Prevention of high fat diet-induced vascular injury

描述(申请人提供)：来自流行病学研究和临床试验的压倒性数据表明，食用鱼油(omega-3多不饱和脂肪酸)可以减少心血管死亡(CVD)，并延缓冠心病患者的动脉粥样硬化进程。然而，omega-3多不饱和脂肪酸发挥保护作用的细胞和分子机制仍然知之甚少。来自申请人实验室的令人兴奋的数据显示，给予omega-3多不饱和脂肪酸显著增加了Thr172处的AMPK的磷酸化和AMPK的活性。AMPK的激活抑制了26S蛋白酶体、核因子kappaB的激活和NAD(P)H氧化酶的表达。在ApoE基因敲除株(Apo E-/-)或低密度脂蛋白受体基因敲除株(LDLR-/-)中，AMPK12的基因缺失显著增加26S蛋白酶体活性、I？B降解、核因子：B反式激活、NAD(P)H氧化酶亚单位过表达、氧化应激、内皮功能障碍和动脉粥样硬化，所有这些都可被长期服用一种有效的细胞通透性蛋白酶体抑制剂MG132抑制。目前应用的中心假设是，omega-3多不饱和脂肪酸选择性激活AMPK可以抑制26S蛋白酶体和NF-B介导的NAD(P)H氧化酶的过度表达，从而减少氧化应激，氧化应激是高脂饮食(HFD)导致血管损伤的关键因素。包括药理学和遗传学手段(siRNA和腺病毒)在内的综合实验手段将被用来(1)确定AMPK激活在omega-3多不饱和脂肪酸抑制内皮细胞NAD(P)H氧化酶异常表达中的重要作用：(2)阐明AMPK和26S蛋白酶体在omega-3多不饱和脂肪酸抑制内皮细胞NAD(P)H氧化酶过度表达中的中心作用；(3)剖析AMPK抑制26S蛋白酶体活性的分子机制；(4)评价AMPK对内皮特异性AMPK缺失小鼠(TG-CRE-AMPK 11或12(FLOX/FLOX，功能丧失)或内皮特异性高表达固有活性AMPK的小鼠(TG-CAAMPK，功能增益))内皮功能和动脉粥样硬化的影响。这种体外和体内技术以及功能获得/丧失方法的强大组合将对omega-3多不饱和脂肪酸如何预防心血管疾病产生重要的见解。重要的是，拟议研究的完成还将为26S蛋白酶体和AMPK是否为对抗与衰老、肥胖、糖尿病和高血压等常见疾病相关的动脉粥样硬化提供新的见解。

DESCRIPTION (provided by applicant): Overwhelming data from epidemiological studies and clinical trials reveal that consumption of fish oils (omega-3 polyunsaturated fatty acids) reduces cardiovascular deaths (CVD) and retards the progression of atherosclerosis in patients with coronary heart diseases. However, the cellular and molecular mechanisms by which omega-3 polyunsaturated fatty acids exert their protective effects remain poorly understood. Exciting data from the applicant's laboratory has revealed that administration of omega-3 polyunsaturated fatty acids significantly increased the phosphorylation of AMPK at Thr172 and AMPK activity. Activation of AMPK suppresses 26S proteasomes, the activation of nuclear factor kappa B, and the expression of NAD(P)H oxidase. Consistently, genetic deletion of AMPK12 in either ApoE knockout (Apo E-/-) or LDL receptor knockout (LDLr-/-) strain markedly increased 26S proteasome activity, I?B degradation, NF:B transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, endothelial dysfunction, and atherosclerosis, all of which were largely suppressed by chronic administration of MG132, a potent and cell permeable proteasome inhibitor. The central hypothesis of the current application is that selective activation of AMPK by omega-3 polyunsaturated fatty acids inhibits 26S proteasomes and NF-?B-mediated overexpression of NAD(P)H oxidase resulting in decreased oxidative stress, a key factor in vascular injury caused by high fat diets (HFD). Comprehensive experimental approaches including pharmacological and genetic means (siRNA and adenoviruses) will be used (1) to establish the essential roles of AMPK activation in omega-3 polyunsaturated fatty acids-induced suppression of NF:B-mediated aberrant expression of NAD(P)H oxidase in endothelial cells; (2) to elucidate the central roles of AMPK and 26S proteasome in omega-3 polyunsaturated fatty acids-induced inhibition on NF:B- mediated overexpression of NAD(P)H oxidase in endothelial cells; (3) to dissect the molecular mechanisms by which AMPK suppresses 26S proteasome activity, and (4) to assess the effects of AMPK on endothelial function and atherosclerosis in mice with the endothelium-specific depletion of AMPK (Tg-Cre-AMPK 11 or 12 (flox/flox), loss-of function) or in mice with the endothelium-specific overexpression of a constitutively active AMPK (Tg-CAAMPK, gain-of-function) in vivo. This powerful combination of in vitro and in vivo techniques and gain-/loss-of-function approaches will yield important insights into how omega-3 polyunsaturated fatty acids protect against cardiovascular diseases. Importantly, completion of the proposed studies will also provide novel insights into whether 26S proteasomes and AMPK are potential therapeutic targets for countering atherosclerosis associated with common diseases including aging, obesity, diabetes, and hypertension.