Abstract
Diabetic retinopathy is a prevalent microvascular complication characterized by apoptotic vascular cell loss in the retina. Previous studies have shown that high glucose (HG)-induced mitochondrial fragmentation plays a critical role in promoting retinal vascular cell apoptosis. Here, we investigated whether downregulation of mitochondrial fission genes, Fis1 and Drp1, which are overexpressed in HG condition, prevents mitochondrial fragmentation, preserves mitochondrial function, and protects retinal endothelial cells from apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (5 mM glucose) or HG (30 mM glucose) medium; in parallel, cells grown in HG medium were transfected with either Fis1 siRNA or Drp1 siRNA, or both siRNAs in combination, or scrambled siRNA as control. Live-cell confocal imaging showed decreased mitochondrial fission in cells transfected with Fis1 siRNA or Drp1 siRNA concomitant with reduced TUNEL-positive cells and a decrease in the expression of pro-apoptotic proteins, Bax and cleaved caspase 3, under HG condition. Importantly, the combined siRNA approach against Fis1 and Drp1 prevented HG-induced changes in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The findings from this study indicate that reducing HG-induced overexpression of mitochondrial fission genes preserves mitochondrial morphology and prevents retinal vascular cell apoptosis associated with diabetic retinopathy.
Highlights
Retinal vascular lesions involving apoptotic cell death represent a prominent characteristic of diabetic retinopathy, the leading cause of blindness in the working age population [1,2,3,4,5,6,7,8]
The findings from this study indicate that reducing high glucose (HG)-induced overexpression of mitochondrial fission genes preserves mitochondrial morphology and prevents retinal vascular cell apoptosis associated with diabetic retinopathy
Cells grown in HG medium exhibited significant upregulation of Drp1 (135 ± 11% of N; p < 0.05, Figure 1A,B) and Fis1 (131 ± 11% of N; p < 0.05, Figure 1A,C) compared with cells grown in N medium
Summary
Retinal vascular lesions involving apoptotic cell death represent a prominent characteristic of diabetic retinopathy, the leading cause of blindness in the working age population [1,2,3,4,5,6,7,8]. Our previous work demonstrated that the HG condition induces mitochondrial fragmentation in retinal vascular cells, and thereby contributes to mitochondrial dysfunction [10,11]. The link between HG-induced mitochondrial morphological changes and its effect on mitochondrial function leading to apoptosis is currently not well understood. Fusion and fission are cellular events that participate in the maintenance of mitochondrial morphology and its network dynamics [9]. Mitochondrial fission is balanced by opposing fusion events [12]; a disruption of the delicate balance between the two events could promote
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