The calcineurin/NFAT signaling axis in diabetic retinopathy pathogenesis

描述（由申请人提供）：细胞钙稳态的破坏是糖尿病引起的高血糖的公认后果。在某些视网膜细胞类型中，细胞内钙升高会刺激致病性反应，导致视网膜白质停滞和血管高通透性——这是非增殖性糖尿病视网膜病变（DR）的两个视力威胁标志。因此，作为关键的细胞内第二信使，Ca2+可能在DR中高糖诱导的血管病理中发挥重要作用，但介导致病性Ca2+信号的下游信号通路尚不清楚。最近的证据表明，活化T细胞核因子（NFAT）在连接高血糖诱导的Ca2+通量和这些病理细胞反应的途径中起关键作用。升高的细胞内钙触发钙调神经磷酸酶（CN）介导的NFAT激活，导致其核易位。NFAT通过与其他转录因子的异二聚体结合，将高血糖诱导的钙通量与其他生化信号整合。这些异二聚体复合物以一种特定于多种生化线索的方式刺激靶基因的转录。例如，在视网膜<s:1> ller细胞中，NFAT通路的激活导致编码HIF-1a、COX-2和VEGF的mRNA的产生，导致视网膜VEGF水平升高，从而诱导血管高通透性和视网膜水肿。我们的初步数据表明，大多数高糖诱导的VEGF依赖于cn。同样，在视网膜微血管内皮细胞（RMEC）中，NFAT转录活性导致炎症细胞因子CXCL2、VCAM-1、ICAM-1、IL-6和IL-8编码mRNA的产生，从而促进白细胞停滞和高通透性。我们的初步数据支持Ca2+， CN， NFAT和炎症介质的产生和分泌之间的联系。为了了解CN/NFAT信号通路在DR中的作用，必须在参与糖尿病反应的特定视网膜细胞中对其信号通路进行表征：<s:1> ller细胞产生生长因子和细胞因子以响应细胞内钙的增加，而RMEC同时产生并响应这些因子。利用这两种细胞类型的原代培养，将系统地研究高血糖诱导的Ca2+通量、CN激活、NFAT激活以及随后的细胞反应之间复杂的相互作用。这些研究将导致识别信号中间体，可能作为适当的治疗靶点。为了更好地了解CN/NFAT信号在视网膜血管疾病中的作用，我们提出以下研究：1)比较高糖和/或VEGF在高选择性CN和NFAT抑制剂存在下对小鼠或人视网膜组织分离细胞的影响；2)高糖对野生分离细胞相关影响的比较

DESCRIPTION (provided by applicant): Disruption of cellular calcium homeostasis is a recognized consequence of diabetes-induced hyperglycemia. In certain retinal cell types, elevated intracellular calcium stimulates pathogenic responses that result in retinal leukostasis and vascular hyperpermeability - two vision-threatening hallmarks of non-proliferative diabetic retinopathy (DR). Thus, as a critical intracellular second messenger, Ca2+ may play an important role in high glucose-induced vascular pathology in DR, but the downstream signaling pathways through which pathogenic Ca2+ signals are mediated remain unknown. Recent evidence points to a critical role for Nuclear Factor of Activated T Cells (NFAT) in the pathway linking hyperglycemia-induced Ca2+ flux to these pathologic cellular responses. Elevated intracellular calcium triggers calcineurin (CN)-mediated activation of NFAT, leading to its nuclear translocation. NFAT integrates hyperglycemia-induced calcium flux with other biochemical signals by heterodimeric binding with other transcription factors. These heterodimeric complexes stimulate the transcription of target genes in a manner specific to multiple biochemical cues. For example, in retinal Müller cells, activation of the NFAT pathway leads to the generation of mRNA coding for HIF-1a, COX-2, and VEGF, resulting in elevated retinal VEGF levels, which induces vascular hyperpermeability and retinal edema. Our preliminary data indicate that the majority of high glucose induction of VEGF is CN-dependent. Likewise, in retinal microvascular endothelial cells (RMEC), NFAT transcriptional activity leads to the production of mRNA coding for the inflammatory cytokines CXCL2, VCAM-1, ICAM-1, IL-6, and IL-8, which promote leukostasis and hyperpermeability. Our preliminary data support the links between Ca2+, CN, NFAT, and production and secretion of inflammatory mediators. In order to understand the role of CN/NFAT signaling in DR, its signaling pathways must be characterized in the specific retinal cells involved in the diabetic response: Müller cells produce growth factor and cytokines in response to increases in intracellular calcium, and RMEC both produce and respond to these factors. Using primary cultures of these two cell types, the complicated interactions between hyperglycemia-induced Ca2+ flux, CN activation, NFAT activation, and consequent cell responses will be studied systematically. These studies will lead to the identification of signaling intermediates that may serve as appropriate therapeutic targets. We propose the following studies, aimed at a better understanding of the role of CN/NFAT signaling in retinal vascular disease: 1) comparisons of the effects of high glucose and/or VEGF in the presence of highly selective CN and NFAT inhibitors on cells isolated from mouse or human retinal tissues; 2) comparisons of the relevant effects of high glucose on cells isolated from wild type and NFAT-/- mice or of the effects of high glucose and/or VEGF on NFAT isoform-directed siRNA- treated cells; and 3) determination of the therapeutic efficacy of highly specific CN and NFAT inhibitors in STZ- treated mice. The STZ model has been selected for its specific relevance to non-proliferative DR.