Endothelial Vasorelaxation Research Articles

Cystathionine γ-Lyase-Produced Hydrogen Sulfide Controls Endothelial NO Bioavailability and Blood Pressure.

Hydrogen sulfide (H2S) and NO are important gasotransmitters, but how endogenous H2S affects the circulatory system has remained incompletely understood. Here, we show that CTH or CSE (cystathionine γ-lyase)-produced H2S scavenges vascular NO and controls its endogenous levels in peripheral arteries, which contribute to blood pressure regulation. Furthermore, eNOS (endothelial NO synthase) and phospho-eNOS protein levels were unaffected, but levels of nitroxyl were low in CTH-deficient arteries, demonstrating reduced direct chemical interaction between H2S and NO. Pretreatment of arterial rings from CTH-deficient mice with exogenous H2S donor rescued the endothelial vasorelaxant response and decreased tissue NO levels. Our discovery that CTH-produced H2S inhibits endogenous endothelial NO bioavailability and vascular tone is novel and fundamentally important for understanding how regulation of vascular tone is tailored for endogenous H2S to contribute to systemic blood pressure function.

Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

BackgroundRoux‐en‐Y gastric bypass (RYGB) reduces obesity‐associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c‐Jun N‐terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity‐induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits.Methods and ResultsAfter 7 weeks of a high‐fat high‐cholesterol diet, obese rats underwent RYGB or sham surgery; sham–operated ad libitum–fed rats received, for 8 days, either the control peptide D‐TAT or the JNK peptide inhibitor D‐JNKi‐1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D‐JNKi‐1 treatment improved endothelial vasorelaxation in response to insulin and glucagon‐like peptide‐1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D‐JNKi‐1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon‐like peptide‐1–mediated signaling. The inhibitory phosphorylation of insulin receptor substrate‐1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability.ConclusionsDecreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity‐induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity.

Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase.

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R−/−), B2- (B2R−/−) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R−/−). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R−/− presented similar level of vascular dysfunction as found in B1R−/− or B2R−/− mice. In accordance, aortic rings from B1R−/− or B2R−/− mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R−/− and B2R−/− mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R−/− or B2R−/− mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.

AMP-activated protein kinase mediates the effects of lipoprotein-associated phospholipase A2 on endothelial dysfunction in atherosclerosis.

The present study aimed to investigate the effects of lipoprotein-associated phospholipase A2 (Lp-PLA2) on endothelial dysfunction in an in vitro cell model of atherosclerosis, and to determine whether AMP-activated protein kinase (AMPK) mediates the effects of Lp-PLA2 on endothelial dysfunction. A total of 392 patients with coronary artery disease (CAD), including various sub-conditions, were recruited, and the plasma concentrations of Lp-PLA2 were evaluated. In addition, an in vitro model of atherosclerosis was established by exposing human umbilical vein endothelial cells (HUVECs) to oxidized low-density lipoprotein (oxLDL). SB-435495 was used to inhibit Lp-PLA2, and compound C was used to suppress AMPK expression. Lp-PLA2, AMPKα and phosphorylated-AMPKα (T172) expression in HUVECs were evaluated using western blot analysis. The concentrations of nitric oxide (NO), endothelin 1 (ET-1), intercellular adhesion molecule 1 (ICAM-1) and platelet/endothelial cell adhesion molecule 1 (PECAM-1) in cell culture supernatant were determined using commercially available ELISA kits. MTT assays were employed to indicate changes in cell viability. The current study found the plasma Lp-PLA2 levels were elevated in the CAD patients with stable angina pectoris, unstable angina pectoris, acute coronary syndromes and acute myocardial infarction, compared with a healthy control population. In addition, the in vitro results showed that Lp-PLA2 expression levels were elevated in oxLDL-exposed HUVECs. Lp-PLA2 suppression could increase cell viability, induce the production of NO and decrease the secretion of ET-1, in addition to suppressing the expression of cell adhesion molecules, including ICAM-1 and PECAM-1 in oxLDL-exposed HUVECs. The expression of AMPKα and phosphorylated-AMPKα (T172) was regulated by Lp-PLA2, and AMPK suppression was able to reverse the effects of Lp-PLA2 with regard to cell viability, endothelial vasorelaxation capacity and the secretion of adhesion molecules in oxLDL-exposed HUVECs. In conclusion, the present study provides initial evidence that Lp-PLA2 is able to cause endothelial dysfunction in an in vitro model of atherosclerosis, and the effects of Lp-PLA2 on endothelial dysfunction was at least partially a result of the downregulation of AMPKα, thus contributing to the progression of atherosclerosis.

Low-Dose Lithium Stabilizes Human Endothelial Barrier by Decreasing MLC Phosphorylation and Universally Augments Cholinergic Vasorelaxation Capacity in a Direct Manner.

Lithium at serum concentrations up to 1 mmol/L has been used in patients suffering from bipolar disorder for decades and has recently been shown to reduce the risk for ischemic stroke in these patients. The risk for stroke and thromboembolism depend not only on cerebral but also on general endothelial function and health; the entire endothelium as an organ is therefore pathophysiologically relevant. Regardless, the knowledge about the direct impact of lithium on endothelial function remains poor. We conducted an experimental study using lithium as pharmacologic pretreatment for murine, porcine and human vascular endothelium. We predominantly investigated endothelial vasorelaxation capacities in addition to human basal and dynamic (thrombin-/PAR-1 receptor agonist-impaired) barrier functioning including myosin light chain (MLC) phosphorylation (MLC-P). Low-dose therapeutic lithium concentrations (0.4 mmol/L) significantly augment the cholinergic endothelium-dependent vasorelaxation capacities of cerebral and thoracic arteries, independently of central and autonomic nerve system influences. Similar concentrations of lithium (0.2–0.4 mmol/L) significantly stabilized the dynamic thrombin-induced and PAR-1 receptor agonist-induced permeability of human endothelium, while even the basal permeability appeared to be stabilized. The lithium-attenuated dynamic permeability was mediated by a reduced endothelial MLC-P known to be followed by a lessening of endothelial cell contraction and paracellular gap formation. The well-known lithium-associated inhibition of inositol monophosphatase/glycogen synthase kinase-3-β signaling-pathways involving intracellular calcium concentrations in neurons seems to similarly occur in endothelial cells, too, but with different down-stream effects such as MLC-P reduction. This is the first study discovering low-dose lithium as a drug directly stabilizing human endothelium and ubiquitously augmenting cholinergic endothelium-mediated vasorelaxation. Our findings have translational and potentially clinical impact on cardiovascular and cerebrovascular disease associated with inflammation explaining why lithium can reduce, e.g., the risk for stroke. However, further clinical studies are warranted.

Morus alba extract modulates blood pressure homeostasis through eNOS signaling.

Morus alba is a promising phytomedicine cultivated in oriental countries that is extensively used to prevent and treat various cardiovascular problems. To date, despite its beneficial effects, the molecular mechanisms involved remain unclear. Thus, we investigate the vascular and haemodynamic effects of Morus alba extract in an experimental model focusing our attention on the molecular mechanisms involved. Through vascular reactivity studies, we demonstrate that Morus alba extract evokes endothelial vasorelaxation through a nitric oxide-dependent pathway. Our molecular analysis highlights an increase in endothelial nitric oxide synthase (eNOS) phosphorylation. In vivo administration of Morus alba extract reduces blood pressure levels exclusively in wild-type mice, whereas it fails to evoke any haemodynamic effects in eNOS-deficient mice. Molecular analyses revealed that its beneficial action on vasculature is mediated by the activation of two important proteins that act as stress sensors and chaperones: PERK and heat shock protein 90. Finally, Morus alba extract exerts antihypertensive action in an experimental model of arterial hypertension. Through its action on eNOS signaling, Morus alba extract could act as a food supplement for the regulation of cardiovascular system, mainly in clinical conditions characterized by eNOS dysfunction, such as arterial hypertension.

JNK INHIBITION IMPROVES OBESITY-INDUCED ENDOTHELIAL DYSFUNCTION AND OXIDATIVE STRESS MIMICKING THE BENEFICIAL EFFECTS OF ROUX-EN-Y GASTRIC BYPASS SURGERY

Roux-en-Y gastric bypass surgery (RYGB) reduces cardiovascular risk in obese patients. We showed that in diet-induced obese (DIO) rats RYGB improves endothelial vasorelaxation 8 days after surgery independently from weight loss via decreased aortic JNK phosphorylation and increased endothelial

Abstract 13780: JNK Inhibition Mimics the Beneficial Effect of Roux-en-Y Gastric Bypass Surgery on Obesity-Induced Endothelial Dysfunction

Introduction: Roux-en-Y gastric bypass surgery (RYGB) reduces weight and long-term cardiovascular risk in obese patients. We previously demonstrated that in diet-induced obese rats RYGB improves aortic endothelial vasorelaxation 8 days after surgery in a weight loss-independent manner. Improved endothelial function was associated with decreased JNK phosphorylation and increased Akt and eNOS phosphorylation in the aorta. Hypothesis: We investigated whether in vivo inhibition of JNK activity in sham-operated ad libitum-fed rats mimics these endothelial effects of RYGB. Methods: Male Wistar rats were fed a high fat (60%) high cholesterol (1.25%) diet for 7 weeks before undergoing either RYGB or sham surgery; sham-operated rats received vehicle (sham_AL) or the JNK-specific inhibitor SP600125 40mg/kg/day s.c. for 8 days post-surgery (sham_SP). Then, thoracic aortic rings were isolated and subjected to ex vivo isometric tension recordings. After submaximal contraction with norepinephrine (10-6mol/L), cumulative relaxation responses were performed to GLP-1 (7-36) amide (10-12 to 10-6mol/L) or insulin (10-12 to 10-5mol/L); tests were repeated after preincubation with the eNOS inhibitor L-NAME (10-4mol/L). Western blot analysis of JNK, Akt and eNOS was also performed on aortic tissue lysates. Results: Body weight did not differ between sham_SP and sham_AL rats, while the weight loss of RYGB rats was significant 8 days after surgery. GLP-1- and insulin-induced vasorelaxation responses improved in RYGB compared to sham_AL rats; interestingly, sham_SP rats had endothelial relaxation similar to RYGB rats. All responses were blocked by preincubation with L-NAME, suggesting an eNOS-dependent effect. JNK protein phosphorylation in aortic lysates was decreased in RYGB and sham_SP rats compared to sham_AL, while Akt phosphorylation was increased. eNOS phosphorylation and dimerization were also increased in RYGB and sham_SP compared to sham_AL rats. Conclusion: JNK inhibition seems to mimic the immediate eNOS-mediated endothelial protective effects of RYGB. Further, our study underlines a crucial role of JNK in obesity-associated endothelial dysfunction, suggesting a novel mechanism for the cardiovascular effects of RYGB.

FGF23 directly impairs endothelium-dependent vasorelaxation by increasing superoxide levels and reducing nitric oxide bioavailability.

Fibroblast growth factor 23 (FGF23) is secreted primarily by osteocytes and regulates phosphate and vitamin D metabolism. Elevated levels of FGF23 are clinically associated with endothelial dysfunction and arterial stiffness in chronic kidney disease (CKD) patients; however, the direct effects of FGF23 on endothelial function are unknown. We hypothesized that FGF23 directly impairs endothelial vasorelaxation by hindering nitric oxide (NO) bioavailability. We detected expression of all four subtypes of FGF receptors (Fgfr1-4) in male mouse aortas. Exogenous FGF23 (90-9,000 pg/ml) did not induce contraction of aortic rings and did not relax rings precontracted with PGF2α. However, preincubation with FGF23 (9,000 pg/ml) caused a ∼36% inhibition of endothelium-dependent relaxation elicited by acetylcholine (ACh) in precontracted aortic rings, which was prevented by the FGFR antagonist PD166866 (50 nM). Furthermore, in FGF23-pretreated (9,000 pg/ml) aortic rings, we found reductions in NO levels. We also investigated an animal model of CKD (Col4a3(-/-) mice) that displays highly elevated serum FGF23 levels and found they had impaired endothelium-dependent vascular relaxation and reduced nitrate production compared with age-matched wild types. To elucidate a mechanism for the FGF23-induced impairment, we measured superoxide levels in endothelial cells and aortic rings and found that they were increased following FGF23 treatment. Crucially, treatment with the superoxide scavenger tiron reduced superoxide levels and also restored aortic relaxation to ACh. Therefore, our data suggest that FGF23 increases superoxide, inhibits NO bioavailability, and causes endothelial dysfunction in mouse aorta. Together, these data provide evidence that high levels of FGF23 contribute to cardiovascular dysfunction.

Abstract 15: Novel Functions of Small GTPase Rap1 in Regulating Endothelial Homeostasis: Control of Nitric Oxide Release, Vascular Function and Blood Pressure

Endothelial dysfunction, resulting from decreased nitric oxide (NO) bioavailability is a pathology linked to endothelial vasomotor dysfunction and hypertension, inflammation and atherosclerosis, perturbed endothelial barrier and progression of diabetes. In blood vessels, NO is produced by the endothelial NO synthase (eNOS), the activity of which is regulated by Ca2+/calmodulin, binding of regulatory cofactors, and posttranslational modifications, including phosphorylation events on Ser1177, which stimulate NO production. Rap1 is a ubiquitously expressed small GTPase implicated in promoting vascular barrier. We have shown that endothelial cell (EC)-specific Rap1 deletion leads to defective angiogenesis in vivo due to faulty VEGFR2 activation and signaling. Importantly, EC-specific Rap1 knockout mice developed hypertension and pathological left ventricular hypertrophy. The objective of the study was to determine the role of small G protein Rap1 in regulating endothelial NO production and endothelial-dependent vasorelaxation in vivo and ex vivo. Using ex vivo myography and tamoxifen-inducible, endothelial-specific Rap1-knockout mice (Cadh5-CreERT2+/0;Rap1f/f), we demonstrate that Rap1 deficiency completely abrogates NO-dependent vasodilation and attenuates NO production. Mechanistically, we show that Rap1 is rapidly activated in response to receptor agonists that activate eNOS via Ca2+/calmodulin- dependent pathway and in response to shear flow, which modules eNOS activity by its phosphorylation. Rap1 deletion in human ECs, in vitro, leads to deficient NO release in response to both these stimuli, and interferes with PI3K/Akt pathway and eNOS Ser1177 phosphorylation. Further, we demonstrate Rap1 is required for transducing signals from the endothelial mechanosensing complex comprising PECAM-1, VE-cadherin and VEGFR2 in response to shear flow, leading to ligand-independent VEGFR2 activation and signaling to stimulate NO production. We conclude that Rap1 in endothelium is critically required for endothelial homeostasis and NO production, thereby affecting vascular tone and regulation of blood pressure. Furthermore, this study establishes Rap1 as a novel regulator of mechanotransduction in response to shear flow.

Obeticholic acid, a farnesoid X receptor agonist, improves portal hypertension by two distinct pathways in cirrhotic rats

The farnesoid X receptor (FXR) is a nuclear bile acid receptor involved in bile acid homeostasis, hepatic and intestinal inflammation, liver fibrosis, and cardiovascular disease. We studied the effect of short-term treatment with obeticholic acid (INT-747), a potent selective FXR agonist, on intrahepatic hemodynamic dysfunction and signaling pathways in different rat models of cirrhotic portal hypertension (PHT). For this, thioacetamide (TAA)-intoxicated and bile-duct-ligated (BDL) rats were used as models. After gavage of two doses of 30 mg/kg of INT-747 or vehicle within 24 hours, in vivo hemodynamics were assessed. Additionally, we evaluated the direct effect of INT-747 on total intrahepatic vascular resistance (IHVR) and intrahepatic vascular tone (endothelial dysfunction and hyperresponsiveness to methoxamine) by means of an in situ liver perfusion system and on hepatic stellate cell contraction in vitro. FXR expression and involved intrahepatic vasoactive pathways (e.g., endothelial nitric oxide synthase [eNOS], Rho-kinase, and dimethylarginine dimethylaminohydrolase [DDAH]) were analyzed by immunohistochemistry, reverse-transcriptase polymerase chain reaction, or western blotting. In both cirrhotic models, FXR expression was decreased. Treatment with INT-747 in TAA and BDL reactivated the FXR downstream signaling pathway and decreased portal pressure by lowering total IHVR without deleterious systemic hypotension. In the perfused TAA and BDL cirrhotic liver, INT-747 improved endothelial vasorelaxation capacity, but not hyperresponsiveness. In both groups, this was associated with an increased eNOS activity, which, in TAA, related to down-regulation of Rho-kinase and in BDL to up-regulation of DDAH-2. FXR agonist INT-747 improves PHT in two different rat models of cirrhosis by decreasing IHVR. This hemodynamic effect relates to increased intrahepatic eNOS activity by pathways that differ depending on the etiology of cirrhosis.

Vasodilator response to <i>Psidium guajave</i> leaves of mesenteric bed vessels from diabetic and non-diabetic rats

Mortality from cardiovascular abnormalities is almost three times more prevalent in the diabetic population than that in the general population. Psidium guajave has been used traditionally for a long time as a medicinal herb to cure diabetes. Endothelial dependent vasorelaxation effect of Psidium guajave extract on normal aortic rings was observed. This study was designed to investigate the endothelium and nitric oxide roles inPGE-induced vasorelaxation in diabetic rat vessel. Diabetes was induced by a single intraperitoneal injection of streptozotocin. Eight weeks later, superior mesenteric arteries of non-diabetic and diabetic groups were isolated and perfused according to the McGregor method. Prepared vascular beds were constricted with phenylephrine to induce 70% - 75% of maximal constriction. Psidium guajave extract at concentrations of 0.1 to 4 mg/100ml added to the medium and perfusion pressure was recorded. Baseline perfusion pressure of diabetic group was significantly higher than non-diabetic rats in both intact and denuded endothelium. Psidium guajave extract caused a significantly dose depended decrease in perfusion pressure in non-diabetic and diabetic groups in both intact and denuded endothelium. In the presence of N (ω)-nitro-L-arginine methyl ester, Psidium guajave ex-tractinduced relaxation in intact mesenteric beds of non-diabetic and diabetic animals was not suppressed. From the results of this study, it may be concluded that vasorelaxatory effect of Psidium guajave extract is not mediated by endothelium and nitric oxide in both diabetic and non-diabetic vessel and supports the use of the plant’s leaves as a natural, adjunct phytomedicine in the management of diabetes vessel complications.

Inwardly rectifying potassium channels 2.x contribute to endothelial hyperpolarization and vasorelaxation to P2Y receptor stimulation in murine cerebral artery

Inwardly rectifying potassium channels (Kir) 2.x are expressed in cerebral endothelial cells (EC) but little is known about their role in vascular tone control. We tested the hypothesis that endothelial Kir amplify membrane hyperpolarization and cerebral vasodilation to ATP and UTP.MethodsExpression of Kir 2.1 and 2.2 was demonstrated in freshly isolated EC from rat middle cerebral artery (MCA) by PCR. Vasorelaxation (pressure myography) and endothelial hyperpolarization (di‐8‐ANEPPS) were measured in pressurized MCA following intraluminal delivery of UTP. Membrane current and voltage changes were recorded from mouse cerebral EC using perforated‐patch voltage clamp and current clamp techniques.ResultsUnstimulated EC showed Kir‐like (sensitive to Ba2+ and Kir 2.x blocker ML 133) and transient outward KCa‐like currents. Stimulation with ATP activated large outward currents that were sensitive to SKCa and IKCa blockers (UCL1684 and TRAM34). The ensuing membrane hyperpolarization was significantly attenuated in cells pretreated with Ba2+ or ML 133. In pressurized MCA, intraluminal application of Ba2+ significantly decreased hyperpolarization and relaxation responses to UTP.ConclusionEndothelial Kir2.x channels play a significant role in cerebral vasorelaxation by maintaining endothelial hyperpolarization in response to P2Y receptor stimulation. Supported by R01HL088435 to SPM

Type I interferons modulate vascular function, repair, thrombosis, and plaque progression in murine models of lupus and atherosclerosis

Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis. Lupus-prone New Zealand mixed 2328 (NZM) mice and atherosclerosis-prone apolipoprotein E- knockout (apoE(-/-) ) mice were compared to mice lacking type I IFN receptor (INZM and apoE(-/-) IFNAR(-/-) mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE(-/-) mice exposed to an IFNα-containing or empty adenovirus. Loss of type I IFN receptor signaling improved endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus-prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE(-/-) IFNAR(-/-) mice, compared to apoE(-/-) mice, while NZM and apoE(-/-) mice exposed to IFNα developed accelerated thrombosis and platelet activation. These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.

S11 * ALCOHOL AND THE HEART * S11.1 * CLINICAL OVERVIEW OF ALCOHOL EFFECTS ON THE HEART

Alcohol consumption is associated with positive beneficial and negative toxic effects on the heart and cardiovascular system. Moderate alcohol consumption has been linked to beneficial effects on arterial atherogenesis and thrombus formation, with increase of HDL cholesterol, endothelial vasorelaxation, reduced platelet aggregation and fibrinogen. The French paradox of relatively low cardiovascular mortality despite high saturated fat intake has sparked research of chronic red wine consumption. Alcoholic heart disease caused by high consumption includes arrhythmias, especially atrial fibrillation and holiday heart syndrome, increased hypertension with LV hypertrophy, heart failure and cardiomyopathy (CMP). Consumption of >90 g/day over 5 years has been associated with development of alcoholic CMP. Alcoholic CMP is accompanied by apoptosis of cardiac myocytes, but importantly shows improved outcome in absolute alcohol abstinence. In chronic alcohol abuse even without overt cardiac disease, we recently reported increased cardiac NT-proBNP plasma levels, decreasing after withdrawal, reflecting early negative cardiac effects. Interestingly, even in patients with previous cardiovascular ischemic events, up to moderate alcohol consumption has been shown to decrease secondary mortality. Overall, alcohol has controversial J-curve dose effects on the heart and cardiovascular system. # S11.2 QUANTITATIVE PROTEOMIC ANALYSIS OF ALCOHOLIC CARDIOMYOPATHY {#article-title-2} Chronic heavy alcohol consumption is responsible for the development of alcoholic cardiomyopathy (ACM), a dilated cardiomyopathy which at early stages may be reversed after withdrawal from alcohol consumption. If untreated, ACM leads often to heart failure. Mitochondrial dysfunction, reduced cardiac contractility, left ventricular (LV) dilation and reduction in LV ejection fraction have been shown to be involved in the pathobiology of ACM. However, there are no biomarkers characteristic of ACM that could be used for early diagnosis. For the discovery of proteomic biomarkers, we developed an animal model of ACM of rats fed long-term a Lieber DeCarli liquid diet and isocalorically fed paired controls. Hearts pre-screened by echocardiography and hemodynamics for cardiac dysfunction were used for protein fractionation, digestion, peptides separation by strong cation ion exchange liquid chromatography (LC) and detection by mass spectrometry (MS) using a Fourier Transform Ion Cyclotron Resonance instrument. Comparison of LC-MS/MS spectra from rats with ACM and paired controls provided over 300 differentially expressed proteins. Quantitative analysis of the datasets by Elucidator software identified 32 downregulated proteins with P < 0.05 that were visualized on IPA pathway maps and DAVID canonical pathways into three pathways: oxidative phosphorylation, ribosome and glutathione. Human heart samples of transplantation quality from heavy alcohol drinkers with or without ACM, and non-alcoholic controls were obtained from the University of Barcelona Hospital Clinic, and processed by the same proteomic methodology as rat samples. Analysis on the datasets identified 117 differentially expressed proteins among the three sample groups, with significant changes in 31 proteins belonging to the oxidative phosphorylation chain, cardiac muscle and citrate cycle pathways. In the oxidative phosphorylation chain, alcohol affected predominantly proteins of the mitochondrial Complex I and III in alcoholics without ACM, which progressed to include proteins of Complex IV and V in alcoholics with clinical symptoms of ACM. Overall, the label-free proteomic and pathway analyses of rat and human heart samples provided potential targets to aid in the early diagnosis of ACM. # S11.3 MOLECULAR MECHANISM OF ALCOHOL-INDUCED CARDIAC DAMAGE {#article-title-3} One-third of alcohol abusers manifest decreased cardiac contractility and muscle strength termed, respectively, alcoholic cardiomyopathy and myopathy. In fact, the most common cause of cardiomyopathy with congestive heart failure is chronic alcoholism. However, the pathogenesis of these conditions still remains obscure. Studies on human alcoholics and animal models have demonstrated that chronic ethanol causes structural and functional derangements of mitochondria. Impairments of mitochondrial bioenergetics and the apoptosis-regulating function of mitochondria are considered to be central for the increased cell death in both alcoholic heart and liver disease. Emerging research indicates the interdependence between the factors that regulate mitochondrial morphology and function. Here, we have studied the effect of chronic alcohol exposure on mitochondrial dynamics in cardiac cells using organelle-targeted fluorescent proteins. Since chronic ethanol treatment results in oxidative stress, not only in liver, but also in heart, we have also investigated the effect of reactive oxygen species (ROS) on mitochondrial fusion–fission and motility. Our studies provide evidence that both chronic alcohol exposure and ROS result in a decrease in mitochondrial motility and fusion activity. ROS produced by a subset of mitochondria resulted in rapid and spatially confined motility inhibition. Although both alcohol and ROS promote mitochondrial permeability transition pore (PTP) opening and the ensuing swelling can contribute to suppression of mitochondrial dynamics, inhibition of motility by ROS was also observed in the presence of cyclosporine A, a drug that inhibits PTP opening. These data indicate that mitochondrial dynamics is suppressed during chronic alcohol exposure, and this may be mediated by increased ROS production through some PTP-dependent and -independent pathways.

Endothelial arginase II and atherosclerosis

Atherosclerotic vascular disease is the leading cause of morbidity and mortality in developed countries. While it is a complex condition resulting from numerous genetic and environmental factors, it is well recognized that oxidized low-density lipoprotein produces pro-atherogenic effects in endothelial cells (ECs) by inducing the expression of adhesion molecules, stimulating EC apoptosis, inducing superoxide anion formation and impairing protective endothelial nitric oxide (NO) formation. Emerging evidence suggests that the enzyme arginase reciprocally regulates NO synthase and NO production by competing for the common substrate L-arginine. As oxidized LDL (OxLDL) results in arginase activation/upregulation, it appears to be an important contributor to endothelial dysfunction by a mechanism that involves substrate limitation for endothelial NO synthase (eNOS) and NO synthesis. Additionally, arginase enhances production of reactive oxygen species by eNOS. Arginase inhibition in hypercholesterolemic (ApoE-/-) mice or arginase II deletion (ArgII-/-) mice restores endothelial vasorelaxant function, reduces vascular stiffness and markedly reduces atherosclerotic plaque burden. Furthermore, arginase activation contributes to vascular changes including polyamine-dependent vascular smooth muscle cell proliferation and collagen synthesis. Collectively, arginase may play a key role in the prevention and treatment of atherosclerotic vascular disease.

TNF‐derived TIP peptide ameliorates High Glucose (HG)‐induced Arginase (ARG) mediated Endothelial Dysfunction (ED) via inhibiting PKC‐α activation

During diabetes, elevated ARG activity causes ED by competing with NO synthase (eNOS) for their common substrate L‐arginine. HG levels activate protein kinase C (PKC)‐α, implicated in impaired endothelial vasorelaxation and hyperpermeability. PKC‐α activates NADPH oxidase (NOX)‐dependent ROS generation and the small GTPase RhoA, upstream to ARG activation. We hypothesize that during diabetes, HG‐induced PKC‐α activation causes reduced NO production and endothelial hyperpermeability via NOX‐RhoA dependent ARG activation, which is prevented using TIP peptide. Indeed, our results show that TIP peptide (50 μg/mL) inhibits HG (30 mM)‐induced PKC‐α activity (7 fold vs ctrl‐5mM glucose, 30 min), ROS generation (160% of ctrl) and p115 Rho GEF membrane translocation (2h) in aortic endothelial cells (AEC). Moreover, HG‐induced ARG activation (24h), impaired NO production (55% of ctrl) and endothelial hyperpermeability, were also prevented by TIP peptide. ARG inhibitor BEC (100 μM) also preserved endothelial barrier function in HG‐treated AEC, indicating a central role for ARG in ED. Compared to conventional PKC‐α inhibitors, TIP peptide does not demonstrate any cytotoxicity in AEC after 24h treatment. In conclusion, these results indicate that substances able to blunt HG‐induced PKC‐α activation, such as TIP peptide, might be useful in treating diabetes‐associated ED. (Supported in part by NHLBI 70215).

Elastin Haploinsufficiency Induces Alternative Aging Processes in the Aorta

Elastin, the main component of elastic fibers, is synthesized only in early life and provides the blood vessels with their elastic properties. With aging, elastin is progressively degraded, leading to arterial enlargement, stiffening, and dysfunction. Also, elastin is a key regulator of vascular smooth muscle cell proliferation and migration during development since heterozygous mutations in its gene (Eln) are responsible for a severe obstructive vascular disease, supravalvular aortic stenosis, isolated or associated to Williams syndrome. Here, we have studied whether early elastin synthesis could also influence the aging processes, by comparing the structure and function of ascending aorta from 6- and 24-month-old Eln+/- and Eln+/+ mice. Eln+/- animals have high blood pressure and arteries with smaller diameters and more rigid walls containing additional although thinner elastic lamellas. Nevertheless, longevity of these animals is unaffected. In young adult Eln+/- mice, some features resemble vascular aging of wild-type animals: cardiac hypertrophy, loss of elasticity of the arterial wall through enhanced fragmentation of the elastic fibers, and extracellular matrix accumulation in the aortic wall, in particular in the intima. In Eln+/- animals, we also observed an age-dependent alteration of endothelial vasorelaxant function. On the contrary, Eln+/- mice were protected from several classical consequences of aging visible in aged Eln+/+ mice, such as arterial wall thickening and alteration of alpha(1)-adrenoceptor-mediated vasoconstriction. Our results suggest that early elastin expression and organization modify arterial aging through their impact on both vascular cell physiology and structure and mechanics of blood vessels.

Characterization of the vasorelaxant mechanisms of the endocannabinoid anandamide in rat aorta

Studies in isolated preparations of vascular tissue (mainly resistance vessels) provide evidence that anandamide exerts vasorelaxation. The aim of the present work was to further characterize the mechanisms involved in the vascular response induced by anandamide in a conduit vessel, rat aorta. Isometric tension changes in response to a cumulative concentration-response curve of anandamide (1 nM-100 micro M) were recorded in aortic rings from male Wistar rats. The involvement of a number of factors in this relaxation was investigated including endothelium-derived vasorelaxant products, cannabinoid and vanilloid receptors (transient potential vanilloid receptor-1 (TRPV1)), release of calcitonin gene-related peptide (CGRP), anandamide metabolism and the membrane transporter for anandamide. Anandamide caused a significant concentration-dependent vasorelaxation in rat aorta. This vasorelaxation was significantly inhibited by Pertussis toxin, by a non-CB1/non-CB2 cannabinoid receptor antagonist, by endothelial denudation, by inhibition of nitric oxide synthesis or inhibition of prostanoid synthesis via cyclooxygenase-2 (COX-2), by blockade of prostaglandin receptors EP4 and by a fatty acid amino hydrolase inhibitor. Antagonists for CB1, CB2, TRPV1 or CGRP receptors, an inhibitor of the release of endothelium-derived hyperpolarizing factor, and an inhibitor of anandamide transport did not modify the vascular response to anandamide. Our results demonstrate, for the first time, the involvement of the non-CB1/non-CB2 cannabinoid receptor and an anandamide-arachidonic acid-COX-2 derived metabolite (which acts on EP4 receptors) in the endothelial vasorelaxation caused by anandamide in rat aorta.

Abstract 232: Vascular Dysfunction Induced By High Pressure Levels Is Mediated By Rac-1

Oxidative stress plays a crucial role in the vascular dysfunction present in hypertension. The molecular mechanism involved are not completely elucidated. One of the principal source of oxidative stress at vascular level is the NADPH oxidase enzyme which needs Rac-1 to be activated. Aim of our study was to evaluate the role of Rac-1 in vascular dysfunction induced by high pressure levels. To test this hypothesis, carotid of mice were placed on pressure system and subjected to increasing levels of pressure (100 and 180 mmHg). In particular, after a period of perfusion (1h) with 100 mmHg vessels were subjected to 180 mmHg for three hours. At the end of perfusion period the vascular reactivity was tested. Our results demonstrate that vessels perfused with 180mmHg (n=8) showed an impaired vasorelaxation to acetylcholine as compared to vessels perfused with 100 mmHg (n=7) (% max vasorelaxation: 47±3 vs 88±7, p&lt;0.01). Vasorelaxation to nitroglycerin was comparable between vessels perfused respectively at 100 and 180 mmHg. Some vessels were used to evaluate oxidative stress (n=5) by dihydroethidium and Rac-1 activity (n=8) by Rac-1/PAK complex. Our results demonstrate that high pressure levels induced an increased oxidative stress and more interesting an increased Rac-1 activity as compared to vessels perfused with 100 mmHg. Thus, to clarify the role of Rac-1 in high pressure evoked oxidative stress and vascular dysfunction, some vessels (n=6) were infected with adenoviral vector containing Rac-1 dominant negative. In this experimental setting vessels perfused at 180 mmHg showed an oxidative stress and endothelial vasorelaxation comparable to that observed at 100 mmHg (% max vasorelaxation: 79±6 vs 85±7, n.s.), thus demonstrating a key role of Rac-1 in oxidative stress and endothelial dysfunction evoked by high pressure levels. Thus, Rac-1 could represent the target of novel therapeutic strategies for reducing vascular damage in hypertension.