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。在某些类型的视网膜细胞中，细胞内钙升高会刺激致病反应，导致视网膜白细胞停滞和血管高通透性--这是非增殖性糖尿病视网膜病变(DR)威胁视力的两个特征。因此，作为重要的细胞内第二信使，Ca~(2+)可能在高糖诱导的糖尿病视网膜病变血管病变中发挥重要作用，但致病Ca~(2+)信号传导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)在高血糖诱导的钙离子流量与这些病理细胞反应之间的联系中起着关键作用。细胞内钙离子升高触发钙调神经磷酸酶(CN)介导的NFAT活化，导致其核 易位。NFAT通过与其他转录因子的异源二聚体结合，将高血糖诱导的钙流与其他生化信号整合在一起。这些异二聚体复合体以多种生化信号特有的方式刺激靶基因的转录。例如，在视网膜Muller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，从而导致血管高通透性和视网膜水肿。我们的初步数据表明，大部分高糖诱导的血管内皮生长因子是CN依赖的。同样，在视网膜微血管内皮细胞(RMEC)中，NFAT转录活性导致编码炎症细胞因子CXCL2、VCAM-1、ICAM-1、IL-6和IL-8的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钙离子、CN、NFAT与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，必须在参与糖尿病反应的特定视网膜细胞中表征其信号通路：Muller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既产生并响应这些因素。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钙离子流量、CN激活、NFAT激活以及随后的细胞反应之间的复杂相互作用。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。我们建议进行以下研究，旨在更好地了解CN/NFAT信号在视网膜血管疾病中的作用：1)比较高糖和/或血管内皮生长因子在高选择性CN和NFAT抑制剂存在下对分离的小鼠和人视网膜组织细胞的影响；2)比较高糖对野生细胞分离的相关影响 研究内容包括：(1)研究NFAT-/-小鼠或高糖和/或血管内皮生长因子对NFAT异构体siRNA处理细胞的影响；3)确定高度特异的CN和NFAT抑制剂在STZ处理的小鼠中的治疗效果。STZ模型之所以被选中，是因为它与非增殖性DR具有特定的相关性。

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 Muller 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: Muller 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.