Abstract
Purpose :
Chronic inflammation has been identified as a pivotal factor in the progression of diabetic retinopathy (DR). Peroxisome proliferator-activated receptor-alpha (PPARα) deficiency in microglia, the resident immune cells in the retina, disturbs microglial metabolic homeostasis and triggers retinal dysfunction. However, the mechanism underlying microglia-induced neurovascular dysfunction remains unclear. We hypothesizes that PPARα deficiency in microglia promotes retinal astrocyte senescence and endfeet loss through the release of extracellular vesicles (EVs).
Methods :
PPARα was knocked out in mouse microglia (Cx3cr1-PCKO), and diabetes was induced by Streptozotocin. Retinal astrocytic morphology and astrocyte-vascular coverage were analyzed using fluorescence microscopy. PPARα was knocked down (KD) in primary human microglia, followed by high glucose (HG, 25mM) treatment. EVs were collected via sucrose cushion ultracentrifugation (100,000xg) after the removal of dead cells and debris. EVs were characterized using exosome markers (CD81, Alix, TSG101) and cell lysis markers (calnexin and cytochrome c). EV quality was monitored with transmission electron microscopy. To confirm astrocytic uptake of microglial EVs, human retinal astrocytes were incubated with PKH26-labeled microglial EVs. Z-stack images were obtained with confocal microscopy. Astrocytes were cultured with microglial EVs. Cell senescence was assessed through β-galactosidase activity and immunoblotting of senescence markers P16INK4a and P21WAF1/CIP1.
Results :
PPARα deletion in microglia resulted in retinal astrocytic morphological alterations under diabetic conditions (n=4, p<0.05). The coverage of retinal vessels by astrocyte endfeet was significantly reduced in diabetic Cx3cr1-PCKO mice (n=4, p<0.001). EVs from PPARα KD microglia exposed to HG induced astrocytic senescence, as evidenced by increased β-galactosidase activity (n=3, p<0.01) and senescence markers (n=3, both P16INK4a and P21WAF1/CIP1 p<0.05).
Conclusions :
These results suggest that microglia-derived EVs in diabetes regulate retinal astrocytic senescence, leading to astrocytic endfeet loss. These findings propose that the exchange of EVs between microglia and astrocytes could be a potential target for investigating pharmaceutical intervention for DR.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.