Role of intercellular communication in pathogenesis of diabetic retinopathy

描述(申请人提供)：这个项目的总体目标是测试假设，即在糖尿病视网膜病变中，高糖/糖尿病引发的Cx43表达减少和随后的细胞-细胞通讯改变导致细胞死亡和视网膜毛细血管内血管动态平衡的破坏。这一假说是基于以下发现：高糖降低微血管内皮细胞和视网膜周细胞中Cx43的表达，并损害缝隙连接细胞间的通讯。我们目前的研究表明，Cx43表达减少会触发细胞凋亡(手稿正在修订中，IOVS)。总体的工作假说是，Cx43的表达减少会触发血管细胞死亡，这是与糖尿病视网膜病变的发展相关的一种显著的早期病变，而糖尿病视网膜病变反过来又减少了视网膜血管细胞内的细胞间通讯，最终破坏了血管的动态平衡。众所周知，视网膜血管细胞死亡是通过细胞凋亡发生的，但在糖尿病视网膜病变的发展过程中，细胞凋亡是如何被触发的还不清楚。初步数据表明，血管细胞，即内皮细胞-内皮细胞、内皮细胞-周细胞和周细胞-周细胞之间的通讯对它们的生存是必不可少的，细胞-细胞通讯的中断可能会触发细胞凋亡，并干扰它们通过细胞连接蛋白(Cx)通道形成功能单位的作用。这一建议的重点是确定(I)视网膜内皮细胞和周细胞Cx43表达减少的影响，(Ii)高糖诱导的过度ECM合成是否调节视网膜内皮细胞和周细胞Cx43的表达，以及(Iii)Cx43表达减少是否导致视网膜内皮细胞紧密连接功能障碍。将利用多种细胞生物学、分子生物学和生化技术，包括反义寡核苷酸介导的基因表达特异性下调、刮擦负载染料转移技术、荧光显微镜和Western印迹方法，研究高糖抑制Cx43表达和细胞-细胞通讯减少对视网膜细胞存活和功能的影响。这些研究结果将有助于更好地了解视网膜血管细胞中Cx43表达改变和缝隙连接通道背后的细胞间通讯，以及它们在糖尿病视网膜病变相关的血管内稳破坏中的作用。该机构的一项重要使命是寻找治疗和预防糖尿病视网膜病变引起的并发症。这项拟议的项目有望提供重要的发现，有助于更好地了解糖尿病视网膜病变的发病机制。与公共卫生相关：目前，糖尿病视网膜病变还没有治愈，这是美国劳动年龄人群失明的主要原因。预计该项目的发现将为测试视网膜血管细胞丢失和毛细血管渗漏的新机制提供有价值的见解，这两个机制是糖尿病视网膜病变发展的两个关键步骤。

DESCRIPTION (provided by applicant): The overall goal of this project is to test the hypothesis that reduced Cx43 expression and subsequent altered cell-cell communication triggered by high glucose/diabetes leads to cell death and breakdown of vascular homeostasis in the retinal capillaries in diabetic retinopathy. The hypothesis is based on findings that high glucose reduces Cx43 expression in microvascular endothelial cells and retinal pericytes and compromises gap junction intercellular communication. Our current study showed reduced Cx43 expression triggers apoptosis (manuscript under revision, IOVS). The overall working hypothesis is that reduced Cx43 expression triggers vascular cell death, a prominent and early lesion associated with the development of diabetic retinopathy, which in turn, reduces cell-cell communication in the retinal vascular cells and ultimately disrupts vascular homeostasis. Retinal vascular cell death is known to occur by apoptosis but it is unknown how apoptosis is triggered during the development of diabetic retinopathy. Preliminary data suggests that communication between vascular cells, that is endothelial cell-endothelial cell, endothelial cell-pericyte, and pericyte-pericyte is essential for their survival, and that disruption in cell-cell communication may trigger apoptosis and interfere with their role to form a functional unit via the connexin (Cx) channels in cell junctions. The specific focus of this proposal is to determine (i) the effect of reduced Cx43 expression in retinal endothelial cells and pericytes, (ii) whether high glucose-induced excess ECM synthesis modulates Cx43 expression in retinal endothelial cells and pericytes, and (iii) whether reduced Cx43 expression induces tight junction dysfunction in the retinal endothelial cells. A variety of cell biological, molecular biological and biochemical techniques including antisense oligo mediated specific downregulation of gene expression, scrape load dye transfer technique, fluorescence microscopy and Western blot method will be used for studying the consequence of high glucose-induced inhibition of Cx43 expression and reduced cell-cell communication on retinal cell viability and function. Findings from these studies will provide a better understanding of cell-cell communication underlying altered Cx43 expression and gap junction channels in retinal vascular cells and their role in breakdown of vascular homeostasis associated with diabetic retinopathy. An important mission of the agency is to find a cure and prevent complications arising from diabetic retinopathy. The proposed project is expected to provide important findings that can help in better understanding the pathogenesis of diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Currently, there is no cure for diabetic retinopathy, the leading cause of blindness in the working age Americans. It is expected that the findings from this project would provide valuable insight towards testing a novel mechanism underlying retinal vascular cell loss and capillary leakage, the two critical steps in the development of diabetic retinopathy.