AMPK调控ALK1-Smad1/5信号通路抑制视网膜新生血管生成的功能研究

Retinal neovascularization (RNV) is a common cause of blindness. Accumulated evidence indicates the AMPK and ALK1 pathway both play an important role in angiogenesis, and ALK1 is closely related to retinal vascular development. Our previous studies have indicated that the activation of AMPK could inhibit angiogenesis induced by ALK1 ligand in HUVECs. And we have found a phosphorylation site modified by AMPK in ALK1 catalytic domain and this site is required for R-Smads coupling, presumably AMPK might regulate ALK1-Smad1/5 pathway through phosphorylating ALK1 and inhibit angiogenesis. To test the hypothesis, we will firstly observe the effects of AMPK on functional indicators such as retinal vascular morphology and ALK1-Smad1/5 signaling pathway by the AMPK constitutively active mutant, AMPK dominant negative mutant and ALK1 constitutively active mutant (ALK1-AAD) in rat retinal vascular endothelial cells and mice model of RNV. And then we will analyze the interaction of AMPK with ALK1 and phosphorylation site of ALK1 by Co-immunoprecipitation and mass spectrometry methods. After determining the phosphorylation site of ALK1, we will explore the effect of ALK1 phosphorylation on AMPK-mediated RNV inhibition using ALK1-AAD and ALK1-AAD with phosphorylation site mutant in vitro and in vivo. The proposed study will clarify the inhibitory roles of AMPK in RNV through regulating ALK1-Smad1/5 signaling pathway, and provide a new idea for the prevention and treatment of RNV-related diseases.

视网膜新生血管生成(RNV)是致盲的重要原因。研究显示AMPK和ALK1-Smad1/5通路在血管生成中有重要作用，且ALK1与视网膜血管发育密切相关。前期发现AMPK抑制ALK1配体诱导的血管生成，且在ALK1找到一个AMPK磷酸化位点，该位点是R-Smads耦联必需的，推测AMPK调控ALK1-Smad1/5通路抑制血管生成。据此，本课题以RNV为病理模型，通过制备AMPK永久活性和显性负性突变体、ALK1永久活性突变体（ALK1-AAD）视网膜血管内皮细胞与RNV小鼠，观察AMPK对视网膜血管形态等功能指标及ALK1-Smad1/5通路的影响；采用免疫共沉淀和质谱等方法确定AMPK与ALK1的相互作用及ALK1磷酸化位点；确定其位点后，在ALK1-AAD基础上制备其磷酸化位点突变体，探讨 ALK1磷酸化在AMPK抑制RNV中的作用。可望从新的视角阐明RNV分子机制，为其防治提供新思路

病理性血管生成与许多疾病密切相关，如肿瘤、糖尿病视网膜病变、老年性黄斑相关的脉络膜血管新生（CNV）等。目前靶向VEGF的药物在血管新生相关性疾病治疗中正显示其局限性。寻找新的抗血管生成药物靶点是该领域的重要科学问题之一。ALK1被证实是抗肿瘤血管生成的重要靶点，AMPK与肿瘤血管生成亦密切相关。本项目旨在探讨AMPK对ALK1介导的信号通路的调控作用及其在血管新生中的作用。本项目结果显示：1）metformin及AMPK其他激活剂均能抑制BMP9和ALK1组成性活化突变体（ALK1-AAD）诱导的Smad1/5的磷酸化和管腔形成，且metformin的效应能被AMPKα1显性负性突变体（AMPK-DN）所阻断；同时，AMPKα1组成性活化突变体（AMPK-CA）能产生AMPK激活剂类似的效应。2）体内实验显示metformin能抑制BMP9介导的血管生成及镭射诱导的CNV，且显著降低ALK1的表达。故本项目体内、外证实AMPK能够抑制ALK1介导的信号通路及血管生成，这将为metformin联合抗VEGF药物用于血管生成相关性疾病的治疗提供实验和理论依据。