Molecular Targets of Corneal Anti-angiogenesis

描述（由申请人提供）：全世界角膜失明的主要原因是血管生成，这是从现有血管系统中致病性生长新血管。角膜中的正常伤口愈合不涉及血管生成。因此，鉴定负责防止自我破坏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。一种新的生物化学特征，防止角膜基质细胞参与致病性纤维化修复过程，最近已被链接到进化保守的泛素蛋白酶体途径（UPP）。UPP调节蛋白质质量控制、炎性转录因子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，UPP在血管内皮细胞中被血管生成刺激物激活时以及在体内血管化角膜的损伤愈合过程中上调。这种UPP激活模式可被有效的小分子血管生成抑制剂withaferin A减弱。为了证明醉茄素A的蛋白质结合靶标介导该药物的抗血管生成机制，我们产生了醉茄素A的生物素化类似物。我们已经成功地利用这种亲和试剂，并分离出的withaferin结合目标。在该R 01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，并验证新鉴定的抗血管生成分子靶标。对于这些研究，醉茄素A将作为药理学试剂和其结合蛋白靶标功能的细胞可渗透探针。具体而言，我们将（1）表征药物对靶向UPP的血管生成激活的抑制作用模式，（2）研究UPP的关键组分作为药物活性的介质，以及（3）研究withaferin-蛋白靶缺陷小鼠模型，以验证WFA的角膜抗血管生成机制中药物-蛋白相互作用的要求。我们相信，这些研究集中在UPP和醉茄素A的目标，将使我们能够达到我们的长期目标，发现新的治疗方法，以控制血管生成和促进其稳态。

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.