Targeting TGFB signaling to treat ocular neovascular disease

项目概要/摘要扎尔卡达 糖尿病视网膜病变和老年性黄斑病变等眼部疾病患者的过度血管生成 退化，导致美国大部分严重视力丧失。目前的治疗方法不适合或 所有患者均获得成功，需要额外的方法来管理眼部新生血管。最近的工作从 Eichmann实验室认为，血管抑制性转化生长因子-β（TGFβ）信号的增加， 内皮细胞（ECs）可以成功地抑制血管生成。虽然以前的研究认为， 下游TGFβ信号传导效应物SMAD 2和SMAD 3在TGFβ受体活化的下游协同作用， 任一SMAD的EC特异性消融对EC行为有不同的影响（候选人未发表的数据）。这 这意味着需要选择性操纵TGFβ下游信号臂，以最大化 治疗潜力在AIM 1中，我们将实施条件小鼠模型来剖析内皮细胞的作用。 SMAD 2和3在视网膜血管生成中的作用。此外，我们将确定SMAD的特定基因靶点， 下一代RNA测序的EC。这项分析将揭示一些新的SMAD转录因子， 对血管功能有潜在影响的靶点，其中一些将被证明适合于治疗 干预目的2将重点研究神经纤毛蛋白1（Neuropilin 1，NRP 1）的功能 EC中的信号。将采用条件小鼠模型和体外方法来描述特定的 NRP 1对TGFβ信号转导的调节作用。最后，AIM 3将整合以前目标的结果 血管性眼病临床前模型的转化实验。这个项目将破译 生理和病理条件下内皮TGFβ信号在萌芽血管生成中的作用， 用眼睛视网膜作为模型。因此，这一建议完全符合国家眼科研究所的使命。 此申请还包括一个详细的培训计划，将促进候选人的过渡到一个校长 调查员职位。候选人在血管生物学方面有扎实的背景，并一直在此基础上发展 在过去两年中，通过学习新的方法和优化所需的协议， 完成这项研究计划。该奖项的K99阶段将在博士的指导下进行。 他是血管形成和视网膜血管生成的公认领导者。额外指引及 Nicoli博士和Adelman博士将提供支持，他们是比较mRNA转录组的专家 脉络膜新生血管形成的分析和转化模型。这项建议将付诸实施 耶鲁大学医学院心血管医学部，提供多种培训机会 以课程、研讨会和讲座的形式，以及跨学科伙伴关系的大量机会。 总的来说，这一建议不仅将加强我们对TGFβ介导的发芽调控的理解， 血管生成，但也将提供一个很好的指导框架，以创造未来的独立研究人员。

PROJECT SUMMARY/ABSTRACT G. Zarkada Excessive angiogenesis in patients with ocular diseases, including diabetic retinopathy and age-related macular degeneration, causes the majority of severe vision loss in the US. Current treatments are not appropriate or successful for all patients, calling for additional means to manage ocular neovascularization. Recent work from the Eichmann lab suggests that increased angio-suppressive transforming growth factor-β (TGFβ) signaling in endothelial cells (ECs) could successfully restrict angiogenic sprouting. While previous studies accept that downstream TGFβ signaling effectors SMAD2 and SMAD3 cooperate downstream of TGFβ receptor activation, EC-specific ablation of either SMAD has different effects on EC behavior (unpublished data by candidate). This implies that selective manipulation of TGFβ downstream signaling arms is required in order to maximize therapeutic potential. In AIM 1 we will implement conditional mouse models to dissect the roles of endothelial SMAD2 and 3 during retinal angiogenesis. In addition, we will identify specific gene targets of either SMAD in ECs by Next-Generation RNA Sequencing. This analysis will reveal a number of new SMAD transcriptional targets with potential influence on vascular function, some of which will prove to be suitable for therapeutic intervention. AIM 2 will focus on the functions of Neuropilin 1 (NRP1), which is a powerful suppressor of TGFβ signaling in ECs. Conditional mouse models and in vitro approaches will be employed to delineate the specific regulatory effects of NRP1 on TGFβ signaling. Finally, AIM 3 will integrate the results from the previous aims with translational experimentation of preclinical models of vascular eye disease. This project will decipher the role of endothelial TGFβ signaling in sprouting angiogenesis during physiological and pathological conditions, using the eye retina as a model. Thus, this proposal is fully aligned with the mission of the National Eye Institute. This application also includes a detailed training program that will facilitate the candidate’s transition to a principal investigator position. The candidate has a solid background in vascular biology and has been building upon it during the past two years by learning new methodologies and by optimizing the protocols required for the fulfillment of this research proposal. The K99 phase of this award will be conducted under the mentorship of Dr. Eichmann, who is an established leader in vascular patterning and retinal angiogenesis. Additional guidance and support will be provided by Dr. Nicoli and Dr. Adelman, who are experts in comparative mRNA transcriptome profiling and translational models of choroidal neovascularization respectively. The proposal will be carried out at the Cardiovascular Medicine Division of Yale School of Medicine, which offers numerous training possibilities in the form of courses, seminars and lectures, as well as plentiful opportunities for cross-disciplinary partnerships. Overall this proposal will not only enhance our understanding on TGFβ-mediated regulation of sprouting angiogenesis, but will also provide an excellent mentoring framework to create a future independent researcher.