Diabetic regulatory T Cells Enhances Suture-induced Corneal Neovascularization (CNV)

Abstract

Purpose : Diabetes mellitus (DM) is associated with a spectrum of systemic complications including pathological angiogenesis.The underlying mechanisms that cause CNV in DM are yet to be elucidated. As regulatory T cells (Tregs) play a critical role in modulating angiogenic processes, we investigate their contribution to regulating angiogenesis in a suture induced CNV in type1DM murine model.

Methods : Type 1 DM was induced by injection of streptozotocin (50mg/kg) in 8-week-old BALB/c mice. After 8 weeks, CNV was induced in DM and non-diabetic (non-DM) mice (N=8) by placing three figure-of-8 sutures. CD4⁺CD25⁺ Tregs from DM mice or non-DM were MACS-sorted and adoptively transferred into non-DM or DM mice respectively. Mice were followed for 7 days and CNV was assessed by CD31 staining and imaged by confocal microscopy. Expression of IL-10 and FOXp3 in DM and non-DM mice was assessed by qRT-PCR.Tregs from DM mice were cocultured with a mouse vascular endothelial cell line (MS1) for 4 hours in Matrigel with or without anti-IL-10 blocking antibody (10ug/ml) and MS1/non-DM Treg co-culture served as control. Under a brightfield microscopy, both tube length and junctions were imaged and analyzed by image J.

Results : We did not observe development of spontaneous CNV in DM mice. Upon suture placement, we observed significant differences in the CNV in DM (26±7.6) compared to non-DM (15±8, p=0.039) mice. The adoptive transfer of non-DM Tregs to DM mice resulted in significant CNV suppression (1.1± 0.8vs 26±7.6; p<0.0001) compared to the controls. However, adoptive transfer of DM Tregs to non-DM mice did not result in significantly higher CNV compared to controls. Among proangiogenic factors, significant upregulation of IL-10 expression levels was observed in DM compared to non-DM Tregs (11842±116 vs 4580±515; p=0.0026).On co-culture with VEC, we observed significantly higher junction frequency (15.8±2.4vs 4.1±1.1; p=0.001) and tube length (3711±407vs 2243±469; p=0.003) with DM Tregs compared to non-DM Tregs. Addition of 10mg/ml of anti-IL-10 to the VEC-DM Treg co-culture resulted in reduction of tube formation: junction frequency (7.8+2.9vs 15.8±2.4) and tube length (2352±344vs 3711±407 p=0.002) compared to VEC-DM Treg co culture.

Conclusions : Our results indicate that Treg undergo functional adaptation (such as increased IL-10 expression) in response to DM, thereby promoting angiogenesis in a suture-induced CNV diabetic model.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.