Decreased eNOS Activity Research Articles

C1q/Tumor Necrosis Factor-Related Protein-9 Is a Novel Vasculoprotective Cytokine That Restores High Glucose-Suppressed Endothelial Progenitor Cell Functions by Activating the Endothelial Nitric Oxide Synthase.

This study investigated whether gCTRP9 (globular C1q/tumor necrosis factor-related protein-9) could restore high-glucose (HG)-suppressed endothelial progenitor cell (EPC) functions by activating the endothelial nitric oxide synthase (eNOS). EPCs were treated with HG (25 mmol/L) and gCTRP9. Migration, adhesion, and tube formation assays were performed. Adiponectin receptor 1, adiponectin receptor 2, and N-cadherin expression and AMP-activated protein kinase, protein kinase B, and eNOS phosphorylation were measured by Western blotting. eNOS activity was determined using nitrite production measurement. Invivo reendothelialization and EPC homing assays were performed using Evans blue and immunofluorescence in mice. Treatment with gCTRP9 at physiological levels enhanced migration, adhesion, and tube formation of EPCs. gCTRP9 upregulated the phosphorylation of AMP-activated protein kinase, protein kinase B, and eNOS and increased nitrite production in a concentration-dependent manner. Exposure of EPCs to HG-attenuated EPC functions induced cellular senescence and decreased eNOS activity and nitric oxide synthesis; the effects of HG were reversed by gCTRP9. Protein kinase B knockdown inhibited eNOS phosphorylation but did not affect gCTRP9-induced AMP-activated protein kinase phosphorylation. HG impaired N-cadherin expression, but treatment with gCTRP9 restored N-cadherin expression after HG stimulation. gCTRP9 restored HG-impaired EPC functions through both adiponectin receptor 1 and N-cadherin-mediated AMP-activated protein kinase /protein kinase B/eNOS signaling. Nude mice that received EPCs treated with gCTRP9 under HG medium showed a significant enhancement of the reendothelialization capacity compared with those with EPCs incubated under HG conditions. CTRP9 promotes EPC migration, adhesion, and tube formation and restores these functions under HG conditions through eNOS-mediated signaling mechanisms. Therefore, CTRP9 modulation could eventually be used for vascular healing after injury.

The Importance of Nitric Oxide and Oxidative Stress in Atrial High-Rate Episodes in Patients with Cardiac Devices.

Atrial High Rate Episodes (AHRE) are subclinical atrial tachyarrhythmias detectable by cardiac implantable electronic devices (CIEDs). AHREs have been associated with an increased risk of developing atrial fibrillation (AF), thromboembolism, cardiovascular and cerebrovascular events, and mortality. Although recent studies have assessed the value of oxidative stress markers in patients with AF, the relationships between AHRE and oxidative stress markers, including nitric oxide, has not yet been elucidated. This study aims to investigate the relationship between these markers and AHRE. This prospective, cross-sectional study comprised 180 patients with CIEDs. The study population was divided into two groups based on the presence (n = 78) and absense (n = 102) of AHRE to analyze its association with biomarkers. The AHRE (+) group was significantly older, had a higher prevalence of hypertension, higher NT-proBNP (508.8 ± 249 pg/mL vs. 415.3 ± 292.1; P = 0.037), MDA levels (20.9 ± 4.1 μmol/L vs. 19.1 ± 3.1 μmol/L; P = 0.006), and iNOS activity (1,935.9 ± 326.1 pg/mL vs. 1,677.4 ± 363.2 pg/mL; P < 0.001). Logistic regression analysis identified age, hypertension, MDA (odds ratio [OR]: 1.131, 95%CI: 1.009 - 1.268, P = 0.035), inducible nitric oxide synthase (iNOS) activity (OR = 1.002, 95% CI = 1.001 - 1.003, P < 0.001), and endothelial nitric oxide synthase (eNOS) activity (OR = 0.990, 95% CI = 0.986 - 0.984, P < 0.001) as independent predictors of AHRE. The study findings indicated that plasma levels of NT-proBNP, MDA, nitric oxide, and the expression of iNOS and eNOS were significantly associated with AHRE. Moreover, elevated plasma MDA concentrations, increased iNOS activity, and decreased eNOS activity were identified as independent predictors of AHRE.

Actions of Esomeprazole on the Maternal Vasculature in Lean and Obese Pregnant Mice with Impaired Nitric Oxide Synthesis: A Model of Preeclampsia.

Preeclampsia is a devastating, multisystem disorder of pregnancy. It has no cure except delivery, which if premature can impart significant neonatal morbidity. Efforts to repurpose pregnancy-safe therapeutics for the treatment of preeclampsia have led to the assessment of the proton pump inhibitor, esomeprazole. Preclinically, esomeprazole reduced placental secretion of anti-angiogenic sFlt-1, improved endothelial dysfunction, promoted vasorelaxation, and reduced maternal hypertension in a mouse model. Our understanding of the precise mechanisms through which esomeprazole works to reduce endothelial dysfunction and enhance vasoreactivity is limited. Evidence from earlier studies suggested esomeprazole might work via the nitric oxide pathway, upregulating endothelial nitric oxide synthase (eNOS). Here, we investigated the effect of esomeprazole in a mouse model of L-NAME-induced hypertension (decreased eNOS activity). We further antagonised the model by addition of diet-induced obesity, which is relevant to both preeclampsia and the nitric oxide pathway. Esomeprazole did not decrease blood pressure in this model, nor were there any alterations in vasoreactivity or changes in foetal outcomes in lean mice. We observed similar findings in the obese mouse cohort, except esomeprazole treatment enhanced ex vivo acetylcholine-induced vasorelaxation. As acetylcholine induces nitric oxide production, these findings hint at a function for esomeprazole in the nitric oxide pathway.

Inflammation of the Human Dental Pulp Induces Phosphorylation of eNOS at Thr495 in Blood Vessels.

The activity of endothelial nitric oxide synthase (eNOS) in endothelial cells increased with the phosphorylation of the enzyme at Ser1177 and decreased at Thr495. The regulation of the phosphorylation sites of eNOS at Ser1177 and Thr495 in blood vessels of the healthy and inflamed human dental pulp is unknown. To investigate this, healthy and carious human third molars were immersion-fixed and decalcified. The localization of eNOS, Ser1177, and Thr495 in healthy and inflamed blood vessels was examined in consecutive cryo-sections using quantitative immunohistochemical methods. We found that the staining intensity of Ser1177 in healthy blood vessels decreased in inflamed blood vessels, whereas the weak staining intensity of Thr495 in healthy blood vessels strongly increased in inflamed blood vessels. In blood vessels of the healthy pulp, eNOS is active with phosphorylation of the enzyme at Ser1177. The phosphorylation of eNOS at Thr495 in inflamed blood vessels leads to a decrease in eNOS activity, contributing to eNOS uncoupling and giving evidence for a decrease in NO and an increase in O2− production. Since the formation of the tertiary dentin matrix depends on intact pulp circulation, eNOS uncoupling and phosphorylation of eNOS at Thr495 in the inflamed pulp blood vessels should be considered during caries therapy.

Morphological and functional adaptation of pancreatic islet blood vessels to insulin resistance is impaired in diabetic db/db mice

The pancreatic islet vasculature is of fundamental importance to the β-cell response to obesity-associated insulin resistance. To explore islet vascular alterations in the pathogenesis of type 2 diabetes, we evaluated two insulin resistance models: ob/ob mice, which sustain large β-cell mass and hyperinsulinemia, and db/db mice, which progress to diabetes due to secondary β-cell compensation failure for insulin secretion. Time-dependent changes in islet vasculature and blood flow were investigated using tomato lectin staining and in vivo live imaging. Marked islet capillary dilation was observed in ob/ob mice, but this adaptive change was blunted in db/db mice. Islet blood flow volume was augmented in ob/ob mice, whereas it was reduced in db/db mice. The protein concentrations of total and phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 were increased in ob/ob islets, while they were diminished in db/db mice, indicating decreased eNOS activity. This was accompanied by an increased retention of advanced glycation end-products in db/db blood vessels. Amelioration of diabetes by Elovl6 deficiency involved a restoration of capillary dilation, blood flow, and eNOS phosphorylation in db/db islets. Our findings suggest that the disability of islet capillary dilation due to endothelial dysfunction impairs local islet blood flow, which may play a role in the loss of β-cell function and further exacerbate type 2 diabetes.

Gestational Intermittent Hypoxia Induces Sex-Specific Impairment in Endothelial Mechanisms and Sex Steroid Hormone Levels in Male Rat Offspring.

Obstructive sleep apnea (OSA) is highly prevalent during gestation and is linked with adverse fetal outcomes. We examined whether gestational intermittent hypoxia (GIH), the main feature of OSA, leads to sex-specific alterations in cardiovascular function and vascular mechanisms in the offspring. Pregnant rats exposed to intermittent hypoxia or ambient air from gestation days 10 to 21 and their offspring were used for the study. GIH exposure did not affect water and food intake in dams. Compared to controls, the male and female offspring born to GIH dams were smaller in weight by 14% and 12%, respectively, and exhibited catch-up growth. Cardiac function was not affected in either GIH males or females. At 12weeks of age, blood pressure was increased in GIH males, but not GIH females, compared to their control counterparts. While mesenteric arterial contractile responses to phenylephrine and endothelin were unaffected in GIH males and females, relaxation response to acetylcholine was reduced in GIH males but not GIH females. Relaxation to sodium nitroprusside was unaffected in both GIH males and females. Total eNOS expression was not affected, but phospho(Ser1177)-eNOS levels were decreased in GIH males. eNOS expression and its phosphorylation status were unaffected in GIH females. Serum testosterone and estradiol levels were higher in GIH males but were unaltered in GIH females. Together, these findings suggest that GIH leads to a sex-specific increase in blood pressure in adult male offspring with blunted endothelium-mediated relaxation, decreased eNOS activity, and elevated sex steroid hormone levels.

Extracellular Endothelial Microvesicles Causative Agents in Obesity‐Related Endothelial Dysfunction

Obesity is associated with increased cardiovascular disease morbidity and mortality. Moreover, obese individuals demonstrate accelerated rates of atherosclerosis, as well as increased incidence of myocardial infarction and stroke. Extracellular microvesicles, particularly endothelial cell-derived microvesicles (EMVs), are seminal functional modulators of vascular health and disease. We have previously demonstrated that EMVs are elevated with obesity and associated with endothelial dysfunction, although the underlying mechanisms are unknown. The experimental aim of this study was to determine the effects of EMVs isolated from obese adults on endothelial cell inflammation, apoptosis and nitric oxide (NO) production. Twenty-four middle-aged adults were studied: 12 normal weight (8M/4F; BMI: 24.4+0.3 kg/m2; EMVs: 85+13 EMV/µL) and 12 obese (6M/6F; BMI: 31.1+0.4 kg/m2; EMVs: 160+18 EMV/µL). All subjects were sedentary, non-smokers, normotensive, normolipidemic, non-diabetic and free of overt coronary artery disease. EMVs (CD31+/CD42b-) were identified, enumerated, and isolated from plasma by flow cytometry. Human umbilical vein endothelial cells were cultured and treated with EMVs from either normal weight or obese adults. EMVs from obese adults induced significantly greater release of both IL-6 (108.2±2.3 vs 90.9±2.9 pg/mL) and IL-8 (74.5±2.9 vs 59.5±3.3 pg/mL) from endothelial cells vs EMVs from normal weight adults. Intracellular expression of total NF-kB p65 was not significantly different in cells treated with EMVs from the normal weight (78.2+9.3 AU) or obese (86.5+8.7 AU) subjects. However, EMVs from obese subjects induced ~25% higher expression of phosphorylated-NF-kB p65 (Ser536; active NF-kB) (145.0+9.8 vs 114.5+8.8 AU). There were no significant differences in cellular expression of total p38-MAPK (190.6+18.4 vs 163.2+9.5 AU) and caspase-3 (250.9±20.9 vs 283.0±19.4 AU) in cells treated with EMVs from normal weight and obese adults. However, EMVs from obese adults induced significantly higher (~50%) expression of phospho-p38-MAPK(15.4+1.6 vs 9.2+0.7 AU) and active caspase-3 (168.2±18.9 vs 107.8±11.7 AU) than EMVs from normal weight adults. Total eNOS expression was not significantly different between cells treated with EMVs from normal weight (110.9+12.0 AU) and obese (112.8+14.4 AU) adults; however, p-eNOS (Ser1177) expression was ~50% lower (P<0.05) in the cells treated with EMVs from obese adults (18.4+3.4 vs 34.6+2.8 AU). Consistent with changes in p-eNOS, NO production was ~20% lower (6.9+0.4 vs 8.7+0.2 mmol/L) in cells treated with EMVs from obese adults. Increased inflammation, apoptosis susceptibility and decreased eNOS activity and NO production renders the vascular endothelium prone to atherosclerosis and thrombosis. Circulating EMVs likely contribute to the proatherogenic endothelial phenotype and, in turn, the increased incidence of cardiovascular disease with obesity.

Abstract 14993: Dysfunctional Extracellular Microvesicles in Andean Highlanders With Excessive Erythrocytosis

Background: Chronic mountain sickness, a maladaptation to high altitude (&gt;2,500 m) characterized by excessive erythrocytosis (EE) and often severe hypoxemia, is prevalent in Andean highlanders. EE increases the risk of cardiovascular events and contributes to vascular dysfunction. Circulating extracellular microvesicles (MVs) are key mediators of cardiovascular health and disease through their interaction with the vascular endothelium. The experimental aim of this study was to determine the effects of MVs isolated from adults with EE on endothelial cell inflammation, oxidative stress, apoptosis and nitric oxide (NO) production. Methods: Twenty-six male residents of Cerro de Pasco, Peru (4,340 m) were studied: 12 highlanders without EE (healthy; age: 40±4 yr; BMI: 26.4±1.7; Hb: 17.4±0.5 g/dL, SpO 2 : 86.9±1.0%) and 14 highlanders with EE (EE: 43±4 yr; 26.2±0.9; 24.4±0.4 g/dL; 79.7±1.6%). MVs were isolated from plasma by flow cytometry. Human umbilical vein endothelial cells were cultured and treated with MVs from either healthy or EE men. Results: MVs from highlanders with EE induced significantly higher release of interleukin (IL)-6 (89.8±2.7 vs 77.1±1.9 pg/mL) and IL-8 (62.0±2.7 vs 53.3±2.2 pg/mL) compared with MVs from healthy highlanders. Although intracellular expression of total NF-κB p65 (65.3±6.0 vs 74.9±7.8.9 AU) was not significantly affected, MVs from EE men resulted in ~25% higher (P&lt;0.05) expression of p-NF-κB p65 (Ser536; active NF-κB) (173.6±14.3 vs 132.8±12.2 AU). Additionally, cell expression of the anti-inflammatory miR-146a and miR-181b were significantly suppressed by EE MVs. Cell oxidative stress and apoptotic susceptibility were not significantly affected by MVs from EE men. However, eNOS activation (231.3±15.5 vs 286.6±23.0 AU) and NO production (8.3±0.6 vs 10.7±0.7 μM/L) were significantly lower in cells treated with MVs from EE vs healthy men. Conclusion: Increased inflammation and decreased eNOS activity and NO production renders the vascular endothelium highly susceptible to atherosclerosis and thrombosis. Andean highlanders with EE exhibit dysfunctional circulating extracellular MVs that induce a proatherogenic endothelial phenotype contributing to their increased cardiovascular risk.

Effects of circulating extracellular microvesicles from spinal cord-injured adults on endothelial cell function.

People with spinal cord injury (SCI) have three- to four-fold greater risk of cardiovascular disease (CVD) compared with those without SCI. Although circulating extracellular microvesicles are key effectors of vascular health and disease, how their functional phenotype might be altered with SCI is unknown. The aim of the present study was to determine the effects of microvesicles isolated from SCI adults on endothelial cell inflammation and oxidative stress as well as endothelial nitric oxide (NO) synthase (eNOS) activation and tissue-type plasminogen activator (t-PA) expression. Eighteen young and middle-aged adults were studied: 10 uninjured (7M/3F; age: 39 ± 3 years) and 8 cervical level spinal cord injured (SCI; 7M/1F; 46 ± 4 years; cervical injury: C3: n=1; C5: n=4; C6: n=3). Circulating microvesicles were isolated, enumerated and collected from plasma by flow cytometry. Human umbilical vein endothelial cells (HUVECs) were cultured and treated with microvesicles from either the uninjured or SCI adults. Microvesicles from SCI adults did not affect cellular markers or mediators of inflammation and oxidative stress. However, microvesicles from the SCI adults significantly blunted eNOS activation, NO bioavailability and t-PA production. Intercellular expression of phosphorylated eNOS at Ser1177 and Thr495 sites, specifically, were ∼65% lower and ∼85% higher, respectively, in cells treated with microvesicles from SCI compared with uninjured adults. Decreased eNOS activity and NO production as well as impaired t-PA bioavailability renders the vascular endothelium highly susceptible to atherosclerosis and thrombosis. Thus, circulating microvesicles may contribute to the increased risk of vascular disease and thrombotic events associated with SCI.

Neurogranin regulates eNOS function and endothelial activation

Endothelial nitric oxide (NO) is a critical mediator of vascular function and vascular remodeling. NO is produced by endothelial nitric oxide synthase (eNOS), which is activated by calcium (Ca2+)-dependent and Ca2+-independent pathways. Here, we report that neurogranin (Ng), which regulates Ca2+-calmodulin (CaM) signaling in the brain, is uniquely expressed in endothelial cells (EC) of human and mouse vasculature, and is also required for eNOS regulation. To test the role of Ng in eNOS activation, Ng knockdown in human aortic endothelial cells (HAEC) was performed using Ng SiRNA along with Ng knockout (Ng −/−) in mice. Depletion of Ng expression decreased eNOS activity in HAEC and NO production in mice. We show that Ng expression was decreased by short-term laminar flow and long-them oscillating flow shear stress, and that Ng siRNA with shear stress decreased eNOS expression as well as eNOS phosphorylation at S1177. We further reveled that lack of Ng expression decreases both AKT-dependent eNOS phosphorylation, NF-κB-mediated eNOS expression, and promotes endothelial activation. Our findings also indicate that Ng modulates Ca2+-dependent calcineurin (CaN) activity, which suppresses Ca2+-independent AKT-dependent eNOS signaling. Moreover, deletion of Ng in mice also reduced eNOS activity and caused endothelial dysfunction in flow-mediated dilation experiments. Our results demonstrate that Ng plays a crucial role in Ca2+-CaM-dependent eNOS regulation and contributes to vascular remodeling, which is important for the pathophysiology of cardiovascular disease.

Corin plays a protective role via upregulating MAPK and downregulating eNOS in diabetic nephropathy endothelial dysfunction.

Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients, but its pathogenesis is unclear. We aimed to study the role of the pro-ANP convertase Corin in the pathogenesis of DN. Corin and ANP expression in DN rat kidneys and high-glucose-treated HK-2 cells was analyzed by real-time PCR, western blotting, and immunohistochemical staining. The effect of Corin-siRNA or ANP-siRNA HK-2 cells on EA.hy926 cell migration was determined by scratch-wound healing assay. The expression of mitogen-activated protein kinase (MAPK) and endothelial NO synthase (eNOS) in EA.hy926 cells treated with conditioned medium from Corin-siRNA- or ANP-siRNA-transfected HK-2 cells was determined by western blotting. We found a significant reduction in Corin and ANP expression in DN rat kidneys. These results were recapitulated in HK-2 cells treated with high glucose. EA.hy926 cells treated with conditioned medium from Corin-deficient HK-2 cells had inhibited migration, increased MAPK activity, and decreased eNOS activity. Similar effects were observed with ANP-siRNA transfection. Finally, adding ANP to the Corin-deficient HK-2 conditioned medium rescued the above defects, indicating that Corin mediates its effects through ANP. In conclusion, Corin plays a renoprotective role through pro-ANP processing, and defects in Corin cause endothelial dysfunction through MAPK and eNOS signaling in DN.

Vanadium compounds induced damage of human umbilical vein endothelial cells and the protective effect of berberine.

This study was conducted to investigate the damage caused by vanadium compounds and to explore the protective effects of berberine (BBR) in human umbilical vein endothelial cells (HUVECs). BBR is a biologically active small molecule found in Coptis rhizome, a remedy used in traditional Chinese medicine to treat diabetes. BBR has also been shown to lower blood glucose in diabetic patients. MTT assay was performed to observe the influence of bis(acetylacetonato)-oxidovanadium [VO(acac)2] or sodium metavanadate (NaVO3) and BBR on viability of HUVECs. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TER). The endothelial nitric oxide synthase (eNOS) activity was detected by ELISA. Flow cytometry was performed to detect the generation of reactive oxygen species (ROS). The results showed that the viability of HUVECs was decreased by treatment with vanadium compounds 50-400μM in a concentration-dependent manner, while 0.01-1μM BBR effectively protected HUVECs from the inhibitory effects of vanadium compounds on cell viability. Also 100 and 200μM VO(acac)2 induced high permeability and decreased eNOS activity in HUVECs. While 0.01-1μM BBR showed no improvement in the permeability, and failed to reverse the VO(acac)2-induced changes of eNOS activity, but BBR treatment increased the eNOS activity in control cells. The addition of 200μM VO(acac)2 significantly induced ROS generation in HUVECs, while 0.01 or 0.1 μM BBR reversed the change of ROS. In summary, BBR has protective effects in HUVECs damage induced by vanadium compounds, which is not mediated by eNOS, but related to reduced intracellular ROS.

Vasoreactivity of isolated aortic rings from dyslipidemic and insulin resistant inducible nitric oxide synthase knockout mice

Recent study from this lab indicated enhanced susceptibility of iNOS KO mice for diet induced obesity (DIO) and systemic insulin resistance (IR) as compared to C57BL/6 (WT) mice. The present study investigates aortic vasoreactivity in high fat diet (HFD) induced insulin resistant iNOS KO mice.WT and iNOS KO mice were fed with 45% HFD/10% LFD for ten weeks. Systemic IR was assessed via measurement of circulating lipids, glucose, and insulin; while phenylephrine (PE)/acetylcholine (ACh) induced responses were monitored in the isolated aortic rings. To understand the mechanism, qPCR or Western blotting experiments were performed in aorta and Ea.hy926 cells. After 10 weeks of HFD feeding, significant increase in the body weight/fat mass, augmented circulating lipids, glucose, insulin and inflammatory cytokines along with impaired acetylcholine induced aortic vasorelaxation and enhanced iNOS expression was observed in the aortic tissue of WT mice. In the aminoguanidine (AG, 20 mg/kg for 4 weeks) treated WT mice and also in iNOS KO mice, acetylcholine induced vasorelaxation was significantly preserved. Further, acetylcholine mediated vasorelaxation correlated with increased eNOS phosphorylation at Ser1177 residue in the iNOS KO mice and same was also observed in the iNOS silenced Ea.hy926 cells. Moreover, treatment of Ea.hy926 cells with palmitic acid or TNFα also caused a significant decrease in eNOS activity, which was reversed in iNOS silenced Ea.hy926 cells suggesting the role of iNOS in the reduction of eNOS activity. The study thus implies a critical role of iNOS in vascular diseases associated with dyslipidemia/IR.

GYY4137 Modulates Renal Remodeling in Hyperhomocysteinemia

IntroductionElevated plasma levels of homocysteine, also known as hyperhomocysteinemia (HHcy), is associated with chronic kidney disease and end‐stage renal failure. HHcy is also a vascular risk factor that causes glomerular remodeling by inducing and accumulating extracellular matrix (ECM) in the glomerular and peri‐glomerular spaces. This remodeling is, in part, due to Hcy‐mediated imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) and disruption of cell‐to‐cell gap junction protein, connexins (Cxs). HHcy also contribute to renal microvascular impairment and vasoconstriction due to decreased endogenous hydrogen sulfide (H2S) production and homocysteinylation of endothelial nitric‐oxide synthase (eNOS). However, the molecular events targeting Caveolin, eNOS, MMPs/TIMPs, Cxs and the role of H2S in HHcy associated ECM turnover and renovascular remodeling remain elusive.HypothesisWe investigated whether GYY4137, slow releasing H2S donor ameliorates HHcy effect and renal remodeling.MethodsCaveolin protein 1 knockout B6.Cg‐Cav1 (Cav1−/ −), C57BL6/J (WT) and hyperhomocysteinemic CBS+/− mice were supplemented without or with Hcy (2% in diet) and treated without or with GYY4137 (0.5 mg/Kg/d, IP) for a period of 8 weeks.ResultsHHcy decreased plasma H2S levels, upregulated Cav1 and decreased eNOS activity in the kidney. HHcy also resulted in the upregulation of mRNA and protein levels of MMPs −1, 2, −9, −8, −13 as well as connexins – 40 and −43 and decreased the activities of (TIMPs) −1, −2, −3, −4. Immunohistochemistry results indicated increased collagen IV deposition and expression of connexins in the glomerular and interstitial areas. GYY4137 treatment mitigated these changes in HHcy. In conclusion, our data suggests that H2S alleviates renovascular remodeling by modulating Cav‐1, eNOS, MMPs/TIMPs and Cxs in HHcy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Disruption of Renal Arginine Metabolism Promotes Kidney Injury in Hepatorenal Syndrome in Mice.

We propose that hyperammonemia may contribute to impaired renal arginine metabolism, leading to decreased eNOS activity, impaired microcirculation, tubular cell death, tubulointerstitial nephritis and fibrosis. Genetic deletion of arginase-2 partially restores microcirculation and thereby alleviates tubular injury. We also demonstrate that BDL in mice is an excellent, clinically relevant model to study the renal consequences of HRS. (Hepatology 2018; 00:000-000).

Insulin resistance disrupts cell integrity, mitochondrial function, and inflammatory signaling in lymphatic endothelium

Lymphatic vessel dysfunction and increased lymph leakage have been directly associated with several metabolic diseases. However, the underlying cellular mechanisms causing lymphatic dysfunction have not been determined. Aberrant insulin signaling affects the metabolic function of cells and consequently impairs tissue function. We hypothesized that insulin resistance in LECs decreases eNOS activity, disrupts barrier integrity increases permeability, and activates mitochondrial dysfunction and pro-inflammatory signaling pathways. LECs were treated with insulin and/or glucose to determine the mechanisms leading to insulin resistance. Acute insulin treatment increased eNOS phosphorylation and NO production in LECs via activation of the PI3K/Akt signaling pathway. Prolonged hyperglycemia and hyperinsulinemia induced insulin resistance in LECs. Insulin-resistant LECs produced less NO due to a decrease in eNOS phosphorylation and showed a significant decrease in impedance across an LEC monolayer that was associated with disruption in the adherence junctional proteins. Additionally, insulin resistance in LECs impaired mitochondrial function by decreasing basal-, maximal-, and ATP-linked OCRs and activated NF-κB nuclear translocation coupled with increased pro-inflammatory signaling. Our data provide the first evidence that insulin resistance disrupts endothelial barrier integrity, decreases eNOS phosphorylation and mitochondrial function, and activates inflammation in LECs.

Ticagrelor Improves Endothelial Function by Decreasing Circulating Epidermal Growth Factor (EGF).

Ticagrelor is one of the most powerful P2Y12 inhibitor. We have recently reported that, in patients with concomitant Stable Coronary Artery Disease (SCAD) and Chronic Obstructive Pulmonary Disease (COPD) undergoing percutaneous coronary intervention (PCI), treatment with ticagrelor, as compared to clopidogrel, is associated with an improvement of the endothelial function (Clinical Trial NCT02519608). In the present study, we showed that, in the same population, after 1 month treatment with ticagrelor, but not with clopidogrel, there is a decrease of the circulating levels of epidermal growth factor (EGF) and that these changes in circulating levels of EGF correlate with on-treatment platelet reactivity. Furthermore, in human umbilical vein endothelial cells (HUVEC) incubated with sera of the patients treated with ticagrelor, but not with clopidogrel there is an increase of p-eNOS levels. Finally, analyzing the changes in EGF and p-eNOS levels after treatment, we observed an inverse correlation between p-eNOS and EGF changes only in the ticagrelor group. Causality between EGF and eNOS activation was assessed in vitro in HUVEC where we showed that EGF decreases eNOS activity in a dose dependent manner. Taken together our data indicate that ticagrelor improves endothelial function by lowering circulating EGF that results in the activation of eNOS in the vascular endothelium.

GYY4137 Mitigates Caveolin‐dependent Renal Remodeling in Hyperhomocysteinemia

IntroductionHyperhomocysteinemia (HHcy), an increase in plasma homocysteine levels induces collagen deposition by causing imbalance between MMPs and TIMPs leading to fibrosis, and predisposing hypertension. HHcy also decreases the endogenous production of hydrogen sulfide (H2S) and homocysteinylates endothelial nitric‐oxide synthase (eNOS) resulting in further increase of vascular resistance. In the contrary, deletion of a membrane protein Caveolin‐1 (Cav‐1) is known to mitigate hypertension. However, the interplay between Caveolin, eNOS and MMPs/TIMPs and the role of H2S in HHcy associated renovascular remodeling and hypertension remains largely unknown. We tested the hypothesis that GYY4137, a H2S donor mitigates hyperhomocysteinemic effect in renal microvasculature and thus normalizes vascular tone.MethodsB6.Cg‐Cav1(Cav‐1) mice representing caveolae protein 1 knock‐out and C57BL6/J (WT) mice were treated without or with GYY4137 (0.5 mg/Kg/d, IP) and supplemented without or with Hcy (2% in diet) for a period of 8 weeks.ResultsHHcy decreased plasma H2S levels, upregulated Cav1 and decreased eNOS activity in kidney. HHcy resulted in the upregulation of mRNA and protein levels of matrix metalloproteinases (MMPs) ‐2,‐9,‐1,‐8,‐13 and decreased the activities of tissue inhibitor of metalloproteinases (TIMPs) ‐1,‐2,‐3,‐4. Immunohistochemistry showed increased collagen IV deposition in the glomerular and interstitial areas. GYY4137 treatment reversed these changes in HHcy. In conclusion, our data suggests that H2S mitigates renovascular remodeling by modulating Cav‐1, eNOS and MMPs/TIMPs in HHcy.Support or Funding InformationThis study was supported by National Institute of Health Grant ‐ DK104653 (to U.S.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential effects of heat shock protein 90 and serine 1179 phosphorylation on endothelial nitric oxide synthase activity and on its cofactors.

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. Previous studies showed that bovine eNOS serine 1179 (Serine 1177 for human eNOS) phosphorylation enhanced NO synthesis. Meanwhile, heat shock protein 90 (Hsp90) plays a critical role in maintenance of eNOS structure and function. However, the regulatory difference and importance between Serine 1179 phosphorylation and Hsp90 on eNOS activity have not been evaluated. In current studies, S1179D eNOS was employed to mimic phospho-eNOS and exhibited markedly increased enzyme activity than wild type eNOS (WT eNOS). Hsp90 showed a dose-dependent increase for both WT eNOS and S1179D eNOS activity at the presence of all eNOS cofactors, such as Calcium/Calmodulin (Ca2+ /CaM), BH4, and NADPH etc. The enhancement effects were abolished by dominant-negative mutant Hsp 90 protein. ENOS-cofactors dynamic assay showed that Hsp90 enhanced WT eNOS affinity to NADPH, L-arginine, and CaM but not to Ca2+ and BH4. The impact of eNOS Serine 1179 phosphorylation and Hsp90 on eNOS affinity to cofactors has also been compared. Different from the effect of Hsp90 on eNOS affinity to specific cofactors, Serine 1179 phosphorylation significantly increased eNOS affinity to all cofactors. Moreover, VEGF-induced eNOS phosphorylation in bovine aortic endothelial cells (BAECs) and more NO generation from eNOS compared to control. Inhibition of Hsp90 by geldanamycin decreased eNOS activity and decreased endothelial viability. In conclusion, by changing eNOS structure, Hsp90 profoundly affected eNOS functions, including change of affinity of eNOS to cofactors like Ca2+, L-arginine, BH4 and further affecting NO generation capability. These specific cofactors regulated by Hsp 90 could become potential therapeutic targets of the eNOS-related diseases in future.

Caveolin 1 and G-Protein–Coupled Receptor Kinase-2 Coregulate Endothelial Nitric Oxide Synthase Activity in Sinusoidal Endothelial Cells

Liver injury leads to a vasculopathy in which post-translational modifications of endothelial nitric oxide synthase (eNOS) lead to impaired nitric oxide synthesis. We hypothesized that caveolin 1 (CAV1), a well-known eNOS interactor, regulates eNOS activity in sinusoidal endothelial cells (SECs) via its interaction with G-protein-coupled receptor kinase-2 (GRK2) that also post-translationally modifies eNOS. Liver injury with portal hypertension was established using bile duct ligation in rats. CAV1 function was modified using a CAV1 scaffolding domain construct and cDNAs encoding wild-type CAV1, and CAV1 phosphorylation was increased in injured SECs, resulting in increased GRK2-CAV1 interaction and decreased eNOS activity. In injured SECs, endothelin-1 blocked CAV1 phosphorylation induced by CAV1 scaffolding domain, indicating that CAV1 interaction with GRK2 is inversely regulated by endothelin-1 and CAV1 scaffolding domain after liver injury. In addition, after transduction with DNA encoding wild-type CAV1 into SECs isolated from Cav1-deficient mice, GRK2 association with CAV1 was evident, whereas transduction with a dominant negative CAV1 mutated at tyrosine 14 reduced the interaction. Finally, isoproterenol-induced GRK2 phosphorylation enhanced CAV1-GRK2 interaction and reduced eNOS activity. Our data suggest a novel mechanism and model in which CAV1 phosphorylation facilitates CAV1 scaffolding and GRK2-CAV1 interaction, thus clustering eNOS within a complex that inhibits eNOS activity. This process takes place in injured, but not in normal, SECs.