Biomarker Identification of Vitreous Fluid for Diabetic Retinopathy

Abstract

For investigating the pathogenesis of DR, 2 main strategies are applied for the analysis of vitreous protein. The first strategy is antibody-based detection of vitreous cytokines. In previous studies of vitreous cytokines in DR, conventional enzyme-linked immunosorbent assay (ELISA) were used [4,5]. Recently, simultaneous analysis of the expression profiles of multiple cytokines and chemokines in the vitreous fluid was performed using an array system of antibody-coated beads [6]. Compared with the control group, interleukin (IL)-6, IL-8, IL-10, IL-13, interferon-inducible 10-kDa protein (IP-10), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 beta (MIP-1β), platelet-derived growth factor (PDGF), and VEGF levels in the vitreous fluid were significantly higher in the DR group. The second strategy is proteomic analysis of vitreous proteins [7-11]. Fluorescence-based difference gel electrophoresis (DIGE) combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has enabled accurate quantitative comparisons of multiple proteins [12]. MALDI-TOF MS analysis of vitreous fluid detected approximately 1300 protein spots, and 25 of these intravitreal proteins were differentially expressed between DME and control groups. Hemopexin, beta-crystallin S, clusterin, and transthyretin were found to be specifically associated with DME. In a study using the same technique, 1242 protein spots were detected containing 19 differentially expressed intravitreal proteins between proliferative DR and control groups [13]. Differences in hemopoxin, clusterin, and pigment epithelium-derived factor (PEDF) levels were also observed between proliferative DR and control groups. Therefore, hemopexin expression was upregulated in DME and proliferative DR, whereas clusterin expression was downregulated in DME and proliferative DR and PEDF expression was downregulated only in proliferative DR. Hemopexin is an acute-phase plasma glycoprotein and is expressed in multiple cell lines derived from different tissues [14]. Importantly, increasing glucose concentrations in vitro increased hemopexin expression and modulated the reactive oxygen species levels in cells; these effects were partially reversed by addition of reduced glutathione. Clusterin is associated with protection from apoptosis of retinal cells [15]. In a mouse model of DR, clusterin reduced the leakage from vessels in the diabetic retina and restored expression of tight junction