Evaluation of Proteoforms of the Transmembrane Chemokines CXCL16 and CX3CL1, Their Receptors, and Their Processing Metalloproteinases ADAM10 and ADAM17 in Proliferative Diabetic Retinopathy

Ahmed M Abu El-Asrar,Ghislain Opdenakker,Alexandra De Zutter,Marfa Blanter,Jo Van Damme,Ajmal Ahmad,Eef Allegaert,Gert De Hertogh,Sofie Struyf,Priscilla W Gikandi,Mohd Imtiaz Nawaz,Mohammad Mairaj Siddiquei

doi:10.3389/fimmu.2020.601639

Abstract

The transmembrane chemokine pathways CXCL16/CXCR6 and CX3CL1/CX3CR1 are strongly implicated in inflammation and angiogenesis. We investigated the involvement of these chemokine pathways and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of proliferative diabetic retinopathy (PDR). Vitreous samples from 32 PDR and 24 non-diabetic patients, epiretinal membranes from 18 patients with PDR, rat retinas, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by enzyme-linked immunosorbent assay, immunohistochemistry and Western blot analysis. In vitro angiogenesis assays were performed and the adherence of leukocytes to CXCL16-stimulated HRMECs was assessed. CXCL16, CX3CL1, ADAM10, ADAM17 and vascular endothelial growth factor (VEGF) levels were significantly increased in vitreous samples from PDR patients. The levels of CXCL16 were 417-fold higher than those of CX3CL1 in PDR vitreous samples. Significant positive correlations were found between the levels of VEGF and the levels of CXCL16, CX3CL1, ADAM10 and ADAM17. Significant positive correlations were detected between the numbers of blood vessels expressing CD31, reflecting the angiogenic activity of PDR epiretinal membranes, and the numbers of blood vessels and stromal cells expressing CXCL16, CXCR6, ADAM10 and ADAM17. CXCL16 induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB and VEGF in cultured Müller cells and tumor necrosis factor-α induced upregulation of soluble CXCL16 and ADAM17 in Müller cells. Treatment of HRMECs with CXCL16 resulted in increased expression of intercellular adhesion molecule-1 (ICAM-1) and increased leukocyte adhesion to HRMECs. CXCL16 induced HRMEC proliferation, formation of sprouts from HRMEC spheroids and phosphorylation of ERK1/2. Intravitreal administration of CXCL16 in normal rats induced significant upregulation of the p65 subunit of NF-κB, VEGF and ICAM-1 in the retina. Our findings suggest that the chemokine axis CXCL16/CXCR6 and the processing metalloproteinases ADAM10 and ADAM17 might serve a role in the initiation and progression of PDR.

Highlights

Ischemia-induced retinal angiogenesis, inflammation and fibrosis are the pathological hallmarks of proliferative diabetic retinopathy (PDR) and are critical mechanisms for PDR initiation and progression
Given the key roles of CXCL16 and CX3CL1 in inflammation and angiogenesis, we investigated the involvement of these chemokines and their processing metalloproteinases ADAM10 and ADAM17 in the pathophysiology of PDR
Our analysis showed that the levels of CXCL16 in the vitreous fluid of patients with PDR were 417-fold higher than those of CX3CL1

Summary

Introduction

Ischemia-induced retinal angiogenesis, inflammation and fibrosis are the pathological hallmarks of proliferative diabetic retinopathy (PDR) and are critical mechanisms for PDR initiation and progression. Increasing evidence supports the causal relationship between persistent inflammation and angiogenesis [14, 15] These observations suggest that the link between inflammation and angiogenesis plays critical roles in PDR initiation and progression. This paradigm is supported by the dysregulated expression of multiple signaling molecules of the inflammatory response with angiogenic activities, such as chemokines [4, 16,17,18,19,20] and matrix metalloproteinases [2, 21] in the ocular microenvironment of patients with PDR. Molecules with roles in inflammation and pathological neovascularization are considered potential targets for treatment of PDR

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