酶促产生的25-OHC对血管壁巨噬细胞增殖凋亡及动脉粥样硬化进程的影响和ORP4L介导作用机制的研究

Macrophage apoptosis and proliferation are the centrol events in atherosclerosis. Oxysterols enhance cell proliferation but the extra increased oxysterols mediate cell apoptosis.So far, no reports are found in the study that the effects of 25-hydroxylase catalyzed 25-OHC in cell proliferation and apoptosis. We have reported that oxysterol binding protein-related protein-4 (ORP4L) regulates calcium signaling, sustains Bcl-XL expression and counteracts the cytotoxicity of exogenous 25-hydroxycholesterol (25-OHC) to protect macrophage from apoptosis (Circ Res. 2016 Dec9; 119(12):1296-1312). Based on the findings above, the editorial suggested that the impact of endogenous 25-OHC on macrophage apoptosis and proliferation should been assessed, which will offer the evidences that how lipids deposited in the subendothelial layer and cell debris cannot be cleaned efficiently by macrophages（Circ Res. 2016;119:1258-1261. In cultured cells, we found that CH25H overexpression promoted cell proliferation whereas attenuated by ORP4L knockdown. Overexpression of CH25H as well as knockdown of oxysterol-7α- hydroxylase (CYP7B1) significantly increased the sensitivity of cells to apoptosis. However, this phenomenon can be abolished by ORP4L overexpression. Thus,we speculate that in the blood vessel wall the enzyme catalyzed 25-oxysterol may enhance macrophages proliferation via ORP4L mediaed-Ca2+ signaling, but the extra increased oxysterol may inactive ORP4L function, resulting cell apoptosis. In this study , we will produce macrophage specific TgCH25H and CYP7B1-/- mice, which will be crossed with ORP4L-/+/LDLr-/- or TgORP4L/LDLr-/-mice to get TgCH25H /ORP4L-/+/LDLr-/- and TgCH25H /TgORP4L/LDLr-/-,or TgCH25H / CYP7B1-/-/ORP4L-/+/LDLr-/- and TgCH25H / CYP7B1-/-/TgORP4L/LDLr-/- ,respectively. Combined the analysis of molecule and cell biology in vitro, we aim to reveal in vivo the effects of the interaction of endogenous enzyme catalyzed 25-OHC and ORP4L in apoptosis and proliferation of macrophages and atherosclerosis. We hope to find out prevention and therapeutic approaches for the direct intervention of atherosclerosis.

巨噬细胞增殖和凋亡是动脉粥样硬化（AS）进程的主要事件。体外低浓度氧化固醇促细胞增殖，高浓度则诱导细胞凋亡。但25-hydroxylase (CH25H)催化产生的25-氧化固醇(25-OHC)对巨噬细胞增殖和凋亡的影响未见报道。我们发现ORP4L调控钙信号并对抗25-OHC诱导的细胞凋亡。培养细胞中过表达CH25H提高其增殖，但敲低ORP4L则降低这个效率；CH25H过表达同时Oxysterol-7α-hydroxylase (CYP7B1)沉默提高细胞凋亡敏感性，但ORP4L过表达消减该现象。因此，我们提出假说:CH25H催化的25-OHC通过ORP4L介导Ca2+信号,促进巨噬细胞增殖；其浓度过高则抑制ORP4L及Ca2+信号,致巨噬细胞凋亡。本课题将在分子、细胞、动物水平等多层次揭示酶促产生的25-OHC在巨噬细胞命运及AS进程中的作用和ORP4L的介导机制，为AS防治提供证据。

巨噬细胞增殖和凋亡是关联一系列重要疾病包括动脉粥样硬化形成及脂肪动员进程的主要事件。我们前期发现体外低浓度氧化固醇促细胞增殖，高浓度则诱导细胞凋亡（Circ. Res. 2016 Dec 9;119(12):1296-1312）。但25-hydroxylase (CH25H)催化产生的内源性25-氧化固醇(25-OHC)对巨噬细胞增殖和凋亡的影响未见研究。我们发现氧化固醇结合蛋白ORP4L调控钙信号并对抗25-OHC诱导的细胞凋亡。培养细胞中过表达CH25H提高其增殖，因此，我们提出假说:CH25H催化的内源25-OHC通过ORP4L介导Ca2+信号,促进巨噬细胞增殖；同时，这些增殖的巨噬细胞改变相关疾病比如动脉粥样硬化和肥胖的发展进程。. 根据上述研究计划，我们执行了一个庞大的模式动物基础的实验计划：我们购买和产生了CH25H巨噬细胞专一的KO和KI小鼠；ORP4L巨噬细胞专一的KO和KI小鼠。在实验过程中，为方便在体观察巨噬细胞的增殖，除动脉粥样硬化斑块外，我们增加了睾丸脂肪组织为观察对象。.经过几年的研究，我们证明了体内存在一个通过免疫细胞相互作用，通过巨噬细胞增殖，控制动脉粥样硬化进程及脂肪组织平衡的机制。.实验证据如下：.1.T细胞分泌的IFN-γ能够提升巨噬细胞CH25H的表达，提高内源25-OHC的水平；.2.CH25H能够提升脂肪组织和动脉粥样斑块中巨噬细胞增殖；.3.巨噬细胞25-OHC驱使的巨噬细胞增殖被ORP4L介导；.4.ORP4L通过钙离子信号介导巨噬细胞增殖；.5.增殖的巨噬细胞在一定阶段可能通过其分泌的THF-α控制脂肪聚集和动脉粥样硬化斑块形成。. 上述发现首次阐明了巨噬细胞增殖的机制，证明内源25-OHC 驱使的巨噬细胞，具有新的功能,其可能对动脉粥样硬化发展和脂肪平衡，发挥重要作用。. 本课题已经交叉支持了ORP4L的其他研究，相关文章已经发表在Science Trans Med., 2022 DOI:10.1126/scitranslmed.abh2548；Blood, 2021, DOI：10.1182/blood.2021013579，Cell Reports, 2019;FASEB J，2019;JBC,2018.等期刊。