SR-BI以HDL荷脂相关方式通过Caveolae/Cav-1协同调节血管内皮细胞S1P1/S1P2受体功能的机制

Vascular endothelial dysfunction is the pivotal early event in the development of atherosclerosis.High density lipoprotein (HDL) possesses the antiatherogenic role thouth its promotion of reverse cholesterol transport and signaling function. The vascular endothelial protection of HDL-associated sphigosine-1-phosphate (HDL-S!P) is attributed to the diverse biological actions of S1P which binds to different subtypes of G protein-coupled receptors. However, HDL subclass HDL3 and HDL2 display differential antiatherogenic effects. HDL-S1P and free S1P exhibits different biological effects to vascular system. S1P receptors, S1P1 and S1P2 are also demonstrated the reciprocal effects on vascular endothelial cells. The mechanisms of the mentioned above is not fully understood. Our previous research data indicate that scavenger receptor class B type I (SR-BI), a high affinity recetor for HDL, and HDL-S1P activated the endothelial nitric oxide synthase (eNOS) and increased the release of NO, and upregulated expression of cyclooxygenase-2 (COX-2) and elevated production of prostaglandin I2 (PGI2), via sharing the same intracelluar signaling pathways. We noticed that SR-BI and given subtypes of G-alpha proteins, which are potentially coupled to S1P receptors, are localized in caveolae, and that caveolae/caveolin-1 (Cav-1) regulates the transmenbrane signal transduction. Considered all above, we raise the hypothesis: SR-BI may modulates the biological actions of S1P1/S1P2 receptors in vascular cells through caveolae/caveolin-1 in an HDL- lipid burden-pattern. To test the hypotheses, we plan to carry out the experiments of, both in vtro, by methods of drug interfering and signaling blocking in endothelial cells, and in vivo, by strategies of endothelial-specfic Cav-1 overexpression or knockdown in SR-BI defficient mice. This studies may provide new insights into the mechanisms of HDL mediated vascular endothelial protection.

HDL-S1P的血管内皮保护作用主要通过S1P与不同亚型的G蛋白偶联受体结合所产生的不同生物学效应。然而，HDL亚类HDL3与HDL2的抗动脉粥样硬化作用不同，HDL-S1P与游离S1P的血管生物学效应也不同，S1P1与S1P2的血管内皮功能基本上是反向的，以上机制尚不十分清楚。本课题组的前期研究工作表明，SR-BI与HDL-S1P通过共同的细胞内信号通路激活eNOS促NO释放，上调COX-2表达促PGI2生成。由于SR-BI和某些亚型G蛋白在小窝相对聚集，并且小窝/Cav-1调节多种跨膜信号功能，本课题提出：SR-BI可能以HDL荷脂相关方式通过小窝/Cav-1协同调节血管内皮细胞S1P1/2受体功能的科学假设。拟通过受体功能干预和信号途径阻断的细胞实验以及内皮特异性表达或沉默Cav-1基因的SR-BI基因缺陷小鼠动物实验验证以上假设。本研究将为认识HDL的血管内皮保护机制提供新视角。

项目的背景： HDL-S1P通过结合其5种膜受体 (S1PR1~S1PR5) 来介导HDL诸多功能。清道夫受体B类I型SR-BI)是HDL的天然高亲和生理性受体，也能结合载脂蛋白A-I (apoA-I)来介导HDL的诸多功能。.主要研究内容：通过分别或同时对膜受体SR-BI、S1P1、S1P2和/或S1P3的表达或功能的干预，观察用S1P、apoA1、HDL-S1P、处理后的细胞保护效应（细胞增殖、凋亡、迁移等）以及信号分子（PI3K、Akt、ERK1/2）的表达及激酶活性的变化。通过siRNA干扰或药物阻断抑制SphK1/2活性，或阻断血管内皮细胞内源性S1P的生成和释放，观察HDL-S1P处理细胞后，SphK1、eNOS和COX-2表达和活性以及S1P和NO释放的变化。通过TGF-beta处理肝细胞观察ApoM表达的变化并探索介导基因表达变化的TAK-1-JNK-c-Jun信号途径。.重要结果及其科学意义：(1) Apo A-I能促进内皮细胞S1P释放增加。Apo A-I介导的S1P释放依赖于ABCA1和SR-BI受体。推测 S1PR1/3-ERK1/2-Sph K1 信号通路为 apo A-I 介导的 S1P 释放提供了正反馈途径。(2) HDL 通过 S1P2-Gi/o-PKC-P38MAPK/ERK-Sph K2-pCREB途径影响 COX-2 的表达。HDL激活内皮细胞核内鞘氨醇激酶和使 CREB 磷酸化，从而上调 COX-2 的表达。(3) S1PR1/3-PI3K-Akt信号转导通路参与S1P对THP-1源性巨噬细胞胆固醇流出的调控作用。S1PR1通过PI3K-Akt-LXR信号转导通路促进THP-1源性巨噬细胞胆固醇流出。(4) S1P、HDL和apoA-I可以促进人脐静脉内皮细胞的迁移。HDL/SR-BI和HDL-S1P /S1P1通过PI3K-Akt信号通路共同调节人脐静脉内皮细胞的迁移。(5) S1P 对内皮细胞的炎症效应主要是通过 S1PR2。SR-BI抑制 S1P/ S1PR2 对血管内皮细胞的炎症效应。SR-BI抑制 S1P/S1PR2 对血管内皮细胞的炎症效应与PI3K–AKT-eNOS 信号通路有关。(6) TGF-β可以以时间依赖性和浓度依赖性的方式通过TAK-1-JNK-c-Jun途径抑制HepG2细胞中apoM的表达。.