Nogo-B在晚期动脉粥样硬化发展及斑块内巨噬细胞凋亡中的作用机制研究

Macrophage endoplasmic reticulum (ER) stress in regulating the pathogenesis of atherosclerosis is not fully understood. The central goal of this proposal is to examine the role of RTN-4B (aka, Nogo-B), an integral membrane protein mainly localized to ER, in macrophage ER stress and atherosclerosis. We have shown that Nogo-B is highly abundant in endothelial cells (EC), vascular smooth muscle cells (VSMC) and monocytes/macrophages with diverse and cell specific function. The genetic loss of Nogo-B (Nogo-/-) results in exaggerated neointima .formation after arterial injury and impaired blood flow recovery post-ischemia. Nogo-B regulates acute inflammatory response in EC, and suppresses hypoxia-induced pulmonary VSMC apoptosis. In human, the expression of Nogo-B is negatively correlated with the severity of atherosclerosis, suggests the local reduction of Nogo-B might contribute to plaque formation and/or instability. In exciting preliminary data, .we show that Nogo-/- mice on an ApoE knockout background (ApoE-/-Nogo-/-) develop larger and more advanced atherosclerotic lesions with increased macrophage apoptosis compared to ApoE-/- mice. Nogo-/- macrophages are much more prone to apoptosis in response to free cholesterol loading compared to those from wild-type mice. To further investigate the cell specific role of Nogo-B in atherosclerosis, we have generated floxed allele of Nogo mouse. We hypothesize that Nogo-B governs macrophage functions to regulate atherosclerotic plaque formation and necrosis. In this proposal, we will: (1) Define the cell specific role of Nogo-B in atherosclerotic plaque progression and necrosis in vivo. (2) Elucidate the mechanisms by which Nogo-B regulates ER stress induced macrophage apoptosis in vitro. The findings of this study will advance the Nogo field; provide insights in understanding how Nogo regulate vascular homeostasis.

内质网应激所引起的晚期动脉粥样硬化斑块巨噬细胞凋亡、斑块坏死和破裂是导致心血管事件并致死的主要促进因素。而动脉粥样硬化发生和发展过程中内质网应激调控机制仍未阐明。内质网膜蛋白Nogo-B 在人动脉粥样硬化斑块内的表达降低可能与斑块形成和/或斑块不稳定性有关。前期实验表明，Nogo-B基因敲除小鼠具有更大的动脉粥样硬化斑块，增加的细胞凋亡和斑块坏死。内质网应激可以调控Nogo-B表达，而Nogo-B基因缺失促进游离胆固醇诱导的巨噬细胞凋亡。据此，我们假设Nogo-B 可以通过控制巨噬细胞凋亡而调节动脉粥样硬化斑块的发展和坏死灶形成。本课题将 1：明确巨噬细胞Nogo-B在体内动脉粥样硬化斑块进展和坏死过程中的作用。2：阐明细胞内源性 Nogo-B调控内质网应激导致的巨噬细胞凋亡机制。本课题将揭示Nogo-B 在动脉粥样硬化进程中的作用；进一步阐述内质网蛋白在调控血管功能稳态中的重要作用。

内质网应激所引起的晚期动脉粥样硬化斑块中的巨噬细胞凋亡、斑块坏死和破裂是导致心血管事件并致死的主要促进因素。而动脉粥样硬化发生和发展过程中内质网应激调控机制函待阐明。内质网膜蛋白Nogo-B在人动脉粥样硬化斑块内的表达降低可能与斑块形成和/或斑块不稳定性有关。前期实验表明，Nogo-B基因敲除小鼠具有更大的动脉粥样硬化斑块，斑块内细胞凋亡增加并显著增大斑块内坏死灶。内质网应激可以调控Nogo-B表达，而Nogo-B基因缺失促进游离胆固醇诱导的巨噬细胞凋亡。据此，本课题以期确证Nogo-B可以通过控制巨噬细胞凋亡而调节动脉粥样硬化斑块的发展和坏死灶形成。通过运用骨髓移植及巨噬细胞特异性敲除小鼠，本研究明确了巨噬细胞Nogo-B在体内动脉粥样硬化斑块进展和坏死过程中的保护作用。通过免疫共沉淀等方法，在离体实验中阐明了细胞内源性 Nogo-B抑制内质网应激导致的巨噬细胞凋亡机制。通过本课题研究，首次发现了Nogo-B是调控动脉粥样硬化斑块稳定性的重要分子。巨噬细胞内源性Nogo-B通过与细胞凋亡调控蛋白Bcl-2的直接结合并调节其内质网膜表达和转位，进而有效调节内质网应激诱导的细胞凋亡。本研究更进一步发现了Nogo-B中RHD结构域中的66个氨基酸片段可以替代全长Nogo-B，与Bcl-2结合。这一重要发现为今后以Nogo-B为靶点，治疗晚期动脉粥样硬化斑块不稳定性，预防冠状动脉和颈动脉疾病所致的急性心梗和脑埂塞提供了明确且重要的理论依据。