Aorta Of ApoE Research Articles

Correlation of Vasa Vasorum Neovascularization, Inflammation and Plaque Progression in Aortas of ApoE-/-/LDL-/- Double Knockout Mice by Micro-CT

Ziele: Because of the very thin arterial wall, vasa vasorum (VV) are generally not present in mice aortas. However, as atherosclerosis in large animals is associated with increase in vasa vasorum density, we investigated whether vasa vasorum neovascularization occurs in the aorta of apoE-/-/LDL-/- deficient mice using 3-D Micro-CT and histology. Methode: Aortas from apoE-/-/LDL-/- mice at the age of 16, 18, 20 or 80 weeks were infused in situ with Microfil, harvested and scanned with micro-CT (5 and 20µm cubic voxel). Using the 3D micro-CT images, we characterized plaque volume, CT “density“ and VV luminal volume along the aorta using Analyze 6.0 software. Immunohistochemistry demonstrated VV and inflammatory cells. VV within the adventitia were confirmed by Masson's trichrome staining. Ergebnis: From 16 to 80 weeks, plaque volume and CT density (index of subresolution VV opacified lumen volume and/or accumulation of calcium or other radiopaque material) increased with age throughout the entire aorta (p<0.001).

Atheroma development in apolipoprotein E-null mice is not affected by partial inactivation of PTEN

PTEN is a dual-specificity phosphatase that has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and migration, two key events in the ethiopathogenesis of atherosclerosis. Adenovirus-mediated PTEN overexpression inhibited the formation of vascular obstructive lesions induced by mechanical injury of the vessel wall. In this study, we investigated whether PTEN protects against atheroma formation in apolipoprotein E-null mice (apoE-/-), a widely used animal model characterized by the development of hypercholesterolemia and atherosclerosis. We examined atheroma development in the aorta of apoE-/- mice with an intact Pten gene and apoE-/- mice lacking one allele of Pten (Pten(+/-)apoE-/-) that were challenged for six weeks with an atherogenic diet. Compared with apoE-/- controls, Western blot analysis of arterial cell lysates from Pten(+/-)apoE-/- mice revealed a decrease in PTEN expression. This correlated with increased phosphorylation of AKT, thus demonstrating that Pten inactivation in Pten(+/-)apoE-/- mice has functional consequences. However, the extent of atherosclerosis was undistinguishable in both groups of fat-fed mice. Likewise, the atheroma of Pten(+/-)apoE-/- and apoE-/- mice displayed similar VSMC content, cellularity and rates of proliferation and apoptosis. Thus, in spite of the cytostatic and antimigratory activities of PTEN, and in contrast to previous studies demonstrating that Pten is haplo-insufficient for tumor suppression, our results demonstrate that atherosclerosis in hypercholesterolemic mice is not aggravated by partial inactivation of Pten.

Alteration of vascular urotensin II receptor in mice with apolipoprotein E gene knockout

Urotensin II (U-II), originally identified as a fish neuropeptide, exerts a broad spectrum of biological functions and is considered to be the most potent vasoconstrictor in mammals. Recently the intense staining of U-II-like immunoreactivity within coronary atheroma and the effect of mitogenic in vascular smooth muscle cells (VSMCs) suggest that U-II is possibly a proatherogenic factor in the pathogenesis of cardiovascular diseases (CVD). In the present study, we analyzed the gene expression of U-II and GPR14, a high-affinity receptor for U-II, in aorta of apolipoprotein E (apoE) −/− mutant mice by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Compared with wild-type mice at the same age group, GPR14 mRNA level is significant increase in aorta of apoE −/− mice at age of 18, 28 and 38 weeks, respectively. The increased GPR14 mRNA level was 54.2, 50.0 and 97.0% in the three age groups, respectively. We did not detect U-II mRNA expression in aorta either in apoE −/− mice or in wild-type mice. In 28-week mice, ligand-binding assay showed that maximum binding capacity ( B max) of 125I-U-II aorta from apoE −/− mice was increased by 64%, while the affinity of the binding did not change compared with that of wild-type mice. These results indicate that the up-regulation of vascular U-II receptor (UT), GPR14 might be involved in the pathogenesis of atherosclerosis.

Inhibition of cyclooxygenase with indomethacin phenethylamide reduces atherosclerosis in apoE-null mice

Non-selective inhibition of cyclooxygenase (COX) has been reported to reduce atherosclerosis in both rabbit and murine models. In contrast, selective inhibition of COX-2 has been shown to suppress early atherosclerosis in LDL-receptor null mice but not more advanced lesions in apoE deficient (apoE −/−) mice. We investigated the efficacy of the novel COX inhibitor indomethacin phenethylamide (INDO-PA) on the development of different stages of atherosclerotic lesion formation in female apoE −/− mice. INDO-PA, which is highly selective for COX-2 in vitro, reduced platelet thromboxane production by 61% in vivo, indicating partial inhibition of COX-1 in vivo. Treatment of female apoE −/− mice with 5 mg/kg INDO-PA significantly reduced early to intermediate aortic atherosclerotic lesion formation (44 and 53%, respectively) in both the aortic sinus and aorta en face compared to controls. Interestingly, there was no difference in the extent of atherosclerosis in the proximal aorta in apoE −/− mice treated from 11 to 21 weeks of age with INDO-PA, yet there was a striking (76%) reduction in lesion size by en face analysis in these mice. These studies demonstrate the ability of non-selective COX inhibition with INDO-PA to reduce early to intermediate atherosclerotic lesion formation in apoE −/− mice, supporting a role for anti-inflammatory approaches in the prevention of atherosclerosis.

Interleukin-1 plays a major role in vascular inflammation and atherosclerosis in male apolipoprotein E-knockout mice

To examine the role of the balance between interleukin (IL)-1 and IL-1 receptor antagonist (IL-1Ra) in atherosclerosis and vascular inflammation. Transgenic (Tg) mice overexpressing either secreted IL-1Ra or intracellular IL-1Ra1 as well as IL-1Ra-deficient mice (IL-1Ra -/-) were crossed with apolipoprotein E-deficient mice (ApoE -/-). In males fed a cholesterol-rich diet for 10 weeks, average atherosclerotic lesion area within aortic roots was significantly decreased in ApoE -/- secreted IL-1Ra Tg (-47%) and ApoE -/- intracellular IL-1Ra1 Tg (-40%) mice as compared to ApoE -/- non-Tg controls. The extent of sudanophilic lesions was reduced within the thoraco-abdominal aorta in ApoE -/- secreted IL-1Ra (-53%) and ApoE -/- intracellular IL-1Ra1 (-67%) Tg mice. In parallel experiments, we observed early mortality and illness among double deficient mice, whereas ApoE -/- IL-1Ra +/+ and ApoE +/+ IL-1Ra -/- mice were apparently healthy. After 7 weeks of diet, ApoE -/- IL-1Ra -/- mice exhibited massive aortic inflammation with destruction of the vascular architecture, but no signs of atherosclerosis. ApoE -/- IL-1Ra +/+ had atherosclerosis and a moderate inflammatory reaction, whereas ApoE +/+ IL-1Ra -/- mice were free of vascular lesions. Macrophages were present in large amounts within inflammatory lesions in the adventitia of ApoE -/- IL-1Ra -/- mice. Our results demonstrate that the IL-1/IL-1Ra ratio plays a critical role in the pathogenic mechanisms leading to vascular inflammation and atherosclerosis in ApoE -/- mice.

Urokinase plasminogen activator receptor promotes macrophage infiltration into the vascular wall of ApoE deficient mice

The urokinase plasminogen activator receptor (uPAR) regulates macrophage adhesion and migration by binding directly to matrix proteins and signaling through integrin complexes. In this study, we examined the role of uPAR on macrophage infiltration into the vascular wall. Stable murine macrophage (Raw264.7) cell lines expressing high levels of human uPAR, human urokinase plasminogen activator (uPA), or both were established using expression vectors driven by the human CD68 promoter. Stimulation with human uPA specifically induced phosphorylation of early response regulated kinase (ERK) in cells expressing human uPAR but not in sham transfected cells. The human uPAR expressing Raw264.7 cells showed increased adhesion to both human uPA and vitronectin (Vn). Raw264.7 cells expressing human uPAR or both human uPAR and uPA, but not uPA alone, were detected in the aortic wall of ApoE(-/-) mice, and no cells were detected in that of age-matched C57BL/6J mice after intravenous infusion of the cells. Blocking of Mac-1/ICAM-1 interaction by anti-alphaM antibody (M1/70) significantly reduced the infiltration of huPAR-expressing Raw264.1 cells into aorta of ApoE(-/-) mice. Treatment of C57BL/6J mice with angiotensin II resulted in infiltration of Raw264.7 cells expressing human uPAR. These data demonstrate that uPAR plays a key role in promoting macrophage infiltration into the arterial wall of ApoE(-/-) mice.

Effects of amlodipine, atorvastatin and combination of both on advanced atherosclerotic plaque in APOE*3-Leiden transgenic mice

Combined treatment of statins and calcium channel blockers has been suggested to be superior to statin therapy alone. We quantified the anti-atherosclerotic potential of amlodipine, atorvastatin and their combination on existing atherosclerotic plaques in the aorta of APOE*3-Leiden transgenic mice. Sixty-two mice were fed a high cholesterol containing diet for 18 weeks. A subgroup of 10 mice was then killed. All other mice received the diet for another 18 weeks, alone (late control group), along with 0.01% atorvastatin, 0.002% w/w amlodipine, or their combination (all groups, n = 13). Atherosclerotic lesions, collagen content and monocyte adherence were quantified using standard histology (aortic root). Raman spectroscopy was used to quantify the content of cholesterol and calcification (aortic arch). Compared to the late control group, treatment with amlodipine, atorvastatin or the combination, reduced atherosclerostic lesion area by, respectively, 25%, 39% and 46% in the aortic root ( P < 0.01) and by 53%, 55% and 60% in the aortic arch ( P < 0.05). Atorvastatin, but not amlodipine reduced the adherence of monocytes in the intima. Lesion severity and plaque contents of collagen, cholesterol and calcification were equal for all treatment groups. Neither treatment resulted in regression of atherosclerotic plaque size. In conclusion, both atorvastatin and amlodipine significantly retard the progression of existing atherosclerotic lesions. No additive effect of the combination of amlodipine and atorvastatin could be observed in this study.

Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice

The apolipoprotein E gene knockout (apoE−/−) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE−/− mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE−/− and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcβ1-4Galβ1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE−/− mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcβ1-4[NeuNAcα2-3]Galβ1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE−/− mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE−/− mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcα2-3Galβ1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE−/− mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE−/− mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.—Garner, B., D. A. Priestman, R. Stocker, D. J. Harvey, T. D. Butters, and F. M. Platt. Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice.

Gelatin intake increases the atheroma formation in apoE knock out mice

The effect of gelatin ingestion on cholesterol metabolism and on atheroma formation was evaluated in both wild type ( n=14) and apoprotein E (apoE) knock out (apoE −/−) ( n=20) C57BL/6 7-week-old mice. Animals were fed a cholesterol-free isoproteic semi-purified diet containing 20% of casein (control diet) or 10% of casein plus 10% of gelatin (gel diet) for 8 weeks. In wild type mice, dietary gelatin caused a reduction in the serum triacylglycerols levels associated with an increase in the fecal excretion. No difference in blood cholesterol was seen at the sixth week of experiment. At the eighth week of experiment, there was a modest but significant reduction of serum total and high density lipoprotein (HDL) cholesterol in apoE −/− mice fed on gel diet compared to the control. Total cholesterol/HDL cholesterol ratio was 2-fold higher in the gel group than that seen in the control group (14.39 and 7.84, respectively). Histological analyzes showed a 2.2-fold increase in the dimension of the atherosclerotic plaques in the proximal aorta in apoE −/− mice fed on a gel diet compared to those fed on a control diet. The gel diet also promoted a reduction in the fecal excretion of bile acids. Hepatic cholesterol was similar in both groups. In conclusion, although gelatin reduced total serum cholesterol, this reduction was associated to a decrease of HDL cholesterol and consequent increase of total cholesterol/HDL cholesterol ratio, resulting in an acceleration of atherogenesis.