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2024 OMIG Abstract

Role of T Regulatory Cells in Modulating Angiogenesis in a Murine Model of Diabetic Corneal Neovascularization

Francesca Kahale, Katayoon Forouzanfar, Akitomo Narimatsu, Shima Dehghani, Seokjoo Lee,
Rohan Bir Singh, Tomas Blanco, Reza Dana

Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Boston, MA

Purpose: Diabetes mellitus (DM) is associated with a spectrum of systemic complications including pathological angiogenesis. The underlying mechanisms leading to corneal neovascularization 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 diabetic murine model of suture induced corneal neovascularization.

Methods: Type 1 DM was induced by injection of streptozotocin (50mg/kg) in 8-week-old BALB/c mice. After 4 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 either 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 scored clinically by measuring percentage of CNV on slit lamp images as well as via CD31 staining and quantification. Expression of IL-10 DM and non-DM mice was assessed by qRT-PCR. In vitro, Tregs from DM mice were cocultured with a mouse vascular endothelial cell line (MS1) for 4 hours on Matrigel with or without anti-IL-10 blocking antibody (10ug/ml) and MS1/non-DM Treg co-culture served as a cell 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 significantly higher CNV scores 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± 0.8 versus 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±407 vs 2243±469; p=0.003) with DM Tregs compared to non-DM Tregs. Addition of 10μg/ml of anti-IL-10 to the VEC-DM Treg co-culture resulted in reduction of tube formation: junction frequency (7.8+2.9 vs 15.8±2.4) and tube length (2352±344vs 3711±407 p=0.002) compared to VEC-DM Treg co culture. CNV scores in DM mice treated with IL-10 KO Treg was significantly lower compared to DM Treg (2.5± 3.5 vs 14.5±8.2; p<0.0083).

Conclusions: Our study demonstrates that Tregs from diabetic mice play a crucial role in promoting heightened angiogenesis in suture-induced corneal neovascularization, primarily through upregulated IL-10 expression. The suppression of CNV upon IL-10 inhibition indicates that targeting IL-10 in Tregs may offer a novel therapeutic strategy for controlling the elevated pathological angiogenesis observed in diabetes.


Disclosure:

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