Abstract
Purpose :
Previous work highlights the detrimental effects of increased ceramide generation in diabetic retinopathy (DR) progression. We have shown that ASM mediated ceramide generation leads to chronic low-grade inflammation in various cell types such as endothelial cells. We hypothesize that underlying mechanism is based on the formation of pro-inflammatory ceramide rich platforms (CRP). The focus of this project is to address the novel concept of functional and highly-specific ceramide inhibition using cutting edge anti-ceramide immunotherapy to prevent early onset diabetes induced pro-inflammatory and pro-apoptotic changes in the retinal vasculature.
Methods :
Ischemia reperfusion (I/R) studies were utilized to mimic acute vascular degeneration in mice as observed in DR. Streptozotocin was utilized to induce hyperglycemia-induced vascular degeneration as a type-1 diabetes model. Anti-ceramide single chain variable fragment (scFv) treatment was administered via a 2 ug single dose into the vitreous. Pro-inflammatory markers (IL-1β, VEGF and ICAM-1) as well as ASM expression levels were analyzed via qRT-PCR. Ceramide species were analyzed via lipidomics (nESI-MS/MS). Vascular degeneration was captured using FITC-albumin technique and confocal microcopy and quantified using MetaMorph software.
Results :
In vivo I/R studies in asm+/+ and asm-/- mice identified significant upregulation of ASM, C16 and C18 ceramide production, as well as IL-1β, VEGF and ICAM-1 gene expression in asm+/+ but not in asm-/- mice (n=6 per group; p<0.05). To determine feasibility of anti-ceramide immunotherapy, intravitreal injection of anti-ceramide scFv was first performed in the mouse I/R model. We found significant upregulation of IL-1β, VEGF and ICAM-1 gene expression in WT untreated retinas injured by I/R. These pro-inflammatory changes were inhibited by a single injection of anti-ceramide scFv 12 hours prior to I/R injury (n=6-8 per group; p<0.05). Impact of intravitreal injection of scFv was next tested in STZ-induced Wistar rat diabetes model. A single injection of scFv at the onset of hyperglycemia prevented diabetes-induced increase in retinal vascular permeability (n=3-10 per group; p=0.08).
Conclusions :
These results suggest that anti-ceramide scFv immunotherapy may represent a novel approach to targeting deleterious ceramide-rich platform mediated pro-inflammatory and pro-apoptotic changes prevalent in the early stages of vasculopathy in the diabetic retina.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.