Use Of Animal Models To Study Lipid Genes That Modulate Atherosclerosis

National Heart, Lung, And Blood Institute

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Abstract

To explore the effect of expressing lipid modifying genes in endothelial cells, three different lines of mice were made, using the Tie2 endothelial specific promoter as described below: (1) ABCA1: ABCA1 transporter is key protein in reverse cholesterol transport and is also responsible for biogenesis of HDL. To evaluate the contribution of endothelial-expressed ABCA1 in HDL generation and in processes of diet induced atherosclerosis, we expressed hABCA1 in endothelial cells with the endothelial specific Tie2 promoter. In isolated lung endothelial cells Tie2hABCA1 was expressed at the level 10+/-3% of expression of mouse ABCA1. No hABCA1 was found to be expressed in peritoneal macrophages. Expression of hABCA1 did not alter the pattern of expression of mouse ABCA1, SR-B1 and ABCG1 in aorta, heart, kidney, liver, lung, muscle, spleen and testis. Despite the modest level of expression of hABCA1 in endothelium, it had a significant impact on plasma levels of total cholesterol (TC) and HDL-C. In females, TC was increased in comparison with normal siblings from 853 to 1013; HDL-C from 572 to 682 (all values in mg/dl, p<0.001); in males TC was changed from 1004 to 1275 (p<0.001), HDL-C from 73.52.6 to 843.5 (p<0.03). This effect of hABCA1 was found only on normal C57Bl background. When Tie2hABCA1 was transferred to ABC1-K/O or apoE-K/O backgrounds no effect of hABCA1 on plasma lipids was revealed. Experiments on cultivated in Transwells polarized lung endothelial cells isolated from Tie2hABCA1 mice on ABCA1-K/O background revealed that Tie2hABCA1 was able to promote cholesterol efflux to apoA1 directed mostly to apical side of the cells. Expression of Tie2hABCA1 in C57Bl mice protected them against diet-induced atherosclerosis. After 6 months on Cocoa Butter Diet (TD90221) control females had 6.50.8% of en face aorta lesions, whereas transgenic animals had 40.5% of the lesions (p<0.02). HDL-C after 6 months on diet was higher in transgenic vs control females (563 vs 40.53.5, p<0.003). There was no protection if hABCA1 was on ABCA1-K/O or apoE K/O backgrounds. In summary, the results support a role of endothelial ABCA1 expression in the generation of HDL and in the protection of diet induced atherosclerosis. (2)Cholesterol homeostasis in endothelium plays significant role in protecting against development of atherosclerosis and in supporting normal function of the vasculature. (2) Arginase I and II: Endothelium is a major regulator of local vascular homeostasis. Its normal function is crucial for prevention of the development of atherosclerosis, hypertension and other cardiovascular disorders. Reduced nitric oxide (NO) bioavailability is one of the earliest and most important markers of endothelial dysfunction. L-arginine is the substrate for nitric oxide synthase (NOS). Arginase (Arg) can compete with eNOS for L-arginine and thus may play a role in endothelial dysfunction. To investigate the role of ArgII in endothelial function and in atherosclerosis we generated transgenic mice with human ArgII (hArgII) gene under control of endothelial-specific Tie2 promoter. Expression of hArgII was measured by RT-PCR in eight tissues of transgenic males and compared with the level of mouse ArgII (mArgII) expression in the kidneys of normal C57Bl mice, which was taken as 100%. hArgII was expressed at very high levels in all tissues, especially in aorta (A, 2700%), heart (H, 3500%), kidney (K, 1600%), lung (9860%) and muscle (M, 2000%). Arginase activity was elevated 4.6-62 fold in all tissues except liver. Lung endothelial cells isolated from hArgII transgenic mice had 4.4-fold greater arginase activity than whole lung. Resident peritoneal macrophages from hArgII transgenic and normal mice had similar levels of arginase activity, confirming endothelial specificity of the Tie2 promoter. Overexpression of hArgII neither led to significant changes in plasma levels of arginine, citrulline, NOHA, ADMA, SDMA and ornithine, nor to changes in plasma lipid levels. However, ArgII overexpression on apoE-knockout background was accompanied by a 10% increase in plasma total cholesterol (p<0.05). hArgII transgenic mice also had blood pressures (mean arterial and diastolic) that averaged 17% higher than controls. Aortic ring segments from hArgII transgenic mice, precontracted with phenylephrine (10-6M), exhibited decreased endothelium-dependent relaxation to increasing concentrations of acetylcholine (10-9 to 10-3.5M), indicating endothelial dysfunction secondary to NO insufficiency. These results reveal the Tie2hArgII transgenic mouse to be a relevant a new model for investigating the role of ArgII in atherosclerosis and other vasculopathies associated with endothelial dysfunction.