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活化，导致其核

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.