Downregulation Of eNOS Research Articles

Alleviation of preeclampsia-like symptoms through PlGF and eNOS regulation by hypoxia- and NF-κB-responsive miR-214-3p deletion

Preeclampsia is caused by placental hypoxia and systemic inflammation and is associated with reduced placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS) levels. The molecular signaling axes involved in this process may play a role in the pathogenesis of preeclampsia. Here, we found that hypoxic exposure increased hypoxia-inducible factor-1α (HIF-1α)/Twist1-mediated miR-214-3p biogenesis in trophoblasts, suppressing PlGF production and trophoblast invasion. TNF-α stimulation increased NF-κB-dependent miR-214-3p expression in endothelial cells, impairing eNOS expression and causing endothelial dysfunction. Synthetic miR-214-3p administration to pregnant mice decreased PlGF and eNOS expression, resulting in preeclampsia-like symptoms, including hypertension, proteinuria, and fetal growth restriction. Conversely, miR-214-3p deletion maintained the PlGF and eNOS levels in hypoxic pregnant mice, alleviating preeclampsia-like symptoms and signs. These findings provide new insights into the role of HIF-1/Twist1- and NF-κB-responsive miR-214-3p-dependent PlGF and eNOS downregulation in the pathogenesis of preeclampsia and establish miR-214-3p as a therapeutic or preventive target for preeclampsia and its complications.

The effects of HIV-1-proteins and antiretroviral therapy on aortic endothelial cells (AECs). A mechanistic in vitro approach

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation Introduction Endothelial dysfunction (ED) is an early precursor of cardiovascular disease characterized by decreased nitric oxide (NO) and increased oxidative stress, creating a pro-inflammatory environment. Increased ED and cardiovascular risk have been observed in HIV-1 infection and antiretroviral therapy (ART). Purpose The United Nations AIDS update indicates that in 2015 17.0 million people worldwide were living with HIV and receiving antiretroviral therapy. Considering the extent of this global pandemic and its comorbidity with CVD, zooming in on HIV’s intersection with antiretroviral therapy and cardiovascular health is of paramount importance. Yet, many of the mechanisms involved are still poorly understood. This knowledge can ultimately assist with the careful selection of antiretroviral-agents in patients with underlying cardiovascular risk factors. Objectives Establishing a simulated model of in vitro HIV-1-infection in aortic endothelial cells and determining the effects of non/nucleoside reverse transcriptase inhibitors (NRTI/NNRTIs) and protease inhibitors (PIs) on markers of endothelial function and the expression/activation of vascular signalling proteins. Methods A simulated HIV-1-model was established by adding recombinant HIV-1 proteins to growth medium in a single cocktail containing Tat, Gp160 and Nef. ART1 (NRTI/NNRTIs) were administered as a single cocktail to growth medium, containing efavirenz (EFV), emtricitabine (FTC) and tenofovir (TDF). ART2 (PIs) were administered as a single cocktail to growth medium, containing lopinavir (LPV) and ritonavir (RTV). All the aforementioned treatments were subject to dose-response investigations and treatment lasted 24 hours. Cell viability, NO-production and oxidative stress were measured using fluorescent probes. Selected dosages from above studies were further used and important vascular signalling proteins in HIV-1 exposed AECs were investigated by western blotting. Results HIV-1 (100ng/ml, 24h) resulted in ↓NO-levels (DAF-fluorescence intensity: 100±1.25% vs. 76.00±3.67%; p<0.05) with no oxidative stress. HIV-1+PI treatment lowered NO levels (DAF-fluorescence: 100±0.72% vs. 94.47±1.91%; p<0.05). HIV-1+ NRTI/NNRTI treatment had no effects. HIV-1+PI treatment ↓IκBα expression (inhibitory signalling protein in pro-inflammatory NF-κB pathway) (1.0 ± 0.15 vs 0.37 ± 0.03; p<0.05) and ↓eNOS expression (1.0 ± 0.21 vs. 0.28 ± 0.04; p<0.05). The same was observed for PKB/Akt (1.0 ± 0.03 vs. 0.68 ± 0.02; p<0.05). No effects on cell viability and oxidative stress were observed. Conclusion Exposure of AECs to combined HIV-1-protein and PI treatment lowered NO-production, which may be linked to underlying PKB/Akt and eNOS downregulation. HIV-1+PI treatment was associated with increased pro-inflammatory NF-κB signalling. Overall, our results suggest that a dysfunctional state was induced in AECs exposed to HIV-1 proteins and PI treatment.

Exploring the Impact of Nitric Oxide and Epidermal Growth Factor Receptor on Vascular Remodeling Induced by Mechanical Stretch

Hypertension is a key risk factor in many cardiovascular diseases like heart failure, kidney failure and vascular hypertrophy. Mechanical stretch/hypertension, promotes vascular smooth muscle cells remodeling by inducing leptin expression and other hypertrophic proteins like ERK1/2, AKT, and cofilin. Also the Epidermal growth factor receptor (EGFR) transactivation has shown to mediate vascular remodeling by enhancing VSMC hypertrophy, migration, and proliferation. Nitric oxide (NO) has been shown to be a critical factor for many cellular physiological function, it has the ability to induce VSMC relaxation leading to reduce hypertension. Moreover NO has received extensive attention as an anti- hypertrophic agent. Several Studies have shown that nitric oxide does not only promote vasodilation but it also inhibits VSMC proliferation. The major aim of this study was to investigate the inhibitory effect of NO and the effect of blocking the EGFR in mechanical stretch induced vascular remodeling. Methods: Rat portal veins (RPVs) were isolated and pretreated with/without NO donor (SNAP) or EGFR kinase blocker (AG1478) and then stretched to study their effects on mechanical stretch induced leptin synthesis and VSMC remolding. Western blot analysis was done to detect protein expression for leptin, adiponectin, eNOS p-ERK1/2 and p-AKT. Immunocytochemistry was performed on rat aortic vascular smooth muscle (RASMC) to detect GATA-4 nuclear translocation. Moreover, we used laser confocal microscopy on frozen RPV sections to detect the level of ROS formation and leptin. Finally, we performed qPCR for adiponectin, adipoR1 and adipoR2 mRNA. Results: In this study we showed the effect of mechanical stretch at different time points (1hour and 24 hours). Mechanical stretch-induced leptin expression was mediated by ERK1/2, AKT activations and ROS formation. These effects were associated with eNOS and adiponectin downregulation. Mechanical stretch had no effect on the expression of adiponectin receptors (adipoR1 and adipoR2). RASMCs pretreated with 3.1 nmol/L for 3 hours had induced GATA-4 nuclear translocation. Pre-incubation the RPV with SNAP significantly decreased mechanical stretch induced leptin expression, ERK1/2 and AKT phosphorylation. Moreover, SNAP significantly inhibited the adiponectin downregulation by mechanical stretch. Leptin-induced GATA-4 nuclear translocation was inhibited by SNAP. The involvement of EGFR transactivation in mechanical stretch-induced leptin expression was investigated using AG1478. Pretreatment of RPVs with AG1478 had no effect on mechanical stretch-induced early leptin expression (1 hour) while it inhibited mechanical stretch-induced late leptin expression (24 hours), ROS formation, and adiponectin and eNOS downregulation. However, treatment with AG1478 showed no effect on leptin-induced GATA-4 nuclear translocation. Conclusion: This study showed that NO has a direct inhibitory effect on mechanical stretch-induced AKT and ERK1/2 pathways and inhibits ROS formation that leads to vascular remodeling. On the other hand, EGFR transactivation was able to mediate mechanical stretch-induced vascular hypertrophy via ROS formation and leptin production, thus making EGFR and NO a promising therapeutic target for hypertension induced hypertrophy. This work was funded by Qatar University grants award UREP27-050-3-020 from Qatar National Research Fund (a member of Qatar Foundation) to AZ. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of long-term inorganic nitrate administration on myocardial ischemia-reperfusion injury in ovariectomized rats.

Introduction: Menopause is associated with reduced nitric oxide (NO) bioavailability and lower tolerance against myocardial ischemia-reperfusion (IR) injury. This study investigated whether long-term nitrate administration provides resistance against myocardial IR injury in ovariectomized (OVX) rats. Method: After ovariectomy, female rats were assigned to the OVX and the OVX + nitrate groups (n = 14/group); the latter group consumed nitrate (100mg/L) for 9months. At month 9, each group was divided into two subgroups (n = 7/subgroup), of which one subgroup was exposed to myocardial IR (IR+ hearts) and the other was not exposed (IR- hearts). The hearts of rats were isolated, and NO metabolite (NOx), oxidative stress indices, and mRNA expressions of endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) NO synthases, as well as markers of apoptosis, were measured in the IR- and IR+ hearts. In the IR+ hearts, cardiac function indices (CFI) and the infarct size were also measured. Results: Nitrate increased catalase activity (97%) and eNOS expression (2.94-fold) in the IR- hearts. In the IR+ hearts, nitrate reduced left ventricular (LV) end-diastolic pressure (11.6%) and infarct size (26.2%) and increased recovery of LV developed pressure (44.0%) and peak rate of positive (28.9%) and negative (15.4%) changes in LV pressure. In addition, in the IR+ hearts, nitrate increased eNOS and B-cell lymphoma-2 (Bcl-2) as well as decreased iNOS, Bcl-2 associated X protein (Bax), caspase-3, caspase-8, caspase-9, and tumor necrosis factor-α (TNF-α) expression. Nitrate increased total antioxidant capacity (TAC) and catalase (CAT) activity and decreased malondialdehyde (MDA) levels at month nine in serum and IR+ hearts. Conclusion: The favorable effects of nitrate against IR injury were associated with higher eNOS and Bcl-2 expression, CAT activity, TAC, and lower iNOS, Bax, caspase-3, caspase-8, caspase-9 and TNF-α expression, and MDA in the heart tissue. Nitrate preconditioning alleviated IR-induced myocardial injury in OVX rats; this effect was associated with eNOS upregulation before IR and the blunting of OVX-induced eNOS downregulation, iNOS upregulation, apoptosis, and oxidative stress in heart tissue after IR.

Nitric Oxide Resistance in Priapism Associated with Sickle Cell Disease: Mechanisms, Therapeutic Challenges, and Future Directions.

Patients with sickle cell disease (SCD) display priapism, a prolonged penile erection in the absence of sexual arousal. The current pharmacological treatments for SCD-associated priapism are limited and focused on acute interventions rather than prevention. Thus, there is an urgent need for new drug targets and preventive pharmacological therapies for this condition. This review focuses on the molecular mechanisms linked to the dysfunction of the NO-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) pathway implicated in SCD-associated priapism. In murine models of SCD, reduced nitric oxide (NO)-cGMP bioavailability in the corpus cavernosum is associated with elevated plasma hemoglobin levels, increased reactive oxygen species levels that inactive NO, and testosterone deficiency that leads to endothelial nitric oxide synthase downregulation. We discuss the consequences of the reduced cGMP-dependent PDE5 activity in response to these molecular changes, highlighting it as the primary pathophysiological mechanism leading to excessive corpus cavernosum relaxation, culminating in priapism. We also further discuss the impact of intravascular hemolysis on therapeutic approaches, present current pharmacological strategies targeting the NO-cGMP-PDE5 pathway in the penis, and identify potential pharmacological targets for future priapism therapies. In men with SCD and priapism, PDE5 inhibitor therapy and testosterone replacement have shown promising results. Recent preclinical research reported the beneficial effect of treatment with haptoglobin and NO donors. SIGNIFICANCE STATEMENT: This review discusses the molecular changes that reduce NO-cGMP bioavailability in the penis in SCD and highlights pharmacological targets and therapeutic strategies for the treatment of priapism, including PDE5 inhibitors, hormonal modulators, NO donors, hydroxyurea, soluble guanylate cyclase stimulators, haptoglobin, hemopexin, and antioxidants.

Human calcified aortic valves release procoagulant microparticles inducing endothelial dysfunction and thrombogenicity: Role of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Background Aortic stenosis (AS) pathogenesis is a dynamic process with interplay between the hemostatic system and the valve leading to inflammation, oxidative stress, endothelial dysfunction, and calcification. Within the pathological valve, mechanical strain induces the release of cell-derived microparticles (MPs) that promote mineralization and carry a pro-oxidant activator signal. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) showed cardiovascular benefit and anti-inflammatory responses in patients with heart failure. Oxidative stress has been shown to trigger SGLT2 expression in endothelial cells (ECs) to induce endothelial dysfunction. Therefore, this study evaluated calcified AS valves and the effect of AS-derived MPs on ECs and, if so, the role of the SGLT2. Methods Pathological valves were collected from 90 AS and 15 aortic insufficiency (AI) patients. MPs were extracted from human pathological valves, and quantified through their prothrombinase activity. Primary cultures of porcine valvular endothelial cells (VECs) were treated with MPs (10 nM) or thrombin (1 U/ml). Protein expression levels were assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-qPCR, oxidative stress using dihydroethidium, and nitric oxide (NO) generation by DAF-FM diacetate. THP1 cells were used to evaluate the adhesiveness of VECs, and the generation of thrombin using a specific chromogenic substrate. Results Calcified AS valves showed increased levels of thrombogenic (TF, PAI-1), adhesive (VCAM-1, ICAM-1) and inflammatory markers (CD68, p-p65 NF-κB) and SGLT2 compared to non-calcified valves. These responses were associated with a higher level of oxidative stress that was reduced by inhibitors of the angiotensin system (ACEi, AT1R) and SGLT2, and an increased release of procoagulant MPs. Exposure of VECs to AS-MPs increased the expression of markers of the angiotensin system (ACE, p67 phox), inflammation (CXCL10, CCL11, CXCL8), thrombogenicity (TF, PAI-1), angiogenesis (VEGFR2, ANGPTL4), matrix remodeling (BMP-2, MMP-1), and SGLT2 and caused the down-regulation of eNOS. These responses were associated with a sustained pro-oxidant response, the nuclear translocation of NF-kB and blunted bradykinin-induced NO formation, which were prevented by inhibition of AT1R, NADPH oxidases, SGLT2, and NF-kB. AS-MPs-treated VECs showed also increased adhesion of THP-1 cells, and generation of thrombin. Conclusions Calcified AS valve is a potent reservoir of MPs, acting as a pro-thrombogenic source per se and prompting valvular endothelial cells dysfunction through activation of the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway. The observed protective effect of empagliflozin suggests SGLT2 inhibitors as a novel therapeutic option to curb valve dysfunction.

Hydroxyurea does not reverse functional alterations of the nitric oxide-cGMP pathway associated with priapism phenotype in corpus cavernosum from sickle cell mouse.

Sickle cell disease (SCD) is a genetic disorder that has been associated with priapism. The role of hydroxyurea, a common SCD therapy, in influencing the nitric oxide (NO)-cGMP pathway and its effect on priapism is unclear. To investigate the effect of hydroxyurea treatment on smooth muscle relaxation of corpus cavernosum induced by stimulation of the NO-cGMP pathway in SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice, which are used as model of priapism associated with the low bioavailability of endothelial NO. Four-month-old wild-type (WT, C57BL/6), SCD transgenic, and eNOS-/- male mice were treated with hydroxyurea (100 mg/Kg/day) or its vehicle (saline) daily for three weeks via intraperitoneal injections. Concentration-response curves for acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS) were generated using strips of mice corpus cavernosum. The SCD mice demonstrated an amplified CC relaxation response triggered by ACh, EFS, and SNP. The corpus cavernosum relaxation responses to SNP and EFS were found to be heightened in the eNOS-/- group. However, the hydroxyurea treatment did not alter these escalated relaxation responses to ACh, EFS, and SNP in the corpus cavernosum of the SCD group, nor the relaxation responses to EFS and SNP in the eNOS-/- group. In conclusion, hydroxyurea is not effective in treating priapism associated with SCD. It is likely that excess plasma hemoglobin and reactive oxygen species, which are reported in SCD, are reacting with NO before it binds to GCs in the smooth muscle of the corpus cavernosum, thus preventing the restoration of baseline NO/cGMP levels. Furthermore, the downregulation of eNOS in the penis may impair the pharmacological action of hydroxyurea at the endothelial level in SCD mice. This study emphasize the urgency for exploring alternative therapeutic avenues for priapism in SCD that are not hindered by high plasma hemoglobin and ROS levels.

Neutralization of excessive CCL28 improves wound healing in diabetic mice.

Introduction: Chronic, non-healing skin wounds such as diabetic foot ulcers (DFUs) are common in patients with type 2 diabetes mellitus (T2DM) and often result in limb amputation and even death. However, mechanisms by which T2DM and inflammation negatively impact skin wound healing remains poorly understood. Here we investigate a mechanism by which an excessive level of chemokine CCL28, through its receptor CCR10, impairs wound healing in patients and mice with T2DM. Methods & Results: Firstly, a higher level of CCL28 was observed in skin and plasma in both patients with T2DM, and in obesity-induced type 2 diabetic db/db mice. Compared with WT mice, adipose tissue from db/db mice released 50% more CCL28, as well as 2- to 3-fold more IL-1β, IL-6, and TNF-α, and less VEGF, as determined by ELISA measurements. Secondly, overexpression of CCL28 with adenovirus (Adv-CCL28) caused elevation of proinflammatory cytokines as well as CCR10 expression and also reduced eNOS expression in the dorsal skin of WT mice as compared with control Adv. Thirdly, topical application of neutralizing anti-CCL28 Ab dose-dependently accelerated wound closure and eNOS expression, and decreased IL-6 level, with an optimal dose of 1 μg/wound. In addition, mRNA levels of eNOS and anti-inflammatory cytokine IL-4 were increased as shown by real-time RT-PCR. The interaction between eNOS and CCR10 was significantly reduced in diabetic mouse wounds following application of the optimal dose of anti-CCL28 Ab, and eNOS expression increased. Finally, enhanced VEGF production and increased subdermal vessel density as indicated by CD31 immunostaining were also observed with anti-CCL28 Ab. Discussion: Taken together, topical application of neutralizing anti-CCL28 Ab improved dorsal skin wound healing by reducing CCR10 activation and inflammation in part by preventing eNOS downregulation, increasing VEGF production, and restoring angiogenesis. These results indicate anti-CCL28 Ab has significant potential as a therapeutic strategy for treatment of chronic non-healing diabetic skin wounds such as DFUs.

PS-B12-12: NOVEL PATHOPHYSIOLOGICAL ROLES OF A-SYNUCLEIN IN AGE-RELATED VASCULAR ENDOTHELIAL DYSFUNCTION AND E NHANCED PERMEABILITY.

Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide (NO) production in ECs. Treatment with rSNCA also suppressed TNF-α and palmitic acid induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β; gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Mechanistically, loss-of-function of SNCA upregulated the expression of β;-site amyloid precursor protein cleaving enzyme 1 (BACE1), which was reported to exacerbate eNOS inactivation and permeability in ECs. Addition of SNAP, NO donor, rescued the increment of BACE1, which indicated eNOS downregulation by loss-of-function of SNCA caused BECE1 increment in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high fat diet, displayed elevated blood pressure compared with wild-type (WT) mice; this could be eNOS dysfunction dependent because of the lower difference caused by L NAME administration. In addition, increased depositions of immunoglobulin were found in the aortas of SNCA KO compared with those of WT mice, which indicated enhanced endothelial permeability by in vivo loss-of-function of SNCA. Thus, these results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction and enhanced permeability might be partially ascribed to loss-of-function of SNCA in ECs.

93 EFFECT OF SACUBITRIL/VALSARTAN ON ENDOTHELIAL DYSFUNCTION AND ARTERIAL STIFFNESS IN PATIENTS WITH CHRONIC HEART FAILURE

Abstract Aim Heart failure (HF) is associated to endothelial dysfunction, a pathological condition characterized by imbalance between the production of vasoconstrictor and vasodilator factors, increase in the production of cytokines, down-regulation of eNOS, platelets activation and increased oxidative stress. Furthermore, endothelial dysfunction promotes the increase of arterial stiffness, augmenting myocardial damage. Sacubitril/Valsartan (sac/val) is used in the treatment of HF reduced ejection fraction (HFrEF) and has been proven effective in reducing CV disease progression and all-cause mortality in HFrEF patients. The aim of the study was to evaluate the effect of sac/val on endothelial dysfunction and arterial stiffness in patients with HFrEF, at baseline and after 6 months of treatment. Moreover, we evaluated the effects of sac/val on oxidative stress levels and platelets activation. Materials Methods We enrolled 46 Caucasian patients (mean age 70.1±7.1), suffering from HFrEF. Inclusion criteria were LVEF<35, functional class NYHA II or III. All clinical evaluation and laboratory tests were performed at baseline and after 6 months of treatment. The serum values of the markers of oxidative stress (8-isoprostane, NOX-2) and platelets activation (Sp-selectin, GPVI) were assessed with ELISA sandwich. Endothelial function was estimated with the measurement of the reactive hyperemia index (RHI); arterial stiffness (AS) was evaluated with the measurement of carotid-femoral pulse wave velocity (PWV), augmentation pressure (AP) and augmentation index (AI). Results The mean dose of sac/val was 180.5±110 mg without serious adverse events. At 6 months, data showed a significant improvement in in hemodynamic and clinical parameters such as heart rate (HR) (p<0.0001), NT-ProBNP (p<0.0001), fasting plasma glucose (FPG) (p<0.0001). Furthermore, there was a significant reduction in oxidative stress, platelets activation and inflammatory indices. We observed a significant improvement in PWV (p<0.0001), AI (p<0.0001), AP (p<0.0001) and RHI (p<0.0001). A linear correlation analysis was performed to assess the association between vascular parameters and different covariates expressed as Δ variation between baseline and follow-up. ΔPWV was directly correlated with ΔHOMA (p=0.037), ΔIL-6 (p=0.034), ΔTNF-α (p=0.001), Δ8-isoprostane (p=0.016), ΔNox-2 (p=0.01), ΔGP6 (p=0.018), ΔSp-selectin (p=0.023); ΔRHI was inversely correlated with ΔHOMA (p=0.003), ΔIL-6 (p=0.004), ΔTNF-α (p=0.023), ΔCRP (p=0.011), Δ8-isoprostane (p=0.012), ΔNox-2 (p=0.01), ΔGP6 (p=0.014), ΔSp-selectin (p=0.015). From stepwise multivariate linear regression model, ΔTNF-α was the stronger predictor of ΔPWV, justifying 48.5% of its variation and ΔSp-selectin was the major predictor of ΔRHI explaining 23.2% of its variation. Conclusion The treatment with sac/val improved endothelial dysfunction and arterial stiffness, due to reduced levels of oxidative stress, platelet activation and inflammation.

Abstract 10442: Protective Effect of Inhibition of Sglt2 on Nanoplastics-Induced Premature Endothelial Senescence and Dysfunction

Background: Nanoplastics (NPs) are now widely acknowledged as a vital part of environmental pollution. However, the effects of NPs on the cardiovascular system are not well understood. We previously reported that NPs induce premature endothelial senescence and dysfunction via a redox-sensitive signaling pathway. Increasing evidence has shown that SGLT2 inhibitors (gliflozins) exhibit protective effects against the development of major cardiovascular diseases. Objective: This study aimed to investigate the role of SGLT2 on NPs-induced premature endothelial senescence and dysfunction, and, if so, to assess the effect of SGLT2 inhibition. Methods: Porcine coronary artery (PCA) rings and cultured endothelial cells (ECs) were exposed to NPs in the presence or absence of the SGLT2 inhibitor, Enavogliflozin (ENA), and the level of SGLTs expression, senescence-associated-beta galactosidase (SA-β-gal) activity, senescence markers, oxidative stress level, and vascular function were evaluated. Results: NPs were internalized in ECs in a time- and concentration-dependent manner. Exposure of PCAECs to NPs significantly up-regulated SGLT2 expression and increased SA-β-gal activity, one of the prominent senescence markers. ENA significantly reduced the NPs-induced increase of SA-β-gal activity in PCA and PCAECs. In addition, ENA prevented the NPs-induced up-regulation of senescence markers, p53, and p21 that promoted the inhibition of ECs proliferation. ENA prevented also the NPs-induced oxidative stress and up-regulation of NOX2 and p22phox in ECs. Exposure of coronary artery rings to NPs blunted endothelium-dependent relaxation and decreased the expression level of endothelial nitric oxide synthase level, and both of these effects were prevented by ENA. Conclusion: The present findings indicate that premature endothelial senescence and dysfunction induced by nanoplastics involve, at least in part, an upregulation of SGLT2, which is associated with increased levels of oxidative stress and the down-regulation of eNOS. They further raise the possibility that SGLT2 may be a potential target for preventing and/or treating environment pollution-associated cardiovascular diseases.

Effect of sacubitril/valsartan on endothelial dysfunction and arterial stiffness in patients with chronic heart failure

Abstract Background Heart failure (HF) is associated to endothelial dysfunction, a pathological condition characterized by imbalance between the production of vasoconstrictor and vasodilator factors, increase in the production of cytokines, down-regulation of eNOS, platelets activation and increased oxidative stress. Furthermore, endothelial dysfunction promotes the increase of arterial stiffness, augmenting myocardial damage. Sacubitril/Valsartan (sac/val) is used in the treatment of HF reduced ejection fraction (HFrEF) and has been proven effective in reducing CV disease progression and all-cause mortality in HFrEF patients. Purpose The aim of the study was to evaluate the effect of sac/val on endothelial dysfunction and arterial stiffness in patients with HFrEF, at baseline and after 6 months of treatment. Moreover, we evaluated the effects of sac/val on oxidative stress levels and platelets activation. Methods We enrolled 46 Caucasian patients (mean age 70.1±7.1), suffering from HFrEF. Inclusion criteria were EF<35, functional class NYHA II or III. All clinical evaluation and laboratory tests were performed at baseline and after 6 months of treatment. The serum values of the markers of oxidative stress (8-isoprostane, NOX-2) and platelets activation (Sp-selectin, GPVI) were assessed with ELISA sandwich. Endothelial function was estimated with the measurement of the reactive hyperemia index (RHI); arterial stiffness (AS) was evaluated with the measurement of carotid-femoral pulse wave velocity (PWV), augmentation pressure (AP) and augmentation index (AI). Results The mean dose of sac/val was 180.5±110 mg without serious adverse events. At 6 months, data showed a significant improvement in in hemodynamic and clinical parameters such as heart rate (HR) (p<0.0001), NT-ProBNP (p<0.0001), fasting plasma glucose (FPG) (p<0.0001). Furthermore, there was a significant reduction in oxidative stress, platelets activation and inflammatory indices. We observed a significant improvement in PWV (p<0.0001), AI (p<0.0001), AP (p<0.0001) and RHI (p<0.0001). A linear correlation analysis was performed to assess the association between vascular parameters and different covariates expressed as Δ variation between baseline and follow-up. ΔPWV was directly correlated with ΔHOMA (p=0.037), ΔIL-6 (p=0.034), ΔTNF-α (p=0.001), Δ8-isoprostane (p=0.016), ΔNox-2 (p=0.01), ΔGP6 (p=0.018), ΔSp-selectin (p=0.023); ΔRHI was inversely correlated with ΔHOMA (p=0.003), ΔIL-6 (p=0.004), ΔTNF-α (p=0.023), ΔCRP (p=0.011), Δ8-isoprostane (p=0.012), ΔNox-2 (p=0.01), ΔGP6 (p=0.014), ΔSp-selectin (p=0.015). From stepwise multivariate linear regression model, ΔTNF-α was the stronger predictor of ΔPWV, justifying 48.5% of its variation and ΔSp-selectin was the major predictor of ΔRHI explaining 23.2% of its variation. Conclusion The treatment with sac/val improved endothelial dysfunction and arterial stiffness, due to reduced levels of oxidative stress, platelet activation and inflammation. Funding Acknowledgement Type of funding sources: None.

Novel pathophysiological roles of α-synuclein in age-related vascular endothelial dysfunction.

Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide production in ECs. Treatment with rSNCA also suppressed TNF-α- and palmitic acid-induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β-gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high-fat diet, displayed elevated blood pressure compared with wild-type mice; this could be eNOS dysfunction-dependent because of the lower difference caused by L-NAME administration. These results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction might be partially ascribed to loss-of-function of SNCA in ECs.

Inhibition of CCL28/CCR10-Mediated eNOS Downregulation Improves Skin Wound Healing in the Obesity-Induced Mouse Model of Type 2 Diabetes.

Chronic, nonhealing skin wounds, such as diabetic foot ulcers (DFUs), are common in patients with type 2 diabetes. Here, we investigated the role of chemokine (C-C motif) ligand 28 (CCL28) and its receptor C-C chemokine receptor type 10 (CCR10) in downregulation of endothelial nitric (NO) oxide synthase (eNOS) in association with delayed skin wound healing in the db/db mouse model of type 2 diabetes. We observed reduced eNOS expression and elevated CCL28/CCR10 levels in dorsal skin of db/db mice and subdermal leg biopsy specimens from human subjects with type 2 diabetes. Further interrogation revealed that overexpression of CCR10 reduced eNOS expression, NO bioavailability, and tube formation of human dermal microvascular endothelial cells (HDMVECs) in vitro, which was recapitulated in mouse dorsal skin. In addition, incubation of HDMVECs with CCL28 led to internalization of the CCR10/eNOS complex and colocalization with lysosome-associated membrane protein 1. Finally, topical application of myristoylated CCR10 binding domain 7 amino acid (Myr-CBD7) peptide prevented CCR10-eNOS interaction and subsequent eNOS downregulation, enhanced eNOS/NO levels, eNOS/VEGF-R2+ microvessel density, and blood perfusion, reduced inflammatory cytokine levels, and importantly, decreased wound healing time in db/db mice. Thus, endothelial cell CCR10 activation in genetically obese mice with type 2 diabetes promotes eNOS depletion and endothelial dysfunction, and targeted disruption of CCR10/eNOS interaction improves wound healing.

Downregulation of eNOS and preserved endothelial function in endothelial-specific arginase 1-deficient mice

Arginase 1 (Arg1) is a ubiquitous enzyme belonging to the urea cycle that catalyzes the conversion of l-arginine into l-ornithine and urea. In endothelial cells (ECs), Arg1 was proposed to limit the availability of l-arginine for the endothelial nitric oxide synthase (eNOS) and thereby reduce nitric oxide (NO) production, thus promoting endothelial dysfunction and vascular disease. The role of EC Arg1 under homeostatic conditions is in vivo less understood. The aim of this study was to investigate the role of EC Arg1 on the regulation of eNOS, vascular tone, and endothelial function under normal homeostatic conditions in vivo and ex vivo. By using a tamoxifen-inducible EC-specific gene-targeting approach, we generated EC Arg1 KO mice. Efficiency and specificity of the gene targeting strategy was demonstrated by DNA recombination and loss of Arg1 expression measured after tamoxifen treatment in EC only. In EC Arg1 KO mice we found a significant decrease in Arg1 expression in heart and lung ECs and in the aorta, however, vascular enzymatic activity was preserved likely due to the presence of high levels of Arg1 in smooth muscle cells. Moreover, we found a downregulation of eNOS expression in the aorta, and a fully preserved systemic l-arginine and NO bioavailability, as demonstrated by the levels of l-arginine, l-ornithine, and l-citrulline as well as nitrite, nitrate, and nitroso-species. Lung and liver tissues from EC Arg1 KO mice showed respectively increase or decrease in nitrosyl-heme species, indicating that the lack of endothelial Arg1 affects NO bioavailability in these organs. In addition, EC Arg1 KO mice showed fully preserved acetylcholine-mediated vascular relaxation in both conductance and resistant vessels but increased phenylephrine-induced vasoconstriction. Systolic, diastolic, and mean arterial pressure and cardiac performance in EC Arg1 KO mice were not different from the wild-type littermate controls. In conclusion, under normal homeostatic conditions, lack of EC Arg1 expression is associated with a down-regulation of eNOS expression but a preserved NO bioavailability and vascular endothelial function. These results suggest that a cross-talk exists between Arg1 and eNOS to control NO production in ECs, which depends on both L-Arg availability and EC Arg1-dependent eNOS expression.

UPREGULATION OF PPET-1 AND DOWNREGULATION OF ENOS ASSOCIATE WITH VASCULAR REMODELING AND INFLAMMATION IN THE HEART AND KIDNEY OF RATS WITH HIGH FAT DIET

Objective: Obesity alters several metabolic activities leading to cardiovascular diseases. The insulin resistance-caused obesity stimulates vasodilator and vasoconstrictor agent disturbances that ended up with cardiac vascular remodeling. This study aims to investigate the effect of obesity to the heart and kidney, focusing on inflammatory mediators associated with endothelial dysfunction. Design and method: Rats (3 months-old, weight 200 g) were divided into control (n = 6), and rats fed on high-fat diet (HFD) for 1 month (n = 6, OB1), 2 months (n = 6, OB2), and 4 months (n = 6, OB4). Then, rats were terminated, and the heart and kidney were harvested for histological quantification and the quantification of mRNA expression of inflammatory mediators, eNOS and ppET-1 using RT-PCR. Sirius Red staining was done to assess vascular remodeling and the immunohistochemistry staining of CD68 protein expression was performed to assess the localization of macrophage. Results: HFD induced high body weight compared to the control group. It was followed by increase of mRNA expression of NF-KB, MCP-1 and CD68 in heart of HFD groups compared to the control group, especially in OB4. Immunostaining revealed positive staining of macrophage (CD68) in the heart of OB groups. Histological staining of the kidney showed slight tubular injury and glomerulosclerosis in OB4 group. Long-term obesity promoted vascular remodeling with increased wall in the heart of OB4 group. OB4 group demonstrated upregulation of ppET-1 with downregulation of eNOS mRNA expression in the heart and kidney. Conclusions: HFD induced upregulation of ET-1 and downregulation of eNOS which may associate with cardiac vascular remodeling, kidney injury and inflammation in the heart and kidney.

Abstract 227: Statins Decrease Endothelial Cell Inflammation On Top Of Maximal Cholesterol Reduction: Findings From The American Heart Association Strategically Focused Cardiometabolic Health And Type 2 Diabetes Mellitus Research Network

Background: Dyslipidemia induces a proinflammatory endothelium and vascular dysfunction. Lipid lowering therapy (LLT) consistently reduces cardiovascular (CV) events. Clinical studies suggest that statin-based LLT has pleiotropic anti-inflammatory effects beyond cholesterol reduction. However, the impact of statins or ezetimibe on top of potent cholesterol reduction is unknown. Thus, on a background of PCSK9 inhibition (with evolocumab), we analyzed the effect of LLT with atorvastatin or ezetimibe on the endothelium using a novel direct brachial vein (BV) endothelial cell (EC) harvesting technique. Methods: CHORD (CHOlesterol lowering and Residual Risk in Diabetes) is an ongoing, prospective study designed to evaluate the effect of maximal LLT on CV risk. Subjects with LDL-C >100mg/dl were treated with evolocumab (140mg/2 weeks) and either atorvastatin (80mg/day) or ezetimibe (10mg/day) for 30 days. In a subset of participants, EC harvesting was performed by inserting a J-wire into the BV, and ECs were isolated and analyzed using next generation RNA sequencing. Results: Among 20 participants (11 atorvastatin, 9 ezetimibe) undergoing EC RNASeq, median LDL-C (130 mg/dl, IQR [110, 147]) decreased by 75% [67, 81] on background PCSK9 inhibition with no significant difference in 30-day LDL-C between atorvastatin (27 mg/dl [20, 32]) and ezetimibe (41 mg/dl [26, 53]) groups (p=0.07). Following cholesterol reduction, 850 genes were upregulated and 2468 were downregulated (p<0.05) with downregulation of eNOS, Endothelin-1, and Senescence pathways by canonical pathway analysis. When stratified by LLT, patients receiving atorvastatin had a significant decrease in inflammation and senescence pathways compared to patients receiving ezetimibe (Figure). Conclusions: Robust lipid lowering with PCSK9 inhibition plus statins, as opposed to ezetimibe has anti-inflammatory effects and suggests a preferred strategy to improve the endothelium and reduce CV risk.

Physiological, Pathological and Pharmacological Interactions of Hydrogen Sulphide and Nitric Oxide in the Myocardium of Rats with Left Ventricular Hypertrophy.

Left ventricular hypertrophy (LVH) is characterized by increased myocardium thickness due to increased oxidative stress and downregulation of cystathione γ lyase (CSE) endothelial nitric oxide synthase (eNOS). Upregulation of CSE by hydrogen sulphide (H2S) and ENOS by L-arginine can arrest the progression of LVH individually. The present study explored the combined treatment of H2S and NO in the progression of LVH, and demonstrated that the response is due to H2S, NO or formation of either new molecule in physiological, pathological, and pharmacological in vivo settings of LVH. Exogenous administration H2S+NO in LVH significantly reduced (all p < 0.05) systolic blood pressure (SBP) and mean arterial pressure (MAP), LV index, heart index and oxidative stress when compared to the LVH group. There was downregulation of CSE mRNA and eNOS in the heart, and exogenous administration of H2S+NO groups upregulated eNOS MRNA while CSE MRNA remained downregulated in the hearts of the LVH group. Similar trends were observed with concentrations of H2S and NO in the plasma and tissue. It can be concluded that combined treatment of LVH with H2S and NO significantly ameliorate the progression of LVH by attenuating systemic hemodynamic and physical indices, and by decreasing oxidative stress. Molecular expression data in the myocardium of LVH depicts that combined treatment upregulated eNOS/NO while it downregulated CSE/H2S pathways in in vivo settings, and it is always eNOS/NO pathways which play a major role.

ENOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

Vitamin D deficiency attenuates endothelial function by reducing antioxidant activity and vascular eNOS expression in the rat microcirculation.

This study examined the effects of vitamin D deficiency on vascular function and tissue oxidative status in the microcirculation; and whether or not these effects can be ameliorated with calcitriol, the active vitamin D metabolite. Three groups (n=10 each) of male Sprague Dawley rats were fed for 10weeks with control diet (CR), vitamin D-deficient diet without (DR), or with oral calcitriol supplementation (0.15μg/kg) for the last four weeks (DSR). After 10weeks, rats were sacrificed; mesenteric arterial rings were studied using wire myograph. Oxidative stress biomarkers malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were measured in the mesenteric arterial tissue. Vascular protein expression of endothelial nitric oxide synthase (eNOS) was determined by Western blotting. Acetylcholine-induced endothelium-dependent relaxation of DR was lower than CR. eNOS expression and SOD activity were lower in mesenteric arterial tissue of DR compared to CR. Calcitriol supplementation to DSR did not ameliorate the above parameters; in fact, augmented endothelium-dependent contraction was observed. Serum calcium was higher in DSR compared to CR and DR. In conclusion, vitamin D deficiency impaired microvascular vasodilation, associated with eNOS downregulation and reduced antioxidant activity. Calcitriol supplementation to vitamin D-deficient rats at the dosage used augmented endothelium-dependent contraction, possibly due to hypercalcaemia.