The calcineurin/NFAT signaling axis in diabetic retinopathy pathogenesis

描述（由申请方提供）：细胞钙稳态的破坏是糖尿病诱导的高血糖症的公认后果。在某些视网膜细胞类型中，升高的细胞内钙刺激致病反应，导致视网膜白细胞停滞和血管通透性过高-非增殖性糖尿病视网膜病变（DR）的两个威胁视力的标志。因此，作为关键的细胞内第二信使，Ca 2+可能在高糖诱导的DR血管病理学中发挥重要作用，但介导致病性Ca 2+信号的下游信号通路仍不清楚。最近的证据表明，活化T细胞核因子（NFAT）在将高血糖诱导的Ca 2+通量与这些病理性细胞反应联系起来的途径中起着关键作用。升高的细胞内钙触发钙调磷酸酶（CN）介导的NFAT活化，导致其核转位。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流与其他生化信号整合。这些异源二聚体复合物以对多种生化信号特异的方式刺激靶基因的转录。例如，在视网膜Müller细胞中，NFAT途径的激活导致编码HIF-1 a、考克斯-2和VEGF的mRNA的产生，导致视网膜VEGF水平升高，其诱导血管通透性过高和视网膜水肿。我们的初步数据表明，大多数高糖诱导的VEGF是CN依赖性的。同样，在视网膜微血管内皮细胞（RMEC）中，NFAT转录活性导致产生编码炎性细胞因子CXCL 2、VCAM-1、ICAM-1、IL-6和IL-8的mRNA，其促进白细胞停滞和高渗透性。我们的初步数据支持Ca 2+，CN，NFAT和炎症介质的产生和分泌之间的联系。为了了解CN/NFAT信号在DR中的作用，其信号通路必须在参与糖尿病反应的特定视网膜细胞中表征：Müller细胞产生生长因子和细胞因子以响应细胞内钙的增加，RMEC产生并响应这些因子。使用这两种细胞类型的原代培养物，高血糖诱导的Ca 2+通量，CN激活，NFAT激活，和随之而来的细胞反应之间的复杂的相互作用将被系统地研究。这些研究将导致识别可能作为适当治疗靶点的信号中间体。我们提出了以下研究，旨在更好地理解CN/NFAT信号传导在视网膜血管疾病中的作用：1）在高选择性CN和NFAT抑制剂存在下，比较高糖和/或VEGF对从小鼠或人视网膜组织分离的细胞的作用; 2）比较高糖对从野生型视网膜组织分离的细胞的相关作用。 型和NFAT-/-小鼠中的细胞毒性或高葡萄糖和/或VEGF对NFAT同种型定向的siRNA处理的细胞的影响;和3）测定高度特异性CN和NFAT抑制剂在STZ处理的小鼠中的治疗功效。选择STZ模型是因为其与非增殖性DR的特定相关性。

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.