Vav家族蛋白差异调控动脉粥样硬化中巨噬细胞NF-κB活化的机制

Atherosclerosis is accounting for over 30% of the global death, and each year around 20 million people die of diseases caused by atherosclerosis. The Vav family proteins are highly conserved in domain organization and all expressed in macrophages. Vav1 has been implicated in CD36 mediated lipid uptake, and genetic ablation of Vav1 resulted in amelioration of atherosclerosis pathological consequences in ApoE deficient mice treated with high fat diet. However very little is known whether Vav family proteins are important for lipid laden macrophage inflammatory response, specifically the activation of NF-κB pathway. The applicant used CRISPR/Cas9 genome editing tools, knocked out each of the 3 Vav family genes, in RAW264.7 cells. All the Vav family genes are expressed in primary and RAW264.7 macrophages, Ox-LDL uptake was found decreased in RAW264.7 cell lines deficient in each of the Vav family genes. Very surprisingly, we found that Vav family genes differentially regulate JNK pathway which was well characterized for CD36 mediated foam cell formation. More importantly, our previous studies found that both Vav1 and Vav2 are required for phosphorylation of NF-κB in RAW264.7 cells following Ox-LDL treatment. In the proposed study, our primary goal is to understand whether the other members Vav2 and Vav3 are important for atherosclerosis development in ApoE deficient mice and how the Vav family proteins differentially regulate NF-κB activation in terms of p65 translocation, phosphorylation and degredation of IκBα, and how Vav1 and Vav2 regulate the NF-κB activation, and what are atherogenic consequences in terms of inflammatory cytokine production and lipid uptake in Vav protein deficient macrophages. Elucidation of such mechanisms will provide new insights for development of novel therapies against atherosclerosis.

全球每年有约2000万人死于动脉粥样硬化，超过总死亡人数的30%。Vav家族蛋白结构域保守、且均在巨噬细胞表达，目前仅Vav1被证明影响CD36介导的脂质摄取，Vav1缺失显著减轻ApoE敲除小鼠动脉粥样硬化，但其它成员还有待研究。Vav家族成员是否影响巨噬细胞NF-κB的活化、炎症细胞因子分泌仍然缺乏研究。申请人在巨噬细胞中分别敲除Vav1、Vav2和Vav3，发现脂质摄取水平均显著降低。令人吃惊的是仅Vav1、Vav2敲除影响Ox-LDL诱导的NF-κB活化。本课题在ApoE敲除小鼠中逐一敲除Vav家族成员，将验证其对动脉粥样硬化形成的影响；在Vav家族蛋白缺失及野生型巨噬细胞中比较p65核转位、IκBα磷酸化与降解、NF-κB通路相互作用蛋白等方面的差异；分析Vav成员缺失对巨噬细胞炎症细胞因子分泌的影响。明确上述机制将为动脉粥样硬化治疗提供新视野。

动脉粥样硬化是与巨噬细胞密切相关的慢性炎症疾病，是心血管疾病的首要致病因素，有报道指出其导致的死亡人数到2030年将超过2360万人。目前对Vav家族蛋白是否参与巨噬细胞炎症反应仍缺乏研究。本课题在ApoE敲除小鼠中逐一敲除Vav家族成员，成功构建了Vav2敲除、Vav1和Vav3巨噬细胞条件性敲除ApoE小鼠。高脂建模后Vav1和Vav2缺失小鼠动脉粥样硬化病变减轻，而Vav3缺失并未对病变产生影响。进一步分析Vav1、Vav2、Vav3单敲小鼠巨噬细胞在Ox-LDL刺激下炎症相关细胞因子的表达，发现Vav3的单敲对巨噬细胞炎症相关细胞因子的表达基本无影响，而Vav1和Vav2单敲后巨噬细胞中促炎因子降低的同时抑炎因子升高。为进一步揭示其差异机制，通过研究巨噬细胞脂质摄取过程中NF-κB信号通路的活化情况，发现Vav1、Vav2敲除后IκBα和NF-κBp65的磷酸化下降，而Vav3敲除对巨噬细胞IκBα的磷酸化无影响。以上结果表明，Vav家族蛋白对NF-κB信号通路的活化水平不同，特别是IκBα活化不同，继而影响巨噬细胞炎症相关细胞因子的表达，最终导致Vav1、Vav2、Vav3单敲ApoE小鼠的病理差异。本研究为全面认识Vav家族蛋白在巨噬细胞炎症反应中的作用及动脉粥样硬化治疗提供新视野。