Human retinal pigment epithelium proteome changes in early diabetes

Abstract

Aims/hypothesis: Diabetic retinopathy is the most common complication of diabetes and a leading cause of blindness among working-age adults. Anatomical and functional changes occur in the retina and retinal pigment epithelium (RPE) prior to clinical symptoms of the disease. However, the molecular mechanisms responsible for these early changes, particularly in the RPE, remain unclear. To begin defining the molecular changes associated with pre-retinopathic diabetes, we conducted a comparative proteomics study of human donor RPE.

Methods: The RPE was dissected from diabetic human donor eyes with no clinically apparent diabetic retinopathy (n=6) and from eyes of age-matched control donors (n=17). Soluble proteins were separated based upon their mass and charge using two-dimensional (2-D) gel electrophoresis. Protein spots were visualised with a fluorescent dye and spot densities were compared between diabetic and control gels. Proteins from spots with significant disease-related changes in density were identified using mass spectrometry.

Results: Analysis of 325 spots on 2-D gels identified 31 spots that were either up- or downregulated relative to those from age-matched control donors. The protein identity of 18 spots was determined by mass spectrometry. A majority of altered proteins belonged to two major functional groups, metabolism and chaperones, while other affected categories included protein degradation, synthesis and transport, oxidoreductases, cytoskeletal structure and retinoid metabolism.

Conclusions/interpretation: Changes identified in the RPE proteome of pre-retinopathic diabetic donor eyes compared with age-matched controls suggest specific cellular alterations that may contribute to diabetic retinopathy. Defining the pre-retinopathic changes affecting the RPE could provide important insight into the molecular events that lead to this disease.

FIGURE 1

RPE proteins resolved by 2-D gel electrophoresis. Representative Flamingo stained-gel (125 μg) indicates identified proteins showing altered expression with diabetes. Boxed spots show increased expression and circled spots show decreased expression. The pI range for separation in the first dimension (pH 5-8) is shown at the top. The position of molecular mass markers is indicated on the left. Numbers correspond with density summaries in Figure 2 and identified proteins listed in Tables 2 and 3.

FIGURE 2

Summary of protein spot density. Results of densitometry for protein spots demonstrating a significant increase (a, b) or decrease (c) in spot density that were identified by mass spectrometry. Numbers correspond with Figure 1 and identified proteins listed in Tables 2 and 3.

FIGURE 3

Summary of the functional groups for proteins with altered expression in pre-retinopathic RPE. The diagram indicates the relative percent of proteins in each functional group. a: Protein degradation, b: Structural, c: Chaperones, d: Membrane dynamics, e: Energy metabolism, f: Protein synthesis , g: Protein transport, h: Retinoid metabolism, i: Oxidoreductases