AGEs通过RAGE-mDia-1-Src信号通路调控“VEGF抵抗”

Vascular endothelial growth factor (VEGF) resistance has been demonstrated as a molecular basis for the impaired angiogenesis in diabetes mellitus. The molecular mechanism underlying VEGF resistance is not fully understood. Advanced glycation end products (AGEs) and receptor for AGEs (RAGE) contribute to the pathogenesis of impaired angiogenesis in diabetes mellitus. However, no previous studies have implicated the role of AGEs in VEGF resistance. Our preliminary results demonstrated that AGEs-BSA significantly decreased VEGF-induced phosphorylation of VEGF receptor-2 (VEGFR-2) and endothelial cell migration, which indicates that AGEs may inhibit VEGF-induced angiogenesis. Furthermore, we found that AGEs-BSA induced RAGE-mDia-1-Src complex formation in endothelial cells. And previous reports have shown that the recruitment of Src to VEGFR-2 is required for VEGF/VEGFR-2 signaling activation. Taken together, we hypothesize that AGEs prevents VEGF signaling and VEGF-induced angiogenesis by sequestering Src through RAGE-mDia-1-Src signaling pathway. In this study, immunoprecipitation, RNA interference and hindlimb ischemia model will be used to investigate the effects of AGEs on VEGF signaling and VEGF-induced angiogenesis. We anticipate that the proposed studies will explain the molecular mechanism of VEGF resistance and provide a novel therapeutic target for treatment of impaired angiogenesis in people with diabetes mellitus.

“VEGF抵抗”是糖尿病血管新生障碍的重要原因，其机制远未阐明。AGEs及其受体RAGE参与了糖尿病血管新生障碍的发生，但AGEs对“VEGF抵抗”的影响尚未见报道。申请人预实验发现：AGEs-BSA显著降低了VEGF介导的VEGFR-2的磷酸化和内皮细胞迁移，这提示AGEs可能抑制了VEGF信号的激活；我们进一步证实AGEs-BSA介导RAGE-mDia-1-Src形成复合物，而VEGFR-2对Src的招募是VEGF信号激活的关键。由此我们假设：AGEs通过RAGE-mDia-1-Src通路剥夺VEGF信号激活时VEGFR-2对Src的招募，从而抑制VEGF信号及其介导的血管新生，导致“VEGF抵抗”。本项目拟利用免疫共沉淀、RNA干扰、下肢缺血模型等手段探讨AGEs对VEGF信号及其介导血管新生的影响，旨在阐明“VEGF抵抗”产生的原因和机理，为糖尿病血管新生障碍的防治提供理论依据。

项目背景：糖尿病时血管内皮生长因子（VEGF）信号激活障碍是糖尿病血管新生不足的重要原因，其机制仍未阐明，晚期糖基化终产物（AGEs）可能在其中发挥重要作用。本研究旨在探讨AGEs对VEGF信号及其介导血管新生的影响及机制。 .研究内容：以体外培养的内皮细胞和整体动物为研究对象，探讨AGEs对VEGF信号激活及其介导血管新生的影响；并从VEGF受体-2（VEGFR-2）和c-Src相互作用的角度，阐明AGEs调控VEGF信号的分子机制。 .重要结果：本研究在细胞水平证实AGEs抑制VEGF介导的内皮细胞迁移和管腔形成，并抑制VEGF介导的VEGFR-2和Akt激活以及VEGFR-2-c-Src复合物形成；AGEs介导RAGE-c-Src复合物的形成，并因此剥夺VEGFR-2对c-Src的招募，抑制VEGFR-2和c-Src的相互作用，而RAGE特异性抗体解除了AGEs对VEGF信号的抑制。在动物水平，利用RAGE基因敲除（Rage-/-）小鼠制备糖尿病模型和下肢缺血模型发现，缺血诱导正常小鼠和糖尿病小鼠VEGF表达都显著增多，但糖尿病WT小鼠缺血侧血管新生显著降低，而与糖尿病WT小鼠相比，糖尿病Rage-/-小鼠缺血侧下肢血流灌注和血管新生显著恢复，提示：糖尿病时，AGEs可能通过RAGE抑制VEGF信号的激活，导致“VEGF抵抗”，从而引起血管新生障碍。.关键数据：AGEs抑制VEGF介导内皮细胞迁移和管腔形成；AGEs抑制VEGF信号激活；AGEs抑制VEGFR-2和c-Src相互作用；AGEs介导RAGE-c-Src复合物形成；抗-RAGE抗体解除AGEs对VEGF信号的抑制；RAGE基因敲除挽救了糖尿病导致的缺血侧下肢血管新生减少。.研究结论：糖尿病时AGEs形成显著增多，AGEs通过介导RAGE-c-Src复合物形成剥夺了VEGFR-2对c-Src的招募，解偶联VEGFR-2和c-Src的相互作用，抑制VEGF信号的激活及VEGF介导的血管新生，导致“VEGF抵抗”。.科学意义：本项目的研究结果提示AGEs可能是导致“VEGF抵抗”的关键因素，这为揭示糖尿病血管新生障碍的机制提供了新的着眼点和突破口，为临床上预防和治疗糖尿病缺血性病变提供理论依据。.