Dysfunction In ApoE Research Articles

Clearance of lipid droplets by chimeric autophagy-tethering compound ameliorates the age-related macular degeneration phenotype in mice lacking APOE

ABSTRACT Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among the elderly, and there is currently no clinical treatment targeting the primary impairment of AMD. The earliest clinical hallmark of AMD is drusen, which are yellowish spots mainly composed of lipid droplets (LDs) accumulated under the retinal pigment epithelium (RPE). However, the potential pathogenic role of this excessive LD accumulation in AMD is yet to be determined, partially due to a lack of chemical tools to manipulate LDs specifically. Here, we employed our recently developed Lipid Droplets·AuTophagy Tethering Compounds (LD∙ATTECs) to degrade LDs and to evaluate its consequence on the AMD-like phenotypes in apoe−/− (apolipoprotein E; B6/JGpt-Apoeem1Cd82/Gpt) mouse model. apoe−/− mice fed with high-fat diet (apoe−/−-HFD) exhibited excessive LD accumulation in the retina, particularly with AMD-like phenotypes including RPE degeneration, Bruch’s membrane (BrM) thickening, drusen-like deposits, and photoreceptor dysfunction. LD·ATTEC treatment significantly cleared LDs in RPE/choroidal tissues without perturbing lipid synthesis-related proteins and rescued RPE degeneration and photoreceptor dysfunction in apoe−/−-HFD mice. This observation implied a causal relationship between LD accumulation and AMD-relevant phenotypes. Mechanically, the apoe−/−-HFD mice exhibited elevated oxidative stress and inflammatory signals, both of which were mitigated by the LD·ATTEC treatment. Collectively, this study demonstrated that LD accumulation was a trigger for the process of AMD and provided entry points for the treatment of the initial insult of AMD by degrading LDs. Abbreviations: AMD: age-related macular degeneration; APOE: apolipoprotein E; ATTECs: autophagy-tethering compounds; BODIPY: boron-dipyrromethene; BrM: Bruch’s membrane; ERG: electroretinogram; HFD: high-fat diet; LD·ATTECs: Lipid Droplets·AuTophagy Tethering Compounds; LDs: lipid droplets; OA: oleic acid; OPL: outer plexiform layer; ROS: reactive oxygen species; RPE: retinal pigment epithelium

Bone morphogenetic protein 4 in perivascular adipose tissue ameliorates hypertension through regulation of angiotensinogen

Perivascular adipose tissue (PVAT), an active endocrine organ, exerts direct effect on vascular tone through paracrine. Activation of PVAT metabolism plays an inhibitory role in atherosclerosis via secreting relaxing factors. The present studies were designed to investigate the role of PVAT metabolism in regulation of hypertension. Apolipoprotein E (ApoE) knockout mice with BMP4 knockout in adipose tissue or brown adipose tissue (aP2-DKO or UCP1-DKO, respectively) were used for exploring the role of impaired PVAT metabolism in hypertension. Vascular function was assessed using wire myography. The potential regulatory factor of vascular function was explored using qPCR and ELISA and further confirmed in perivascular fat cell line. Knockout of BMP4 either in adipose tissue or specifically in BAT aggravates high-fat diet (HFD, 40% fat)-induced hypertension and endothelial dysfunction in ApoE-/- mice. In the meanwhile, deficiency of BMP4 also aggravates Ang II (angiotensin II) -induced hypertension and vascular remodeling in ApoE-/- mice. Moreover, deficiency of BMP4 inhibits NO release and induces ROS production. In vitro system, aortic rings pretreated with PVAT extracts from BMP4-DKO mice showed increased vasoconstriction and reduced endothelial-dependent relaxation compared with the controls. We further demonstrated that PVAT of BMP4-DKO mice expressed higher level of angiotensinogen (AGT) and Ang II compared with the controls. Impaired PVAT metabolism aggravates hypertension, and this effect is dependent on the activation of local renin-angiotensin-aldosterone system (RAAS). The results of this study first demonstrate the regulatory role of PVAT metabolism in hypertension.

E-selectin-targeted copolymer reduces atherosclerotic lesions, adverse cardiac remodeling, and dysfunction

Endothelial activation with up-regulation of E-selectin adhesion molecules mediates leukocyte rolling along the vascular wall and controls inflammation in many diseases including atherosclerosis and heart failure. Therefore, we aimed to test the hypothesis that inhibition of E-selectin-mediated interactions by a new E-selectin-targeted copolymer could inhibit the progression of atherosclerosis. To target E-selectin on activated endothelium, we developed a new N-(2-hydroxypropyl)methacrylamide (HPMA)-based E-selectin binding copolymer with or without dexamethasone (Dex) (designated P-(Esbp)-Dex and P-Esbp, respectively). To determine the effect of P-(Esbp)-Dex and P-Esbp on atherosclerosis, we allocated ApoE (−/−) mice on a high fat diet, to weekly intra-peritoneal injections of either 1) P-Esbp; 2) P-(Esbp)-Dex; 3) free Dex (1 mg/kg) or 4) saline, for four weeks. Aortic atherosclerosis and left ventricular (LV) remodeling and function were assessed by serial ultrasound studies and histology. Monocytes and macrophages were characterized by flow cytometry.After four weeks of treatment, P-Esbp effectively targeted aortic atherosclerotic lesions. Both P-Esbp and P-(Esbp)-Dex reduced wall thickening of the ascending aortas. However, only the drug-free copolymer (P-Esbp) significantly decreased the areas of necrotic core in the plaques and switched spleen macrophages toward an anti-inflammatory (M2) phenotype. Furthermore, P-Esbp attenuated adverse LV remodeling and dysfunction in ApoE (−/−) mice.In summary, P-Esbp copolymer targets activated endothelial cells, regresses and stabilizes atherosclerotic plaques, and prevents adverse LV remodeling and dysfunction in ApoE (−/−) mice. Our results suggest a new, drug-free macromolecular therapy to treat vascular inflammation.

Chronic Cadmium Exposure Accelerates the Development of Atherosclerosis and Induces Vascular Dysfunction in the Aorta of ApoE-/- Mice.

Cadmium exposure is related to cardiovascular diseases, including hypertension, atherosclerosis, increased oxidative stress, endothelial dysfunction, and specific biochemical changes induced by this metal. Thus, we aimed to investigate whether cadmium exposure induces endothelial dysfunction, accelerates atherosclerotic plaque formation in the aorta, and enhances oxidative stress in apolipoprotein E knockout (ApoE-/-) mice. Experiments were performed in 14-week-old male wild-type and ApoE-/- mice. ApoE-/- mice received cadmium (CdCl2 100mg/L in drinking water for 28days) or vehicle (distilled water). After treatment, vascular reactivity to phenylephrine, acetylcholine, and sodium nitroprusside was analyzed using isolated aorta. Bone marrow cells were isolated to assess the production of nitric oxide and reactive oxygen and nitrogen species. ApoE-/- cadmium-treated mice had higher cholesterol levels than non-exposed mice. Cadmium exposure decreased the vasodilatation response to acetylcholine in aortic ring of ApoE-/- mice, though no changes in phenylephrine or sodium nitroprusside responses were observed. L-NAME reduced vasodilator responses to acetylcholine; this effect was lower in ApoE-/- cadmium-treated mice, suggesting reduction in nitric oxide (NO) bioavailability. Moreover, in bone marrow cells, cadmium decreased cytoplasmic levels of NO and increased superoxide anions, hydrogen peroxide, and peroxynitrite in ApoE-/- mice. Morphological analysis showed that cadmium exposure increased plaque deposition in the aorta by approximately 3-fold. Our results suggest that cadmium exposure induces endothelial dysfunction in ApoE-/- mice. Moreover, cadmium increased total cholesterol levels, which may promote the early development of atherosclerosis in the aorta of ApoE-/- mice. Our findings support the hypothesis that cadmium exposure might increase the risk of atherosclerosis.

β-Elemene attenuates atherosclerosis in apolipoprotein E-deficient mice via restoring NO levels and alleviating oxidative stress

β-Elemene is a major bioactive sesquiterpenoids compound isolated from the essential oils of Curcuma Wenyujin, a Chinese medicinal herb that treats tumor in clinics. However anti-atherosclerotic effects of β-elemene have not been fully investigated in vivo. The objective of this study is to further elucidate the anti-atherosclerotic activities of β-elemene in ApoE−/− mice. Staining techniques and immunohistochemistry were used to validate atherosclerosis. Serum lipids, plasma nitrite and nitrate were analyzed by colorometric methods. ROS and antioxidative enzymes were measured through kits. Proteome profiler array was performed to analyze atherosclerosis-related inflammatory Cytokine. Western blot was used for measuring various proteins expressions. These results revealed that β-elemene inhibited atherosclerotic lesion size and increased stability of plaques in ApoE−/− mice by alleviating levels of vascular oxidative stress and preventing pro-inflammatory cytokine production. In addition β-elemene maintained endothelial function by significantly improving plasma nitrite and nitrate levels and expression of phosphorylation-eNOS in vivo. β-elemene also increased the production of the nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) and promoted phosphorylation of eNOSser1177 and Akt in vitro. In Conclusive, data revealed that β-elemene attenuated atherosclerosis and enhanced stability of plaques at least partially through its antioxidative and anti-inflammatory features and protected against endothelial dysfunction in ApoE−/− mice.

366 - Serum Amyloid A Stimulates Atherogenesis and Renal Dysfunction in Apolipoprotein E-Deficient Mice Fed a Normal Chow Diet

Elevated serum amyloid A (SAA) can cause endothelial dysfunction via enhanced oxidative stress that advances inflammation/thrombosis in cardiovascular and renal pathology. We investigated the impact of SAA on aortic vessels and renal function in apolipoprotein E-deficient (ApoE -/- ) mice. Male ApoE -/- mice received vehicle-(control), low-level lipopolysaccharide (LPS) or recombinant human SAA by i.p . injection every third day (over 2-weeks). Heart, aorta and kidney were harvested after 3 or 5 days, or after a total of 4 weeks (designated as young) or 18 weeks (old) mice. SAA-mediated-renal injury was evident in young mice manifesting as increased plasma blood nitrogen products, and urinary levels of protein and kidney injury molecule-1 (KIM-1), tissue content of oxidized lipids and several cytokines/chemokines, relative to controls. SAA stimulated NFκB activation and macrophage recruitment in kidneys as early as 3 days after administration. SAA increased vascular cell adhesion molecule (VCAM)-1 in aortic vessels and plasma monocyte chemotactic protein (MCP)-1 levels in young mice relative to their respective controls. Aortic gene expression of nuclear-factor-kappa-beta (NFκB-p50) and tissue factor (TF) also increased in young mice confirming that SAA stimulated pro-inflammatory/pro-thrombotic responses. These data suggest that SAA promotes renal impairment and endothelial dysfunction in ApoE -/- mice in the absence of a high-fat diet.

Modulation of the sympathetic nervous system by renal denervation prevents reduction of aortic distensibility in atherosclerosis prone ApoE-deficient rats.

BackgroundApolipoprotein E-deficient (ApoE−/−) rodents spontaneously develop severe hypercholesterolemia and increased aortic stiffness, both accepted risk factors for cardiovascular morbidity and mortality in humans. In patients with resistant hypertension renal denervation (RDN) may improve arterial stiffness, however the underlying mechanisms are incompletely understood. This study investigates the impact of RDN on aortic compliance in a novel atherosclerosis prone ApoE−/−-rat model.MethodsNormotensive, 8 weeks old ApoE−/− and Sprague–Dawley (SD) rats were subjected to bilateral surgical RDN (n = 6 per group) or sham operation (n = 5 per group) and fed with normal chow for 8 weeks. Compliance of the ascending aorta was assessed by magnetic resonance imaging. Vasomotor function was measured by aortic ring tension recordings. Aortic collagen content was quantified histologically and plasma aldosterone levels were measured by enzyme-linked immunosorbent assay (ELISA).ResultsAfter 8 weeks, ApoE−/−-sham demonstrated a 58 % decrease in aortic distensibility when compared with SD-sham (0.0051 ± 0.0011 vs. 0.0126 ± 0.0023 1/mmHg; p = 0.02). This was accompanied by an impaired endothelium-dependent relaxation of aortic rings and an increase in aortic medial fibrosis (17.87 ± 1.4 vs. 12.27 ± 1.1 %; p = 0.006). In ApoE−/−-rats, RDN prevented the reduction of aortic distensibility (0.0128 ± 0.002 vs. 0.0051 ± 0.0011 1/mmHg; p = 0.01), attenuated endothelial dysfunction, and decreased aortic medial collagen content (12.71 ± 1.3 vs. 17.87 ± 1.4 %; p = 0.01) as well as plasma aldosterone levels (136.33 ± 6.6 vs. 75.52 ± 8.4 pg/ml; p = 0.0003). Cardiac function and metabolic parameters such as hypercholesterolemia were not influenced by RDN.ConclusionApoE−/−-rats spontaneously develop impaired vascular compliance. RDN improves aortic distensibility and attenuated endothelial dysfunction in ApoE−/−-rats. This was associated with a reduction in aortic fibrosis formation, and plasma aldosterone levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0914-9) contains supplementary material, which is available to authorized users.

Nutritional depletion in n-3 PUFA in apoE knock-out mice: A new model of endothelial dysfunction associated with fatty liver disease.

Western diets are characterized by low intake of n-3 PUFA compensated by constant amounts of n-6 PUFA. Reduced intake of n-3 PUFA is associated with increased cardiovascular risk, as observed in nonalcoholic fatty liver disease patients. The study aimed to evaluating the impact of dietary n-3 PUFA depletion on endothelial function, an early key event of cardiovascular diseases. C57Bl/6J or apolipoprotein E knock-out (apoE-/- ) were fed control (CT) or n-3 PUFA-depleted diets (DEF) for 12 wks. Mice fed n-3 DEF diet developed a hepatic steatosis, linked to changes in hepatic expression of genes controlled by Sterol Regulatory Element Binding Protein-1 and -2. Vascular function was assessed on second- and third-order mesenteric arteries and n-3 PUFA-depleted apoE-/- mice presented endothelial dysfunction characterized by decreased vasorelaxation in response of acetylcholine. The presence of a nitric oxide synthase (NOS) inhibitor blunted the relaxation in each groups and heme-nitrosylated hemoglobin blood (Hb-NO) level was significantly lower in n-3 PUFA-depleted apoE-/- mice. Twelve weeks of n-3 DEF diet promote steatosis and accelerate the process of endothelial dysfunction in apoE-/- mice by a mechanism involving the NOS/NO pathway. We propose n-3 PUFA-depleted apoE-/- mice as a new model to study endothelial dysfunction related to hepatic steatosis independently of obesity.

Abstract 269: Xanthine Oxidase Inhibition by Febuxostat Attenuates Experimental Atherosclerosis in Mice

Objective: Atherosclerosis is a chronic inflammatory disease of the vascular wall. Multiple mechanisms are implicated in the inflammatory process, including oxidative stress. Xanthine oxidase (XO), which is converted from xanthine dehydrogenase (XDH), is a major enzymatic source of reactive oxygen species (ROS) and has been reported to be involved in atherosclerosis, but the underlying mechanisms remain unclear. We have examined whether XO contributes to atherosclerosis in mice and whether XO mediates cholesterol crystal (CC)-induced inflammation. Approach and Results: ApoE -/- mice fed a high cholesterol diet for 12 weeks (ApoE -/- ) developed atherosclerotic plaques, increased ROS generation, increased tissue activities of XO/XDH, and endothelial dysfunction. Immunohistochemical analysis revealed that XO/XDH were increased in macrophages in atherosclerotic plaques and in aortic endothelial cells. Febuxostat, a highly potent XO/XDH inhibitor, significantly attenuated plaque formation, decreased ROS generation, and improved endothelial dysfunction in ApoE -/- mice without affecting plasma cholesterol levels. Additionally, in aorta from febuxostat-treated group, MCP-1 mRNA was significantly decreased whereas eNOS mRNA was increased. Furthermore, we examined the effects of febuxostat on CC-induced inflammation in macrophages. Febuxostat significantly inhibited CC-induced ROS formation as well as MCP-1, IL-1α, IL-1β and IL-6 production and its effects could be recapitulated by ROS scavengers. Conclusions: These results demonstrated that increased XO leads to ROS formation and contributes to the progression of atherosclerosis via endothelial dysfunction and macrophage inflammation, and that febuxostat may be a potential therapeutic agent to target inflammation in atherosclerosis.

Xanthine Oxidase Inhibition by Febuxostat Attenuates Experimental Atherosclerosis in Mice

Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE−/− mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE−/− mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis.

Angiotensin-(1–7) Modulates Renal Vascular Resistance Through Inhibition of p38 Mitogen-Activated Protein Kinase in Apolipoprotein E–Deficient Mice

Apolipoprotein E-deficient (apoE(-/-)) mice fed on Western diet are characterized by increased vascular resistance and atherosclerosis. Previously, we have shown that chronic angiotensin (Ang)-(1-7) treatment ameliorates endothelial dysfunction in apoE(-/-) mice. However, the mechanism of Ang-(1-7) on vasoconstrictor response to Ang II is unknown. To examine Ang-(1-7) function, we used apoE(-/-) and wild-type mice fed on Western diet that were treated via osmotic minipumps either with Ang-(1-7) (82 μg/kg per hour) or saline for 6 weeks. We show that Ang II-induced renal pressor response was significantly increased in apoE(-/-) compared with wild-type mice. This apoE(-/-)-specific response is attributed to reactive oxygen species-mediated p38 mitogen-activated protein kinase activation and subsequent phosphorylation of myosin light chain (MLC(20)), causing renal vasoconstriction. Here, we provide evidence that chronic Ang-(1-7) treatment attenuated the renal pressor response to Ang II in apoE(-/-) mice to wild-type levels. Ang-(1-7) treatment significantly decreased renal inducible nicotinamide adenine dinucleotide phosphate subunit p47phox levels and, thus, reactive oxygen species production that in turn causes decreased p38 mitogen-activated protein kinase activity. The latter has been confirmed by administration of a specific p38 mitogen-activated protein kinase inhibitor SB203580 (5 μmol/L), causing a reduced renal pressor response to Ang II in apoE(-/-) but not in apoE(-/-) mice treated with Ang-(1-7). Moreover, Ang-(1-7) treatment had no effect in Mas(-/-)/apoE(-/-) double-knockout mice confirming the specificity of Ang-(1-7) action through the Mas-receptor. In summary, Ang-(1-7) modulates vascular function via Mas-receptor activation that attenuates pressor response to Ang II in apoE(-/-) mice by reducing reactive oxygen species-mediated p38 mitogen-activated protein kinase activity.

Abstract 299: Remodeling of Endothelial Function in Atherosclerotic Mice Overexpressing Endothelin-1 Restricted to Endothelium

Background: In human atherosclerosis , which is associated with elevated plasma and coronary endothelin (ET)-1 levels, ET type A receptor antagonists improved coronary endothelial function. Mice overexpressing ET-1 specifically in the endothelium (eET-1) crossed with apolipoprotein E knockout mice (Apoe-/-) exhibited exaggerated high-fat diet (HFD)-induced atherosclerosis. Since endothelial dysfunction often precedes development of atherosclerosis, we investigated whether endothelium-specific ET-1 overexpression causes endothelial dysfunction in Apoe-/- mice. Methods: Eight-week male old eET-1, Apoe-/-, eET-1/Apoe-/- and wild-type (WT) mice were fed a regular diet or HFD for 8 weeks. Endothelial function was assessed in mesenteric arteries by pressurized myography. Results: In HFD-fed mice, acetylcholine-induced endothelium-dependent relaxation (EDR) was reduced in Apoe-/- and eET-1 compared to WT (Emax: 29.2±2.9% and 51.8±7.9%, respectively, vs. 88.0±2.6%, P<0.05). Surprisingly, EDR was not impaired in eET-1/Apoe-/- (70.9±9.8%) compared to WT. Endothelium-independent relaxation to the nitric oxide (NO) donor sodium nitroprusside and contractile responses to norepinephrine were unaffected. Similar results were observed in regular diet-fed mice. In the presence of inhibitors of either NO synthase (NOS)-mediated relaxation, Nω-nitro-L-arginine methyl ester, or endothelium-dependent hyperpolarization (EDH)-mediated relaxation, apamin plus Tram34, EDR was blunted in WT (Emax: 2.8±0.4% and 35.7±8.4 respectively, P<0.01), whereas relaxation was weakly reduced or unaffected in eET-1/Apoe-/- (Emax: 68.6±9.9, P<0.05 and 80.6±8.0, respectively). However, the concomitant inhibition of NOS- and EDH-mediated relaxation strongly reduced EDR in eET-1/Apoe-/- (Emax: 43.0±8.2%, P<0.05). Conclusions: These results show an interdependence of NOS and EDH pathways in EDR in WT mice. ET-1 overexpression induced development of compensatory mechanisms in pre-atherosclerotic arteries of Apoe-/- mice, which permits either NOS or EDH pathway to mediate independently EDR. Investigation of mechanisms involved in the remodeling of EDR in eET-1/Apoe-/- mice will allow a better understanding of the ET-1 role in atherosclerosis.

Divergent effects of a CLA-enriched beef diet on metabolic health in ApoE−/− and ob/ob mice

Conjugated linoleic acid (CLA) is found naturally in meat and dairy products, and represents a potential therapeutic functional nutrient. However, given the discrepancies in isomer composition and concentration, controversy surrounds its proposed antidiabetic, antiobesity effects. This study focused on the effects of CLA-enriched beef (composed predominantly of c9, t11-CLA) in two separate models of metabolic disease: proatherosclerotic ApoE−/− mice and diabetic, leptin-deficient ob/ob mice. Animals were fed CLA-enriched beef for 28 days, and markers of the metabolic syndrome and atherosclerosis were assessed. Comprehensive hepatic transcriptomic analysis was completed to understand divergent metabolic effects of CLA. CLA-enriched beef significantly reduced plasma glucose, insulin, nonesterified fatty acid and triacylglycerol and increased adiponectin levels in ob/ob mice. In contrast, plasma lipid profiles and glucose homeostasis deteriorated and promoted atherosclerosis following the CLA-enriched beef diet in ApoE−/− mice. Hepatic transcriptomic profiling revealed divergent effects of CLA-enriched beef on insulin signaling and lipogenic pathways, which were adversely affected in ApoE−/− mice. This study demonstrated clear divergence in the effects of CLA. CLA-enriched beef improved metabolic flexibility in ob/ob mice, resulting in enhanced insulin sensitivity. However, CLA-enriched diet increased expression of lipogenic genes, resulting in inefficient fatty acid storage which increases lipotoxicity in peripheral organs, and led to profound metabolic dysfunction in ApoE−/− mice. While CLA may have potential health effects, in some circumstances, caution must be exercised in presenting this bioactive lipid as a potential functional food for the treatment of metabolic disease.

Decreased production of neuronal NOS‐derived hydrogen peroxide contributes to endothelial dysfunction in atherosclerosis

Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)-derived H(2)O(2) is an important endothelium-derived relaxant factor in the mouse aorta. The role of H(2)O(2) and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS-derived H(2)O(2) contributes to the impaired vasodilatation in apolipoprotein E-deficient mice (ApoE(-/-)). Changes in isometric tension were recorded on a myograph; simultaneously, NO and H(2)O(2) were measured using carbon microsensors. Antisense oligodeoxynucleotides were used to knockdown eNOS and nNOS in vivo. Western blot and confocal microscopy were used to analyse the expression and localization of NOS isoforms. Aortas from ApoE(-/-) mice showed impaired vasodilatation paralleled by decreased NO and H(2)O(2) production. Inhibition of nNOS with L-Arg(NO2) -L-Dbu, knockdown of nNOS and catalase, which decomposes H(2)O(2) into oxygen and water, decreased ACh-induced relaxation by half, produced a small diminution of NO production and abolished H(2)O(2) in wild-type animals, but had no effect in ApoE(-/-) mice. Confocal microscopy showed increased nNOS immunostaining in endothelial cells of ApoE(-/-) mice. However, ACh stimulation of vessels resulted in less phosphorylation on Ser852 in ApoE(-/-) mice. Our data show that endothelial nNOS-derived H(2)O(2) production is impaired and contributes to endothelial dysfunction in ApoE(-/-) aorta. The present study provides a new mechanism for endothelial dysfunction in atherosclerosis and may represent a novel target to elaborate the therapeutic strategy for vascular atherosclerosis.

Chronic treatment with angiotensin‐(1‐7) improves renal endothelial dysfunction in apolipoproteinE‐deficient mice

ApolipoproteinE-deficient [apoE (-/-)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin-(1-7) [Ang-(1-7)], acting through its specific GPCR, the Mas receptor, has endothelium-dependent vasodilator properties. Here we have investigated if chronic treatment with Ang-(1-7) improved endothelial dysfunction in apoE (-/-) mice. ApoE (-/-) mice fed on a lipid-rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang-(1-7) (82 µg·kg(-1) ·h(-1) ) or the same dose of Ang-(1-7) together with D-Ala-Ang-(1-7) (125 µg·kg(-1) ·h(-1) ) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys. Ang-(1-7)-treated apoE (-/-) mice showed improved renal endothelium-dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (-/-) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium-dependent vasorelaxation in kidneys of saline-treated apoE (-/-) mice whereas no effect was observed in Ang-(1-7)-treated mice. Chronic treatment with D-Ala-Ang-(1-7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang-(1-7) on endothelium-dependent vasorelaxation. Renal endothelium-independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang-(1-7)-treated mice, compared with untreated mice, whereas eNOS expression was increased. Chronic infusion of Ang-(1-7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO.

Protective Effects of PARP-1 Knockout on Dyslipidemia-Induced Autonomic and Vascular Dysfunction in ApoE−/− Mice: Effects on eNOS and Oxidative Stress

The aims of this study were to investigate the role of poly(ADP-ribose) polymerase (PARP)-1 in dyslipidemia-associated vascular dysfunction as well as autonomic nervous system dysregulation. Apolipoprotein (ApoE)−/− mice fed a high-fat diet were used as a model of atherosclerosis. Vascular and autonomic functions were measured in conscious mice using telemetry. The study revealed that PARP-1 plays an important role in dyslipidemia-associated vascular and autonomic dysfunction. Inhibition of this enzyme by gene knockout partially restored baroreflex sensitivity in ApoE−/− mice without affecting baseline heart-rate and arterial pressure, and also improved heart-rate responses following selective blockade of the autonomic nervous system. The protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction was associated with preservation of eNOS activity. Dyslipidemia induced PARP-1 activation was accompanied by oxidative tissue damage, as evidenced by increased expression of iNOS and subsequent protein nitration. PARP-1 gene deletion reversed these effects, suggesting that PARP-1 may contribute to vascular and autonomic pathologies by promoting oxidative tissue injury. Further, inhibition of this oxidative damage may account for protective effects of PARP-1 gene deletion on vascular and autonomic functions. This study demonstrates that PARP-1 participates in dyslipidemia-mediated dysregulation of the autonomic nervous system and that PARP-1 gene deletion normalizes autonomic and vascular dysfunctions. Maintenance of eNOS activity may be associated with the protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction.

Identification of HPA axis dysfunction in ApoE targeted replacement mice

The association of allele four of apoE (apoE4), a cholesterol transport protein, as a genetic risk factor for AD has been well established. However, despite knowing for over a decade that apoE4 is a risk factor for AD, the molecular mechanism behind the risk-factor activity of apoE4 remains elusive. To gain a better understanding of apoE4 biology we chose to examine the transcriptional differences associated with apoE4 expression compared to apoE3 expression in human apoE knock-in mice. Using transcriptional analysis we have investigated gene expression levels in the hippocampus of human-targeted replacement apoE3 and apoE4 knock-in mice. Results from the transcriptional profiling study showed that apoE4 mice express increased mRNA levels of glucocorticoid responsive genes and decreased levels of pituitary hormones genes compared to their apoE3 expressing counterparts. Given that glucocorticoids are known to have a negative feedback effect on the pituitary, these data indicate that apoE4 mice may have a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis. To investigate this further we examined levels of the circulating glucocorticoid hormone, corticosterone, in plasma samples taken from apoE3 and apoE4 animals. In agreement with the transcriptional profiling data, levels of plasma corticosterone were significantly up-regulated in apoE4 mice compared to apoE3 mice. Furthermore, this increase in corticosterone was accompanied by a down-regulation of plasma adrenocorticotropic hormone (ACTH), an anterior pituitary hormone. Interestingly, dysregulation of the HPA axis has been reported early in AD and disturbances of this neuroendocrine pathway are associated with hippocampal atrophy, memory impairments and neurodegeneration. It is therefore possible that the apoE4 genotype may increase the risk of AD by inducing a state of long-term chronic exposure to glucocorticoids. Taken together our results suggest that expression of apoE4 may lead to disturbances of the HPA axis and that this dysregulation may contribute to the cognitive decline and neurodegeneration that occurs in AD.

Native C-reactive protein induces endothelial dysfunction in ApoE −/− mice: Implications for iNOS and reactive oxygen species

Objective In addition to being a risk marker for cardiovascular disease, recent data suggests that C-reactive protein (CRP) induces endothelial dysfunction and promotes oxidative stress. We evaluated the effects of two conformers of CRP (pentameric, or native [nCRP], versus monomeric, or modified [mCRP]) on vessel function and production of reactive oxygen species (ROS) in an in-vivo model of atherosclerosis. Methods and Results Female ApoE −/− mice, fed a “western-type” diet, were treated with either human nCRP or mCRP (2.5 mg/kg s.c., weekly) or saline for 8 weeks. Endothelium-dependent and endothelium-independent vascular functions were assessed in isolated aortic rings under isometric conditions. Production of ROS in aortic rings was measured by electron spin resonance (ESR). Endothelium-dependent relaxation was impaired in nCRP-treated but not in mCRP-treated ApoE −/− mice. This impairment was reversed by preincubation with an inhibitor of inducible nitric oxide synthase (iNOS). Endothelium-independent relaxation, and iNOS and endothelial NOS (eNOS) protein expressions were similar among all groups. ESR experiments revealed lesser amounts of superoxide in the nCRP group as compared to the saline group, which is consistent with an increased transformation of NO to peroxynitrite. Conclusions nCRP can facilitate cardiovascular disease through impairment of endothelium-dependent vasoreactivity, in a manner that involves increased iNOS activity and a potential for increased peroxynitrite formation.

Thrombin Stimulates Human Endothelial Arginase Enzymatic Activity via RhoA/ROCK Pathway

Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate l-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis. In human endothelial cells isolated from umbilical veins, thrombin concentration- and time-dependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (P<0.001) at 1 U/mL for 24 hours. The effect of thrombin was prevented by C3 exoenzyme or the HMG-CoA reductase inhibitor fluvastatin, which inhibit RhoA, or by the ROCK inhibitors Y-27632 and HA-1077. Adenoviral expression of constitutively active RhoA or ROCK mutants enhanced arginase activity (approximately 3-fold, P<0.001), and the effect of active RhoA mutant was inhibited by the ROCK inhibitors. Neither thrombin nor the active RhoA/ROCK mutants affected arginase II protein level, the only isozyme detectable in the cells. Moreover, a significantly higher arginase II activity (1.5-fold, not the protein level) and RhoA protein level (4-fold) were observed in atherosclerotic aortas of apoE-/- compared with wild-type mice. Interestingly, l-arginine (1 mmol/L), despite a significantly higher eNOS expression in aortas of apoE-/- mice, evoked a more pronounced contraction, which was reverted to a greater vasodilation by the arginase inhibitor l-norvaline (20 mmol/L) compared with the wild-type animals (n=5, P<0.001). Thrombin enhances arginase activity via RhoA/ROCK in human endothelial cells. Higher arginase enzymatic activity is involved in atherosclerotic endothelial dysfunction in apoE-/- mice. Targeting vascular arginase may represent a novel therapeutic possibility for atherosclerosis.

Plaque-associated endothelial dysfunction in apolipoprotein E-deficient mice on a regular diet. Effect of human apolipoprotein AI.

Apolipoprotein E-deficient mice (apoE(-/-)) on a regular diet become hypercholesterolemic and develop atherosclerosis, but endothelium-dependent relaxation remains undisturbed for up to 6 months. We investigated whether vasomotor dysfunction develops in aged apoE(-/-), whether the defect was systemic (hypercholesterolemia-dependent) or focal (plaque-related), and the effect of human apolipoprotein AI transgenesis (apoAI/E(-/-)). Arteries of apoE(-/-) (n=5), apoAI/E(-/-) (n=6) and C57Bl/6J (WT, n=4) mice (18 months) were systematically dissected for isometric tension recording and subsequent morphometry. Acetylcholine (ACh)-induced relaxation was impaired (P<0.01) in atherosclerotic segments of apoE(-/-) (26+/-14%) as compared to WT mice (93+/-2%). Similar reduced (P<0.01) responses to adenosine 5'-triphosphate (apoE(-/-) 38+/-14, WT 94+/-3%) and the calcium ionophore A23187 (apoE(-/-) 19+/-6%, WT 97+/-2%) pointed to a post-receptor defect. Indeed, responses to exogenous nitric oxide were impaired in atherosclerotic segments as well (apoE(-/-) 71+/-7%, WT 92+/-1%, P<0.05). Furthermore, relaxations inversely correlated with plaque size (ACh r(s)=-0.74, P<0.01). In adjacent plaque-free segments however, responses to ACh (apoE(-/-) 92+/-3%, WT 97+/-1%) and all other agents were preserved, despite the prolonged hypercholesterolemia. ApoAI improved vasomotor responses in atherosclerotic segments. However, negative correlations between maximal relaxation and plaque area remained in apoAI/E(-/-) mice (ACh r(s)=-0.67, P<0.01). Indeed, covariate analysis of variance did not point to direct protection of vasomotor function by apoAI when the smaller lesions were taken into account. Endothelial dysfunction in apoE(-/-) mice is not affected by hypercholesterolemia alone, but is strictly associated with plaque formation. Human apoAI transgenesis-known to raise HDL-attenuated atherogenesis, thereby indirectly improving relaxation responses in apoE(-/-) mice.