Methods to enable cholesterol catabolism in human monocyte derived macrophages

描述(申请人提供)：动脉粥样硬化性心血管疾病(CVD)是美国主要的死亡原因。CVD起源于正常脂代谢的异常(一些基因，一些生活方式的选择)，导致血浆脂蛋白(主要是低密度脂蛋白)升高和/或高密度脂蛋白(HDL)水平降低。对许多人来说，心血管疾病是一种年龄相关的进行性疾病，在疾病的后期发生事件(如心肌梗死或中风)之前，基本上没有被发现或被忽视。因此，目前的治疗方法侧重于通过降低低密度脂蛋白的循环水平和/或增加低密度脂蛋白来预防第二次事件(或高危个体的主要事件)。然而，在生化水平上，巨噬细胞不能降解胆固醇的胆烷环是心血管疾病的基本成分。如果巨噬细胞具有降解胆固醇的能力，它们就不会被胆固醇/胆固醇酯充斥，并引发导致动脉粥样硬化发生和发展的非适应性免疫反应。最近，关于分枝杆菌在人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，而胆固醇则位于巨噬细胞的吞噬小体中。重要的是，已经确定了两种催化胆烷开环的酶。我们计划测试这样一个假设，即在细菌中发现的编码酶的基因可以人源化，并用于转化人类单核细胞来源的巨噬细胞，从而能够降解吞噬小体胆固醇。主要目标是：1)将编码关键开环酶的细菌基因人源化，2)开发一种创新的表达系统来调节这些基因的表达，以响应细胞内胆固醇水平的变化，以及3)表征B环开环后产生的化合物的产生和命运。如果这一挑战范式的假说是真的，拟议的研究应该会导致开发一种全新的心血管疾病医疗管理方法。

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.