Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response

Abstract

BackgroundDespite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes.ResultsUsing the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals.ConclusionThese changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.

The PKCβ/HuR/VEGF pathway in diabetic retinopathy

“…Studies of retinal VEGF mRNA expression in STZ-induced diabetic rats have produced conflicting results, with VEGF mRNA concentrations reported to be increased [34,53,54], decreased [55,56] or unchanged [57] in diabetic rats compared with levels in control rats. The present findings show that, following diabetes, there is a higher phosphorylation of the HuR protein by PKCβ resulting in the activation of HuR itself that then targets VEGF mRNA, finally leading to an increased amount of the correspondent VEGF protein (Fig.…”

Neural inflammation and the microglial response in diabetic retinopathy

“…With the exception of CCL2, none of the confirmed genes can be considered to be conventional inflammatory mediators. Notably, CCL2 mRNA was increased as much as 80-fold in diabetic rat retinas [41].…”

“…In a genome-wide analysis of gene expression in STZ-induced diabetic rats after 3 months of diabetes, the confirmed gene changes associated with inflammation did not include interleukins and cytokines most often associated with tissue inflammation [41]. Instead, the confirmed inflammatory genes diabetes-altered retinal expression included MCP-1/CCL2, chemokine C-C motif receptor 5 (CCR5), cell surface glycoprotein CD44, janus kinase 3, signal transducer and activator of transcription 3 (STAT3), laminin, tissue inhibitor of metalloproteinase-1 and 27-kDa heat shock protein 1, chitinase 3-like 1, galactose-binding soluble lectin 3 and its binding protein, complement component 1 inhibitor, as well as PDGF.…”

A multistep validation process of biomarkers for preclinical drug development

“…In these instances, implementation of tests using molecular biomarkers can aid drug development as an initial high-throughput assessment of therapeutic interventions. Numerous changes in gene and protein expression have been reported in DR animal models 14,15,[17][18][19] that share important commonalities with clinical features of human DR, 14,20,21 but no previous reports have developed these changes into validated biomarkers for use in preclinical drug development studies.…”

“…The time course of pathophysiology was determined from our previous studies. 19,22,23 In Step 2, potential biomarkers were identified from data in our genome-wide gene expression database. 19 The reproducibility of gene expression changes across multiple independent animal experiments was then assessed to determine the most consistent changes in Step 3.…”