Abstract
BackgroundAs a leading cause of adult blindness, diabetic retinopathy is a prevalent and profound complication of diabetes. We have previously reported duration-dependent changes in retinal vascular permeability, apoptosis, and mRNA expression with diabetes in a rat model system. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies.Methodology/Principal FindingsA multi-modal proteomic approach of antibody (Luminex)-, electrophoresis (DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to maximize coverage of the retinal proteome. Transcriptomic profiling through microarray analysis was included to identify additional targets and assess potential regulation of protein expression changes at the mRNA level. The proteomic approaches proved complementary, with limited overlap in proteomic coverage. Alterations in pro-inflammatory, signaling and crystallin family proteins were confirmed by orthogonal methods in multiple independent animal cohorts. In an independent experiment, insulin replacement therapy normalized the expression of some proteins (Dbi, Anxa5) while other proteins (Cp, Cryba3, Lgals3, Stat3) were only partially normalized and Fgf2 and Crybb2 expression remained elevated.Conclusions/SignificanceThese results expand the understanding of the changes in retinal protein expression occurring with diabetes and their responsiveness to normalization of blood glucose through insulin therapy. These proteins, especially those not normalized by insulin therapy, may also be useful in preclinical drug development studies.
Highlights
97% of patients with Type 1 diabetes develop some degree of diabetic retinopathy (DR) within 25 years of diagnosis [1]
Insulin treatment reduced HbA1c levels significantly compared to untreated diabetic animals but HbA1c levels remained slightly elevated compared to controls (p,0.001, SNK post-hoc, in both comparisons)
Fgf2 protein and mRNA expression were not normalized by insulin treatment, with levels remaining significantly elevated compared to controls (Figure 7)
Summary
97% of patients with Type 1 diabetes develop some degree of diabetic retinopathy (DR) within 25 years of diagnosis [1]. Even with improvements in therapies for glycemic control, DR remains a leading cause of new cases of adult blindness worldwide [2,3,4]. With both manifest and subclinical DR, visual impairment in hue discrimination and contrast sensitivity, delayed dark adaptation, abnormal visual fields and decreased overall visual acuity are observed [5,6,7,8,9]. We have identified retinal transcriptomic changes with diabetes associated with vascular dysfunction, a pro-inflammatory state, and neuronal compromise [12,14,15]. The aim of this study was to identify retinal proteomic alterations associated with functional dysregulation of the diabetic retina to better understand diabetic retinopathy pathogenesis and that could be used as surrogate endpoints in preclinical drug testing studies
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