Endothelial Nitric Oxide Synthase Phosphorylation Research Articles

Ghrelin ameliorates impaired angiogenesis of ischemic myocardium through GHSR1a-mediated AMPK/eNOS signal pathway in diabetic rats

ObjectivesGhrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), has been found to stimulate angiogenesis in vivo and in vitro. However, the effect and the corresponding mechanisms of ghrelin on impaired myocardial angiogenesis in diabetic and myocardial infarction (MI) rat model are still unknown. MethodsIn the present study, adult SD rats were randomly divided into 4 groups: control, DM, DM+ghrelin, DM+ghrelin+[D-Lys3]-GHRP-6 groups. DM was induced by streptozotocin (STZ) 60mg/kg body weight. 12 weeks post STZ injection all groups were subjected to MI, which was induced by ligation left anterior descending artery (LAD). Ghrelin and [D-Lys3]-GHRP-6 were administered via intraperitoneal injection at the doses 200μg/kg and 50mg/kg for 4 weeks, respectively. Left ventricular function, microvascular density (MVD), myocardial infarct size, the expression of hypoxia-inducible factor (HIF1α), vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1), AMPK and endothelial nitric oxide synthase (eNOS) phosphorylation were examined. ResultsCompared with the DM group, left ventricular ejection fraction (LVEF), fractional shortening (FS), and MVD were increased, whereas myocardial infarct size decreased remarkably in DM+ghrelin group. For the mechanism study, we found that ghrelin promoted the HIF1α, VEGF, Flk-1 and Flt-1 expression, AMPK and eNOS phosphorylation in diabetic rats. However, the above biochemical events in ghrelin treated diabetic rats were completely inhibited by GHSR-1a blocker [D-Lys3]-GHRP-6. ConclusionsThese results suggest that administration of ghrelin ameliorates impaired angiogenesis in diabetic MI rats. And these beneficial effects derive from regulating GHSR1a-mediated AMPK/eNOS signal pathway by upregulating of HIF1α, VEGF and its receptors Flk-1, Flt-1 expressions.

Antihypertensive effects of probiotics Lactobacillus strains in spontaneously hypertensive rats.

The cardiovascular effects of probiotics Lactobacillus fermentum CECT5716 (LC40), or L. coryniformis CECT5711 (K8) plus L. gasseri CECT5714 (LC9) (1:1) in spontaneously hypertensive rats (SHR) were evaluated. Ten Wistar Kyoto rats (WKY) and 30 SHR were randomly assigned to four groups (n = 10): a control WKY group, a control SHR groups, an SHR group treated with LC40, and an SHR treated with K8/LC9 group for 5 weeks (at a dose of 3.3 × 10(10) colony-forming units/day in drinking water). Long-term administration of probiotics reduced systolic blood pressure. The consumption of K8/LC9 mixture significantly reduced the cardiac and renal hypertrophy. Both groups of probiotics reversed the impaired aortic endothelium-dependent relaxation to acetylcholine observed in SHR. They also abolished the increased aortic superoxide levels by reducing the increased toll-like receptor-4 mRNA levels and NADPH oxidase activity found in SHR. K8/LC9 consumption also increased endothelial nitric oxide synthase phosphorylation. Probiotic treatments induced a change in the cecum microbiota of SHR, with higher counts of the Lactobacillus spp. cluster, and lower counts of Bacteriodes spp. and Clostridium spp. Probiotics exert cardiovascular protective effects in genetic hypertension related to the improvement of vascular pro-oxidative and pro-inflammatory status.

Chronic peroxisome proliferator-activated receptorβ/δ agonist GW0742 prevents hypertension, vascular inflammatory and oxidative status, and endothelial dysfunction in diet-induced obesity.

Endothelial dysfunction plays a key role in obesity-induced risk of cardiovascular disease. The aim of the present study was to analyze the effect of chronic peroxisome proliferator-activated receptor (PPAR)β/δ agonist GW0742 treatment on endothelial function in obese mice fed a high-fat diet (HFD). Five-week-old male mice were allocated to one of the following groups: control, control-treated (GW0742, 3 mg/kg per day, by oral gavage), HFD, HFD + GW0742, HFD + GSK0660 (1 mg/kg/day, intraperitoneal) or HFD-GW0742-GSK0660 and followed for 11 or 13 weeks. GW0742 administration to mice fed HFD prevented the gain of body weight, heart and kidney hypertrophy, and fat accumulation. The increase in plasma levels of fasting glucose, glucose tolerance test, homeostatic model assessment of insulin resistance, and triglyceride found in the HFD group was suppressed by GW0742. This agonist increased plasma HDL in HFD-fed mice and restored the levels of tumor necrosis factor-α and adiponectin in fat. GW0742 prevented the impaired nitric oxide-dependent vasodilatation induced by acetylcholine in aortic rings from mice fed HFD. Moreover, GW0742 increased both aortic Akt and endothelial nitric oxide synthase phosphorylation, and inhibited the increase in caveolin-1/endothelial nitric oxide synthase interaction, ethidium fluorescence, NOX-1, Toll-like receptor 4, tumor necrosis factor-α, and interleukin-6 expression, and IκBα phosphorylation found in aortae from the HFD group. GSK0660 prevented all changes induced by GW0742. PPARβ/δ activation prevents obesity and exerts protective effects on hypertension and on the early manifestations of atherosclerosis, that is, endothelial dysfunction and the vascular pro-oxidant and pro-inflammatory status, in HFD-fed mice.

Abstract P198: Differential Regulation of Endothelial Nitric Oxide Synthase Phosphorylation by Protease-Activated Receptors in Adult Human Endothelial Cells

Protease-activated receptors (PARs) have been shown to regulate endothelial nitric oxide synthase (eNOS) through the activation of specific sites on the enzyme. It has been established that phosphorylation of eNOS-Ser-1177 leads to the production of the potent vasodilator nitric oxide (NO), and is associated with PAR-2 activation; while phosphorylation of eNOS-Thr-495 decreases NO production, and is coupled to PAR-1 activation. In this study, we demonstrate a differential regulation of the eNOS/NO pathway by the PARs using primary adult human coronary artery endothelial cells (HCAEC). Thrombin and the PAR-1 activating peptide, TFLLR, which are known to phosphorylate eNOS-Thr-495 in bovine and human umbilical vein endothelial cells, phosphorylated eNOS-Ser-1177 in HCAECs, and increased NO production. The PAR-1 responses were blocked using SCH-79797, a PAR-1 inhibitor, and L-NAME was used to inhibit NO production. A PAR-2 specific ligand, SLIGRL, which has been shown to phosphorylate eNOS-Ser-1177 in bovine and human umbilical vein endothelial cells, primarily regulated eNOS-Thr-495 phosphorylation and suppressed NO production in the HCAECs. PAR-3, known for its non-signaling potential, was activated by TFRGAP, a PAR-3 mimicking peptide, and only induced phosphorylation of eNOS-Thr-495 with no effect on NO production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was calcium-dependent using the calcium chelator, BAPTA, and eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632. These data suggest a vascular bed specific differential coupling of PARs to the signaling pathways that regulate eNOS and NO production that may be responsible for the modulation of endothelial function associated with cardiovascular disease.

β1-Adrenergic Inhibition Improves Cardiac and Vascular Function in Experimental Septic Shock.

Preliminary experimental data suggest that selective β1-blockers may improve ex vivo cardiac function in animal sepsis. Currently, the effects of esmolol on in vivo cardiac function and on vascular function are unknown. The present study was designed to examine the effects of the β1-selective blocker esmolol on myocardial and vascular function in peritonitis-induced septic rats and to explore the inflammatory pathways involved in this process. Randomized animal study. University research laboratory. Male Wistar rats. Four hours after cecal ligation and puncture, Wistar rats were randomly allocated to the following groups: control, esmolol, norepinephrine (started at 18 hr after the surgery), and esmolol (started at 4 hr after the surgery) + norepinephrine (started at 18 hr after the surgery). Assessment at 18 hours after surgery was focused on cardiac contractility and vascular ex vivo function. Cardiac and vascular protein expressions of nuclear factor κB and endothelial nitric oxide synthase/Akt/inducible nitric oxide synthase pathways were assessed by Western blotting. When compared with sham-operated animals, cecal ligation and puncture animals developed hypotension, cardiac depression, and vascular hyporesponsiveness to vasopressor treatment. Esmolol infusion increased cardiac contractility and restored mesenteric vasoreactivity. This effect was associated with a decrease in nuclear factor κB activation, an increase in Akt and endothelial nitric oxide synthase phosphorylation, and a decrease in inducible nitric oxide synthase expression both at the cardiac and vessel level. Esmolol infusion was also associated with an up-regulation in α1-vascular adrenoreceptors. Adjunction of selective β1-blockade to standard septic shock management enhances intrinsic cardiac contractility and vascular responsiveness to catecholamines. These protective cardiovascular effects are likely predominantly attributed to the anti-inflammatory effect of esmolol.

Aging effects on pedicled fasciocutaneous flap survival in rats.

Poorer surgical outcomes in older patients undergoing locoregional head and neck reconstruction have raised questions about tolerance of aging tissue to iatrogenic ischemic insults. We examined the effects of aging on viability of pedicled composite flaps in 2-month and 6-month old Sprague-Dawley male rats and correlated flap survival with vascular endogenous growth factor (VEGF) and VEGF receptor 2-mediated signaling events. Flap segments were assessed for gross/cellular necrosis by optical microscopy and for proangiogenic, apoptotic, and proliferative protein-marker content. Flap necrosis significantly increased with age (4.2% in young vs 49.17% in old), correlating with reduced expression of VEGF, inhibition of signal transducer and activator of transcription 3 (STAT3), and Akt activation, impaired Akt-dependent endothelial nitric oxide synthase (eNOS) phosphorylation, elevated Bax/Bcl-2 ratio, activation of Caspase-3, upregulated nuclear poly (ADP-ribose) polymerase-1 (PARP-1) cleavage and lower proliferating cell nuclear antigen (PCNA) levels. Pedicled flap survival is higher in younger rats in part because of unhindered expression of VEGF and enhanced activity of cell survival and promigratory signaling pathways. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1152-E1162, 2016.

Adrenocorticotropic Hormone and PI3K/Akt Inhibition Reduce eNOS Phosphorylation and Increase Cortisol Biosynthesis in Long-Term Hypoxic Ovine Fetal Adrenal Cortical Cells.

This study was designed to determine the role of the MEK/ERK1/2 and PI3K/Akt pathways in cortisol production and endothelial nitric oxide synthase (eNOS) phosphorylation (peNOS) in the ovine fetal adrenal in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for the last 100 days of gestation (dGa). At 138 to 142 dGa, fetal adrenal cortical cells (FACs) were collected from LTH and age-matched normoxic fetuses. Cortisol production and peNOS were measured in response to pretreatment with the MEK/ERK1/2 pathway inhibitor UO126 (UO) and adrenocorticotropic hormone (ACTH) stimulation. UO126 reduced ACTH-stimulated cortisol in both normoxic and LTH FACs. UO126 alone or in combination with ACTH reduced peNOS in the normoxic group, while ACTH alone or ACTH + UO inhibited peNOS in LTH FACs. Additionally, cortisol was measured in response to pretreatment with UO and treatment with 22R-hydroxycholesterol (22R-OHC) or water-soluble cholesterol (WSC) with and without ACTH stimulation. UO126 had no effect on 22R-OHC-treated cells, but reduced cortisol in cells treated with WSC and/or ACTH. Cortisol and peNOS were also measured in response to pretreatment with PI3K/Akt pathway inhibitor Wortmannin (WT) and ACTH stimulation. Wortmannin further increased cortisol under ACTH-stimulated conditions and, like ACTH, reduced peNOS in LTH but not normoxic FACs. Together, these data suggest that in LTH FACs MEK/ERK1/2 does not regulate peNOS but that UO acts downstream from eNOS, possibly at cholesterol transport, to affect cortisol production in LTH FACs, while the PI3K/Akt pathway, along with ACTH, regulates peNOS and plays a role in the fetal adaptation to LTH in FACs.

Angiotensin I-converting enzyme (ACE) inhibition and nitric oxide (NO)-mediated antihypertensive effect of octaphlorethol A isolated from Ishige sinicola: In vitro molecular mechanism and in vivo SHR model

Angiotensin I-converting enzyme (ACE) inhibition and nitric oxide (NO) production are important factors that regulate blood pressure. In this study, the effects of octaphlorethol A (OPA) isolated from Ishige sinicola on ACE inhibition and NO production, the molecular mechanism underlying ACE inhibition, as well as its antihypertensive effect in spontaneously hypertensive rats (SHRs) were investigated. IC50 value of OPA against ACE was 59 µM. Molecular modelling studies indicated that the compound interacts with Cys370, Glu162, Glu376, Glu403, Glu411, Asp377, His383, His387, Tyr520, Arg522, Tyr523, and Lys511. In human endothelial cells, OPA increased endothelial nitric oxide synthase (eNOS) phosphorylation. We also demonstrated that these OPA-induced effects essentially depended on protein kinase B (Akt) and AMP-activated protein kinase (AMPK) activation. Furthermore, systolic blood pressure was reduced (21.9 mmHg in 6 h) by administration of the compound in SHRs. The results of this study suggested that OPA could be developed as a therapeutic agent for hypertension.

The F-BAR Protein NOSTRIN Dictates the Localization of the Muscarinic M3 Receptor and Regulates Cardiovascular Function.

Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear. We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice. The levels of NO and cGMP were significantly reduced in mice with endothelial cell-specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca(2+) in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished. NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure.

Computational modeling of shear forces and experimental validation of endothelial cell responses in an orbital well shaker system

Vascular endothelial cells are continuously exposed to hemodynamic shear stress. Intensity and type of shear stress are highly relevant to vascular physiology and pathology. Here, we modeled shear stress distribution in a tissue culture well (R = 17.5 mm, fill volume 2 ml) under orbital translation using computational fluid dynamics with the finite element method. Free surface distribution, wall shear stress, inclination angle, drag force, and oscillatory index on the bottom surface were modeled. Obtained results predict nonuniform shear stress distribution during cycle, with higher oscillatory shear index, higher drag force values, higher circular component, and larger inclination angle of the shear stress at the periphery of the well compared with the center of the well. The oscillatory index, inclination angle, and drag force are new quantitative parameters modeled in this system, which provide a better understanding of the hydrodynamic conditions experienced and reflect the pulsatile character of blood flow in vivo. Validation experiments revealed that endothelial cells at the well periphery aligned under flow and increased Kruppel-like Factor 4 (KLF-4), cyclooxygenase-2 (COX-2) expression and endothelial nitric oxide synthase (eNOS) phosphorylation. In contrast, endothelial cells at the center of the well did not show clear directional alignment, did not induce the expression of KLF-4 and COX-2 nor increased eNOS phosphorylation. In conclusion, this improved computational modeling predicts that the orbital shaker model generates different hydrodynamic conditions at the periphery versus the center of the well eliciting divergent endothelial cell responses. The possibility of generating different hydrodynamic conditions in the same well makes this model highly attractive to study responses of distinct regions of the same endothelial monolayer to different types of shear stresses thereby better reflecting in vivo conditions.

Role of the bradykinin B2 receptor in a rat model of local heart irradiation

Purpose: Radiation-induced heart disease (RIHD) is a delayed effect of radiotherapy for cancers of the chest, such as breast, esophageal, and lung. Kinins are small peptides with cardioprotective properties. We previously used a rat model that lacks the precursor kininogen to demonstrate that kinins are involved in RIHD. Here, we examined the role of the kinin B2 receptor (B2R) in early radiation-induced signaling in the heart.Materials and methods: Male Brown Norway rats received the B2R-selective antagonist HOE-140 (icatibant) via osmotic minipump from 5 days before until 4 weeks after 21 Gy local heart irradiation. At 4 weeks, signaling events were measured in left ventricular homogenates and nuclear extracts using western blotting and real-time polymerase chain reaction. Numbers of CD68-positive (monocytes/macrophages), CD2-positive (T-lymphocytes), and mast cells were measured using immunohistochemistry.Results: Radiation-induced c-Jun phosphorylation and nuclear translocation were enhanced by HOE-140. HOE-140 did not modify endothelial nitric oxide synthase (eNOS) phosphorylation or alter numbers of CD2-positive or mast cells, but enhanced CD68-positive cell counts in irradiated hearts.Conclusions: B2R signaling may regulate monocyte/macrophage infiltration and c-Jun signals in the irradiated heart. Although eNOS is a main target for kinins, the B2R may not regulate eNOS phosphorylation in response to radiation.

Ultrasound stimulates formation and release of vasoactive compounds in brain endothelial cells.

Stroke outcome is improved by therapeutic ultrasound. This benefit is presumed to be principally from ultrasound-mediated thrombolysis. We hypothesized that the therapeutic benefit of ultrasound in stroke may, in part, be mediated by the release of beneficial vasoactive substances. Accordingly, we investigated the effect of ultrasound on levels of cytochrome P-450, lipoxygenase, and cyclooxygenase metabolites of arachidonic acid as well as adenosine release and endothelial nitric oxide synthase (eNOS) phosphorylation in primary brain endothelial cells in vitro. Brain endothelial cells were exposed to 1.05-MHz ultrasound at peak rarefactional acoustic pressure amplitudes of 0.35, 0.55, 0.90, and 1.30 MPa. Epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraenoic acids (HETEs), PGE2, adenosine, nitrate/nitrite, and eNOS phosphorylation were measured after ultrasound exposure. Levels of 8,9-EET, 11,12-EET, and 14,15-EET increased by 230 ± 28%, 240 ± 30%, and 246 ± 31% (P < 0.05), respectively, whereas 5-HETE and 15-HETE levels were reduced to 24 ± 14% and 10 ± 3% (P < 0.05), respectively, compared with cells not exposed to ultrasound. PGE2 levels were reduced to 56 ± 14% of control. Adenosine increased more than sixfold after ultrasound exposure compared with unstimulated cells (1.36 ± 0.22 vs. 0.37 ± 0.10 ng/ml, P < 0.05), nitrate/nitrite was below levels of quantification, and eNOS phosphorylation was not altered significantly. Our results suggest that ultrasound may enhance tissue perfusion during stroke by augmenting the generation of vasodilator compounds and inhibiting that of vasoconstrictors. Such regulation supports a beneficial role for therapeutic ultrasound in stroke independent of its effect on the occlusive thrombus.

Arginase inhibition: a new treatment for preventing progression of established diabetic nephropathy

Our previous publication showed that inhibition of arginase prevents the development of diabetic nephropathy (DN). However, identification of targets that retard the progression of established DN-which is more clinically relevant-is lacking. Therefore, we tested the hypothesis that arginase inhibition would prevent the progression of established DN. Effects of arginase inhibition were compared with treatment with the angiotensin-converting enzyme inhibitor captopril, a current standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) or captopril starting at 6 wk of age for 12 wk (early treatment) or starting at 12 wk of age for 6 wk (late treatment). Early and late treatment with BEC resulted in protection from DN as indicated by reduced albuminuria, histological changes, kidney macrophage infiltration, urinary thiobarbituric acid-reactive substances, and restored nephrin expression, kidney nitrate/nitrite, kidney endothelial nitric oxide synthase phosphorylation, and renal medullary blood flow compared with vehicle-treated Ins2(Akita) mice at 18 wk of age. Interestingly, early treatment with captopril reduced albuminuria, histological changes, and kidney macrophage infiltration without affecting the other parameters, but late treatment with captopril was ineffective. These findings highlight the importance of arginase inhibition as a new potential therapeutic intervention in both early and late stages of diabetic renal injury.

Calorie Restriction Attenuates Monocrotaline-induced Pulmonary Arterial Hypertension in Rats.

:Calorie restriction (CR) is one of the most effective nonpharmacological interventions protecting against cardiovascular disease, such as hypertension in the systemic circulation. However, whether CR could attenuate pulmonary arterial hypertension (PAH) is largely unknown. The PAH model was developed by subjecting the rats to a single subcutaneous injection of monocrotaline. CR lowered mean pulmonary arterial pressure (mPAP) and reduced vascular remodeling and right ventricular hypertrophy in PAH rats. Meanwhile, CR attenuated endothelial dysfunction as evidenced by increased relaxation in response to acetylcholine. The beneficial effects of CR were associated with restored sirtuin-1 (SIRT1) expression and endothelial nitric oxide synthase (eNOS) phosphorylation and reduced eNOS acetylation in pulmonary arteries of PAH rats. To further clarify the role of SIRT1 in the protective effects of CR, adenoviral vectors for overexpression of SIRT1 were administered intratracheally at 1 day before monocrotaline injection. Overexpression of SIRT1 exhibited similar beneficial effects on mPAP and endothelial function, and increased eNOS phosphorylation and reduced eNOS acetylation in the absence of CR. Moreover, SIRT1 overexpression attenuated the increase in mPAP in hypoxia-induced PAH animals. Overall, the present data demonstrate that CR may serve as an effective treatment of PAH, and targeting the SIRT1/eNOS pathway may improve treatment of PAH.

Protective Effects of Glucagon-like Peptide 1 on Endothelial Function in Hypertension.

Vascular endothelial cells play a major role in maintaining cardiovascular homeostasis. Endothelial dysfunction, characterized by reduced endothelium-dependent relaxations or accompanied by enhanced endothelium-dependent contractions, is a hallmark of and plays a pivotal role in the pathogenesis of hypertension. Endothelial dysfunction in hypertension has been linked to decreases in nitric oxide (NO) bioavailability, reflecting the impaired generation of NO and/or the enhanced inactivation of NO by reactive oxygen species. Many of these conditions can be improved by glucagon-like peptide 1 (GLP-1), a proglucagon-derived hormone secreted by intestinal endocrine L-type cells, which is rapidly inactivated by an enzyme dipeptidyl peptidase 4 in circulation. On one hand, GLP-1 analogues or dipeptidyl peptidase 4 inhibitors upregulate endothelial nitric oxide synthase expression and increase endothelial nitric oxide synthase phosphorylation, resulting in improved production of NO and thus endothelium-dependent relaxations. On the other hand, GLP-1 and related agents attenuate endothelium-dependent contractions by reducing reactive oxygen species generation and cyclooxygenase-2 expression. GLP-1 elevating agents and GLP-1 receptor agonists improve endothelial function in hypertension, suggesting that GLP-1 signaling could be a therapeutic target in hypertension-related vascular events.

Abstract 452: Hyperglycemia and Oxidative Stress Impair Fluid Shear Stress-mediated Endothelial Nitric Oxide Synthase Phosphorylation and Activation via Disrupting Adherens Junctions

Endothelial dysfunction, characterized by a decrease of nitric oxide (NO) bioavailability in the vessel wall, plays a crucial role in the pathogenesis of atherosclerosis. Oxidative stress due to increased reactive oxygen species (ROS) is implicated in endothelial dysfunction associated with diabetes. However the molecular mechanisms by which oxidative stress causes endothelial dysfunction remain incompletely understood. Blood flow, which generates fluid shear stress acting on endothelium, is the most potent physiological stimulus for NO production through endothelial NO synthase (eNOS) activation. Here we report that hyperglycemia and oxidative stress impairs fluid shear stress signal transduction and eNOS activation in endothelium. We found that the exposure of endothelial cells (ECs) to ROS generators such as menadione and xanthine/xanthine oxidase inhibited laminar flow-mediated Akt and eNOS phosphorylation and activation in human endothelial cells. Moreover, ECs pre-exposed to high glucose that generates ROS production in ECs, failed to respond to laminar flow for Akt/eNOS phosphorylation and activation. Consequently NO production from ECs in response to laminar flow was attenuated by high glucose treatment. Mechanistically, we observed that hyperglycemia and oxidative stress altered endothelial adherens junction integrity, manifested by the alternation of the localization of cell-cell junction molecules vascular endothelial cadherin (VE-cadherin) and beta-catenin. Silencing VE-cadherin or beta-catenin with small interference RNA also inhibited laminar flow-mediated signaling for eNOS activation, which mimics the effects of oxidative stress, suggesting that cell-cell junction integrity is critical for fluid shear stress signal transduction and eNOS activation. Collectively, our results demonstrate that hyperglycemia and oxidative stress impair laminar flow-mediated eNOS activation through the alternation of endothelial junction integrity and fluid shear stress signal transduction. Our findings suggest a novel mechanism whereby oxidative stress induces diabetes-associated endothelial dysfunction.

Abstract 479: Orotic Acid Induces Hypertension Associated With Impaired Endothelial Nitric Oxide Synthesis

Orotic acid (OA) is an intermediate of pyrimidine nucleotide biosynthesis. Hereditary deficiencies in some enzymes associated with pyrimidine synthesis or the urea cycle induce OA accumulation, resulting in orotic aciduria. A link between patients with orotic aciduria and hypertension has been reported; however, the molecular mechanisms remain elusive. In this study, to elucidate the role of OA in vascular insulin resistance, we investigated whether OA induced endothelial dysfunction and hypertension. OA inhibited insulin- or metformin-stimulated nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation in human umbilical vein endothelial cells (HUVEC). A decreased insulin response by OA was mediated by impairment of the insulin-stimulated phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling pathway in cells overexpressing the p110-PI3K catalytic subunit. Impaired effects of metformin on eNOS phosphorylation and NO production were reversed in cells transfected with constitutively active AMP-activated protein kinase. Moreover, experimental induction of orotic aciduria in rats caused insulin resistance, measured as a 125% increase in the homeostasis model assessment, and hypertension, measured as a 25% increase in systolic blood pressure. OA increased the plasma concentration of endothelin-1 by 201% and significantly inhibited insulin- or metformin-induced vasodilation. A compromised insulin or metformin response on the Akt/eNOS and AMPK/eNOS pathway was observed in aortic rings of OA-fed rats. Taken together, we showed that OA induces endothelial dysfunction by contributing to vascular and systemic insulin resistance that affects insulin- or metformin-induced NO production, leading to the development of hypertension.

Cross‐talk Between Autophagy and Mitophagy Regulates Shear‐induced Nitric Oxide Production in Endothelial Cells

Aging is associated with suppressed endothelial cell (EC) autophagy and EC nitric oxide (NO) bioavailability. A link might exist among autophagy, mitophagy, and stimulated NO production in ECs. ECs treated with scrambled siRNA exposed to shear stress (3 h x 20 dyn/cm2) exhibit: ↑LC3‐II:LC3‐1, ↓p62, ↑LC3‐GFP puncta formation (indices of increased autophagy); ↓VDAC, ↓m‐aconitase, ↑TOM20:LAMP1 colocalization, ↓ mitochondrial pH indicating lysosomal compartmentalization (indices of increased mitochondrial turnover), ↑ROS generation, ↑endothelial NO synthase (eNOS) phosphorylation, and ↑NO production (p&lt;0.05 for all). ECs transfected with AuTophaGy‐related protein 3 (Atg3) siRNA did not exhibit shear‐induced autophagy, mitochondrial turnover, eNOS phosphorylation, or NO production, but ROS accumulation and cytokine production (ICAM‐1, MCP‐1, IL‐8, E‐selectin) were exaggerated (all p&lt;0.05). Shear‐induced increases in oxygen consumption rate, extracellular acidification rate, and ATP production (all p&lt;0.05) in ECs were prevented by autophagy suppression, indicating impaired mitochondrial function. Dysfunctional mitochondria might be the source of ROS when autophagy is compromised because: (i) shear‐induced increases in NO generation were restored, and ROS production and inflammation were normalized, when ECs +Atg3 siRNA were treated with a mitochondrial‐targeted O2•‐ scavenger; and (ii) the EC phenotype +Atg3 siRNA could be recapitulated using approaches that limit mitophagy per se. A crosstalk exists among autophagy, mitophagy, and NO production in ECs.

Shear‐Induced Extracellular Regulated Kinase Signaling to eNOS is Increased When Autophagy is Compromised in Endothelial Cells

Frictional forces (i.e., “shear stress”) exerted by flowing blood along the surface of the endothelium are potent stimuli for endothelial nitric oxide (NO) synthase (eNOS) phosphorylation and NO production. Recently we reported that shear‐induced increases in p‐eNOSS1177 (a positive eNOS regulatory site) and NO generation are prevented when the process of autophagy is disrupted in ECs via knockdown of AuTophaGy‐related protein 3 (Atg3) using siRNA. We sought to determine whether shear‐induced kinase stimulation / inhibition of eNOS is altered when autophagy is compromised in ECs. In ECs transfected with scrambled siRNA, shear stress (3 h x 20 dyn/cm2) increased (p&lt;0.05) autophagy (e.g., ↑LC3‐II:LC3‐1, ↓p62, ↑LC3‐GFP puncta formation), p‐eNOSS1177, and NO production (assessed via DAF, EPR), but p‐eNOST495 (a negative eNOS regulatory site) was unchanged. In ECs with &gt;85% knockdown of Atg3 protein expression via siRNA, shear–induced increases in autophagy, p‐eNOSS1177, and NO generation were prevented, but p‐eNOST495 increased (p&lt;0.05). As such, shear‐induced increases (p&lt;0.05) in p‐eNOSS1177 : p‐eNOST495 occur in ECs with intact but not compromised autophagy. Extracellular regulated kinase 1/2 (ERK1/2), which phosphorylates p‐eNOST495 in ECs, increased (p&lt;0.05) in response to shear stress only after Atg3 siRNA. Interestingly, when ERK1/2 activity was inhibited (FR180204) after Atg3 siRNA, shear‐induced increases p‐eNOSS1177 : p‐eNOST495 were restored. Work in progress is exploring mechanisms whereby autophagy suppression might alter kinase signaling to eNOS.

Endocannabinoid GPR18 Receptor Activation Confers Cardiovascular Protection in Diabetic Rats

The endocannabinoid receptor GPR18 lowers BP by reducing oxidative stress in brainstem nuclei that control sympathetic activity. We hypothesized that GPR18 activation confers cardiovascular protection in diabetes by reversing myocardial oxidative stress and dysfunction. Four weeks after diabetes induction (Streptozotocin; STZ, 55 mg/kg; i.p), diabetic rats received daily (100 ug/kg;i.p) of the GPR18 agonist, abnormal cannabidiol (Abn‐cbd) or antagonist, O‐1918, while control rats received vehicle, for 2 weeks before hemodynamic measurements in conscious rats. Abn‐cbd reversed the reduction in myocardial function by increasing left ventricular contractility index +dp/dtmax while maintaining the hypotensive response in diabetic rats, which inferred Abn‐cbd mediated reduction in vascular resistance. Ex vivo molecular findings demonstrated Abn‐cbd reversal of the diabetes‐induced: (i) reductions in plasma and myocardial nitrate /nitrite (NOX) levels; (ii) reductions in plasma adiponectin and increase in myocardial adiponectin receptor‐1 (Adipo R1) levels; (iii) reductions in myocardial protein kinase B (AKT), extracellular signaling kinase (ERK) and endothelial nitric oxide synthase (eNOS) phosphorylation; (iv) elevations in myocardial malondialdehyde (MDA) and antioxidant catalase levels; the latter might serve a counterbalancing role against diabetes‐evoked oxidative stress. The findings that Abn‐cbd enhanced, while the GPR18 antagonist O‐1918 reduced, myocardial GPR18 expression implicate GPR18 signaling in the Abn‐cbd evoked molecular and hemodynamic effects. These findings are the first to demonstrate a favorable cardiovascular role of GPR18 in diabetes and to elucidate the molecular mechanisms implicated in these clinically relevant effects.