DOT1L调控血管内皮功能在血管重构中的作用及表观机制研究

Endothelial dysfunction is the initial step of vascular remodeling and homeostasis imbalance. Our previous results have demonstrated that the upregulation of histone methyltransferase DOT1L was observed in atherosclerotic arteries of patients, which was accompanying with activation of endothelial dysfunction phenotype. The similar results were also observed in oxidized low density lipoprotein (oxLDL)-stimulated rat aortic endothelial cells. All the events were reversed by DOT1L knockdown strategy. However, the role and epigenetic mechanisms of DOT1L in endothelial dysfunction and vascular remodeling remain unclear. Herein, we propose the following hypothesis that DOT1L promotes the development of endothelial dysfunction and vascular pathological remodeling by histone methylation. In our follow-up study, we will elucidate that DOT1L accelerates the development of endothelial dysfunction and vascular remodeling and underlying epigenetic mechanism. 1) Rat aortic endothelial dysfunction induced by DOT1L and artery endothelial cells isolated from atherosclerotic arteries of patients, endothelial function and apoptosis are measured to elucidate that DOT1L accelerates endothelial dysfunction. Lentiviral DOT1L overexpression or knockdown will be used. 2) To elucidate the role of DOT1L in vascular remodeling, vascular dysfunction was induced by renal hypertension in endothelium-specific DOT1L knockout mice. Vascular function and endothelial apoptosis et al. in artery are compared by combining lentiviral-mediated DOT1L overexpression/knockdown. We would confirm the events in atherosclerotic arteries of patients; 3) Atherosclerosis is induced by high fat diet in ApoE-/- mice and the plaque size，fibrous cap stability, endothelial function in atherosclerotic vascular are measured. A positive correlation of DOT1L expression with atherosclerosis and a key role of DOT1L in high fat diet-induced atherosclerosis are identified by combining lentiviral DOT1L overexpression/knockdown strategy. 4) In above-mentioned cell and animal models by various molecular biology techniques, we further verify that the epigenetic mechanism is that DOT1L regulates FoxO transcriptional activity, which promotes endothelial dysfunction and vascular remodeling. These findings will suggest that DOT1L serves as a novel therapeutic target for treating endothelial dysfunction-mediated cardiovascular diseases.

血管内皮细胞（VEC）功能紊乱是血管重构和功能失衡的始动环节。前期已发现动脉斑块中组蛋白甲基转移酶DOT1L上调并伴随VEC功能紊乱；在氧化低密度脂蛋白（oxLDL）诱导的VEC功能紊乱和血管内膜损伤中同样伴有DOT1L增加，沉默或抑制DOT1L可逆转其功能紊乱表型。但DOT1L在VEC功能紊乱及血管重构中的作用和表观调控机制仍不明确，据此提出“DOT1L通过组蛋白甲基化修饰引起VEC功能紊乱表型，促进血管重构的发生发展”的假说。本项目拟用体外VEC功能紊乱和临床动脉粥样硬化血管、体内VEC特异性DOT1L-/-小鼠上建立高血压模型及ApoE-/-小鼠动脉粥样硬化模型，结合慢病毒DOT1L沉默/过表达，阐明DOT1L促进VEC功能紊乱及血管重构；并明确DOT1L调控FoxO转录活性引起VEC功能紊乱的表观调节机制，为心血管疾病的防治提供新的思路。

血管内皮细胞（VEC）功能紊乱是血管重构和功能失衡的始动环节。前期已发现动脉斑块中组蛋白甲基转移酶DOT1L上调并伴随VEC功能紊乱；在氧化低密度脂蛋白（oxLDL）诱导的VEC 功能紊乱和血管内膜损伤中同样伴有DOT1L增加，沉默或抑制DOT1L可逆转其功能紊乱表型。但 DOT1L在VEC功能紊乱及血管重构中的作用和表观调控机制仍不明确，据此提出“DOT1L通过组蛋白甲基化修饰引起VEC功能紊乱表型，促进血管重构的发生发展”的假说。在项目研究过程中探索了DOT1L对内皮功能影响，但基于体内研究的表型没有明显改善，因此我们探究了DOT1L在心脑血管疾病的主要作用，主要研究内容：1）发现DOT1L参与脑缺血引起的神经元凋亡，缺血后DOT1L表达增加，然后激活RIPK1/Caspase 8依赖性凋亡信号通路，导致神经元死亡。抑制或敲低DOT1L能够通过抑制缺血中风后小鼠脑中RIPK1介导的细胞凋亡来防止缺血性神经元死亡；2）明确DOT1L参与缺血引起的心肌纤维化反应，发现在心肌缺血后增加DOT1L的表达上调，抑制和敲低DOT1L可明显改善心功能，并减轻心肌纤维化反应，进一步的研究发现在Ang II诱导的心肌成纤维细胞上，DOT1L表达的上调可以使H3K79me3结合在转录因子FoxO3a启动子区上，促进其转录表达；3）发现DOT1L是肺纤维化发病机制中的表观遗传修饰因子，揭示了通过调节Jag1启动子处的H3K79三甲基化、激活Notch信号并影响促纤维化蛋白的表达以加速肺纤维化。本项目明确了DOT1L在心脑血管疾病中，包括脑缺血和心肌纤维化的主要作用，并拓展了DOT1L在肺纤维化中的作用，为心脑血管疾病的防治提供新的治疗策略理论基础。