Aorta Of Apolipoprotein E-deficient Mice Research Articles

The phosphatase activity of soluble epoxide hydrolase regulates ATP-binding cassette transporter-A1-dependent cholesterol efflux.

The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E‐deficient mice, primarily in macrophage foam cells. Oxidized low‐density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH −/−) in macrophages markedly exacerbated oxLDL‐induced lipid accumulation and decreased the expression of ATP‐binding cassette transporters‐A1 (ABCA1) and apolipoprotein AI‐dependent cholesterol efflux following oxLDL treatment. The down‐regulation of ABCA1 in sEH −/− macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH‐ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL‐induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL‐induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.

Systems Biology Research into Cardiovascular Disease: Contributions of Lipidomics-based Approaches to Biomarker Discovery.

Atherosclerosis is a progressive inflammatory thickening of the arterial wall resulting from increased cellularity and the accumulation of lipids, cellular debris, and extracellular matrix. Conventional determinations of plasma lipoproteins have resulted in a wealth of clinical data documenting the correlation between low- and high-density lipoproteins (LDL and HDL) and cardiovascular disease (CVD) risk. Current mass spectrometry methodologies allow the detection and quantification of multiple molecular lipid species with various structural and functional roles. The opportunities provided by lipidomics for lipid-based biomarker discovery are prominent in disease diagnostics, monitoring of drug efficacy, and translational model development. For example, the analysis of human plasma samples assessing the effects of statins has shown correlative effects between the LDL/HDL ratio and sphingomyelin and phosphatidylcholine. Additionally, at the vascular tissue level, lipids from different classes are enriched in human plaques of coronary arteries and in the aortas of apolipoprotein E-deficient mice exposed to cigarette smoke, highlighting a set of lipid biomarkers for translational research. Molecular lipidomics will provide insights in which other lipids may play important roles in vascular disease progression and will serve as novel markers for preventive as well as therapeutic purposes.

Antigen presenting cell phenotype is altered in the aorta of apolipoprotein-E deficient mice during atherosclerosis

Antigen presenting cell phenotype is altered in the aorta of apolipoprotein-E deficient mice during atherosclerosis

Activation of TRPV1 Prevents OxLDL-Induced Lipid Accumulation and TNF-α-Induced Inflammation in Macrophages: Role of Liver X Receptorα

The transient receptor potential vanilloid type 1 (TRPV1) is crucial in the pathogenesis of atherosclerosis; yet its role and underlying mechanism in the formation of macrophage foam cells remain unclear. Here, we show increased TRPV1 expression in the area of foamy macrophages in atherosclerotic aortas of apolipoprotein E-deficient mice. Exposure of mouse bone-marrow-derived macrophages to oxidized low-density lipoprotein (oxLDL) upregulated the expression of TRPV1. In addition, oxLDL activated TRPV1 and elicited calcium (Ca2+) influx, which were abrogated by the pharmacological TRPV1 antagonist capsazepine. Furthermore, oxLDL-induced lipid accumulation in macrophages was ameliorated by TRPV1 agonists but exacerbated by TRPV1 antagonist. Treatment with TRPV1 agonists did not affect the internalization of oxLDL but promoted cholesterol efflux by upregulating the efflux ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. Moreover, the upregulation of ABC transporters was mainly through liver X receptor α- (LXRα-) dependent regulation of transcription. Moreover, the TNF-α-induced inflammatory response was alleviated by TRPV1 agonists but aggravated by the TRPV1 antagonist and LXRα siRNA in macrophages. Our data suggest that LXRα plays a pivotal role in TRPV1-activation-conferred protection against oxLDL-induced lipid accumulation and TNF-α-induced inflammation in macrophages.

Recombinant Human Erythropoietin Suppresses Endothelial Cell Apoptosis and Reduces the Ratio of Bax to Bcl-2 Proteins in the Aortas of Apolipoprotein E–Deficient Mice

Recent clinical trials have raised concern that therapy with recombinant human erythropoietin (EPO) may increase cardiovascular disease risk, event rate, and mortality. Endothelial cell apoptosis has been implicated in both atherogenesis and in the destabilization and rupture of atheromatous plaques. In the current study, we observed that EPO and the EPO-mimetic peptide EMP-1 markedly suppressed lipopolysaccharide-induced apoptosis in endothelial cell monolayers. Therapeutic concentrations of EPO upregulated Bcl-2 expression and concurrently diminished expression of Bax, resulting in a net decrease in the ratio of Bax to Bcl-2 protein concentrations. In vivo studies demonstrated that EPO receptor is abundantly expressed in murine aorta and that EPO treatment for 10 weeks markedly decreased the ratio of Bax to Bcl-2 protein in the aortas of apolipoprotein E-deficient mice fed a high-fat diet. To our knowledge, these data are the first to reveal a modulation of regulators of the apoptotic pathway in murine aorta by chronic EPO treatment. These observations imply that long-term administration of EPO may have the potential to affect plaque stability.

Abstract 5195: Forced Angiogenesis in Adventitia Promotes Plaque Formation in Abdominal Aorta of Apolipoprotein E-deficient Mice

Background: Accumulating evidence suggests that exaggerated formation of vasa vasorum plays an important role in the pathogenesis of atherosclerotic plaque progression. It was reported that enhanced angiogenesis in adventitia promoted lesion progression after mechanical vascular injuries. However, it remains unclear whether augmented angiogenesis in adventitia could promote hyperlipidemia-induced atherosclerotic lesion formation. Methods: We investigated whether local administration of slow-releasing form (30 days) of basic fibroblast growth factor (bFGF) promotes plaque progression with enhanced perivascular angiogenesis. bFGF (100 μ g/body) incorporated in acid gelatin hydrogel microspheres (AGHMs) (bFGF+AGHM group, n=4), AGHMs (AGHM group, n=7), or PBS (control group, n=9) was administrated into periaortic areas of retroperitoneal space of 10 to 11-week-old male ApoE-deficient mice. The mice were fed on western type diet. The abdominal aorta was harvested with perivascular soft tissues at 13 weeks after the operation. Staining with Lycopersicon Esculentum (Tomato) lectin was performed to visualize micro-vessels at 5 different sections from each aorta. Results : Larger lesions were observed only in the bFGF+AGHMs group ( bFGF+AGHM: 3.5×10 4 ±1.2 × 10 4 μ m 2 , AGHM:0.8 × 10 4 ±0.7×10 4 μ m 2 , control 0 μ m 2 , p<0.005). Plaque formation was associated with increased neovascularization in the adventitia. When the aortic section were divided into 16 radial areas, there was a relation between the number of capillaries and the plaque size (r=0.77, p<0.001). In time course study, proliferation of vasa vasorum and accumulation of Mac3-positive macrophages were observed prior to atherosclerotic lesion formation. Conclusions: Local administration of bFGF in adventita induces development of vasa vasorum, which potentially accelerates plaque lesion progression in ApoE-deficient mice.

High resolution magnetic resonance imaging in atherosclerotic mice treated with ezetimibe

High field magnetic resonance imaging (MRI) was performed to investigate the long-term effect of ezetimibe (eze), a cholesterol resorption blocker, on atherosclerotic lesion formation in the thoracic aorta of apolipoprotein E-deficient mice (apoE ( -/- )) in comparison to wild type mice (WT). Fifteen-month-old apoE ( -/- ) (Western type diet), apoE ( -/-eze ) (Western type diet with eze) which received eze (5 mc/kg/day) continuously, and age-matched WT (normal chow) were studied using contrast-enhanced 3D turbo-spin-echo sequences (RARE factor 2) on a 7 Tesla scanner. Vessel parameters were analyzed in the aortic root (AR) and aortic arch (AA) and compared to those found in histology. Plasma cholesterol levels were reduced at 15 months by 71% (P < 0.01) in apoE ( -/-eze ) compared to apoE ( -/- ). Vessel wall thickness was increased in the AR and AA in apoE ( -/- ) by 189.1 and 147.2%, respectively compared to WT. ApoE ( -/-eze ) showed reduced wall thickness in the AR (127.4%) and AA (102.8%, both P < 0.05 vs. apoE ( -/- )). A significant increase in total aortic vessel area was determined in the AR and AA in apoE ( -/- ) by 134.7 and 118.3%, respectively, compared to WT. This effect was inhibited in apoE ( -/-eze ) (AR: 126.7%, AA: 86.4%, both P < 0.05). Histological analysis confirmed the effect of eze observed by MRI and demonstrated a significant correlation between the two techniques (P < 0.001). MRI demonstrates that ezetimibe significantly reduces atherosclerotic disease in apoE ( -/- ). MRI is therefore a useful technique to perform in vivo interventional studies in experimental atherosclerosis.

Altered Ca 2+ handling of smooth muscle cells in aorta of apolipoprotein E-deficient mice before development of atherosclerotic lesions

To study the effect of hypercholesterolemia on vascular smooth muscle cell (VSMC) function, atherosclerosis-prone but plaque-free endothelium-denuded aortic rings (width 2 mm) from C57Bl6 Wild Type (WT) and apolipoprotein E-deficient (apoE −/−) mice (age 4 months) were mounted in a myograph and loaded with Fura-2 AM to simultaneously measure free Ca 2+ ([Ca 2+] i) and force development. In comparison with WT, apoE −/− mice displayed higher basal [Ca 2+] i. Moreover, the time constant of the second phase of the biphasic high K +-induced [Ca 2+] i response was significantly increased in apoE −/− compared to WT mice. This phase was abolished by treatment with cyclopiazonic acid (CPA), depleting sarcoplasmic reticulum (SR). Further investigation of SR dependent [Ca 2+] i handling with CPA and caffeine revealed no alteration of maximal SERCA or ryanodine receptor function. Inositol (1,4,5)-triphosphate receptor (IP 3R)-mediated [Ca 2+] i release was, however, significantly increased in apoE −/− mice compared to WT mice as established with phenylephrine and ATP. In Ca 2+-free conditions the ATP-induced [Ca 2+] i was not altered. The ATP-induced store-operated Ca 2+ entry was, however, significantly increased in apoE −/− compared to WT mice. The results demonstrate that basal [Ca 2+] i levels and IP 3R-mediated store-operated [Ca 2+] i release over the plasma membrane were elevated in hypercholesterolemic but plaque-free apoE −/− mice.

Effect of combination of calcium antagonist, azelnidipine, and AT1 receptor blocker, olmesartan, on atherosclerosis in apolipoprotein E-deficient mice

Angiotensin II type 1 receptor blockers (ARB) are widely recognized to have a vasculoprotective effect. Accumulating data have revealed that calcium antagonists also retard atherosclerosis. We examined the possibility that combination therapy of ARB and calcium antagonists could more effectively prevent atherosclerosis than monotherapy. We observed a marked increase in the atherosclerotic area, associated with the exaggerated expression of nicotinamide adenine dinucleotide (phosphate), reduced form [NAD(P)H] oxidase subunits (p22 and p47) and superoxide anion production, in the aorta of apolipoprotein E-deficient mice maintained on a 1.25% high-cholesterol diet for 10 weeks. A calcium antagonist, azelnidipine, at a dose of 1 mg/kg a day or an ARB, olmesartan, at a dose of 3 mg/kg a day, significantly inhibited these parameters, with no change in systolic blood pressure and the blood cholesterol level. Moreover, the co-administration of lower doses of azelnidipine (0.1 mg/kg a day) and olmesartan (1 mg/kg a day) significantly inhibited the atherosclerotic area and oxidative stress, whereas azelnidipine or olmesartan alone at these doses did not affect these parameters. Furthermore, we observed similar inhibitory effects of azelnidipine or olmesartan on angiotensin II-induced NADPH oxidase activity and Akt activity in cultured vascular smooth muscle cells. These results suggest that the co-administration of calcium antagonists and ARB synergistically blunts oxidative stress at least partly through the inhibition of Akt activity and enhances the beneficial effects of these drugs on atherosclerosis compared with monotherapy.

Reduced β 3-endonexin levels are associated with enhanced urokinase-type plasminogen activator receptor expression in ApoE −/− mice

Proteolysis of extracellular matrix components is required for cell migration occurring in atherosclerotic lesion formation. In the present study, gene expression of the urokinase plasmingen activator receptor (uPAR) and underlying mechanisms were analyzed during the development of atherosclerosis in the aorta of apolipoprotein E-deficient mice (apoE −/−). A significant increase of uPAR expression was detected in the atherosclerotic tissue with advancing plaque-dimension. As uPAR gene transcription involves the transcription factor nuclear factor-κB (NF-κB), we analyzed nuclear NF-κB activity in vascular tissue of apoE-deficient mice. Congruent to uPAR, we could detect an increase in NF-κB activity, which underlines the chronic inflammatory component of the disease. Recently we reported that β 3-endonexin, a protein that modulates β 3-integrins, regulates uPAR expression through direct interaction with subunits of the NF-κB-complex. Herein we could show that β 3-endonexin protein is expressed in aortic tissue of mice. Moreover, in contrast to uPAR or NF-κB, the expression of β 3-endonexin was reduced in extracts of advanced atherosclerotic aortic tissue. The cytoplasmic protein β 3-endonexin regulates function of β 3-integrins. We revealed that integrin stimulation of endothelial cells led to an enhanced NF-κB activity and secretion of the NF-κB dependent chemokine monocyte chemoattractant protein-1 (MCP-1). The β 3-integrin dependent increase in MCP-1 was notedly reduced in cells that overexpressed β 3-endonexin. These results provide strong evidence that β 3-endonexin acts as a regulating factor in the integrin-mediated signal transduction and the present findings imply a pathophysiological role of β 3-endonexin in atherogenesis.

Expression of CYR61, an angiogenic immediate early gene, in arteriosclerosis and its regulation by angiotensin II.

The renin-angiotensin system is thought to be involved in development and progression of arteriosclerosis, thereby contributing to adverse cardiovascular events. To elucidate the role of angiotensin II (Ang II) at a cellular level, we analyzed the Ang II-induced gene expression profile. Genes induced on Ang II stimulation (10(-7) mol/L, 45 minutes) in rat smooth muscle cells were analyzed by polymerase chain reaction selected subtraction. In addition to known genes, such as interleukin 6, leukemia inhibitory factor, and c-fos, we identified CYR61, an angiogenic immediate early gene. Northern blot analysis revealed a rapid 2.5-fold increase of CYR61 transcript levels by Ang II, peaking at 30 minutes, which was blunted by Ang II type 1 receptor blockade. Exposure of rat aortic rings to Ang II (30 minutes) revealed a 2-fold, and intraperitoneal injection of Ang II (30 minutes) in mice a 3-fold, increase of aortic CYR61 transcripts. In arteriosclerotic aortas of apolipoprotein E-deficient mice, CYR61 transcripts confirmed by in situ hybridization and proteins shown by immunohistochemistry were elevated, whereas they were hardly detectable in wild types. In human carotid atherectomies and arteriosclerotic coronary arteries, immunohistochemical analysis revealed expression of CYR61 within connective tissue in neointima, adventitia, and surrounding small capillaries and blood vessels, colocalized with ACE and Ang II. Normal human arteries showed no significant staining for CYR61. CYR61, an angiogenic factor, is induced by Ang II in vascular cells and tissue. The expression of CYR61, colocalized with Ang II and ACE, in small vessels of human arteriosclerotic lesions is consistent with the notion that the activated renin-angiotensin system may contribute to plaque neovascularization by enhancing regulators of microvessel formation and cell proliferation.

Dietary restriction reduces atherosclerosis and oxidative stress in the aorta of apolipoprotein E-deficient mice

Dietary restriction (DR) has been shown to inhibit almost all the age-related diseases, e.g. cardiomyopathy and cancers, in rodents. However, there is little information for the effect of DR on atherosclerosis. In the present study, we examined the effect of DR on the development of atherosclerosis in mice homozygous knockout for apolipoprotein E gene ( ApoE −/−). The ApoE −/− mice were fed either ad libitum (AL) or 60% of the diet consumed by the mice fed AL. Atherosclerotic lesions in the proximal aorta of these mice were measured. Our results showed that ApoE −/− mice fed the calorie-restricted diet had smaller and relatively early stages of atherosclerotic lesions (e.g. foam cells and free lipids) when compared to ApoE −/− mice fed AL, who developed more advanced lesions (e.g. fibrous caps and acellular areas). In addition, ApoE −/− mice fed the calorie-restricted diet showed a significant decrease in the level of lipid hydroperoxides and the production of superoxide and hydrogen peroxide in the aorta as compared to ApoE −/− mice fed AL. These observations suggest that reduction of oxidative stress in the arterial wall may contribute to the anti-atherogenic effect of DR in ApoE −/− mice.

Selective induction of apoptosis in the atherosclerotic aortas of apolipoprotein-E deficient mice by short-term thermal therapy Tarun Tewatia, Conjun Wu, XuegingXu, Ki-soo Kil, James T. Willerson, S. Ward Casscells, Yong-Jian Geng. The University of Texas Health Sciences Center at Houston, Houston, TX, The Texas Heart Institute, Houston, TX

Selective induction of apoptosis in the atherosclerotic aortas of apolipoprotein-E deficient mice by short-term thermal therapy Tarun Tewatia, Conjun Wu, XuegingXu, Ki-soo Kil, James T. Willerson, S. Ward Casscells, Yong-Jian Geng. The University of Texas Health Sciences Center at Houston, Houston, TX, The Texas Heart Institute, Houston, TX

Corresponding distributions of increased endothelin-B receptor expression and increased endothelin-1 expression in the aorta of apolipoprotein E-deficient mice with advanced atherosclerosis.

Endothelin (ET)-1 causes proliferation of vascular smooth muscle cells (VSMC). Although it has been reported that stimulation of ET(B) receptors as well as ET(A) receptors promote proliferation of VSMC, the precise distribution of each receptor subtype in atherosclerotic vessels is unknown. Previous studies demonstrated that apolipoprotein E (apoE)-deficient mice have hypercholesterolaemia and develop severe atherosclerosis. To investigate the pathophysiological roles of vascular ET system in atherosclerosis, we examined preproET-1 messenger ribonucleic acid expression in the aorta of apoE-deficient mice, and performed immunohistochemical staining for ET-1 and each ET receptor subtype (ET(A) and ET(B) receptors) in the atherosclerotic lesions of these mice. The level of preproET-1 mRNA in the aorta was significantly higher in the apoE-deficient mice than in the control mice. Strong ET-1 staining was observed in the macrophage-foam cells, intimal and medial VSMC in the atherosclerotic lesions of the apoE-deficient mice. In addition, in the atherosclerotic lesions, strong ET(B) receptor staining was observed in the macrophage-foam cells, intimal and medial VSMC, which distribution corresponded closely to that of ET-1. ET(A) receptor staining was observed in the medial VSMC of both groups, but not in the macrophage-foam cells of the apoE-deficient mice. ET(A) receptor staining in the medial VSMC was stronger in the apoE-deficient mice than in the control mice. These results suggest that the vascular ET system, including ET-1 and ET receptors, is activated in the atherosclerotic lesions of apoE-deficient mice. Since the distribution of strong ET(B) receptor staining corresponded closely to that of ET-1, it is suggested that the ET system, mediated by ET(B) receptors, has an important role in the pathophysiology of the atherosclerotic lesions of apoE-deficient mice.

Decreased sensitivity to nitric oxide in the aorta of severely hypercholesterolemic apolipoprotein E-deficient mice.

A normal response to nitric oxide donors has been cited as evidence that impaired endothelium-dependent vasodilation during hypercholesterolemia is due to decreased synthesis of nitric oxide. This tenet was examined by determining responses to nitric oxide gas as well as to acetylcholine and sodium nitroprusside in the isolated aorta of apolipoprotein E-deficient mice fed normal or Western-type cholesterol-rich diet until 21 or 35 weeks of age. In mice fed normal chow, relaxation to all agents remained comparable to that obtained in wild-type mice. In mice fed Western diet, the relaxation to acetylcholine as well as to nitric oxide was decreased at 35 weeks of age. At 21 weeks of age, decreased sensitivity to nitric oxide was observed despite a normal response to acetylcholine. The response to sodium nitroprusside was normal in all groups. A decrease in aortic superoxide dismutase activity as well as an increase in aortic superoxide anion generated in the presence of NADH as measured by lucigenin chemiluminescence was observed in the group fed Western diet at 35 weeks. This provides evidence that altered superoxide anion could contribute to the deterioration in nitric oxide sensitivity that underlies the impaired endothelium-dependent relaxation. These data indicate that decreased sensitivity to nitric oxide may contribute to the development of impaired endothelium-dependent relaxation in hypercholesterolemia. The response to sodium nitroprusside appears not to reflect the decreased sensitivity of vascular smooth muscle to authentic nitric oxide.

Altered expression of multiple genes in the aorta of apolipoprotein-E deficient mice with atherosclerosis: Determination by hybridization of arrayed cDNA clones

Altered expression of multiple genes in the aorta of apolipoprotein-E deficient mice with atherosclerosis: Determination by hybridization of arrayed cDNA clones