Proteome Analysis of Human Vitreous Proteins

Abstract

Purpose: Various protein contents such as enzymes, growth factors, and structural components are responsible for biological activities in organs. We have created a map of vitreous proteins and developed a proteome analysis of human vitreous samples to understand the underlying molecular mechanism and to provide clues to new therapeutic approaches in eyes with proliferative diabetic retinopathy (PDR). Methods: Vitreous and serum samples were obtained from subjects with idiopathic macular hole (MH, 26 cases) and PDR (33 cases). The expressed proteins in the samples were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Protein spots were visualized by silver staining, and their expression patterns were analyzed. Some protein spots of concern were excised from the 2-D gels, digested in situ with trypsin, and analyzed by mass spectrometry. Results: More than 400 spots were detected on 2-D gels of MH cases, of which 78 spots were successfully analyzed. The spots corresponded to peptide fragments of 18 proteins, including pigment epithelium-derived factor, prostaglandin-D2 synthase, and interphotoreceptor retinoidbinding protein. These were not identified in the corresponding serum samples. These proteins were also expressed in PDR samples, with no distinct tendency to increase or decrease compared with the MH samples. More than 600 spots were detected on 2-D gels of PDR cases, of which 141 spots were successfully analyzed. The spots corresponded to peptide fragments of 38 proteins. Enolase and catalase were identified among four detected spots. Neither was found in MH vitreous or in PDR serum samples. Conclusion: A map of protein expression was made in human vitreous from eyes with MH and PDR. In the PDR eyes, the increased protein expression observed was due to barrier dysfunction and/or production in the eye. Proteome analysis was useful in systematic screening of various protein expression in human Hyperglycemia induces many abnormal changes, which are observed as diabetic retinopathy, in vascular and retinal cells in eyes with diabetes mellitus. The breakdown of the bloodretina barrier and new vessel formation are caused by hyperglycemia. In these processes, many hyperglycemia-induced biochemical changes occur, which cause vascular dysfunction. According to previous reports, various factors, including many kinds of proteins, are involved in the pathological processes of diabetic retinopathy. A breakdown of the bloodretina barrier is caused by an intraocular increase of vascular endothelial growth factor (VEGF) 1 (1-7), interleukin-6, angiotensin II, and many other cytokines and/or growth factors. New vessel formation is a very complex multistep process and is regulated by many proteins including cytokines and/or growth factors. In addition to the factors mentioned, basic fibroblast growth factor (bFGF) (8, 9), insulin-like growth factor-1 (4), hepatocyte growth factor (HGF) (6, 7), and others are known to be involved during the destructive processes of endogenous ocular tissue. Changes in the...

Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy

“…Although its biological functions are incompletely understood, it seems to be a novel adipokine that may be involved in the local regulation of adipose tissue function [20][21][22][23]. ZAG has been recently identified by proteomic analysis in vitreous fluid from PDR patients [8,9]. In the present study we have confirmed this finding.…”

“…However, few proteome analyses in human vitreous fluid have been performed in the setting of diabetic eye disease [7][8][9][10][11], and no previous quantitative proteomic comparison has been reported. The aim of the present study was to compare the protein profile of human vitreous fluid from diabetic patients with PDR with that of vitreous fluid obtained from nondiabetic patients with idiopathic macular holes (MH), a condition in which, in contrast to PDR, the retina is not affected by neovascularisation.…”

Proteomics in ocular fluids

“…A 2-D study of vitreous from patients with proliferative diabetic retinopathy and idiopathic macular hole showed characteristic protein patterns in these patients and led to the identification of numerous proteins as for example pigment epithelium-derived factor, prostaglandin-D2 synthase, and interphotoreceptor retinoid-binding protein [50]. These results confirm the findings of another study that analyzed the vitreous humor of diabetic retinopathy and macular hole patients by means of 2-DE.…”

“…Albumin and Ig make up 80% of the protein concentration in the vitreous. Most proteins that have been identified so far in vitreous humor, like albumin, transferrin, apolipoprotein, IgG, amino acid, and transthyretin [50,51], are also found in serum [52]. The vitreous contains a group of enzymes known as matrix metalloproteinases (MMPs) [53].…”

“…which are zincdependent enzymes that mediate the structure and function of the extracellular matrix [54]. Vitreous fluid samples for proteomic analysis can be collected during surgery, e.g., vitrectomy [50,55,56]. An analysis of vitreous proteins can be very interesting in diseases like diabetic macular edema [57] or retinopathy [58], where vitreous is the closest fluid to the sight of damage.…”