ROLE OF VEGF SIGNALING IN CONE AND ROD PHOTORECEPTOR SURVIVAL

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 A.拟议的具体目标是： 1：研究VEGF信号在视锥细胞存活中的作用。 2：检测VEGF信号在视杆细胞存活中的作用。 B。研究和结果 1)视锥细胞系中VEGF信号传导的体外分析。 由于视锥细胞仅由3%的光感受器组成，因此不可能使用整个视网膜匀浆来研究生物化学事件。因此，我们一直使用现有的视锥细胞系（661 W）。661 W细胞表达VEGF、VEGF受体1（R1）和VEGF受体2（R2）。由于在急性缺氧的实验模型中观察到显著的（90%）视网膜感光细胞死亡，我们使用缺氧模型，并用缺氧的化学诱导剂氯化钴（CoCl 2）和受体特异性抑制剂处理细胞。虽然CoCl 2诱导细胞死亡，但进一步抑制VEGFR 2导致细胞死亡增加。我们还观察到VEGFR 1水平轻微但显著增加。VEGFR 1的抑制挽救了缺氧诱导的细胞死亡。我们的研究结果还表明，VEGFR 2和AKT（及其下游靶点）在缺氧条件下被激活，表明VEGFR 2/AKT存活途径是缺氧条件下视锥细胞存活所必需的，而VEGFR 1的激活对视锥细胞存活起负调控作用。这些结果形成了一个工作模型，我们的机制调查锥生存。 2)糖尿病诱导的光感受器死亡的急性模型研究。 因为糖尿病引起的视网膜细胞的变化是缓慢的。我们决定使用急性模型，在小鼠中用氯化钴产生不同程度的视网膜缺血。使用不同浓度的氯化钴来确定在小鼠中产生缺血诱导的光度计死亡的合适条件。对动物进行视网膜电图（ERG）功能分析和视网膜切片的形态学分析。我们已经确定了不同程度的光感受器损失的条件。该模型模拟了糖尿病的晚期阶段，可以被认为是“急性糖尿病动物模型”，这将使我们能够在2周内解决体内问题。因此，这些测定将用于我们的视杆或视锥光感受器特异性VEGFR 2敲除小鼠的体内分析。 3)条件性VEGFR 2敲除小鼠 为了进行Cre/lox为基础的条件基因表达的研究，在锥和杆光感受器，我已经产生了锥或杆特异性Cre小鼠。它们已成功地用于一些研究。将floxed VEGF-R2小鼠与视杆或视锥光感受器特异性Cre小鼠交配以产生条件性VEGF-R2敲除小鼠。我们正在生产足够的动物进行表型分析。少数视杆细胞特异性VEGF-R2敲除小鼠经受缺血。我们的初步研究结果表明，在缺血条件下，VEGF-R2的损失加速视杆细胞死亡。 C.意义 抗VEGF治疗已成为糖尿病视网膜病变血管并发症的主要治疗策略。然而，VEGF也可能是视网膜神经元（包括光感受器）的生存因素。揭示VEGF在视网膜神经元存活中的作用对于长期抗VEGF治疗的安全性至关重要。此外，重要的是要确定VEGF信号传导中间体和它们的亚型，可能只在神经元存活中发挥作用，但在血管渗漏中不起作用，这将对糖尿病患者的视力保护非常有用。这些分子将成为糖尿病视网膜神经元存活的潜在药物。 D.未来计划 在接下来的一年里，我们将专注于确定其他VEGF信号成分的作用，如VEGF-B及其下游VEGF共受体，神经纤毛蛋白1和2在光感受器的生存。我们还将在视杆细胞或视锥细胞感光细胞特异性VEGFR 2敲除小鼠中进行功能分析。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A. The proposed Specific Aims are: 1: To examine the role of VEGF signaling in cone photoreceptor survival. 2: To examine the role of VEGF signaling in rod photoreceptor survival. B. Studies and Results 1) In vitro analysis of VEGF signaling in a cone cell line. Since cones only consist of 3 percent of photoreceptors, it is not possible to investigate the biochemical events using whole retinal homogenates. Therefore, we have been using an existing cone cell line (661W). 661W cells expressed VEGF, VEGF receptor 1 (R1), and VEGF receptor 2 (R2). As significant ( 90%) retinal photoreceptor cells death was observed in experimental model of acute hypoxia, we used a hypoxia model and treated the cells with chemical inducer of hypoxia cobalt chloride (CoCl2) and receptor-specific inhibitors. While CoCl2 induces cell death, further VEGFR2 inhibition leads to an increased cell death. We also observed a slight but significant increase of VEGFR1 levels. Inhibition of VEGFR1 rescued hypoxia-induced cell death. Our results also indicated that VEGFR2 and AKT (and its down stream target) were activated under hypoxic conditions, suggesting that VEGFR2/AKT survival pathway is required for cone photoreceptor survival under hypoxia, which is negatively regulated by VEGFR1 activation. These results formed a working model for our mechanistic investigation for cone survival. 2) An acute model for studying diabetes-induced photoreceptor death. As diabetes-induced alternations in retinal cells are slow. We decided to use an acute model that produces various degree of retinal ischemia with cobalt chloride in mice. Various concentrations of cobalt chloride were used to identify suitable conditions to produce ischemia-induced photometer death in mice. The animals were subjected to functional analysis with electroretinography (ERG) and morphological analysis in retinal sections. We have identified conditions for various degrees of photoreceptor loss. This model mimics late stage of diabetes and can be regarded as "acute diabetic animal model" which will allows us to address questions in vivo within 2 weeks. Therefore, these assays will be used in our in vivo analysis of our rod or cone photoreceptor-specific VEGFR2 knockout mice. 3) Conditional VEGFR2 knockout mice In order to perform Cre/lox-based conditional gene expression studies in cone and rod photoreceptors, I have generated cone- or rod-specific Cre mice. They have been successfully used in a number of studies. The floxed VEGF-R2 mice were mated with rod or cone photoreceptor-specific Cre mice to generate the conditional VEGF-R2 knockout mice. We are in the process of generating sufficient animals for phenotypic analysis. A few rod-specific VEGF-R2 knockout mice were subjected to ischemia. Our preliminary results indicated that loss of VEGF-R2 accelerated rod photoreceptor death under ischemic conditions. C. Significance Anti-VEGF treatments have become a major therapeutic strategy for vascular complication in diabetic retinopathy. However, VEGF may also be a survival factor for retinal neurons, including photoreceptors. Revealing the role of VEGF in retinal neuron survival is paramount to the safety of long-term anti-VEGF therapies. In addition, it is important to identify VEGF signaling intermediates and their isoforms that may play a role only in neuron survival but not in vascular leakage will be very useful for preserving vision in diabetes. These molecules will be potential agents for retinal neuron survival in diabetes. D. Future Plan In the next year, we will focus on identifying the role of other VEGF signaling components, such as VEGF-B and its downstream VEGF co-receptor, neuropilin 1 and 2 in the survival of photoreceptors. We will also perform functional analysis in rod or cone-photoreceptor-specific VEGFR2 knockout mice.