“失功能”HDL调节巨噬细胞脂噬通路的新机制

Our previous studies with atherosclerosis mouse models have shown that the HDL particles are dysfunctional under various pathological conditions. The dysfunctional HDL is characterized by increased oxidation and pro-inflammatory properties. It is noteworthy that the dysfunctional HDL could down-regulate the expression of scavenger receptor BI (SR-BI), induce an increase in autophagic markers LC3 II/I ratio, while suppress the expression of transcription factor EB (TFEB), a master gene for lysosomal biogenesis, contributing to lipid accumulation in lysosomes of macrophages. Furthermore, we found that overexpression of antioxidase PON1, methionine sulfoxide reductase A (MsrA), or an HDL-binding apoE-derived peptide EpK could restore the anti-oxidative capacity of the dysfunctional HDL, and revealed that the autophagy activity is correlated with the lipid homeostasis in macrophages. Since HDL functions are carried out through direct interacting with its receptors localized on the cell surface followed by generation of intracellular signals, and SR-BI is a specific HDL receptor which recognizes both native and dysfunctional HDL and mediates cholesterol transport by a dual-flux feature, we aim to explore the relationship between receptor-mediated cellular signaling pathways and lipophagy, an autophagy selectively involved in lipid degradation. We propose to examine the role of SR-BI in mediating different signaling pathways by native and dysfunctional HDLs in macrophages and determine the underlying mechanisms. We will also test if lipophagy lysosomal pathway is improved by overexpression of TFEB in bone marrow-derived macrophages, and examine the impact on lipid transport in macrophages and atherosclerotic lesion formation in mice. The proposed studies will elucidate new molecular mechanisms for atherogenesis and suggest new drug targets for Atherosclerosis-related cardiovascular diseases.

我们前期在动脉粥样硬化(As)小鼠中证实，病理条件下高密度脂蛋白(HDL)表现为抗As 的“失功能”。值得关注的是，这种氧化炎性的HDL下调巨噬细胞清道夫受体BI (SR-BI)表达的同时，能诱发细胞自噬标志LC3 II/I增高，而控制溶酶体生成的转录因子TFEB表达下调，使胞内脂质积聚于溶酶体；进一步发现体内高表达抗氧化酶或多肽可重塑“失功能”HDL的抗氧化能力，并与巨噬细胞自噬和脂质水平的调节相关。鉴于HDL功能均涉及细胞受体依赖的信号途径，而SR-BI可介导正常及氧化HDL的脂质转运，本课题拟关注一条溶酶体依赖的脂质降解途径——脂噬，从细胞受体信号与脂噬通路的联系——这一新角度，探讨天然及“失功能”HDL对巨噬细胞产生的信号差异及机制，并尝试用高表达TFEB手段改善脂噬溶酶体通路的顺应性，探讨其对巨噬细胞脂质流动及As斑块的调节，此将有助于揭示相关心血管疾病的新分子机制及药物靶点。

我们前期发现，“失功能”高密度脂蛋白(HDL)可下调巨噬细胞清道夫受体BI(SR-BI)表达并表现自噬通路异常，使胞内脂质积聚于溶酶体。由于胞膜受体SR-BI可识别正常及氧化HDL，推测SR-BI依赖的信号途径可能涉及不同HDL致细胞脂质转运差异的机制。本课题关注溶酶体依赖的脂质降解途径——脂噬，从细胞SR-BI受体信号与脂噬通路可能关联的角度，探讨SR-BI介导天然及失功能HDL对巨噬细胞产生的效应信号差异及作用机制，并在体尝试高表达TFEB对脂噬溶酶体通路顺应性及动脉粥样硬化(As)斑块的调节。.本研究首先从野生型(WT)和SR-BI缺陷小鼠血浆分离正常HDL和失功能HDL，作用于WT小鼠巨噬细胞，发现SR-BI-/- HDL可致细胞炎症因子、应激相关蛋白水平增高，酸性溶酶体数量减少、溶酶体酸酯酶LAL和转录因子TFEB表达水平降低，而自噬标志蛋白LC3、Beclin 1、P62均升高，细胞脂质蓄积于溶酶体；尤其对SR-BI缺陷巨噬细胞表现更明显，说明SR-BI参与不同HDL对细胞的信号效应与脂噬溶酶体通路的调节有关。其二，构建TFEB慢病毒载体并转染小鼠巨噬细胞，结果发现，TFEB高表达的细胞ROS与炎症水平降低，溶酶体和自噬体数量增多，脂质沉积减少，其与细胞LAMP1、LAL、ABCA1水平增加，而P62降低有关。转染肝细胞有相似作用，说明TFEB可调节细胞脂噬溶酶体途径协调性而降低脂质沉积与炎症。进一步利用Lv-TFEB慢病毒注射感染apoE敲除小鼠，观察到肝脏TFEB高表达可调节相关基因表达，显著降低肝脂肪变及As斑块，其机制与调节溶酶体功能、增强细胞脂噬溶酶体通路顺应性密切相关。我们也进行感染病毒骨髓干细胞移植入SR-BI缺陷小鼠，在体观察髓源性巨噬细胞自噬溶酶体通路顺应性的调控对血管壁As的作用效果，此将有助于揭示相关心血管疾病的新的分子机制及药物靶点。