decreased the protein expressions of NRF-2 (↓p = 0.1226) and pNRF-2 (↓p = 0.0203). Interestingly, HAART-SP induced an NRF-2 pathway response through upregulating NRF-2 (except for FTC-SP) (↑p < 0.0001), CAT (↑p < 0.0001), and NQO-1 (except for FTC-SP) (↑p < 0.0001) mRNA expression. In addition, NRF-2 (↑p = 0.0085) and pNRF-2 (↑p < 0.0001) protein expression was upregulated in the HepG2 cells post-exposure to HAART-SP. The results, therefore, allude to the fact that Spirulina platensis has the potential to mitigate HAART-adverse drug reactions (HAART toxicity) through the activation of antioxidant response in HepG2 cells. We hereby recommend further studies on Spirulina platensis and HAART synergy.

Cytokine Adsorber Use during DCD Heart Perfusion Counteracts Coronary Microvascular Dysfunction.

Microvascular dysfunction (MVD) in cardiac allografts is associated with an impaired endothelial function in the coronary microvasculature. Ischemia/reperfusion injury (IRI) deteriorates endothelial function. Hearts donated after circulatory death (DCD) are exposed to warm ischemia before initiating ex vivo blood perfusion (BP). The impact of cytokine adsorption during BP to prevent MVD in DCD hearts is unknown. In a porcine DCD model, we assessed the microvascular function of hearts after BP with (DCD-BPCytoS, n = 5) or without (DCD-BP, n = 5) cytokine adsorption (CytoSorb®). Microvascular autoregulation was assessed by increasing the coronary perfusion pressure, while myocardial microcirculation was measured by Laser-Doppler-Perfusion (LDP). We analyzed the immunoreactivity of arteriolar oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal (HNE), endothelial injury indicating cell adhesion molecules CD54, CD106 and CD31, and eNOS. We profiled the concentration of 13 cytokines in the perfusate. The expression of 84 genes was determined and analyzed using machine learning and decision trees. Non-DCD hearts served as a control for the gene expression analysis. Compared to DCD-BP, relative LDP was improved in the DCD-BPCytoS group (1.51 ± 0.17 vs. 1.08 ± 0.17). Several pro- and anti-inflammatory cytokines were reduced in the DCD-BPCytoS group. The expression of eNOS significantly increased, and the expression of nitrotyrosine, HNE, CD54, CD106, and CD31, markers of endothelial injury, majorly decreased in the DCD-BPCytoS group. Three genes allowed exact differentiation between groups; regulation of HIF1A enabled differentiation between perfusion (DCD-BP, DCD-BPCytoS) and non-perfusion groups. CAV1 allowed differentiation between BP and BPCytoS. The use of a cytokine adsorption device during BP counteracts preload-dependent MVD and preserves the microvascular endothelium by preventing oxidative stress and IRI of coronary arterioles of DCD hearts.

Optimization of an Ex-Vivo Human Skin/Vein Model for Long-Term Wound Healing Studies: Ground Preparatory Activities for the 'Suture in Space' Experiment Onboard the International Space Station.

This study is preliminary to an experiment to be performed onboard the International Space Station (ISS) and on Earth to investigate how low gravity influences the healing of sutured human skin and vein wounds. Its objective was to ascertain whether these tissue explants could be maintained to be viable ex vivo for long periods of time, mimicking the experimental conditions onboard the ISS. We developed an automated tissue culture chamber, reproducing and monitoring the physiological tensile forces over time, and a culture medium enriched with serelaxin (60 ng/mL) and (Zn(PipNONO)Cl) (28 ng/mL), known to extend viability of explanted organs for transplantation. The results show that the human skin and vein specimens remained viable for more than 4 weeks, with no substantial signs of damage in their tissues and cells. As a further clue about cell viability, some typical events associated with wound repair were observed in the tissue areas close to the wound, namely remodeling of collagen fibers in the papillary dermis and of elastic fibers in the vein wall, proliferation of keratinocyte stem cells, and expression of the endothelial functional markers eNOS and FGF-2. These findings validate the suitability of this new ex vivo organ culture system for wound healing studies, not only for the scheduled space experiment but also for applications on Earth, such as drug discovery purposes.

Influenza A(H1N1)pdm09 Virus Alters Expression of Endothelial Factors in Pulmonary Vascular Endothelium in Rats.

Influenza virus infection may cause endothelial activation and dysfunction. However, it is still not known to what extent the influenza virus can dysregulate the expression of various endothelial proteins. The aim of the study is to identify the level of expression of endothelial nitric oxide synthase (eNOS), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (tPA) in the pulmonary vascular endothelium, as well as the concentration of PAI-1 and tPA in the blood plasma in Wistar rats. Animals were intranasally infected with rat-adapted influenza A(H1N1)pdm09 virus. The expression of eNOS, PAI-1 and tPA in the pulmonary vascular endothelium was determined by immunohistochemistry; the concentration of PAI-1 and tPA was analyzed by ELISA at 24 and 96 h post infection (hpi). Thus, the expression of eNOS in the pulmonary vascular endothelium decreased by 1.9-fold at 24 hpi and increased by 2-fold at 96 hpi. The expression of PAI-1 in the pulmonary vascular endothelium increased by 5.23-fold and 6.54-fold at 24 and 96 hpi, respectively. The concentration of PAI-1 in the blood plasma of the rats decreased by 3.84-fold at 96 hpi, but not at 24 hpi. The expression of tPA in the pulmonary vascular endothelium was increased 2.2-fold at 96 hpi. The obtained data indicate the development of endothelial dysfunction that is characterized by the dysregulation of endothelial protein expression in non-lethal and clinically non-severe experimental influenza virus infection.

The effect of varied exercise intensity on antioxidant function, aortic endothelial function, and serum lipids in rats with non-alcoholic fatty liver disease.

This study aimed to compare the effects of diet and exercise of different intensities on antioxidant function, aortic endothelial cell function and serum lipids in NAFLD (nonalcoholic fatty liver disease) rats. Fifty Sprague-Dawley (SD) rats (180-220g) were randomly divided into two experimental groups and fed either a standard rodent chow diet (CON; n=10) or a high-fat diet (HFD; n=40). After 16 weeks, the animals that received the HFD were randomly separated into a high-fat control group (HFC; n=10) or three ex-ercise training groups: HFD and low-intensity exercise (LE; n=10), HFD and moderate-intensity exercise (ME; n=10), and HFD and incremental intensity exercise (IE; n=10). These experimental rats keep sedentary or trained for the next six weeks. A detection kit was used to detect nitric oxide synthase (NOs), nitric oxide (NO), malondialdehyde (MDA) and other markers of aor-tic oxidative stress. The expression levels of endothelial nitric oxide synthase (e-NOS) and endothelin-1 (ET-1) were detected by immunohistochemistry. TC, TG, and other lipid metabolism parameters were detected by an auto-matic analyzer. Exercise with different intensities could improve lipid me-tabolism, enhance antioxidant function, reduce MDA (P<0.01), increase NO (P<0.01), and improve the expression of e-NOS and ET-1 (P<0.01) protein levels in NAFLD rats. Decreased blood lipids were exhibited in all exercise groups. Notably, the moderate-intensity exercise demonstrated more effecton increasing glutathione (GSH) contents (P<0.01) and decreased the ex-pression of ET-1protein levels (P<0.01). The results showed that exercise at different intensities improved lipid metabolism and enhanced anti-oxidation function. Moderate exercise could improve the function of aortic endothelial cells.

Abstract 9578: Chronic Phosphodiesterase Type 5 Inhibition by Sildenafil Causes Regression of Heart Failure: Insights into Molecular and Cellular Mechanisms

Background: Sildenafil (SIL), a selective inhibitor of PDE5 has been shown to exert profound beneficial effects in heart failure (HF). However, whether and by what mechanisms SIL can stop the progression of HF, or maybe even reverse the clinical route remain unknown. We tested the hypothesis that chronic SIL would improve intrinsic myocyte function and [Ca 2+ ] i regulation and reverse HF-induced detrimental alterations on cardiac SERCA2a, β-adrenergic receptors (AR) and nitric oxide synthase (NOS) isoforms, thus playing a salutary role in HF. Methods: We evaluated LV and myocyte function and the protein levels of myocyte β 1 - and β 3 - AR, SR Ca 2+ -ATPase (SERCA2a), phospholamban (PLB), and 3 NOS in 3 groups of male rats (8/group) for a period of 3 months (M): 1) HF , 3 M after receiving isoproterenol (ISO) (170 mg/kg sq for 2 days); 2) HF/SIL , 2 M after ISO, then SIL (70 μg/kg/day sq via mini pump) was initiated and given for 1 M; and 3) Controls . Results: Versus C, ISO-treated rats progressed to severe HF at 3 M with significantly reduced LV contractility (E ES , 0.7 vs 1.1 mmHg/μl), and slowed LV relaxation. LV myocyte contraction (dL/dt max , 77 vs 136 μm/sec), relaxation (dR/dt max , 62 vs 104 μm/sec) and [Ca 2+ ] iT (0.15 vs 0.24) were significantly decreased accompanied by a diminished myocyte inotropic response to ISO (10 -8 M) superfusion. These abnormalities were associated with concomitant significant decreases in myocyte protein levels of β 1 -AR (0.23 vs 0.64), SERCA2a (0.46 vs 0.80), PLB Ser16 /PLB ratio, and eNOS (0.28 vs 0.46), but increased protein levels of β 3 -AR (0.23 vs 0.10) and iNOS (0.18 vs 0.08). Chronic SIL treatment prevented HF-caused reduced E ES (1.3 mmHg/μl) and EF (62%) and retained normal dL/dt max (131μm/s), dR/dt max (106 μm/s), and [Ca 2+ ] iT (0.24), and restored normal myocyte contractile response to ISO stimulation. With SIL, protein levels of myocyte β 1 - and β 3 -AR, SERCA2a were normalized to control values, but eNOS was significantly elevated (0.77). Conclusions: Chronic SIL prevents HF-caused downregulation of cardiac β 1 -AR and reverses contrast changes between iNOS and β 3 -AR with SERCA2a and eNOS expression, restores normal myocyte function, [Ca 2+ ] iT , and β-adrenergic reserve, and leads to regression of LV dysfunction in a rat model of progressive HF.

Abstract 15377: Investigating the Role of Breast Cancer Susceptibility Gene 2 in High Glucose-Induced Endothelial Dysfunction

Introduction and Hypothesis: Mutations in the breast cancer susceptibility gene 2 (BRCA2) impair DNA damage repair which can eventually culminate in cancer, inflammation and/or apoptosis. Hyperglycemia-induced oxidative stress triggers DNA damage, inflammation and apoptosis, which are common contributors to endothelial dysfunction in diabetes. Here, we assessed the hypothesis that BRCA2 plays critical role in hyperglycemia-induced endothelial dysfunction. Methods and Results: Human umbilical vein endothelial cells (HUVECs) were exposed to high- (HG; 25 mM) or normal glucose (NG; 5 mM) and BRCA2 expression was measured. Our data showed significant BRCA2 upregulation in HG-treated HUVECs. Next, we successfully silenced BRCA2 in HUVECs, and measured the expression levels of p53 and p21 in HG- and NG-treated BRCA2-silenced HUVECs. We observed a significant upregulation of p53 and p21 in BRCA2-deficient HUVECs, which was further augmented by HG. Next, we measured the migratory and angiogenic potential of control and BRCA2-seilenced HUVECs under NG and HG. Our preliminary data showed that HG and loss of BRCA2 together inhibited the migratory capacity and angiogenic potential of HUVECs. To understand the mechanism behind the effect of HG on BRCA2-deficient ECs, we measured expression levels of endothelial nitric oxide synthase (eNOS). Both expression and activation of eNOS was reduced in HG-treated BRCA2-deficient HUVECs. To understand the effect of loss of BRCA2 on overall endothelial transcriptome and to understand mechanisms, we performed RNA sequencing on scrambled control- and siBRCA2-transfected HUVECs. Interestingly, a total of 3150 genes were differentially expressed in BRCA2-deficient HUVECs compared to the control group. Conclusion: Collectively, these findings reveal a novel role for BRCA2 as a regulator of endothelial function in context of HG treatment in vitro . Our RNA sequencing data shows theimportance of BRCA2 in regulating endothelial transcriptome and overall endothelial function. Further studies are underway; however, these results may provide important implications concerning the susceptibility of BRCA2 mutation carriers to HG-induced endothelial dysfunction as well as other CVDs.

Abstract 10537: A Comparison of Endothelial Nitric Oxide Synthase(eNOS)-Based Platelet Subpopulation Ratios and Platelet VEGF Content Within Covid-19

Background: Nitric oxide (NO) regulates both thrombosis and vascular leakage, two of the major complications of severe Covid19. Platelet-derived NO inhibits adhesion/aggregation and within microvascular endothelial cells NO-signalling mediates vascular endothelial growth factor (VEGF/VPF)-induced permeability. Recently, we identified novel platelet subpopulations with a differential ability to produce NO and the presence/absence of endothelial nitric oxide synthase (eNOS). We showed that eNOS-negative (eNOS neg ) platelets initiate adhesion and aggregation, while eNOS-positive (eNOS pos ) platelets limit aggregate growth. As inflammatory cytokines are known to counter-regulate eNOS and VEGF expression, we hypothesized that the ratio of eNOS-negative to -positive platelets may be increased in COVID-19 patients along with their VEGF content. Aims: To determine the eNOS-positive to -negative platelet ratio and platelet VEGF levels within the blood of hospitalized COVID-19 patients. Methods: Platelets were isolated from age- and sex-matched COVID-19 patients (ICU and non-ICU) and COVID-negative healthy controls. Isolated platelets were intracellularly stained for eNOS, VEGF, and for surface CD62P and analyzed using flow cytometry. A multiplex assay was used to measure plasma cytokines. Results: The percentage of eNOS neg platelets within the blood of COVID-19 patients (ICU and non-ICU) was higher than that within healthy controls and their levels correlated with disease severity (81.2±2.8% ICU vs. 66.0 ±3.1% non-ICU vs. 6.1± 1.3% controls, P -value < 0.0001). Accordingly, the percentage of eNOS pos platelets in COVID-19 patients was lower compared to controls, while the platelet VEGF content and surface CD62P of COVID-19 patients in ICU was higher than that of healthy controls. Further, COVID-19 patients demonstrated higher TNFα, IL-6, and IL-1β plasma concentrations than controls. Conclusion: Preliminary data suggests that eNOS-negative to -positive platelet ratios and VEGF content increase with COVID-19 severity, potentially predisposing patients to thrombosis and enhanced vascular permeability upon platelet activation. These platelet changes may be due to the actions of inflammatory cytokines on megakaryocytes.

Abstract 15352: Lung Endothelial Transport in Glypican 1 Knockout Mice

Introduction: Pulmonary edema is a life-threatening condition that can result from the acute increase in hemodynamic forces in the lung vasculature. The mechanisms whereby pulmonary edema develops are unresolved but increased lung endothelial permeability may be important. The endothelial glycocalyx is a surface layer known to mediate the effects of mechanical forces in the lung vasculature by eNOS-dependent mechanisms. Among the components of the glycocalyx, glypican 1 (Gpc1) is considered a potential mechano-transducer due to its physical interaction with the cellular membrane. Herein we sought to investigate the mechanisms whereby Gpc1 regulates pulmonary permeability. Hypothesis: We hypothesize that Gpc1 regulates endothelial transport by eNOS-dependent mechanisms. Methods: Mouse lung endothelial cells (MLEC) isolated from male and female wild-type (WT) and Gpc1 knockout mice (Gpc1KO, 6-8 weeks old) were used. Endothelial transport was assessed by dextran permeability in vitro, lung barrier stability was assessed by TEER, eNOS pathway was assessed by immunoblotting. To assess the role of eNOS to Gpc1 effects on endothelial transport, MLEC were exposed to an eNOS decoy peptide. Results: Gpc1KO MLEC formed a tighter barrier in vitro when compared to WT MLEC (3.18±0.11 [Gpc1KO] vs 2.34±0.11 [WT], normalized TEER, n=3/group). This was preserved during high pressure conditions. High pressure increased transcellular but not paracellular transport in both WT and Gpc1KO MLEC (1.8 fold in WT and 1.2 fold in Gpc1KO, n=6/group). Gpc1KO showed decreased eNOS expression (0.3 fold, n=4/group ) and activity during (1.947±0.32 [WT] vs 1.316±0.13 [Gpc1KO], n=4/group). The eNOS decoy peptide increased transcellular transport only in Gpc1KO MLEC (3 fold, n=5/group). Conclusions: In conclusion, these data suggest that intracellular transport may be the main mechanism for pressure-induced changes in endothelial permeability in MLEC . The mechanisms whereby Gpc1 regulates transcellular transport seem to be associated with eNOS signaling. This may be important for the management of acute changes in pressure seen in clinical settings when NO donors are used as vasodilators to decrease blood pressure.

Abstract 15321: MicroRNA Mir-378-3p is a Novel Regulator of Endothelial Autophagy and Function

Introduction: Autophagy maintains cellular homeostasis by degradation of unnecessary proteins for their recycling and reuse. MicroRNAs (miRNAs) regulate various biological processes including autophagy. MiRNA and autophagy both regulate endothelial function, however, role of miRNAs in endothelial autophagy and function is unknown. Methods and Results: To understand the role of miRNAs in endothelial autophagy, autophagy was genetically inhibited via ATG7 (autophagy-related gene 7) silencing in Human Umbilical Vein Endothelial Cells (HUVECs) and then a miRNA array was performed, which showed miR-378-3p as the most up-regulated miRNA. Pharmacological (via chloroquine treatment) inhibition of autophagy in HUVECs resulted in significant upregulation of miR-378a-3p. Autophagy activation via starvation in HUVECs significantly reduced miR-378a-3p expression. Next, we over-expressed miR-378a-3p (via mimic) in HUVECs and measured autophagy and endothelial function. MiR-378a-3p overexpression was associated with impaired autophagy (reduced LC3-II/LC3-I ratio), reduced proliferation, and increased migration in HUVECs. At the molecular level, miR-378a-3p overexpression was associated with reduced eNOS expression at both transcript and protein levels in HUVECs. MiR-378a-3p inhibition (via antagomir) led to reduced cell migration and upregulation of eNOS in HUVECs. In-silico study identified PDIA4 as a potential target of miR-378-3p, and our qPCR data and immunoblotting data confirmed increased and reduced PDIA4 expression in mimics and antagomir-transfected HUVECs, respectively. We then inhibited autophagy using chloroquine (50uM) and observed significantly reduced PDIA4 expression. Inhibition of PDIA4 expression via silencing inhibited autophagic flux indicated by reduced LC3-II/LC3-I in HUVECs. Conclusions: Our findings, for the first time, show an inverse relationship between miR-378a-3p expression and endothelial autophagy and endothelial function. PDIA4 was identified as a molecular target for miR-378a-3p in endothelial cells, which revealed PDIA4’s potential role in endothelial autophagy. Our data demonstrate that miR-378-3p regulates endothelial autophagy and function via modulating PDIA4 expression.

2′–5′ oligoadenylate synthetase‑like 1 (OASL1) protects against atherosclerosis by maintaining endothelial nitric oxide synthase mRNA stability

Endothelial nitric oxide synthase (eNOS) decreases following inflammatory stimulation. As a master regulator of endothelial homeostasis, maintaining optimal eNOS levels is important during cardiovascular events. However, little is known regarding the mechanism of eNOS protection. In this study, we demonstrate a regulatory role for endothelial expression of 2′–5′ oligoadenylate synthetase-like 1 (OASL1) in maintaining eNOS mRNA stability during athero-prone conditions and consider its clinical implications. A lack of endothelial Oasl1 accelerated plaque progression, which was preceded by endothelial dysfunction, elevated vascular inflammation, and decreased NO bioavailability following impaired eNOS expression. Mechanistically, knockdown of PI3K/Akt signaling-dependent OASL expression increased Erk1/2 and NF-κB activation and decreased NOS3 (gene name for eNOS) mRNA expression through upregulation of the negative regulatory, miR-584, whereas a miR-584 inhibitor rescued the effects of OASL knockdown. These results suggest that OASL1/OASL regulates endothelial biology by protecting NOS3 mRNA and targeting miR-584 represents a rational therapeutic strategy for eNOS maintenance in vascular disease.

Resveratrol improves vascular endothelial dysfunction in the unpredictable chronic mild stress model of depression in rats by reducing inflammation

Chronic psychological stress may cause depression and it is a risk factor for vascular endothelial dysfunction. Inflammation may contribute to endothelial dysfunction. Resveratrol, which has antiinflammatory and vasculoprotective properties, has been reported its beneficial effects on endothelial dysfunction induced by hypertension, diabetes and, aging. The effects of resveratrol on stress-induced endothelial dysfunction is not investigated yet. This study aimed to investigate the efficacy of resveratrol on vascular function in the unpredictable chronic moderate stress (UCMS) model of rats and to examine the possible mechanisms of resveratrol by assessment of proinflammatory markers. Male rats were assigned to 4 groups (n = 8 for each group): Control, Control+Resveratrol, UCMS, UCMS+Resveratrol. UCMS and UCMS+Resveratrol groups were exposed to the UCMS procedure for 12 weeks. Resveratrol (20 mg/kg/day, i.p., during 12 weeks) was given to the Control+Resveratrol and UCMS+Resveratrol groups.Then depressive-like behaviors were evaluated by forced swimming test. After behavioral tests, systolic blood pressure was recorded. Endothelial function of the thoracic aorta was evaluated by isolated organ bath system. Vascular eNOS expression and inflammatory markers such as TNF-α, IL-1β, IL-6, CRP, ICAM1, MCP in serum and vascular tissue were analyzed to explore the mechanisms of resveratrol. UCMS resulted in depressive-like behavior, endothelial dysfunction and increased inflammatory cytokines in both serum and tissue samples. Resveratrol treatment improved depressive-like behavior, ameliorated vascular dysfunction, and reversed stress-induced inflammation. Our findings suggest that resveratrol exerted antidepressant-like effect and prevented vascular endothelial dysfunction by reducing systemic and peripheral inflammation in UCMS-induced depression in rats. Therefore, resveratrol may be a therapeutic option with a vasculoprotective effect in depression.

ODP435 11-Ketotestosterone is One of the Major Androgens in Pigs

Abstract 11-ketotestosterone (11-KT) is one of the active androgens, which belongs to the novel 11-oxygenated class of androgens. Although 11-KT has been regarded as a teleost-specific androgen, it was recently demonstrated that 11-KT is a major human androgen. However, the details of its functions and dynamics remain unknown. Deficiency of proper animal model is one of the reasons for this. Although rodent models have contributed to reveal synthesis and functions of steroid hormones, only small amounts of 11-KT are synthesized in mice and rats (Yazawa et al. Endocrinology, 2008). In mammal, 11-KT is abundant in limited animal species, such as non-human primates, pig and guinea pig. This suggest that these animal species are valuable models for determining the functions of 11-KT in mammals. In the present study, we evaluated the production and potential functions of 11-KT in pig. In previous studies, we studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs, Yazawa et al. Endocrinology 2006, 2009; Mol Endo, 2010; Endocrine J 2016). Therefore, we first induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. With the aid of cAMP, adenovirus-mediated introduction of SF-1/Ad4BP induced the differentiation of PSPA cells into 11-KT-producing cells. Using PSPA cell-derived steroidogenic cells and our previously established method (Yazawa et al. JSBMB, 2020), we found that porcine AKR1C1 can convert androstenedione into testosterone (T) and is expressed in adrenal, testis and ovary. In addition, we showed that HSD11B2, one of the essential genes to produce 11-KT from T was expressed in testicular Leydig cells and the adrenal cortex. 11-KT was present in the plasma of both immature male and female pigs, with slightly higher levels in males than in females. T levels were much higher in male pigs. It is noteworthy that 11-KT levels were >10-fold higher than T levels in female pigs. However, castration altered the plasma profiles of 11-KT and T in male pigs to the female-like profiles. 11-KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11-KT is produced in porcine adrenal glands and testes. It can potentially be involved in the regulation of cardiovascular functions through eNOS expression. In addition, these findings indicate that pigs (especially female) represent an ideal model for analyzing the functions of 11-KT in mammal. Presentation: No date and time listed

Cerium dioxide nanoparticles modulate antioxidant defences and change vascular response in the human saphenous vein.

Nanoparticles have a promising future in biomedical applications and knowing whether they affect ex vivo vascular reactivity is a necessary step before their use in patients. In this study, we have evaluated the vascular effect of cerium dioxide nanoparticles (CeO2NPs) on the human saphenous vein in response to relaxing and contractile agonists. In addition, we have measured the protein expression of key enzymes related to vascular homeostasis and oxidative stress. We found that CeO2NPs increased expression of both SOD isoforms, and the consequent reduction of superoxide anion would enhance the bioavailability of NO explaining the increased vascular sensitivity to sodium nitroprusside in the presence of CeO2NPs. The NOX4 reduction induced by CeO2NPs may lead to lower H2O2 synthesis associated with vasodilation through potassium channels explaining the lower vasodilation to bradykinin. In addition, we showed for the first time, that CeO2NPs increase the expression of ACE2 in human saphenous vein, and it may be the cause of the reduced contraction to angiotensin II. Moreover, we ruled out that CeO2NPs have effect on the protein expression of eNOS, sGC, BKca channels and angiotensin II receptors or modify the vascular response to noradrenaline, endothelin-1 and TXA2 analogue. In conclusion, CeO2NPs show antioxidant properties, and together with their vascular effect, they could be postulated as adjuvants for the treatment of cardiovascular diseases.

Metformin Prevents Endothelial Dysfunction in Endometriosis through Downregulation of ET-1 and Upregulation of eNOS.

This study aimed to evaluate if the treatment with metformin affects the morphologic structure, endothelial function, angiogenesis, inflammation and oxidation-responsive pathways in the heart of mice with surgically induced endometriosis. B6CBA/F1 mice (n = 37) were divided into four groups; Sham (S), Metformin (M), Endometriosis (E) and Metformin/Endometriosis (ME). The cross-sectional area of cardiomyocytes was assessed after Hematoxylin-Eosin staining and fibrosis after Picrosirius-Red staining. ET-1, nitric oxide synthases-iNOS and eNOS, and VEGF and VEGFR-2 were detected by immunofluorescence. Semi-quantification of ET-1, eNOS, VEGF, NF-kB, Ikβα and KEAP-1 was performed by Western blotting. MIR199a, MIR16-1, MIR18a, MIR20a, MIR155, MIR200a, MIR342, MIR24-1 and MIR320a were quantified by Real-Time qPCR. The interaction of endometriosis and metformin effects was assessed by a two-way ANOVA test. Compared with the other groups, M-treated mice presented a higher cross-sectional area of cardiomyocytes. Heart fibrosis increased with endometriosis. Treatment of endometriosis with metformin in the ME group downregulates ET-1 and upregulates eNOS expression comparatively with the E group. However, metformin failed to mitigate NF-kB expression significantly incremented by endometriosis. The expression of MIR199a, MIR16-1 and MIR18a decreased with endometriosis, whereas MIR20a showed an equivalent trend, altogether reducing cardioprotection. In summary, metformin diminished endometriosis-associated endothelial dysfunction but did not mitigate the increase in NF-kB expression and cardiac fibrosis in mice with endometriosis.

EFFECTS OF PALM OIL DERIVED TOCOTRIENOL RICH FRACTION AND VITAMIN E ISOMERS ON BIOMARKERS OF EARLY ATHEROGENESIS IN STIMULATED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

This study was conducted to investigate the effects of tocotrienol rich fraction (TRF), α-TOC, and pure TCT isomers (α-. γ- & δ-TCT) on inflammation, endothelial activation, nuclear factor kappa B (NFκB), endothelial nitric oxide synthase (eNOS) and monocyte binding activity (MBA) in vitro. Human umbilical vein endothelial cells (HUVECs) were incubated with various concentrations of α-TOC, pure TCT isomers and TRF (0.3-10 µM) together with lipopolysaccharides (LPS) for 16 h. Culture medium and cells were collected and measured for the protein and gene expression of IL-6, TNF-α, NFκB, ICAM-1, VCAM-1, e-selectin, and eNOS. Monocyte binding activity (MBA) was measured by Rose Bengal staining. Area under the curve (AUC) analysis revealed that TRF and pure TCT particularly γ- and δ- isomers, showed better inhibition of inflammation and endothelial activation, MBA and greater eNOS increment than α-TOC. These suggest that TRF and pure TCT isomers have potential as preventive anti-atherogenic agents by attenuating the release of early biomarkers of atherogenesis which is better than α-TOC in LPS-stimulated human endothelial cells.

Characterizing nuclear morphology and expression of eNOS in vascular endothelial cells subjected to a continuous range of wall shear stress magnitudes and directionality

Complex patterns of hemodynamic wall shear stress occur in regions of arterial branching and curvature. Areas within these regions can be highly susceptible to atherosclerosis. Although many studies have characterized the response of vascular endothelial cells to shear stress in a categorical manner, our study herein addresses the need of characterizing endothelial behaviors over a continuous range of shear stress conditions that reflect the extensive variations seen in the vasculature. We evaluated the response of human umbilical vein endothelial cell monolayers to orbital flow at 120, 250, and 350 revolutions per minute (RPM) for 24 and 72 h. The orbital shaker model uniquely provides a continuous range of shear stress conditions from low and multidirectional at the center of each well of a culture plate to high and unidirectional at the periphery. We found distinct patterns of endothelial nuclear area, nuclear major and minor diameters, nuclear aspect ratio, and expression of endothelial nitric oxide synthase over this range of shear conditions and relationships were fit with linear and, where appropriate, power functions. Nuclear area was particularly sensitive with increases in the low and multidirectional WSS region that incrementally decreased as WSS became higher in magnitude and more unidirectional over the radius of the cell layers. The patterns of all endothelial behaviors exhibited high correlations (positive and negative) with metrics of shear stress magnitude and directionality that have been shown to strongly associate with atherosclerosis. Our findings demonstrate the exquisite sensitivity of these endothelial behaviors to incremental changes in shear stress magnitude and directionality, and provide critical quantitation of these relationships for predicting the susceptibility of an arterial segment to diseases such as atherosclerosis, particularly within complex flow environments in the vasculature such as around bifurcations.