Abstract
Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood–retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1β, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 μM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.
Highlights
Diabetic retinopathy is the leading cause of blindness among working-age adults, representing a large socioeconomic burden on society
We used an streptozotocin (STZ)-induced diabetic rat model to determine whether upregulation of acid sphingomyelinase (ASM) expression and activity in cells comprising the inner and outer blood–retinal barriers (BRBs) [15] contributed to the mitochondrial ceramide accumulation that further leads to cell damage [25]
Samples were normalized based on total mitochondrial protein, and sphingolipid peaks were compared to synthetic sphingolipid internal standards incorporated in each run
Summary
Diabetic retinopathy is the leading cause of blindness among working-age adults, representing a large socioeconomic burden on society. The retinal and choroidal vessels, supplying the inner and outer retina, respectively, regulate molecular exchange across the inner and outer blood–retinal barriers. Breakdown of these barriers results in clinically observable lesions, such as microaneurysms and hemorrhages, leading to retinal hypoxia or ischemia and disease progression [1]. The inner blood–retinal barrier, consisting of non-fenestrated retinal endothelial cells and pericytes, has been the focus of many studies, but the outer barrier has received comparatively less attention [1,2,3]. The choriocapillaris, a vascular layer supplying circulation to the outer third of the retina, consists of a fenestrated endothelium separated by Bruch’s membrane from the retinal pigment epithelium (RPE). RPE dysfunction can contribute to the hypoxic conditions common in DR, and to the fluid and ion fluxes thought to cause diabetic macular edema [6,7,8]
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