Abstract
Purpose :
Our previous transcriptomic studies on retinal endothelial cells under hyperglycemic conditions have shown the significant upregulation of the chemokine CXCL1. The purpose of this present study is to show the effect of CXCL1 on recruitment of neutrophils and monocytes in the alteration of blood retinal barrier in an in vivo model of diabetic retinopathy.
Methods :
The experiment design included the following groups: A) Streptozotocin induced diabetic mice (3 months) vs. diabetic mice treated with neutrophil depleting Ly6G-1A8 antibody (50ug) for 15 days (n=10); B) Wildtype C57BL/6 J mice intravitreally (IVT) injected with recombinant CXCL1 (100 ng/eye) vs. animals pretreated with Ly6G-1A8 antibody (50ug) followed by IVT with recombinant CXCL1 (n=10). Flow cytometry analysis for immune cell infiltration (neutrophils and monocytes) in the retinas. qPCR analysis was used to determine mRNA expression of chemokines and its receptors, proteases and adhesion molecules. Western Blot analysis was used to evaluate the expression of albumin to detect retinal vascular permeability. ELISA was used to measure serum levels of CXCL1 levels in patients with human diabetic retinopathy.
Results :
Retinas of both diabetic and intravitreally CXCL1 injected mice showed increased neutrophils (p = 0.01) and monocytes (p = 0.01) and mRNA expression of proinflammatory chemokines (Ang2, CCL2, CCL5, CCL7, VEGF), chemokine receptors (CCR2, CCR5, CXCR2), proteases (Cathepsin-B,K,L,S, MMP2 and MMP9) and adhesion molecules (ICAM1 and VCAM1). The Ly6G-1A8 antibody treatment had significantly decreased infiltration of both neutrophils and monocytes (p=0.05) and decreased expression of these chemokines and proteases. Likewise, western blot analysis confirmed increased extravasation of albumin in retinas of diabetic mice and CXCL1 injected mice (p=0.01) and the Ly6G-1A8 antibody treatment significantly reduced the extravasation (p=0.01). Also, the CXCL1 level was significantly increased in the serum samples of diabetic retinopathy patients compared to non-diabetic subjects (p=0.001).
Conclusions :
Overall, we have shown evidence that neutrophil infiltration triggered by CXCL1 plays an important role in the alteration of the BRB in diabetes. A combination therapy of a CXCL1 inhibitor and the anti-VEGF drugs may be a potential therapeutic approach in the management for diabetic macular edema.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.