Abstract
Purpose :
Diabetic corneal changes are frequent, but are often underdiagnosed complications in patients with diabetes mellitus (DM). Although corneal neovascularization (CNV) associated with DM is not commonly observed in the uninjured cornea; however, diabetics are more prone to develop CNV after surgery or inflammation. We have previously shown that naïve regulatory T cell (Treg) exert a potent anti-angiogenic effect on suture-induced inflamed CNV. Herein, we study this effect in the diabetic mouse model.
Methods :
DM was induced by daily injection of streptozotocin (STZ) (50mg/kg) for 5 days in 8-week-old BALB/c mice. 8 weeks post-induction, CD4+CD25+ Treg from either naïve or diabetic mice were MACS-sorted and cocultured with a mouse vascular endothelial cell line (MS1 VEC) in triplicates for 4 hours in Matrigel. VEC tube formation was imaged by brightfield microscopy and both tube length and junctions were analyzed by image J. CNV was induced in diabetic and non-diabetic mice by placing a suture in cornea. Non-diabetic mice were treated with subconjunctival injection of either 1) saline, 2) Treg (5x105) from diabetic mice or 3) Treg from non-diabetic mice. In addition, diabetic mice were treated with 4) saline or 5) Treg from non-diabetic mice. On day 7, CNV was scored (0-4). The corneas were excised and immunostained with anti-CD31, imaged by confocal microscopy, and analyzed with Image J. Expression of anti-angiogenic factors in non-diabetic and diabetic Treg was evaluated by RT-PCR.
Results :
Diabetic mice had a higher CNV score (p<0.001) and CD31 positivity (P<0.001) vs. non-diabetic mice. Adoptive transfer of non-diabetic Treg to diabetic mice led to decreased CNV (p=0.001) while adoptive transfer of diabetic Treg to non-diabetic mice failed to reduce CNV. Compared to VECs alone, co-culture of VECs with non-diabetic Treg suppressed tube length (3161±346 vs. 5655±306, p=0.001) and the number of junctions (6.6±2 vs.24.6±4.2, p=0.015), while when co-cultured with diabetic Treg the changes were less significant for both tube length (4300±333, p=0.032) and number of junctions (12.25±2.6, p=0.050). Among all anti-angiogenic factors, PDL1 was significantly downregulated in diabetic Treg compared to non-diabetic Treg (p=0.005).
Conclusions :
The diabetic state enhances CNV in response to inflammation and that correlates with the downregulation of antiangiogenic factors by Treg in diabetic mice.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.