ORMDL3基因调控内皮间充质转化在动脉粥样硬化易损斑块发生发展中的作用机制研究

The ORMDL3 gene (ORMDL Sphingolipid Biosynthesis Regulator 3) maps to Chromosome 17q21 and encodes an endoplasmic reticulum (ER)-anchored protein. A growing body of evidence has suggested the genetic association of this gene with a diverse set of inflammatory disorders that include bronchial asthma, inflammatory bowel disease and ankylosing spondylitis. Gene functional investigations have revealed the particular relevance of ORMDL3 in ER stress, lipid metabolism and inflammatory reaction. We have previously confirmed the genetic association of ORMDL3 gene with atherosclerosis (AS) risk in Chinese Han population and uncovered a role of this gene in regulating oxidized-low density lipoprotein (ox-LDL)-induced autophagy, apoptosis and inflammation in endothelial cells (ECs). . Endothelial-to-mesenchymal transition (EndMT), a complex biological process in which the ECs lose their specific markers and acquire the mesenchymal phenotypic and functional property, has been linked with carcinoma, embryogenesis and cardiovascular disorders. It has recently gained increasing attention in the initiation and progression of vulnerable plague in AS. It is becoming clear that EC-derived mesenchymal cells via EndMT account for a majority of AS-associated fibroblasts in the atherosclerotic lesions and the extent of EndMT is positively correlated with the instability of human plagues and the incidence of major ischemic events. In vivo and in vitro evidence suggested that EndMT might destruct the integrity of endothelial barrier, exacerbate the inflammatory cascades in the plague, promote the growth of plague and facilitate the conversion from stable to unstable plagues. However, the detailed mechanisms involved have to be further illuminated. . Our prior work has demonstrated that manipulating the expression of ORMDL3 led to the alterations of expression of EndMT markers and related transcription markers in ECs. Down-regulation of ORMDL3 impeded the migration of ECs. Besides, the stimulation with ox-LDL promoted the activity of EndMT in ECs, which could be reversed by the silencing of ORMDL3. Additionally, ORMDL3 appeared to regulate EndMT via the TGF-β-Smad-Snail and Wnt-catenin pathways. By using the cell models and Ormdl3-/-and ApoE-/- double knockout mouse model, we aim to in vitro and in vivo explore the hypothesis that ORMDL3 gene could fine-tune the evolution of vulnerable plague via EndMT and also the underlying molecular mechanisms. Our study will shed a light on a novel explanation of AS pathogenesis and a promising therapeutic target in the near future.

前期我们证实：ORMDL3基因为中国汉族人群动脉粥样硬化（AS）的易感基因；该基因参与调控氧化低密度脂蛋白（ox-LDL）引起的血管内皮细胞（EC）自噬、凋亡和炎症反应。近年研究发现内皮间充质转化（EndMT）与AS易损斑块的演变关系密切，但详细分子调控机制不明确。最近我们发现在EC中：过表达或干扰该基因表达可引起EndMT相关标志物表达水平变化；干扰该基因可抑制EC迁移能力；ox-LDL刺激有促EndMT作用，而干扰该基因可逆转之；该基因调控EndMT过程可能与TGF-β-Smad-Snail通路及Wnt-catenin通路有关。课题组提出假说：ORMDL3基因通过调控EndMT进而参与AS易损斑块的发生发展。本课题在前期基础上，拟利用细胞模型及Ormdl3-/-与ApoE-/-双敲除小鼠模型，在体外及体内水平上证实上述假说，并探索其分子调控机制，以期为明确AS发病机制奠定理论基础。

1.背景：1）课题组前期研究发现ORMDL3基因与中国汉族人群动脉粥样硬化（Atherosclerosis，AS）的发病风险呈显著相关。2）该基因可能参与血管内皮细胞（EndothelialCell，EC）炎症反应调控。3）内皮间充质转化（EndMT）与AS易损斑块的演变关系密切，在EC中过表达或干扰ORMDL3基因表达可引起EndMT相关标志物表达变化。.2.主要研究内容：1）通过构建细胞模型及Ormdl3敲除的小鼠斑块模型，在体内及体外水平上明确ORMDL3基因在AS斑块发生发展中的作用。2）利用上述模型，明确该基因在EC炎症反应及EndMT过程中的角色，证实其是否通过调控EC炎症反应及EndMT过程进而参与AS病理生理过程。3）探索ORMDL3基因调控EC炎症反应及EndMT过程的分子机制，发现其中的信号通路或网络，筛选并鉴定关键调控分子。.3.重要结果、关键数据：1）（体外水平）在EC中炎症刺激因子可上调ORMDL3基因表达；而（体内水平）高脂喂养的ApoE-/-小鼠中，ORMDL3基因表达较对照小鼠亦明显增高。2）（体外水平）在EC中干扰ORMDL3基因表达可抑制EC炎症反应和EndMT，而过表达ORMDL3基因可促进EC炎症反应和EndMT；（体内水平）AS模型小鼠中（高脂饮食+腺相关病毒造模）敲除ORMDL3基因可使斑块表型及EndMT明显缓解;3）ORMDL3基因主要通过ATF6-NF-κB信号通路调控EC炎症反应;基因调控EndMT过程可能与TGF-β-Smad-Snail通路及Wnt-catenin通路有关。.4.科学意义：1）明确了ORMDL3基因通过促进EC炎症发展和EndMT进而推动AS斑块发生发展;2)阐释ORMDL3基因调控EC炎症反应和EndMT的分子机制和关键调控因子;3）丰富AS发病机制的理解，以期为临床治疗提供新靶点。