Molecular Targets of Corneal Anti-angiogenesis

描述(申请人提供)：全球范围内角膜失明的主要原因是血管生成，这是现有血管系统中新血管的病理性生长。角膜的正常伤口愈合不涉及血管生成。因此，确定防止自毁血管生成过程的分子机制对角膜的稳态、再生和移植具有重要意义。最近，一种新的生物化学特征被认为与进化保守的泛素蛋白酶体途径(UPP)有关，该特征可防止角膜基质细胞参与病理性纤维化修复过程。UPP调控蛋白质质量控制、炎性转录因子核因子-kappaB的激活、细胞周期、分化和抗原提呈。我们研究了一种假说，即DPP介导的遗传程序控制角膜的血管生成表型。我们发现，在体内血管形成的角膜中，当血管生成刺激物激活和损伤愈合时，UPP在血管内皮细胞中上调。这种UPP激活模式可被有效的小分子血管生成抑制剂With aferin A减弱。为了证明With aferin A的蛋白结合靶点(S)介导了该药物的抗血管生成机制，我们合成了Witaferin A的生物素化类似物。我们成功地开发了这种亲和剂，并分离了Wifaferin结合靶点。在这份R01提案中，我们计划利用创伤愈合的血管生成和非血管生成模型来定义角膜的UPP驱动的愈合机制(S)，并验证新发现的抗血管生成的分子靶点。在这些研究中，With aferin A将既作为药理学试剂，又作为其结合蛋白靶标功能的细胞渗透性探针。具体地说，我们将(1)表征药物在靶向UPP血管生成激活上的抑制作用模式，(2)研究UPP的关键成分作为药物活性的介体，以及(3)利用Ferin-Protein靶向缺陷小鼠模型来验证WFA的角膜抗血管生成机制中药物-蛋白质相互作用的必要性。我们相信，这些针对UPP和维甲素目标的研究将使我们能够实现我们的长期目标，即发现控制血管生成和促进其动态平衡的新治疗方法。

DESCRIPTION (provided by applicant): The leading cause of corneal blindness world wide is from angiogenesis, which is the pathogenic growth of new blood vessels from an existing vasculature. Normal wound healing in the cornea does not involve angiogenesis. Therefore, identifying the molecular mechanisms that are responsible for preventing self- destructive angiogenic processes have important bearing on corneal homeostasis, regeneration, and transplantation. A novel biochemical feature preventing corneal stromal cells from participating in pathogenic fibrotic repair processes has recently been linked to the evolutionary conserved ubiquitin proteasome pathway (UPP). The UPP regulates protein quality control, inflammatory transcription factor NF-kappa B activation, cell cycle, differentiation and antigen presentation. We have investigated the hypothesis that a DPP-mediated genetic program controls the angiogenic phenotype of the cornea. We show that the UPP is upregulated in vascular endothelial cells when activated by angiogenic stimulators and during injury healing in vascularized corneas in vivo. This pattern of UPP activation is attenuated by the potent small molecule angiogenesis inhibitor, withaferin A. To demonstrate that a protein binding target(s) of withaferin A mediates this drug's anti-angiogenic mechanism, we generated a biotinylated analog of withaferin A. We have successfully exploited this affinity reagent and isolated the withaferin binding target. In this R01 proposal, we plan to exploit angiogenic and nonangiogenic models of wound healing to define the UPP-driven healing mechanism(s) of the cornea and validate newly identified molecular targets for anti-angiogenesis. For these investigations, withaferin A will serve both as a pharmacological agent and a cell permeable probe of its binding-protein target's function. Specifically, we will (1) characterize the drug's inhibitory mode of action on targeting the angiogenic activation of the UPP, (2) investigate key components of the UPP as mediators of the drugs activity, and (3) investigate withaferin-protein target-deficient mouse models to validate the requirement for drug-protein interaction in the corneal anti-angiogenic mechanism of WFA. We believe these investigations focused on the UPP and the target of withaferin A will enable us to reach our long-term objectives of discovering new therapeutic approaches to control angiogenesis and promote its homeostasis.