Endothelial Dysfunction In Mice Research Articles

Chronic intermittent hypoxia causes endothelial dysfunction in a mouse model of diet-induced obesity

BackgroundObstructive sleep apnea (OSA) is a common disorder characterized by chronic intermittent hypoxia (CIH). OSA is prevalent in obese subjects and is associated with endothelial dysfunction and cardiovascular disorders. We tested the hypothesis that the deleterious effects of IH could be further modulated by diet-induced obesity. DesignThirty adult (8–10weeks) male C57BL/6J mice were divided into four groups. Mice were subjected to CIH or intermittent air (IA) for 12h a day and fed either a high fat (HF) or a low fat control diet (CD) for 6weeks. We analyzed endothelial function using a wire myograph, and measured markers of oxidative stress (plasma malondialdehyde (MDA) and total antioxidant capacity (TAC)) using colorimetrical assays. We also measured C-reactive protein (CRP) using ELISA and endothelial nitric oxide (eNOS) gene expression using real time PCR. ResultsStimulated endothelial dependent dilation was significantly impaired only in the group fed high fat diet and subjected to CIH (Emax: HFIH 78±2%, p<0.0001) when compared to the other groups (Emax: HFIA 95±0.7%, CDIH 94±2%, CDIA 97±1%). Also basal endothelial dependant dilation was attenuated in the HFIH group compared to the HFIA group (Emax: HFIH: 179±10% vs. HFIA: 149±11% in the presence of L-NAME). Levels of MDA were elevated in the CDIH group when compared to CDIA (0.68±0.04 vs. 0.41±0.03μM, p<0.05) but were greatest in the HFIH group (0.83±0.08μM, p<0.05). However, there was no significant increase in MDA levels in the HFIA group (0.45±0.03μM, p=NS) when compared to all other groups. Similar effects were observed with CRP levels; CRP levels were significantly higher in the CDIH group compared with intermittent air (10.39±0.38 vs. 8.70±0.21μg/ml, p<0.05) but the HFIH had the greatest levels of CRP (11.87±0.31μg/ml, p<0.05). In the HFIA group, CRP levels were not elevated (9.96±0.37μg/ml, p=NS). Nevertheless, total antioxidant capacity and eNOS gene expression were not significantly different in the groups. ConclusionCIH caused endothelial dysfunction in mice fed an obesogenic diet. Inflammation and oxidative stress were increased in CIH and an obesogenic diet exacerbated these effects.

Sonic Hedgehog Carried by Microparticles Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice

Microparticles are small fragments of the plasma membrane generated after cell stimulation. We recently showed that Sonic hedgehog (Shh) is present in microparticles generated from activated/apoptotic human T lymphocytes and corrects endothelial injury through nitric oxide (NO) release. This study investigates whether microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in mice. Male Swiss mice were implanted with osmotic minipumps delivering angiotensin II (0.5 mg/kg/day) or NaCl (0.9%). Systolic blood pressure and heart rate were measured daily during 21 days. After 7 day of minipump implantation, mice received i.v. injections of microparticles (10 µg/ml) or i.p. Shh receptor antagonist cyclopamine (10 mg/kg/2 days) during one week. Angiotensin II induced a significant rise in systolic blood pressure without affecting heart rate. Microparticles reversed angiotensin II-induced hypertension, and cyclopamine prevented the effects of microparticles. Microparticles completely corrected the impairment of acetylcholine- and flow-induced relaxation in vessels from angiotensin II-infused mice. The improvement of endothelial function induced by microparticles was completely prevented by cyclopamine treatment. Moreover, microparticles alone did not modify NO and O2 . - production in aorta, but significantly increased NO and reduced O2 . - productions in aorta from angiotensin II-treated mice, and these effects were blocked by cyclopamine. Altogether, these results show that microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in aorta through a mechanism associated with Shh-induced NO production and reduction of oxidative stress. These microparticles may represent a new therapeutic approach in cardiovascular diseases associated with decreased NO production.

Aging compounds western diet-associated large artery endothelial dysfunction in mice: Prevention by voluntary aerobic exercise

We tested the hypothesis that aging will exacerbate the negative vascular consequences of exposure to a common physiological stressor, i.e., consumption of a “western” (high fat/high sucrose) diet (WD), by inducing superoxide-associated reductions in nitric oxide (NO) bioavailability, and that this would be prevented by voluntary aerobic exercise. Incremental stiffness and endothelium-dependent dilation (EDD) were measured in the carotid arteries of young (5.4±0.3mo, N=20) and old (30.4±0.2mo, N=19) male B6D2F1 mice fed normal chow (NC: 17% fat, 0% sucrose) or a western diet (40% fat, 19% sucrose) and housed in either standard cages or cages equipped with running wheels for 10–14weeks. Incremental stiffness was higher in old NC (P<0.05) and both young (P<0.01) and old (P<0.01) WD fed mice compared with young NC mice, but WD did not further increase stiffness in the old mice. In cage control mice, maximal EDD was 17% lower in both NC fed old mice and young WD fed mice (P<0.05). Consumption of WD by old mice led to a further 20% reduction in maximal EDD (P<0.05). Incremental stiffness was 28% lower and maximal EDD was 38% greater in old WD fed mice with access to running wheels vs. old WD fed control mice (P<0.05) and not different from young NC fed controls. Wheel running also tended to improve maximal EDD (+9%, P=0.11), but not incremental stiffness in young WD fed mice. Ex vivo treatment with the superoxide scavenger TEMPOL and NO inhibitor l-NAME abolished these respective effects of age, WD and voluntary running on EDD. Ingestion of a WD induces similar degrees of endothelial dysfunction in old and young adult B6D2F1 mice, and these effects are mediated by a superoxide-dependent impairment of NO bioavailability. However, the combination of old age and WD, a common occurrence in our aging society, results in a marked, additive reduction in endothelial function. Importantly, regular voluntary aerobic exercise reduces arterial stiffness and protects against the adverse influence of WD on endothelial function in old animals by preventing superoxide suppression of NO. These findings may have important implications for arterial aging and the prevention of age-associated cardiovascular diseases.

Palmitate induces apoptosis in mouse aortic endothelial cells and endothelial dysfunction in mice fed high-calorie and high-cholesterol diets

AimsObesity is associated with hypertriglyceridemia and elevated circulating free fatty acids (FFA), resulting in endothelial dysfunction. Endoplasmic reticulum (ER) stress has been implicated in many of these processes. To determine if ER stress participates in palmitate-induced apoptosis, we investigated the effects of diet-induced obesity and palmitate on mouse aortic endothelial cells (MAEC) in vivo and in vitro. Main methodsMale C57BL/6 mice were fed standard chow diets (SCD) or high-calorie and high-cholesterol diets (HCD) for 3months. Insulin resistance was detected, and the serum, including proinflammatory indices and markers of endothelial function, was also analyzed. The ultrastructure and apoptosis of the endothelial cells in the thoracic aorta were observed. The primary MAEC were separated and treated with palmitate at different concentrations or different times respectively to observe any changes in cellular proliferation, intracellular reactive oxygen species (ROS) levels and apoptosis. Finally, the ER stress markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were analyzed. Key findingsHCD-fed obese mice became inflammation-activated and insulin-resistant. Swollen mitochondria, expanded ER and apoptosis in the endothelial cells of the thoracic aorta were observed in HCD-fed mice. Palmitate inhibited cell proliferation, increased production of ROS and induced apoptosis in MAEC. CHOP was overexpressed and shifted into the nucleus (mainly), while the expression of GRP78 was upregulated in the palmitate-treated MAEC. SignificanceOur results indicate that diet-induced obesity results in endothelial dysfunction in vivo, and that oxidative and ER stress may be involved in apoptosis induced by the palmitate in vitro.

Chronic intermittent hypoxia induces endothelial dysfunction in mice fed a high fat diet but not in mice fed a normal diet

ObjectiveObstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (IH). OSA is prevalent in obese subjects, and is associated with endothelial dysfunction and cardiovascular disorders. We evaluated whether the effects of IH could be modulated by diet.MethodsAdult (8–10 wk) male C57BL/6J mice were divided into 4 groups: IH or intermittent air (IA) and fed either a high‐fat (HF) or regular chow (RC) diet for 6 weeks. Endothelial dependent vasodilatation of the aorta was assessed using a wire myograph. Serum malondialdehyde (MDA) and C‐reactive protein (CRP) were also measured.ResultsEndothelial function was impaired in the HFIH group (Emax: 78.0 ±2.4% of induced tone) compared to the other three groups (P&lt;0.0001). Basal NO activity was also reduced in HFIH when compared to HFIA group as measured by Emax constriction before and after L‐NAME (Emax: 145.6±9.9% vs. 183.7±7.7%P&lt;0.01). MDA and CRP levels were elevated in IH (MDA: 0.67±0.05 vs. 0.42±0.04 μM, P&lt;0.05)(CRP: 10.39±0.38 vs. 8.7±0.22 μg/ml, P&lt;0.01), and were more pronounced in the HFIH (MDA: 0.83±0.08 μM ‐ CRP: 11.87±0.32 μg/ml).ConclusionIH causes endothelial dysfunction in mice fed high fat diet but not normal chow. Inflammation and oxidative stress are increased in IH and is exacerbated by a high fat diet. (CIHR Team Grant)

Nano‐metal oxide aspiration exposure induces endothelial dysfunction in mice.

Metal oxide nanoparticles (nMO) are widely used in many diverse industries. We evaluated the effect of aspiration exposure to three nMO: SiO2 (length 51.9 ± 16.2 nm, aspect ratio 1.1 ± 0.1), Al2O3 (84.6 ± 20 nm, 1.4 ± 0.2) and CeO2 (52.5 ± 19.1 nm, 1.5 ± 0.4). Anesthetized (isoflurane) mice (25g) aspirated 20ug of SiO2, Al2O3 or CeO2 in 100ul of 1g% albumin in saline. 24 h later bronchial‐alveolar lavage showed and increase in total cells of 80% SiO2, 85% Al2O3, and 60% CeO2. Only Al2O3 caused weight loss 3.3% overnight. Using intravital microscopy, vasoactive responses were obtained in the cremaster m. model. Compared to controls, baseline diameters were unaltered for the same class of arteriole. Dilation to adenosine (10–4M) was attenuated for each nMO. Constriction to phenylephrine (10–6M) was attenuated for only Al2O3. Exposure to SiO2 (N=4) did not alter the dilation to acetylcholine (ACH 10–7 – 10–4M), but attenuated dilation to bradykinin (BK 10–8 – 10–5M) and isoproterenol (ISO 10–8 – 10–4M). Exposure to Al2O3 (N=4) or CeO2 (N=4) eliminated dilation to BK and attenuated dilation to ACH or ISO. Further, for SiO2 and Al2O3, a significant dilation to ISO was uncovered in the pM range (10–14 – 10–10M) that is mimicked by blocking clathrin endosome formation with dynasore (80uM) in control mice (N=6). Thus, aspiration of 20ug nMO induced moderate to severe endothelial dysfunction that was MO‐type specific. AHA 0655908T

Apelin elevates blood pressure in ICR mice with L-NAME-induced endothelial dysfunction

Apelin is the endogenous ligand of APJ, which belongs to the family of G protein-coupled receptors. Apelin and APJ are highly expressed in various cardiovascular tissues, including the heart, kidney and vascular endothelial and smooth muscle cells. Although apelin exerts hypotensive effects via activation of endothelial nitric oxide synthase (eNOS), the ability of apelin to regulate blood pressure under pathological conditions is poorly understood. In the current study, NG-nitro-L-arginine methyl ester (L-NAME), a potent NOS inhibitor, was administered chronically, to induce peripheral vascular damage in mice. L-NAME-treated mice exhibited hypertension, increased vascular cell adhesion molecule-1 and plasminogen activator inhibitor-1 mRNA levels in the aorta and impaired vasodilatation associated with decreased aortic eNOS expression, consistent with endothelial damage. Three days following withdrawal of L-NAME treatment, the blood pressure response to apelin stimulation was assessed. Although apelin reduced blood pressure in non-treated mice, it was found to transiently elevate blood pressure in L-NAME-treated mice. These results indicate that apelin functions as a vasopressor peptide under pathological conditions, including vascular endothelial dysfunction in mice.

Abstract 173: Depletion of CLIP+ Immune Cells Prevents Toll-Like Receptor-Induced Preeclampsia in Mice

Preeclampsia (PE) is a pregnancy-specific disorder of vascular endothelial dysfunction and inflammation which manifests as hypertension and proteinuria and may result from maladaptation of the maternal immune system. Activation of the dsRNA receptor Toll-like receptor 3 (TLR3) or the ssRNA receptor TLR7 elicits pregnancy-dependent hypertension and endothelial dysfunction in mice in part by increasing pro-inflammatory immune cells. Polyclonal activation of immune cells is associated with expression of Class II-Associated Invariant Peptide Chain (CLIP) in MHC class II which prevents T cell-dependent death leading to persistent immune cell activation. We hypothesized that removal of CLIP reduces pro-inflammatory immune cells and prevents the development of PE in mice. Pregnant mice were given ip injections of normal saline (P), poly I:C (TLR3 agonist; P-PIC), or R837 (TLR7 agonist; P-R837) with or without TPP, a custom-targeted peptide that removes CLIP from MHC class II, on gestational days 13, 15, and 17 and euthanized on day 18. TPP treatment significantly decreased pro-inflammatory CD3+/γδ T cells and increased anti-inflammatory CD4+/CD25+ regulatory T cells in TLR3-induced PE mice as well as TLR7-induced PE mice. TPP treatment also prevented the hypertension (GD17 SBP in mmHg: P=102±3, P+TPP=105±4, P-PIC=147±4*, P-PIC+TPP=103±2, P-R837=133±2*, P-R837+TPP=106±4; *p&lt;0.05 vs. P), proteinuria, and endothelial dysfunction in TLR3-induced and TLR7-induced PE mice while having no detrimental effects in normal pregnant mice. Based on our results, the TPP-mediated decrease in detrimental immune cells positively modulated T cell subsets and prevented the development of PE-like symptoms in mice. CLIP removal from MHC class II with TPP may be a promising therapy for women with PE.

Increased Atherosclerosis and Endothelial Dysfunction in Mice Bearing Constitutively Deacetylated Alleles of Foxo1 Gene

Complications of atherosclerosis are the leading cause of death of patients with type 2 (insulin-resistant) diabetes. Understanding the mechanisms by which insulin resistance and hyperglycemia contribute to atherogenesis in key target tissues (liver, vessel wall, hematopoietic cells) can assist in the design of therapeutic approaches. We have shown that hyperglycemia induces FoxO1 deacetylation and that targeted knock-in of alleles encoding constitutively deacetylated FoxO1 in mice (Foxo1(KR/KR)) improves hepatic lipid metabolism and decreases macrophage inflammation, setting the stage for a potential anti-atherogenic effect of this mutation. Surprisingly, we report here that when Foxo1(KR/KR) mice are intercrossed with low density lipoprotein receptor knock-out mice (Ldlr(-/-)), they develop larger aortic root atherosclerotic lesions than Ldlr(-/-) controls despite lower plasma cholesterol and triglyceride levels. The phenotype is unaffected by transplanting bone marrow from Ldlr(-/-) mice into Foxo1(KR/KR) mice, indicating that it is independent of hematopoietic cells and suggesting that the primary lesion in Foxo1(KR/KR) mice occurs in the vessel wall. Experiments in isolated endothelial cells from Foxo1(KR/KR) mice indicate that deacetylation favors FoxO1 nuclear accumulation and exerts target gene-specific effects, resulting in higher Icam1 and Tnfα expression and increased monocyte adhesion. The data indicate that FoxO1 deacetylation can promote vascular endothelial changes conducive to atherosclerotic plaque formation.

Vascular Remodeling in Hypertension

Over the past few years noninvasive and invasive techniques have allowed a better appreciation of vascular changes in hypertensive humans and experimental animals.1 Large arteries undergo outward hypertrophic remodeling and increased stiffness with aging,2–4 and in hypertension there may be an acceleration of this process leading to enhanced pulse pressure. A reduced aortic diameter in middle-aged hypertensive subjects may also play a role in increases in pulse pressure through increased specific impedance,5 contradicting the classic hypertensive aortic phenotype characterized by vascular wall degeneration and calcification and increased aortic diameter. In advanced hypertension, however, the elastic laminae undergo duplication and fragmentation, with increased deposition of collagen and fetal (EIIIA) fibronectin, contributing to increased stiffness. Classically the remodeling of small arteries in hypertension has been associated with increased media thickness, but recent studies have demonstrated that 2 types of remodeling are found, inward eutrophic or inward hypertrophic remodeling, depending on whether the media cross-sectional area is enlarged (true hypertrophy).6–9 Although eutrophic remodeling is usually found in essential (primary) hypertension in humans and spontaneously hypertensive rats (SHRs); in secondary hypertension such as in renovascular hypertension, primary aldosteronism, or in pheochromocytoma10; in hypertension associated with diabetes mellitus11,12; and in acromegaly,13 hypertrophic remodeling has been described. In mineralocorticoid hypertension in rodents14,15 and in salt-sensitive Dahl rats,16 in both of which the endothelin (ET) system is activated, remodeling of small arteries is also hypertrophic (see below). Thus, when the renin-angiotensin system is even mildly activated (primary hypertension and SHRs), remodeling is usually eutrophic. In salt-dependent hypertension, diabetes mellitus, and malignant hypertension, all conditions in which the ET system is activated, remodeling is hypertrophic.17 Hyperplasia of vascular smooth muscle cells (VSMCs) is found in small arteries of …

Abstract P241: Calpain-1 Is Increased in Mitochondria and Contributes to Mitochondrial ROS Generation in High Glucose--Stimulated Endothelial Cells and Endothelial Dysfunction in Mouse Models of Diabetes

Objectives: Elevated levels of reactive oxygen species (ROS) are the initial source of endothelial dysfunction in diabetes. Calpain has been implicated in diabetic vascular complications. The present study was to investigate the role of calpain in mitochondrial ROS generation in endothelial cells and vascular dysfunction in diabetic mice. Methods: Endothelial cells cultured from human umbilical vein (HUVEC) were stimulated with high glucose. Calpain activity and protein were determined in mitochondria of HUVEC. Intracellular and mitochondrial ROS generation as well as apoptosis were measured. Type I diabetic OVE 26 mice and type II diabetic db/db mice with calpastatin over-expression (OVE26/CAST and db/db-CAST) were generated, respectively. Type I diabetes was also induced in both wild-type and Tg-CAST mice by injection of streptozocin (STZ). The endothelium-dependent relaxation of aortic ring was measured. Results: High glucose significantly increased calpain-1 protein, calpain activity and ROS generation in mitochondria of HUVEC. Pharmacological inhibition of calpain or over-expression of calpastatin abrogated high glucose-induced intracellular ROS production, mitochondrial ROS generation and apoptosis in HUVEC. Incubation of isolated mitochondria with calpain-1 protein significantly induced its ROS generation and the membrane potential. In diabetic mice, calpain activity was induced in aortic vessels, which correlated with an increase in ROS production and protein tyrosine nitration. Over-expression of calpastatin prevented calpain activity, reduced ROS production and inhibited protein tyrosine nitration in diabetic mice. Aortic ring segments from diabetic mice exhibited a significant reduction in vascular relaxation to acetylcholine, which was reversed by over-expression of calpastatin in Tg-CAST, OVE26/CAST and db/db-CAST mice. Conclusions: This study has demonstrated a novel role of calpain in mitochondrial ROS generation, which contributes to apoptosis in endothelial cells during hyperglycemia. Thus, over-expression of calpastatin inhibits reduces ROS production and ameliorates endothelium-dependent vascular dysfunction in mouse models of diabetes.

Abstract 8710: A Crucial Role of Nox2 In Middle-Age Obese-Related Hyperglycaemia and Endothelial Dysfunction in Mice

Middle-age obese is often associated with multiple organ oxidative stress and is a major risk factor for the development of cardiovascular diseases. In this study, we investigated the role of Nox2, a superoxide-generating enzyme, in a mouse model of high fat diet-induced middle-age metabolic disorders and vascular dysfunction. Littermates of C57BL/J6 wild-type and Nox2 knockout mice (7 m old, n=10) were fed with high fat diet (HFD, 45% kcal fat, 20% kcal protein and 35% kcal carbohydrate) or normal chow diet (NCD, 12% kcal fat, 28% kcal protein and 60% kcal carbohydrate) for 16 weeks, and were used at 11 m of age Compared to age-matched NCD-fed mice, wild-type mice fed with HFD showed significant increases in body weight (NCD 42±2.3 vs HFD 56±3.2g) and the levels of fasting glucose (NCD 7.4±3.3 vs HFD 8.8±2.2 mM). These metabolic changes were accompanied by significant increases (42±5%) in NADPH-dependent ROS production by aortas as measured by lucigenin-chemiluminescence, and a decrease in endothelium-dependent vessel relaxation to acetylcholine (Emax NCD 80.45±7.39% vs HFD 74.41±1.74%) as assessed by an organ bath. The systolic BP was also significantly elevated (NFD 127±8 vs HFD 142±12mmHg) (all P&lt;0.05). Compared to WT mice, Nox2 knockout mice fed with HFD had similar scale of increase in body weight (NCD 38±3.6 vs HFD 49±2.6g, P&lt;0.05). However, there was no significant increase in the levels of fasting glucose (NCD 7.5±0.8 vs HFD 6.7±1.5 mM). The aortic NADPH-dependent ROS production remained unchanged and the endothelium- dependent vessel relaxation to acetylcholine was well preserved (Emax NCD 80.53±11.3 vs HFD 78.18±5.6%). There was no significant increase in systolic BP (NCD 123±14 vs HFD130±9mmHg) in HFD-fed Nox2 knockout mice. In conclusion, Nox2 and Nox2-derived ROS play a key role in mediating dietary obesity-related hyperglycaemia and endothelial dysfunction after middle-age, and Nox2 may present a therapeutic target for the prevention and treatment of these diseases.

Soluble epoxide hydrolase inhibition prevents coronary endothelial dysfunction in mice with renovascular hypertension

The study addresses the hypothesis that endothelial dysfunction in experimental arterial hypertension can be related to an alteration in epoxyeicosatrienoic acids (EETs) pathway and can be prevented by the inhibition of EETs degradation by soluble epoxide hydrolase (sEH). Arterial hypertension was induced in FVB/N mice by renal artery stenosis ('two-kidney-one-clip', 2K1C). Seven weeks after surgery, increased aortic pressures (Millar tonometer; Millar Instruments, Houston, Texas, USA) and cardiac hypertrophy (echocardiography) were present in 2K1C mice as compared with control mice. Left coronary artery endothelium-dependent relaxations to acetylcholine were decreased in 2K1C mice without modification in the relaxing responses to NS309 and NS1619, the openers of calcium-activated potassium channels mediating the hyperpolarizing effect of EETs. The inhibitors of the EET-synthesizing enzymes cytochrome P450 epoxygenases, fluconazole and N-methylsulfonyl-6-(2-propargyloxyphenyl)-hexanamide (MSPPOH), reduced the coronary relaxations to acetylcholine in control but not in 2K1C mice. The sEH expression was increased in 2K1C mice. The sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid administered for 2 weeks starting 5 weeks after surgery in 2K1C mice (25 mg/l in drinking water) reduced aortic pressures and cardiac hypertrophy, improved the coronary relaxations to acetylcholine and restored the inhibitory effect of fluconazole and MSPPOH on acetylcholine-induced relaxations, without modifying the relaxations to NS309 and NS1619. These results demonstrate that a reduced EET-mediated relaxations related to an increased degradation by sEH contributes to coronary endothelial dysfunction in 2K1C hypertensive mice. Inhibiting sEH prevents endothelial dysfunction by restoring EET-mediated relaxations and thus, could represent a promising pharmacological intervention to limit cardiovascular morbidity and mortality in arterial hypertension.

Soluble epoxide hydrolase inhibition prevents coronary endothelial dysfunction in mice with renovascular hypertension

Soluble epoxide hydrolase inhibition prevents coronary endothelial dysfunction in mice with renovascular hypertension

Oxidative stress and inflammation in the endothelial dysfunction of obesity: a role for nuclear factor kappa B?

Oxidative stress and inflammation in the endothelial dysfunction of obesity: a role for nuclear factor kappa B?

Tyrosine Nitration of PA700 Activates the 26S Proteasome to Induce Endothelial Dysfunction in Mice With Angiotensin II–Induced Hypertension

The ubiquitin-proteasome system has been implicated in oxidative stress-induced endothelial dysfunction in cardiovascular diseases. However, the mechanism by which oxidative stress alters the ubiquitin-proteasome system is poorly defined. The present study was conducted to determine whether oxidative modifications of PA700, a 26S proteasome regulatory subunit, contributes to angiotensin II (Ang II)-induced endothelial dysfunction. Exposure of human umbilical vein endothelial cells to low concentrations of Ang II, but not vehicle, for 6 hours significantly decreased the levels of tetrahydro-l-biopterin (BH4), an essential cofactor of endothelial NO synthase, which was accompanied by a decrease in GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis. In addition, Ang II increased both tyrosine nitration of PA700 and the 26S proteasome activity, which were paralleled by increased coimmunoprecipitation of PA700 and the 20S proteasome. Genetic inhibition of NAD(P)H oxidase or administration of uric acid (a peroxynitrite scavenger) or N(G)-nitro-l-arginine methyl ester (nonselective NO synthase inhibitor) significantly attenuated Ang II-induced PA700 nitration, 26S proteasome activation, and reduction of GTP cyclohydrolase I and BH4. Finally, Ang II infusion in mice decreased the levels of both BH4 and GTP cyclohydrolase I and impaired endothelial-dependent relaxation in isolated aortas, and all of these effects were prevented by the administration of MG132, a potent inhibitor for 26S proteasome. We conclude that Ang II increases tyrosine nitration of PA700 resulting in accelerated GTP cyclohydrolase I degradation, BH4 deficiency, and consequent endothelial dysfunction in hypertension.

Interaction of protease‐activated receptor 2 with chronic angiotensin II‐induced endothelial dysfunction in mice

Protease‐Activated Receptor 2 (PAR‐2) knockout mice exhibit a different time course in blood pressure response to angiotensin II (AngII). In this study we measured endothelium‐dependent relaxations of mesenteric arteries from PAR‐2 knockout (PAR‐2 ‐/‐) and C57BL/6 mice using wire myographs. Our goal was to test the influence of PAR‐2 ‐/‐ on AngII‐induced endothelial dysfunction. Chronic AngII infusion of C57BL/6 and PAR‐2 ‐/‐ decreased acetylcholine (ACh)‐induced relaxations by ~20 % whereas PAR‐2 agonist (2fly) and sodium nitroprusside relaxations were unchanged relative to saline infusion groups. ACh‐induced relaxations of AngII‐PAR‐2 ‐/‐ arteries, but not AngII‐C57BL/6 arteries, were rescued in the presence of the cyclooxygenases inhibitor indomethacin. In contrast to these effects, indomethacin decreased the sensitivity to ACh in saline‐PAR‐2 ‐/‐ and did not affect either ACh‐ or 2fly‐induced relaxations in saline‐C57BL/6. The sensitivity to 2fly of arteries from AngII‐C57BL/6 was decreased in the presence of indomethacin. A combination of PAR‐2 gene knockout and indomethacin rescued ACh‐induced relaxations of vascular smooth muscle after chronic AngII infusion in mice. Cyclooxygenases contribute to the mechanism of persistent PAR‐2 vasodilation despite endothelial dysfunction and to the mechanism of action for ACh in PAR‐2 ‐/‐ mice. Funding sources: CIHR, IRIF, CFI.

HDL, lipid peroxidation, and atherosclerosis

Genetic, clinical, and pharmacological studies implicate elevated levels of LDL in the pathogenesis of atherosclerotic vascular disease, the leading cause of death in industrialized societies (1). Paradoxically, native LDL fails to exert potentially atherogenic effects in vitro, suggesting that it must be modified to promote vascular disease. Indeed, many lines of evidence support the LDL oxidation hypothesis, which suggests that oxidative damage to LDL is one important mechanism for rendering lipoproteins atherogenic (as reviewed in Refs. 2, 3).

H021 Genetic disruption of protein tyrosine phosphatase 1B attenuates both myocardial and endothelial dysfunction in mice with heart failure

We have shown previously that chronic, in vivo pharmacological inhibition of protein tyrosine phosphatase 1B (PTP1B) prevented both endothelial and cardiac dysfunction in mice with chronic heart failure (CHF). The present study was designed to test whether similar cardiovascular protective effects are present in mice genetically deficient for PTP1B. CHF was induced by coronary ligation, either in wild type (WT) or PTP1B deficient (PTP1B-/-) BALB/c mice. After 2 months of ligation, echocardiographic analysis of left ventricular (LV) function and remodelling was performed, after which flow-mediated, NO-dependent vasodilatation (FMD) of mesenteric resistance arteries was evaluated. In PTP1B-/- mice with CHF (n = 13), compared to CHF WT mice (n = 15) LV end diastolic diameter (LVEDD) and LV end systolic diameter (LVESD) were reduced (LVEDD: CHF WT 6.1 ± 0.2; CHF PTP1B-/- 5.2 ± 0.2 mm, p < 0.01; LVESD: CHF WT 5.5 ± 0.2; CHF PTP1B-/- 3.9 ± 0.3 mm, p<0.01), while fractional shortening (FS) and cardiac output (CO) were increased (FS: CHF WT 10.9 ± 1.6; CHF PTP1B-/- 22.7 ± 2.5 %, p < 0.01; CO: WT 16.3 ± 1.0; PTP1B-/- 22.2 ± 1.1 ml/min, p < 0.05). Genetic disruption of PTP1B was also associated with decreased collagen density. These hemodynamic and structural effects were observed in the context of identical infarct sizes. Vascular studies showed that compared to CHF WT mice (n=15), CHF PTP1B-/- mice (n = 16) displayed an increased FMD (CHF WT 5 ± 1, CHF PTP1B-/- 19 ± 4 %, p < 0.05). Additionally, in vitro downregulation of PTP1B (by a 3 day incubation with shRNA) also increased FMD in arteries isolated from CHF mice (max FMD; untreated: 6 ± 2; scrambled shRNA: 7 ± 2; PTP1B shRNA 27 ± 2 %, p < 0.01). Thus, genetic disruption of PTP1B prevent endothelial and cardiac dysfunction in CHF mice, suggesting that this enzyme may be a new interesting target for the treatment of CHF.

F022 Healing of coronary endothelium by chronic 17beta-estradiol after ischemia/reperfusion injury in mice

Endothelial protective actions of estrogens have been observed in various cardiovascular pathophysiological states. In particular, we showed that chronic 17beta-estradiol (E2) treatment prevents acute ischemia/reperfusion-induced coronary endothelial dysfunction in mice and accelerates reendothelialization after carotid injury. Whether such an endothelial healing effect occurs in a context of chronic reperfusion is unknown. Figure options Download full-size image Download as PowerPoint slide Thus, ischemia was induced by a left anterior descending coronary ligation for 30 minutes followed by one month of reperfusion (IR) in ovariectomized (OVX) mice, implanted or not 24 hours after ischemia (in order to avoid any effect on ischemic and/or early reperfusion injury) with an E2-releasing pellet (80 μg/kg/day). Coronary segments (internal diameter 180 to 220 μm) were removed distal to the site of occlusion and mounted in a wire myograph to study the relaxing response to acetylcholine (Ach). At 24 hours reperfusion (i.e. immediately before the onset of E2 treatment), OVX mice displayed a marked endothelial dysfunction (maximal relaxation to Ach: sham 55+/-4; IR 31+/-4 %; p<0.05). One month after reperfusion, untreated OVX mice showed a persistent decreased relaxation, whereas chronic E2 markedly increased the relaxing responses in IR mice (to a level similar to that of untreated sham mice) (Figure). E2 also slightly increased the relaxing responses in sham mice. In vitro NO Synthase inhibition abolished the differences between mice, suggesting that E2 restored the decreased NO production induced by IR. We thus demonstrate for the first time a marked beneficial effect of E2 on coronary endothelial healing after chronic reperfusion, leading to a restoration of coronary endothelial function.