生物钟基因CLOCK调控血管内皮细胞自噬参与斑块稳定性转变的机制研究

With the aging of the population, stroke has become an enormous public health problem. The mechanisms of vascular remodeling and stable-to-vulnerable plaque transformation have been difficult. Recent studies have found that Clock is involved in carotid plaque vulnerability, but the underlying mechanism is still not clear. Our previous study found the significant expression changes of circadian clock genes in human plaque-derived cells, up-regulation of expression of Clock gene as well as the downstream adhesion related genes and inflammatory cytokines. The autophagy was enhanced and the migration of inflammatory cells increased. Accordingly, we propose the hypothesis that the up-regulation of Clock expression in plaque endothelial cells can cause endothelial dysfunction via the excessive activation of autophagy, which could lead to the increasing migration of inflammatory cells into the plaque and releasing various inflammatory mediators, triggering apoptosis, fibrous cap thinning, and ultimately cause plaque stability change and rupture. To verify this hypothesis, we plan to collect plaque specimens, culture Human Umbilical Vein Endothelial Cells (HUVEC) and the vascular endothelial cell-specific Clock knockout mice, then using a variety of experimental methods to verify that Clock plays an important role in the plaque stability shift in vivo and in vitro. Furthermore, we will clarify the mechanism of how Clock regulates the autophagy-mediated endothelial function in the vulnerable plaque. This study will provide a new perspective on mechanisms of plaque stability shift. It also provides a therapeutic way to prevent plaque rupture base on a new theory.

随着人口老龄化，脑卒中已成为重大的公共卫生问题。血管病理性重塑及斑块稳定性转变机制成为研究难点。新近研究发现，钟基因CLOCK参与颈动脉斑块稳定性转变但具体机制不明。预实验发现：斑块来源细胞中生物钟基因表达紊乱；易损斑块中CLOCK、下游粘附相关基因和炎症因子表达上调；使血管内皮细胞自噬增强、炎症细胞跨膜迁移增多。因此，我们提出假说：斑块内皮细胞CLOCK表达上调，可使细胞自噬过度激活，引起内皮细胞功能损害，炎症细胞迁移至斑块增多，释放各种炎症介质，引发细胞凋亡、纤维帽变薄，最终导致斑块稳定性转变和破裂。我们将通过收集斑块标本、培养HUVEC、构建血管内皮细胞CLOCK条件性敲除小鼠模型，在临床样本、细胞及动物模型验证CLOCK通过调控内皮细胞自噬参与斑块稳定性转变的重要作用，并阐明具体机制。本项目将对斑块稳定性转变研究提供全新的视角，为今后抑制和预防斑块破裂的研究提供理论基础及治疗靶点。

生物钟基因CLOCK可通过调控下游钟控基因参与颈动脉斑块形成及稳定性转变，此作用具有器官特异性且具体机制不明。在本课题中，我们通过收集斑块标本、培养人脐静脉内皮细胞株（HUVESs）、构建CLOCK敲除小鼠模型，从细胞、组织和活体水平验证CLOCK通过调控内皮细胞自噬及内皮-间质转化（EndMT），参与斑块稳定性转变的重要作用和机制。课题组研究发现：（1）体外实验中使用HUVESs，通过hCLOCK过表达/敲除，结果提示NF-κB信号通路激活是缺氧环境下hCLOCK诱导炎症因子表达的关键；（2）斑块来源血管平滑肌细胞（PVSMCs）中脂质含量较正常血管平滑肌细胞（NVSMCs）明显升高，PVSMCs中的总胆固醇含量显著升高，总甘油三酯的含量在两组细胞中无明显差别。体外实验证实，核心生物钟基因表达节律紊乱至少部分由于细胞内高胆固醇含量所致；（3）人主动脉内皮细胞（HAECs）中BMAL1的高表达能够抑制氧化低密度脂蛋白（oxLDL）诱导的细胞内ROS积累以及随后发生的EndMT，人颈动脉斑块中钟基因BMAL1表达减弱与EndMT和斑块易损性密切相关；（4）人颈动脉斑块的易损性与CLOCK表达的丧失、TGF-β/ ROCK1信号传导的激活以及EndMT的程度密切相关，体外实验证实，内皮细胞CLOCK表达缺失加重了TGF-β/ ROCK1调控的EndMT进展；（5）颈动脉狭窄患者血浆中炎症因子TNF-a的表达水平升高是斑块内出血（易损斑块的特征之一）及患者支架术后新发脑缺血病灶的独立风险因素，因此TNF-a或有成为预测斑块内出血及支架术后新发脑缺血病灶的生物标记物的可能；（6）人颈动脉斑块富含脂质的坏死核心中，CLOCK mRNA水平低于斑块移行部，CLOCK mRNA水平降低与人颈动脉斑块狭窄率增加，斑块内出血和斑块纤维帽缺损有关。体内实验表明，ClockΔ19/Δ19小鼠的EndMT和内质网应激显着增加，抑制IRE1-XBP1轴可以减轻由ClockΔ19/Δ19突变和低剪切应力引起的EndMT升高。（7）体外实验证实，缺氧可诱导HUVESs自噬并抑制细胞增殖和迁移，通过应用ROS清除剂Trion，hCLOCK诱导细胞自噬的作用显著减弱，而细胞增殖和迁移增强，结果证实ROS是其中关键因素。本项目对斑块的稳定性转变研究提供了新的视角，为预防斑块破裂的研究提供新的治疗方向。