Methods to enable cholesterol catabolism in human monocyte derived macrophages

描述（由申请人提供）：动脉粥样硬化性心血管疾病（CVD）是美国的主要死亡原因。CVD源于正常脂质代谢的异常（一些遗传，一些生活方式选择），导致血浆脂蛋白（主要是LDL）升高和/或高密度脂蛋白（HDL）水平降低。对于许多人来说，CVD是一种年龄依赖性的进行性疾病，在疾病后期发生事件（即心肌梗死或中风）之前，很大程度上未被发现或被忽视。因此，目前的治疗集中于通过降低LDL的循环水平和/或增加HDL来预防第二事件（或高风险个体中的主要事件）。然而，在生化水平上，巨噬细胞不能降解胆固醇的胆甾烷环是CVD的基本组成部分。如果巨噬细胞具有降解胆固醇的能力，它们就不会充满胆固醇/胆固醇酯并引发导致动脉粥样硬化发作和进展的适应不良免疫应答。最近，分枝杆菌在人类巨噬细胞中存活的研究揭示了一个令人惊讶的观察结果。分枝杆菌以胆固醇为食，同时包含在巨噬细胞的吞噬体中。重要的是，已经鉴定了两种催化胆甾烷开环的酶。我们计划测试这样的假设，即编码细菌中鉴定的酶的基因可以被人源化并用于转化人单核细胞衍生的巨噬细胞，从而使吞噬体胆固醇降解。主要目标是：1）人源化编码关键开环酶的细菌基因，2）开发创新的表达系统以调节这些基因的表达以响应细胞胆固醇水平的变化，以及3）表征B环开环后产生的化合物的产生和命运。如果这一范式挑战性的假设是真的，拟议的研究应该导致一个全新的方法的发展，为心血管疾病的医疗管理。

DESCRIPTION (provided by applicant): Atherosclerotic cardiovascular disease (CVD) is the leading cause of death in the United States. CVD originates from aberrations in normal lipid metabolism (some genetic, some lifestyle choices) that result in elevated plasma lipoproteins (principally LDLs) and/or low levels of high-density lipoproteins (HDLs). For many people, CVD is an age dependent, progressive disease that is largely undetected or ignored until an event (i.e. myocardial infarction or stroke) occurs in the later stages of disease. Therefore, current therapies focus on preventing a second event (or a primary event in high risk individuals) by reducing the circulating levels of LDLs and/or increasing HDLs. However, at a biochemical level the inability of macrophages to degrade the cholestane ring of cholesterol is a fundamental component of CVD. If macrophages had the ability to degrade cholesterol, they would not become engorged with cholesterol/cholesterol esters and elicit the maladaptive immune response that leads to the onset and progression of atherosclerosis. Recently, studies of Mycobacteria survival in human macrophages revealed a surprising observation. Mycobacteria feed on cholesterol while contained in the phagosomes of macrophages. Importantly, two enzymes that catalyze cholestane ring opening have been identified. We plan to test the hypothesis that genes encoding enzymes identified in bacteria can be humanized and used to transformation human monocyte derived macrophages, enabling the degradation of phagosome-cholesterol. The main objectives are to: 1) humanize bacterial genes encoding key ring opening enzymes, 2) develop an innovative expressions systems to regulate the expression of these genes in response to changes in cellular levels of cholesterol, and 3) characterize the production and fate of compounds generated following B-ring opening. If this paradigm-challenging hypothesis is true, the proposed studies should lead to the development of an entirely new approach for the medical management of CVD.