Molecular Targets of Corneal Anti-angiogenesis

描述（由申请人提供）：世界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致病性生长。正常角膜创面愈合不涉及血管生成。因此，确定防止自毁性血管生成过程的分子机制对角膜稳态、再生和移植具有重要意义。一种新的生化特征阻止角膜基质细胞参与致病性纤维化修复过程，最近被认为与进化保守的泛素蛋白酶体途径（UPP）有关。UPP调节蛋白质质量控制、炎症转录因子nf - κ B活化、细胞周期、分化和抗原呈递。我们研究了dpp介导的遗传程序控制角膜血管生成表型的假设。我们发现，在血管生成刺激物激活和血管化角膜损伤愈合过程中，血管内皮细胞中的UPP上调。这种UPP激活模式被有效的小分子血管生成抑制剂减弱，withaferin a。为了证明withaferin a的蛋白质结合靶点介导了这种药物的抗血管生成机制，我们生成了withaferin a的生物素化类似物。我们已经成功地利用了这种亲和试剂并分离了withaferin a的结合靶点。在这个R01提案中，我们计划利用伤口愈合的血管生成和非血管生成模型来定义upp驱动的角膜愈合机制，并验证新发现的抗血管生成分子靶点。在这些研究中，withaferin A既可以作为药理学试剂，也可以作为其结合蛋白靶功能的细胞渗透性探针。具体来说，我们将(1)表征药物针对UPP血管生成激活的抑制作用模式，(2)研究UPP的关键成分作为药物活性的介质，以及(3)研究aferin-protein靶标缺陷小鼠模型，以验证WFA角膜抗血管生成机制中药物-蛋白相互作用的必要性。我们相信这些研究集中在UPP和withaferin A的目标上，将使我们能够实现发现新的治疗方法来控制血管生成并促进其稳态的长期目标。

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.