重塑HDL抗氧化策略对动脉粥样硬化的干预研究

Plasma High-density lipoprotein (HDL) is a well-established athero-protective factor. However, under conditions such as oxidative stress or inflammation, athero-protective HDL may instead become pro-atherogenic. Our previous study showed that dysfunction of HDL is attributed to modification of apolipoprotein AI (apoAI) and significant decrease of HDL-associated anti-oxidative paraoxonase 1 (PON1) activity. In particular, Methionine sulfoxide (MetO) formed in oxidative apoAI has been reported as one of the early features. Our experimental evidence suggested that oxidation of HDL and MetO of oxidative apoAI could be repaired in vitro by a specific enzyme called methionine sulfoxide reductase A (MsrA). This project is aimed at restoring the anti-oxidative capacity of dysfunctional HDLs to attenuate atherogenesis. We will generate lentiviral vectors for overexpression of endogenous antioxidase PON1 of HDL or exogenous antioxidase MsrA with MsrA-EpK form (MsrA fused with an HDL-binding apoE-derived peptide, EpK). Mouse hematopoietic progenitor cells (HPCs) will be transduced with these lentiviruses and transplanted into scavenger receptor BI gene knockout (SR-BI-/-) mice using bone marrow transplantation. We propose to test if monocytes/macrophages derived from the transduced HPCs will express and secrete functional levels of PON1 or MsrA-EpK, confer HDL potent anti-oxidative activity, and protect the development of atherosclerosis in SR-BI-/- mice.

血浆高密度脂蛋白(HDL)是公认的动脉粥样硬化(As)防御因素。氧化应激、炎症等条件可引起HDL病理性修饰，导致HDL从抗As向致As转变。前期研究我们发现"失功能性"HDL以apoAI的氧化修饰、对氧磷酯酶1(PON1)水平和活性降低为突出特征。鉴于体外实验证实氧化的HDL及apoAI中Met氧化产物MetO可被一种特异的甲硫氨酸亚砜还原酶A（MsrA）修复，本课题拟针对性选择HDL内在的PON1和外源的抗氧化酶MsrA，借助慢病毒载体及骨髓造血干细胞（HPCs）移植技术，并利用我们曾合成一种结合HDL的apoE拟肽(EpK) 结构，实现特定单核巨噬细胞中长效、稳定表达分泌性PON1或EpK-MsrA融合蛋白，以期从内、外源性抗氧化酶角度特异重塑"失功能"HDL的抗氧化状态，从细胞水平和基因敲除小鼠（SRBI-/-鼠）在体水平探讨HDL抗氧化的靶向性调控策略对As的防治价值。

血浆高密度脂蛋白(HDL)是公认的动脉粥样硬化(As)防御因素。但氧化应激、炎症等条件可产生致As的“失功能性”HDL。基于前期研究我们证实“失功能性” HDL的氧化修饰特征及一种特异甲硫氨酸亚砜还原酶A(MsrA)对氧化apoAI的修复作用，本课题针对性选择HDL内在的对氧磷酯酶1(PON1)和外源的抗氧化酶MsrA，并借助我们曾合成一种结合HDL的apoE拟肽(EpK) 结构，成功构建慢病毒重组载体：pWPI-PON1、pWPI-Sp-EpK-hMsrA、pWPI-EpK等，从细胞水平及在体基因敲除(apoE-/-和SRBI-/-)小鼠模型上，实现长效、稳定表达分泌性PON1、EpK或EpK-MsrA蛋白，观察对脂质代谢、血管壁局部和整体氧还状态的调节及对As斑块形成的干预效果。结果：①分泌性PON1、EpK或EpK-MsrA改善apoE-/-及SRBI-/- HDL的致炎性，并降低巨噬细胞内脂质积聚；② 肝脏分泌表达EpK，不改变apoE-/-小鼠血脂及脂蛋白分布，但降低血中SAA水平、抑制肝脏炎症因子TNFa和IL6的表达，显著降低小鼠主动脉As斑块；对EpK结构进行改造后合成一新的apoE拟肽(hEp)，则表现改善血脂水平和抗炎作用而显著降低小鼠As斑块；③肝脏hMsrA高表达可通过不同作用机制调节胆固醇转运蛋白apoAI、ABCA1/G8、SR-BI、LDLR等水平、胆固醇酯化相关酶CEH、ACAT的表达水平、胆固醇胆道外排通路以及抑制TG合成，不同程度改善apoE-/-和SRBI-/-小鼠肝脏及循环的氧还状态及炎症水平，从而显著降低肝脏脂质沉积，阻抑小鼠As斑块进程；④ 利用骨髓移植进行apoE-/-小鼠和SRBI-/-小鼠的Lv-EpK-MsrA的骨髓造血干细胞(HPCs)感染与高表达，实现EpK-MsrA在单核巨噬细胞源性的分泌高表达，虽未见小鼠血脂变化，但可观察到对血管壁细胞组成的影响及减轻As斑块的发展。结论：通过高表达内、外源性抗氧化蛋白(MsrA、EpK、PON1等)靶向性重塑HDL的抗氧化能力，可显著调节在体循环及血管壁的氧化状态，为As、脂肪肝等HDL异常相关的重大代谢性疾病的防治提供新的思路与策略。