Abstract
Purpose :
Activation and secretion of acid sphingomyelinase followed by excessive ceramide production in the plasma membrane of retinal endothelial cells (RECs) occurs at the early stages of diabetic retinopathy and critically contributes to chronic inflammation and disease progression. This study aims to address the novel concept of ceramide rich macro-signaling platform formation within the plasma membrane of RECs and their contribution to inflammatory and pro-apoptotic changes in REC in the diabetic retina.
Methods :
Ceramide rich platforms were quantified in bovine REC (BRECs) by confocal imaging. BREC apoptosis was assessed via bisbenzimide staining. Rat streptozotocin-induced diabetes and mouse retinal ischemia reperfusion (I/R) were used as diabetic retinopathy animal models. Anti-ceramide antibody (mAb) or short chain variable fragment (scFv) treatment were administered via a single 6/2 μg intravitreal injection. Inflammatory markers were analyzed via qRT-PCR. Vascular permeability was accessed using FITC-albumin technique.
Results :
In vitro studies in BRECs show generation of ceramide rich platforms upon acute stimulation with 20ng/mL TNFa or 20ng/mL IL-1β. This was ameliorated when treated with anti-ceramide scFv. 1-20ng/mL TNFa and 1-20ng/mL IL-1β dose-dependently induced apoptosis in BRECs over 24 hrs and was fully mitigated when treated with anti-ceramide scFv. To determine feasibility of anti-ceramide immunotherapy in vivo, intravitreal injection of anti-ceramide scFv (2μg) and mAb (6μg) was performed in a mouse I/R model. We found significant upregulation of TNFa, IL-1β, IL-6 and ICAM-1 expression and vascular permeability in WT retinas injured by I/R. A single injection of scFv and mAb at the time of I/R injury inhibited these inflammatory changes and increase in permeability (n=8 -12 per group; p<0.05). Next, intravitreal injection of scFv was tested in a STZ-induced rat diabetes model. A single 4μg injection of scFv at the onset of hyperglycemia prevented diabetes-induced increase in retinal TNFa expression and vascular permeability (n=10-18 per group; p<0.001).
Conclusions :
Functional inhibition of ceramide rich platform formation via anti-ceramide immunotherapy prevents pro-inflammatory and pro-apoptotic changes in the retinal endothelium and may provide a novel approach for prevention and treatment of diabetic retinopathy.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.