495-P: Diabetic Retinopathy Is a Ceramidopathy Reversible by Systemic Anticeramide Therapy

Introduction: Diabetic retinopathy (DR) is characterized by retinal endothelial cell (REC) death and inadequate vascular repair by dysfunctional circulating angiogenic cells (CACs). We have previously demonstrated an increase in ASM-mediated C16 ceramide and ceramide-rich platform (CRP) formation as a major pathogenic mechanism in the development of REC damage and CAC dysfunction in DR. Using anti-ceramide Abs, we aimed to prevent REC damage and restore CAC-mediated repair by dispersing CRPs, thus restoring retinal microvascular health and preventing DR. Methods: Anti-ceramide Abs were administered in vitro or ex vivo at 10 (short chain fragment antibody; scFv) /100 (monoclonal antibody; mAb) μg/mL and in vivo (subcutaneously) at 60μg/g (mAb) bodyweight in STZ induced diabetic mouse model. Migration of CACs was assessed using trans-well assays ex vivo. In vivo vascular degeneration was captured using FITC-albumin technique. In vivo migration of GFP+ CACs (JAX:006567) was examined via confocal imaging. Results: In vitro studies in BRECs show generation of vast ceramide rich platforms followed by apoptosis upon acute stimulation with 20ng/mL TNFa or 10ng/mL IL-1β. This phenotype was fully ameliorated when treated with anti-ceramide scFv. STZ mice showed a significant increase (42% CRP pos. cells) in CRP formation on day 3, with gradual increases on day 7, 14, 42, and 180 (70% pos.) post-hyperglycemia (n=9-12, p<0.0001. Anti-ceramide treatment reduced CRP formation on CACs and protected from retinal vascular permeability 6-weeks post hyperglycemia in vivo (n=10-12; p<0.01. In vivo migration and vascular repair of CACs was impaired in 6-month diabetic STZ mice compared to their age matched controls and rescued following anti-ceramide Abs intervention (scFv and mAb) in vivo (n=4-7; p<0.08). Conclusion: Anti-ceramide Abs restore microvascular health via normalization of both REC and CACs homeostasis to halt or reverse DR. Disclosure J.V. Busik: Consultant; Ceramedix, Inc. T.F. Dorweiler: None. A. Singh: None. M.B. Grant: None. R. Kolesnick: Stock/Shareholder; Ceramedix Holding LLC. Funding R01EY030766R01EY025383R01EY016077