Mitochondrial fragmentation and dysfunction in diabetic retinopathy

这个项目的总体目标是测试高糖(HG)诱导 线粒体分裂基因上调和线粒体融合基因抑制， 伴随自噬和线粒体连接蛋白43(MtCx43)减少 下调，促进线粒体碎裂和功能障碍；改善 这些事件将保护线粒体功能，从而阻止视网膜血管 糖尿病视网膜病变(DR)中的细胞丢失。这一假说是基于线粒体的发现 分裂/融合、自噬和mtCx43在维持线粒体形态中起关键作用 和功能。我们之前的研究表明，HG诱导线粒体碎裂和 线粒体网络的破坏导致线粒体膜电位的增加 异质性，减少氧气消耗，改变细胞外酸化，增加 细胞色素c的释放，最终导致视网膜血管细胞的凋亡。此外，HG 减少这些细胞的自噬和mtCx43的表达。我们的初步数据显示 HG增加Fis1和Drp1的表达，降低融合基因的表达 视网膜血管细胞中的OPA1和Mfn2基因。此外，HG可减少自噬/有丝分裂吞噬，a 去除包括线粒体片段在内的功能失调的细胞成分的过程，以及 从而促进碎片化线粒体的积累。我们还观察到HG减少 MtCx43的表达以及HG诱导的mtCx43通道抑制改变线粒体 大鼠视网膜内皮细胞的形态和细胞色素c的释放。此外，线粒体 糖尿病大鼠视网膜血管细胞出现碎裂，新数据显示减少 糖尿病Drp1/-小鼠视网膜毛细血管脱细胞和周细胞样影的数量 而OPA1/-小鼠提示线粒体碎裂的减弱可能是有益的。 根据信息和初步数据，我们提出了三个具体目标来确定1) 抑制HG诱导的线粒体断裂是否能阻止细胞凋亡 体外培养的视网膜内皮细胞和周细胞，以及糖尿病大鼠的视网膜；2) HG诱导的自噬减少是否促进线粒体积聚 碎片与视网膜血管细胞丢失；3)mtCx43表达是否改变 在实验性DR中促进视网膜血管病变的发展 提出的项目有望确定视网膜血管细胞丢失的新机制(S) 涉及线粒体异常，从而提供了对潜在的预防策略的洞察 这些异常与DR的血管细胞死亡有关。

The overall goal of this project is to test the hypothesis that high glucose (HG)-induced upregulation of mitochondrial fission genes and inhibition of mitochondrial fusion genes, concomitant with decreased autophagy and mitochondrial connexin 43 (mtCx43) downregulation, promotes mitochondrial fragmentation and dysfunction; amelioration of these events would protect mitochondrial function and thereby prevent retinal vascular cell loss in diabetic retinopathy (DR). The hypothesis is based on findings that mitochondrial fission/fusion, autophagy, and mtCx43 play critical roles in maintaining mitochondrial morphology and function. Our previous studies show HG induces mitochondrial fragmentation and breakdown of the mitochondrial networks resulting in increased mitochondrial membrane potential heterogeneity, decreased oxygen consumption, altered extracellular acidification, increased cytochrome c release, and ultimately apoptosis of retinal vascular cells. Furthermore, HG decreases autophagy and mtCx43 expression in these cells. Our preliminary data indicates that HG increases expression of fission genes, Fis1 and Drp1, and decreases expression of fusion genes, Opa1 and Mfn2, in retinal vascular cells. Additionally, HG reduces autophagy/mitophagy, a process that removes dysfunctional cellular components including mitochondrial fragments, and thereby contributes to accumulation of fragmented mitochondria. We also observed HG reduces mtCx43 expression, and that HG-induced mtCx43 channel inhibition alters mitochondrial morphology and cytochrome c release in rat retinal endothelial cells. Furthermore, mitochondrial fragmentation was noted in retinal vascular cells of diabetic rats, and new data indicate decreased number of acellular capillaries and pericyte ghosts in retinal capillaries of diabetic Drp1+/- mice and Opa1+/- mice suggesting attenuation of mitochondrial fragmentation could be beneficial. Based on the information and preliminary data, we propose three Specific Aims to determine 1) whether inhibition of HG-induced mitochondrial fragmentation prevents apoptosis in retinal endothelial cells and pericytes in vitro, as well as in retinas of diabetic rats; 2) whether HG-induced decreased autophagy promotes accumulation of mitochondrial fragments and retinal vascular cell loss; and 3) whether altered mtCx43 expression contributes to the development of retinal vascular lesions in experimental DR. The proposed project is expected to identify novel mechanism(s) underlying retinal vascular cell loss involving mitochondrial abnormalities, and thus, provide insight into potential strategies to prevent these abnormalities related to vascular cell death in DR.