Reduced NO Bioavailability Research Articles

The Potential Impact of Succinic Acid Treatment on Age Associated Vascular and Vascular Mitochondrial Dysfunction

A decline in vascular function is associated with aging. While the age‐related vascular dysfunction is multifactorial, vascular mitochondrial dysfunction is thought to contribute. Succinic acid (SA) has been shown to improve mitochondrial function by increasing mitochondrial biogenesis, though the vascular effects of SA are not well known.PURPOSETo determine the effect of SA treatment on vascular function of young and old mice.METHODSVascular function was measured in the aorta of 32 mice (15 young, 17 aged; 7 and 9 treated with succinic acid (0.75 mg/ml in drinking water), respectively). Using a wire myograph and Phenylephrine (PE), Acetylcholine (ACh), Potassium Chloride (KCl), and Sodium Nitroprusside (SNP) concentration response curves were generated, with and without the nitric oxide synthase (NOS) inhibitor, L‐NAME. In vitro measurements of vascular mitochondrial function were performed using permeabilized tissue respirometry using a substrate inhibitor protocol.RESULTSACh‐induced vasorelaxation was reduced with aging (60±32 v. 37±34 %relaxation). This endothelium dependent vasorelaxation was largely NO‐mediated, in both young (60±32 v. – 2±7 %relaxation) and old (37±34 v. 8±21 %relaxation), control v. L‐NAME, respectively. The aortas of SA‐treated mice had impaired ACh‐induced vasodilation in both young (60±32 v. 38±16 %relaxation) and old (37±34 v. 27±11 %relaxation), vehicle v. SA, respectively. The ACh‐induced vasorelaxation in SA treated mice, while reduced, was abolished with L‐NAME, (38±16 v. −7±2 %relaxation) and old (27±11 v. −8±8 %relaxation), control v. L‐NAME, respectively. The endothelium independent SNP‐induced vasorelaxation was not different between young and old (113±18 v. 98±14 %relaxation). The SNP responses were not different between vehicle and SA in either young (113±18 v. 84±18 %relaxation) or old (98±14 v. 108±9 %relaxation), vehicle v. SA treated, respectively. SNP relaxation was unaffected by L‐NAME in either group. Complex II driven respiration vascular mitochondrial respiration was ~50% lower with age (3.6 v. 7.6 pmol/mg/s, old v. young), which was improved with SA in both groups, but more so in the old (5.8 v. 2.2 Δpmol/mg/s, old v. young). Respiratory control ratio was not different between groups (1.47 vs. 1.43, old v. young), but tended to decrease with SA treatment (1.11 vs. 1.24, old v. young).CONCLUSIONSuccinic Acid reduces endothelium dependent vasorelaxation in both young and old mice, which appears to be due, at least in part, to reduced NO bioavailability. While SA appears to restore age‐related complex II mitochondrial dysfunction, it might come at a cost of reduced respiratory control ratio, perhaps increasing ROS production and abrogating endothelium derived NO. SA, an FDA approved compound, which has been used as an adjuvant therapy and is also often used in drug and food formulations, should be reconsidered given the potential effects of SA on the vasculature.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mercury-induced vascular dysfunction is mediated by angiotensin II AT-1 receptor upregulation

Low doses of mercury (Hg) promote deleterious effects on cardiovascular system, but the mechanisms implicated remain unclear. This study analyzed whether angiotensin II AT-1 receptors are involved in the vascular dysfunction caused by chronic exposure to low HgCl2 doses. For this, rats were divided into four groups and untreated (saline by im injections and tap water by gavage) or treated for 30 days as follows: Mercury (HgCl2im, first dose of 4.6 µg kg−1 and subsequent doses of 0.07 µg kg−1 day−1, and tap water by gavage); Losartan (saline im and losartan, 15 mg kg−1 day−1, by gavage); Losartan-Mercury (HgCl2im and Losartan by gavage). Systolic blood pressure was measured by tail plethysmography, vascular reactivity in aorta by isolated organ bath, oxidative stress by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and antioxidant capacity (FRAP) and protein expression of AT-1 receptors by Western Blot. As results, co-treatment with losartan prevented the increased aortic vasoconstrictor responses to phenylephrine (Phe), the involvement of ROS and prostanoids on the response to Phe and the reduced negative endothelial modulation by nitric oxide on these responses. Moreover, this co-treatment avoided the increase in plasmatic and vascular oxidative stress and AT-1 protein expression in aorta. In conclusion, these results suggest that AT-1 receptors upregulation might play a key role in the vascular damage induced by Hg exposure by increasing oxidative stress and probably by reducing NO bioavailability.

Xanthine Oxidase Activation Modulates the Endothelial (Vascular) Dysfunction Related to HgCl2 Exposure Plus Myocardial Infarction in Rats.

Heavy metal exposure is associated with cardiovascular diseases such as myocardial infarction (MI). Vascular dysfunction is related to both the causes and the consequences of MI. We investigated whether chronic exposure to low doses of mercury chloride (HgCl2) worsens MI-induced endothelial dysfunction 7days after MI. Male Wistar rats were divided into four groups: Control (vehicle), HgCl2 (4weeks of exposure), surgically induced MI and combined HgCl2-MI. Morphological and hemodynamic measurements were used to characterize the MI model 7days after the insult. Vascular reactivity was evaluated in aortic rings. Chronic HgCl2 exposure did not cause more heart injury than MI alone in terms of the morphological or hemodynamic parameters. Vascular reactivity increased in all groups, but the combination of HgCl2-MI increased the vasorelaxation induced by ACh compared with the HgCl2 and MI groups. Results showed reduced endothelial nitric oxide synthase (eNOS) protein expression in the MI group; increased iNOS activity in the HgCl2-MI group, although without enough magnitude to reverse the reduction in NO bioavailability; and increased phenylephrine response in the HgCl2-MI group due to an increase in ROS production, notably via xanthine oxidase (XO). Results suggest that the combination of 1month pre-exposure of HgCl2 before MI changed the endothelial generation of oxidative stress induced by mercury exposure from NADPH oxidase pathway to XO (xanthine oxidase)-dependent ROS production.

Markers of nitric oxide are associated with sepsis severity: an observational study

BackgroundNitric oxide (NO) regulates processes involved in sepsis progression, including vascular function and pathogen defense. Direct NO measurement in patients is unfeasible because of its short half-life. Surrogate markers for NO bioavailability are substrates of NO generating synthase (NOS): L-arginine (lArg) and homoarginine (hArg) together with the inhibitory competitive substrate asymmetric dimethylarginine (ADMA). In immune cells ADMA is cleaved by dimethylarginine-dimethylaminohydrolase-2 (DDAH2). The aim of this study was to investigate whether concentrations of surrogate markers for NO bioavailability are associated with sepsis severity.MethodThis single-center, prospective study involved 25 controls and 100 patients with surgical trauma (n = 20), sepsis (n = 63), or septic shock (n = 17) according to the Sepsis-3 definition. Plasma lArg, hArg, and ADMA concentrations were measured by mass spectrometry and peripheral blood mononuclear cells (PBMCs) were analyzed for DDAH2 expression.ResultslArg concentrations did not differ between groups. Median (IQR) hArg concentrations were significantly lower in patient groups than controls, being 1.89 (1.30–2.29) μmol/L (P < 0.01), with the greatest difference in the septic shock group, being 0.74 (0.36–1.44) μmol/L. In contrast median ADMA concentrations were significantly higher in patient groups compared to controls, being 0.57 (0.46–0.65) μmol/L (P < 0.01), with the highest levels in the septic shock group, being 0.89 (0.56–1.39) μmol/L. The ratio of hArg:ADMA was inversely correlated with disease severity as determined by the Sequential Organ Failure Assessment (SOFA) score. Receiver-operating characteristic analysis for the presence or absence of septic shock revealed equally high sensitivity and specificity for the hArg:ADMA ratio compared to the SOFA score. DDAH2 expression was lower in patients than controls and lowest in the subgroup of patients with increasing SOFA.ConclusionsIn patients with sepsis, plasma hArg concentrations are decreased and ADMA concentrations are increased. Both metabolites affect NO metabolism and our findings suggest reduced NO bioavailability in sepsis. In addition, reduced expression of DDAH2 in immune cells was observed and may not only contribute to blunted NO signaling but also to subsequent impaired pathogen defense.

232 Endothelial ship2 knockout causes nox2 nadph oxidase-dependent oxidative stress and endothelial dysfunction

IntroductionInsulin-resistant type 2 diabetes mellitus (DM) leads to premature death and disability, primarily as a consequence of accelerated vascular disease. Shc homology 2-containing inositol 5 phosphatase-2 (SHIP2) is a lipid...

NOS Blockade Reveals No Sex Difference in Contracting Muscle O2 Delivery-to-Utilization Matching in Rats

Estrogen has been proposed to enhance nitric oxide synthase (NOS) expression and NO bioavailability in females. Importantly, flow-mediated dilation (FMD) is reduced post-menopause when estrogen levels decrease. Thus, FMD in females may rely on the effects of estrogen (mediated via NO) when compared to age-matched males. PURPOSE: Where potentially incomplete blockade of NOS may not show sex differences in pre-menopausal FMD, we tested the hypothesis that complete NOS blockade via L-arginine methyl ester (L-NAME) in female rats would exhibit a greater reduction in muscle O2 delivery-to-utilization matching (assessed via PO2 in the muscle interstitial space (PINTO2)) and speed PINTO2 kinetics following the onset of muscle contractions when compared to age-matched males. METHODS: In Sprague Dawley rats (n = 5 male, 5 female), the spinotrapezius muscle was surgically exposed and electrically stimulated (∼6 V, 1 Hz) for 180 s. Prior to contractions, Oxyphor G4 was injected into the muscle to measure PINTO2. PINTO2 was recorded at rest and during contractions in control (CON) and following NOS blockade (intra-arterial (IA) infusion of L-NAME (10 mg kg-1)) conditions. RESULTS: NOS blockade revealed no differences in resting PINTO2 within and between sexes (Male CON: 20 ± 1 vs Male L-NAME: 21 ± 2 mmHg; Female CON: 17 ± 2 vs Female L-NAME: 17 ± 3 mmHg; p > 0.05). Additionally, there were no differences in kinetics (mean response time) following the onset of contractions (Male CON: 18 ± 2 vs Male L-NAME: 12 ± 4 s; Female CON: 15 ± 2 vs Female L-NAME: 15 ± 2 s; p > 0.05). CONCLUSION: Contrary to our hypothesis, reducing NO bioavailability via NOS blockade did not have any different effect in females versus males with respect to resting PINTO2 or PINTO2 kinetics. These results suggest that estrogen via NO bioavailability does not play a significant role in resting PINTO2 or PINTO2 kinetics during muscle contractions in female rats.

The Role of O-GlcNAcylation in Perivascular Adipose Tissue Dysfunction of Offspring of High-Fat Diet-Fed Rats

Perivascular adipose tissue (PVAT), which reduces vascular contractility, is dysfunctional in the male offspring of rats fed a high-fat diet (HFD), partially due to a reduced NO bioavailability. O-GlcNAcylation of eNOS decreases its activity, thus we investigated the role of O-GlcNAcylation in the prenatal programming of PVAT dysfunction. Female Sprague-Dawley rats were fed either a control (10% fat) or an obesogenic HFD (45% fat) diet for 12 weeks prior to mating, and throughout pregnancy and lactation. Offspring were weaned onto the control diet and were killed at 12 and 24 weeks of age. Mesenteric arteries from the 12-week-old offspring of HFD dams (HFDO) contracted less to U46619; these effects were mimicked by glucosamine in control arteries. PVAT from 12- and 24-week-old controls, but not from HFDO, exerted an anticontractile effect. Glucosamine attenuated the anticontractile effect of PVAT in the vessels from controls but not from HFDO. AMP-activated protein kinase (AMPK) activation (with A769662) partially restored an anticontractile effect in glucosamine-treated controls and HFDO PVAT. Glucosamine decreased AMPK activity and expression in HFDO PVAT, although phosphorylated eNOS expression was only reduced in that from males. The loss of anticontractile effect of HFDO PVAT is likely to result from increased O-GlcNAcylation, which decreased AMPK activity and, in males, decreased NO bioavailability.

Mathematical relationships and their consequences between rat pulse waveform parameters and blood pressure during decreasing NO bioavailability.

What is the central question of this study? We wanted to find out whether the relationship between rat arterial pulse waveform (APW) parameters and blood pressure could be described by known mathematical functions and find mathematical parameters for conditions of hypertension resulting from decreased NO bioavailability. What is the main finding and its importance? We found mathematical functions and their parameters that approximate the relationships of 12 APW parameters to systolic and diastolic blood pressure in conditions of decreased NO bioavailability. The results may assign APW parameters to decreased NO bioavailability, which may have predictive or diagnostic value. Information obtained from the arterial pulse waveform (APW) is useful for characterization of the cardiovascular system in particular (patho)physiological conditions. Our goal was to find out whether the relationships between rat APW parameters could be described by simple mathematical functions and to find mathematical parameters for conditions of hypertension resulting from decreased NO bioavailability. Therefore, we explored details of 14 left carotid APW parameters of anaesthetized male Wistar rats and mathematically characterized their relationship to systolic and diastolic blood pressure (BP) in conditions of a gradual reduction in NO bioavailability after administration of l-NAME. The right jugular vein of anaesthetized Wistar rats was cannulated for l-NAME administration. The left carotid artery was cannulated to detect the APW at high resolution. Here, we show the time-dependent parallel changes of 14 APW parameters before and after i.v. administration of l-NAME and present mathematical functions that approximate the relationships of 12 APW parameters to systolic and diastolic BP. Some APW parameters had minor (e.g. heart rate) or biphasic dependence on BP (e.g. relative level of the maximum rate of ventricular pressure decrease (dP/dtmin )), but all relationships, within a particular range of BP, could be approximated by known regression functions, as a linear function (e.g. pulse BP), exponential decay (e.g. relative level of the maximum rate of ventricular pressure increase (dP/dtmax )), exponential growth (systolic area), exponential rise to a maximum (relative augmentation index) or sigmoid function (e.g. increase of relative level of dP/dtmin ). The mathematical functions may assign APW parameters to decreased NO bioavailability. This may have predictive or diagnostic value.

Abstract P346: Endothelial Function and NO Bioavailability are Improved in Angiotensin Ii-treated Transglutaminase-2 Knock-out Mice

We hypothesized that transglutaminase-2 (TG2) may contribute to the impaired functional properties of resistance arteries from angiotensin-II-treated mice. TG2-knockout mice (TG2-K/O, 12 weeks old, n=6) and wild type (WT) mice were treated or not with angiotensin-II (400ng/kg/min) for 14 days. Blood pressure (BP) and heart rate (HR) were measured by tail-cuff method. Endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (1nM-to-100μM)±L-NAME (100μM) and sodium nitroprusside (10nM-to-1mM) respectively, in mesenteric arteries pre-contracted with norepinephrine (10μM). The expression of p-eNOS-(S1177)/eNOS, NOSIP (the negative modulator of eNOS), NOX-1, and its positive modulator ERp72 were evaluated in aorta by immunoblotting. Reactive oxygen species (ROS) production in aorta was evaluated by dihydroethidium staining. Plasma nitrate/nitrate were measured by ELISA. BP and HR were higher in TG2-K/O mice compared to WT (116.8±0.9 mmHg vs 89.6±1.5 mmHg, P&lt;0.001; and 595.0±15.0 bpm vs 467.1±14.7 bpm, P&lt;0.001, respectively). In both groups, angiotensin-II increased significantly BP (+28% in WT, and +21% in TG2-K/O) and HR (+33% in WT, and +9% in TG2-K/O). Acetylcholine-induced relaxation was preserved in WT and TG2-K/O and it was significantly impaired by angiotensin-II only in WT (-28%). L-NAME blunted this response in all the groups, although this effect was less evident in angiotensin-II-treated WT. Endothelium-independent relaxation was similar in all the groups. Plasma nitrates/nitrates and p-eNOS-(S1177)/eNOS were similar in WT and TG2-K/O, and they were reduced by angiotensin-II significantly only in WT (-37% and -44%, respectively). NOSIP expression was similar in both WT and TG2-K/O and was significantly increased by angiotensin-II only in WT (+40%). ROS production was similar in WT and TG2-K/O and significantly increased by angiotensin-II only in WT (+9%). NOX-1 and ERp72 were similar in WT and TG2-K/O and were significantly increased by angiotensin-II only in WT (+23% and +29%, respectively). In conclusion TG2 may contribute to endothelial dysfunction through the modulation of ROS production and the reduction of NO bioavailability in angiotensin-II infused mice.

Abstract 379: Silencing of Fxyd1 Uncouples Enos in Human Umbilical Vein Endothelial Cells

Introduction/Aims: FXYD1 has been shown to be a master regulator of the sodium potassium pump activity in the caveolae of heart tissue, protecting the pump from glutathionylation and oxidative inhibition. However, very little is known about its expression and function in the endothelium, particularly in regard to its potential interaction with other caveolae proteins. Given the molecular mechanism of eNOS uncoupling is via glutathionylation, we examined whether FXYD1 was expressed in endothelial cells, and whether it had a functional partnership with eNOS. Methods: Co-immunoprecipitation was used to study the interaction of FXYD1 and eNOS. GSH antibody was used for immunodetection of eNOS glutathionylation. Silencing of FXYD1 using siRNA followed by DAF staining or spin trap were used to study the effects of FXYD1 on eNOS function. Enzyme-linked immunosorbent assay was also employed to quantify eNOS glutathionylation level in human erythrocyte samples post exposure to angiotensin II (0 to 500 nM) ex vivo. Results: FXYD1 co-immunoprecipitated with eNOS in human umbilical vein endothelial cells (HUVECs). Silencing of FXYD1 resulted in significantly increased eNOS glutathionylation, associated with significantly reduced NO bioavailability under baseline and acetylcholine stimulated conditions (1μM, 20mins). Furthermore, a dose-dependent increase in eNOS glutathionylation level was also observed in human erythrocyte exposed to angiotensin II. Discussion: Our findings demonstrated a novel functional partnership of FXYD1 with eNOS, protecting this vital enzyme from glutathionylation-mediated uncoupling. This has important implications for our understanding of ROS-signalling in the vasculature. Furthermore, FXYD1 expression/eNOS glutathionylation may serve as important biomarkers for oxidative stress.

Effects of High-LET Radiation Exposure and Hindlimb Unloading on Skeletal Muscle Resistance Artery Vasomotor Properties and Cancellous Bone Microarchitecture in Mice.

Weightlessness during spaceflight leads to functional changes in resistance arteries and loss of cancellous bone, which may be potentiated by radiation exposure. The purpose of this study was to assess the effects of hindlimb unloading (HU) and total-body irradiation (TBI) on the vasomotor responses of skeletal muscle arteries. Male C57BL/6 mice were assigned to control, HU (13-16 days), TBI (1 Gy (56)Fe, 600 MeV, 10 cGy/min) and HU-TBI groups. Gastrocnemius muscle feed arteries were isolated for in vitro study. Endothelium-dependent (acetylcholine) and -independent (Dea-NONOate) vasodilator and vasoconstrictor (KCl, phenylephrine and myogenic) responses were evaluated. Arterial endothelial nitric oxide synthase (eNOS), superoxide dismutase-1 (SOD-1) and xanthine oxidase (XO) protein content and tibial cancellous bone microarchitecture were quantified. Endothelium-dependent and -independent vasodilator responses were impaired in all groups relative to control, and acetylcholine-induced vasodilation was lower in the HU-TBI group relative to that in the HU and TBI groups. Reductions in endothelium-dependent vasodilation correlated with a lower cancellous bone volume fraction. Nitric oxide synthase inhibition abolished all group differences in endothelium-dependent vasodilation. HU and HU-TBI resulted in decreases in eNOS protein levels, while TBI and HU-TBI produced lower SOD-1 and higher XO protein content. Vasoconstrictor responses were not altered. Reductions in NO bioavailability (eNOS), lower anti-oxidant capacity (SOD-1) and higher pro-oxidant capacity (XO) may contribute to the deficits in NOS signaling in skeletal muscle resistance arteries. These findings suggest that the combination of insults experienced in spaceflight leads to impairment of vasodilator function in resistance arteries that is mediated through deficits in NOS signaling.

Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells

Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN–LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

Effects of Huang Qi Decoction on Endothelial Dysfunction Induced by Homocysteine.

Vascular endothelial dysfunction can be induced by homocysteine (Hcy) through promoted oxidative stress. Huang Qi decoction (HQD) is a traditional Chinese medical formula and its components possess antioxidant effect. The study herein was therefore designed to investigate the effects of HQD at different dosage on endothelial dysfunction induced by Hcy. Tempol and apocynin were used to investigate whether antioxidant mechanisms were involved. Endothelium-dependent relaxation of rat aortas was investigated by isometric tension recordings. Reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs) was determined by DHE staining. The assessment related to oxidative stress and NO bioavailability was performed by assay kits and western blot. In isometric tension experiment, HQD at the dose of 30 or 100 μg/mL, tempol, or apocynin prevented impaired endothelium-dependent relaxation in isolated aortas elicited by Hcy. In cellular experiments, substantial enhancement in NADPH oxidase and ROS generation and reduction in NO bioavailability triggered by Hcy were reversed by pretreatment of HQD at the dose of 100 μg/mL, tempol, or apocynin. The results proved that HQD at an appropriate dosage presented favorable effects on endothelial dysfunction initiated by Hcy through antioxidant mechanisms. HQD can act as a potent prescription for the treatment of endothelium related vascular complications.

The acute prothrombotic effect of aldosterone in rats is partially mediated via angiotensin II receptor type 1

IntroductionWe showed previously that the prothrombotic effect of one hour aldosterone (ALDO) infusion in rats was only partially mediated by the mineralocorticoid receptor (MR). Bearing in mind that ALDO potentiates the effects of angiotensin II (Ang II), in the present study we investigated the role of Ang II receptor type 1 — AT1 in acute ALDO prothrombotic action. Materials and methodsThe experiments were performed in a stasis-induced venous thrombosis model in male Wistar, normotensive rats. ALDO (30μg/kg) was infused for 1h. Valsartan (VAL; 10mg/kg), a selective AT1 receptor antagonist, was administered in a single bolus injection before ALDO infusion. Eplerenone (EPL, 100mg/kg), a selective MR receptor antagonist, was administered per os before ALDO. Thrombus weight and incidences of thrombosis were assayed. Bleeding time and platelet adhesion to collagen were evaluated as primary hemostasis parameters. The plasma levels of some coagulation and fibrinolysis parameters, and plasma NO metabolite levels were assayed. ResultsAT1 blockade with valsartan significantly reduced ALDO-induced thrombosis expressed as a reduced thrombus mass (p<0.05 vs ALDO) and diminished the incidence of thrombosis. Valsartan reduced the ALDO-induced changes in bleeding time and platelet adhesion, as well as in coagulation, fibrinolysis, and NO metabolite levels. The effect of AT1 blockade in ALDO-induced thrombosis was similar to the effect of MR blockade. However, dual blockade of AT1 and MR showed no additional benefit. ConclusionsALDO prothrombotic action is partially mediated via AT1 receptor in the mechanism involving enhanced platelet activation, induced coagulation, impaired fibrinolysis and reduced NO bioavailability.

Abstract 16718: Energy Induced Vasodilation in a Model of Endothelial Dysfunction Requires Nitric Oxide

Introduction: Over 8 million Americans are treated for peripheral artery disease (PAD), i.e. intermittent claudication, impaired wound healing, and critical limb ischemia. Endothelial dysfunction of the microcirculation is a potential mechanism for the pathogenesis of PAD because risk factors for PAD- age, hypertension, atherosclerosis, diabetes mellitus, and active tobacco use, have been directly related to a reduction in NOS expression and NO production. These reductions in NO bioavailability impair flow mediated dilation of the brachial artery and acetylcholine induced vasodilation. We have shown energy (670 nm (R/NIR)) can increase NO independent of NOS through release of NO from nitrosyl-heme stores. We hypothesized R/NIR can stimulate vasodilation, and serve as a potential therapy to recover vasodilation in a model of endothelial dysfunction (db/db mice). Methods: Human dermal microvascular endothelial cells (HMVEC) loaded with DAF-2 were stimulated with R/NIR 100mW/cm2 up to 6J. NO was measured using HPLC of DAF-2T. To test vasodilation, facial arteries from C57Bl/6 and db/db mice were isolated, pressurized to 60mmHg, and pre-constricted with U46619. The vessels were irradiated with 670 nm light (10mW/cm2) for 5 min with 10 min dark period between irradiation. Results: HMVEC stimulated with R/NIR increased NO 28.3% (±11.2, p&lt;0.05). The NO scavenger c-PTIO inhibited R/NIR (17.0%±5.9, p&lt;0.05) but was unchanged by pre-incubation with NOS inhibitor L-NAME (1mM) (13.1% ± 5.6). Vessel diameter increased up to 17.8% (±3, p&lt;0.05) after 5 min with R/NIR. L-NAME did not affect dilation (13.74% ±2.2), however c-PTIO (100μM) could attenuate dilation (-.51% ± 1.1, p&lt;0.001). R/NIR vasodilation was abolished with vessel denudation (1.2%± 2.21, p&lt;0.001), suggesting R/NIR dilation was endothelial dependent. Impaired dilation in db/db mice was reversed, as R/NIR could significantly dilate db/db vessels after 5 min [(13.7% ± 2.8) vs. control (17.8% ±3)]. Conclusion: R/NIR vasodilation requires intact endothelium and NO, but does not require NOS. In an obesity model of endothelial dysfunction, R/NIR can significantly restore vasodilation. Therefore, R/NIR as a source for therapeutic NO should be strongly considered in the treatment of ischemic wounds.

Augmented endothelial l-arginine transport ameliorates pressure-overload-induced cardiac hypertrophy.

What is the central question of this study? What is the potential role of endothelial NO production via overexpression of the l-arginine transporter, CAT1, as a mitigator of cardiac hypertrophy? What is the main finding and its importance? Augmentation of endothelium-specific l-arginine transport via CAT1 can attenuate pressure-overload-dependent cardiac hypertrophy and fibrosis. Our findings support the conclusion that interventions that improve endothelial l-arginine transport may provide therapeutic utility in the setting of myocardial hypertrophy. Such modifications may be introduced by exercise training or locally delivered gene therapy, but further experimental and clinical studies are required. Endothelial dysfunction has been postulated to play a central role in the development of cardiac hypertrophy, probably as a result of reduced NO bioavailability. We tested the hypothesis that increased endothelial NO production, mediated by increased l-arginine transport, could attenuate pressure-overload-induced cardiac hypertrophy. Echocardiography and blood pressure measurements were performed 15 weeks after transverse aortic constriction (TAC) in wild-type (WT) mice (n = 12) and in mice with endothelium-specific overexpression of the l-arginine transporter, CAT1 (CAT+; n = 12). Transverse aortic constriction induced greater increases in heart weight to body weight ratio in WT (by 47%) than CAT+ mice (by 25%) compared with the respective controls (P ≤ 0.05). Likewise, the increase in left ventricular wall thickness induced by TAC was significantly attenuated in CAT+ mice (P = 0.05). Cardiac collagen type I mRNA expression was greater in WT mice with TAC (by 22%; P = 0.03), but not in CAT+ mice with TAC, compared with the respective controls. Transverse aortic constriction also induced lesser increases in β-myosin heavy chain mRNA expression in CAT+ mice compared with WT (P ≤ 0.05). Left ventricular systolic pressure after TAC was 36 and 39% greater in WT and CAT+ mice, respectively, compared with the respective controls (P ≤ 0.001). Transverse aortic constriction had little effect on left ventricular end-diastolic pressure in both genotypes. Taken together, these data indicate that augmenting endothelial function by overexpression of l-arginine transport can attenuate pressure-overload-induced cardiac hypertrophy.

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction.

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

PTP1B Deletion Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes‐Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ). After 28 days of T1DM, body weight reduction, decrease in plasma insulin levels and increased in glycemia, cholesterol and triglycerides, were similar in WT and KO mice. T1DM increased PTP1B expression and impaired aortic endothelial NO-dependent relaxation. PTP1B deletion did not affect baseline endothelial NO-dependent relaxation, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice. L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. These data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

Metabolic Syndrome Eliminates Vascular Protection Against Chronic Stress in Female Rats

Metabolic syndrome (MetSyn) is a risk factor for depression, yet the comorbid consequences of chronic stress/depression and MetSyn are not well understood. Using the Unpredictable Chronic Mild Stress (UCMS) model of depression, we previously characterized disparity in disease profile between male and female rats. Specifically, although females were more susceptible to UCMS induced depressive symptoms, they exhibited greater vasodilator responses versus males. However, protection from stress induced vasculopathy is not observed in UCMS females with underlying MetSyn. To interrogate potential mechanisms governing this loss of vascular protection, vascular reactivity was assessed in aortic rings and skeletal muscle arteries of lean and obese female Zucker rats (LZR-F, OZR-F) following 8 weeks of UCMS. Plasma levels of inflammatory markers were measured by electrochemiluminescence (Meso Scale Diagnostics). Impairment of the endothelial-dependent vasodilator responses to pharmacological stimuli in both conduits and resistance arterioles was significantly greater in OZR-F versus LZR-F. OZR-F also exhibited increased adrenergic sensitivity. Compared to LZRs, the dependence on nitric oxide as a vasodilator was significantly attenuated in OZR-F, which exhibited greater dependence on PGI2 as a compensatory mechanism for reduced NO bioavailability. Plasma TNFα, IL-6, and IL-10 were elevated following UCMS, although significantly greater levels were observed in OZR-F versus LZR-F. These results suggest that MetSyn increases the inflammatory response to chronic stress, which may overwhelm the mechanisms imparting vascular protection in lean females. Comorbid MetSyn may alter the pathophysiology of stress-induced vascular disease.

Microvascular Dysfunction is Evident in the Cutaneous Circulation of Psoriatic Patients

Vascular dysfunction in conduit and resistance vessels is evident in adults with plaque psoriasis, a chronic systemic inflammatory disease. However, potential impairments of cutaneous microvascular function in psoriatic patients remain incompletely understood. We hypothesized that microvascular function would be impaired in the cutaneous circulation (non‐lesional) of psoriatic patients. One intradermal microdialysis fiber was placed in the forearm skin of 5 healthy adults (NP: 1M, 4F; 49 ± 1 yr) and 5 adults with moderate (&gt;5% body surface area affected) plaque psoriasis (P: 1M, 4F; 48 ± 1 yr). The site was perfused with lactated Ringer's while red blood cell flux was measured using laser‐Doppler flowmetry during standardized local heating (42°C) to elicit eNOS‐dependent vasodilation. NO‐dependent vasodilation was further quantified after the perfusion of 20 mM L‐NAME (nonspecific NO‐synthase inhibitor). Data were expressed as a percentage of maximum cutaneous vascular conductance (%CVCmax; 28 mM SNP). The local heating‐induced plateau was attenuated in psoriatic patients (NP: 99 ± 1 %CVCmax vs. P: 75 ± 9 %CVCmax; p&lt;0.05). Quantified eNOS‐dependent vasodilation appears to be reduced in the psoriatic group (NP: 57 ± 8 %CVCmax vs. P: 41 ± 8 %CVCmax; p=0.15). These preliminary results suggest that microvascular dysfunction is evident in psoriatic patients and may be mediated by reduced NO bioavailability. Further, the assessment of cutaneous vascular function has particular relevance for this clinical population because the skin‐related symptomology represents the outward manifestation of this systemic inflammatory disease process.FUNDING: Penn State College of HHD Grant