TLR4/p300通路介导的MAP4K4表观遗传修饰在糖尿病视网膜病变中的作用及机制研究

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population. Despite the benefits gained by anti-VEGF therapy, still a considerable part of patients fail to acquire satisfactory response to it, demonstrating the underlying existence of VEGF-independent pathways.. The imperative role of TLR4 in DR has been illustrated by previous study. TLR4 regulates the expression of downstream genes by recruiting acetyltransferase and transcription factors. A great number of GC sites are found in the promoter region of MAP4K4, which can be activated by inflammation-related pathway and regulate endothelial cells migration and intercellular tight junction formation. Our preliminary work indicated that MAP4K4, up-regulated by TLR4 pathway, promoted the migration and angiogenesis of Human Primary Retinal Microvascular Endothelial Cells (HRMECs), which was probably through epigenetic modifications without the involvement of VEGF.. Therefore, we hypothesize that activated TLR4 pathway may promote the expression of MAP4K4 through the increased H3K9ac modification and decreased DNA methylation level in the promoter region as the result of the recruitment of p300 and NF-κB, thus leading to the functional changes in HRMECs and contributing to the pathogenesis of macular edema and angiogenesis in DR.. Using cell co-culture and animal models, we will investigate the function and mechanisms of MAP4K4 in DR via various techniques including pyrosequencing and ChIP, thereby providing new treatment targets to DR.

糖尿病视网膜病变（DR）是工作人群首位致盲眼病，抗VEGF治疗已取得很好疗效，但部分患者治疗反应不佳，提示有不依赖VEGF的通路存在。文献显示TLR4在DR发展中起重要作用，可通过富集乙酰基转移酶和转录因子调控下游基因表达。MAP4K4启动子区含大量CG位点，可由炎症通路激活，调节血管内皮细胞迁移及细胞间紧密连接。我们前期研究显示，人视网膜微血管内皮细胞内MAP4K4可由TLR4介导，调节细胞迁移、成管能力，且其表达受表观遗传修饰酶类调控，但不受VEGF影响。据此提出假设：TLR4可通过富集p300和NF-κB，增加MAP4K4启动子区H3K9ac修饰，降低DNA甲基化水平，促进MAP4K4表达，引起视网膜血管内皮细胞功能变化，在DR黄斑水肿及新生血管生成中起作用。本项目将建立细胞共培养模型、动物模型，运用焦磷酸测序，CHIP等技术，研究MAP4K的功能及调控机制。为DR提供新的治疗策略。

糖尿病视网膜病变（DR）是工作人群首位致盲眼病，抗VEGF治疗已取得很好疗效，但部分患者治疗反应不佳，提示有不依赖VEGF的通路存在。.本课题通过体外实验证实，在人视网膜微血管内皮细胞（HRMECs）中，TLR4的激活能加强HRMECs的增殖、形变、迁移、成管能力，减弱细胞间连接，增加血管内皮细胞单分子层的渗透性，但对细胞凋亡没有影响。TLR4抑制剂TAK-242能抑制脂多糖（LPS）诱导的TLR4表达增加，拮抗LPS引起的HRMECs功能变化。丝裂原活化蛋白激酶4（MAP4K4）对HRMECs增殖，迁移、成管能力，及细胞间粘附连接，内皮细胞单分子层渗透性有调节作用，但对细胞凋亡无影响。在HRMECs中，MAP4K4的表达及功能受Toll样受体4（TLR4）调节。TLR4-MAP4K4 为非VEGF依赖通路。MAP4K4基因干扰和抗VEGF药物ranibizumab在抑制异常血管新生和改善屏障功能方面具有相加或协同作用，这种协同作用在病理条件下更明显。.课题进一步通过体内实验证实，TLR4和MAP4K4在氧诱导视网膜病变（OIR）模型小鼠及db/db糖尿病小鼠视网膜内表达增加，TAK-242玻璃体腔内注射可逆转这一增加。TAK-242和Ranibizumab对减少小鼠OIR模型中视网膜异常血管新生，减少无血管区面积，增加血管密度均有治疗作用，且TAK-242与Ranibizumab在治疗小鼠OIR模型中有联合效果。db/db糖尿病小鼠视网膜中TLR4被抑制后，MAP4K4蛋白表达水平也随之明显下降；同时小鼠视网膜中紧密、粘附和缝隙连接蛋白表达水平上调，分布完整性提高，进而iBRB功能增强。同时，抑制TLR4-MAP4K4信号通路后对糖尿病状态下视网膜新生血管形成也具有抑制作用。.综上，本课题证实TLR4-MAP4K4通路为非VEGF依赖通路，此通路在糖尿病视网膜病变中被激活，在视网膜异常血管新生中及血管内皮间连接破坏中起到关键作用，为糖尿病视网膜病变的治疗提供新的治疗靶点。