Subrata Chakrabarti

The University of Western Ontario, Chief of Pathology and Laboratory Medicine, LHSC/SJHC, 339 Winderemere Rd., London, ON, Canada N6A 5A5

Abstract:

Chronic diabetes leads to the activation of a number of signaling proteins. These signaling cascades are activated in response to hyperglycemia-induced oxidative stress. Such aberrant signaling leads to activation of transcription factors, transcriptional co-activators and cause microRNA (miRNA) alteration. miRNAs are endogenous, ~20-25 nucleotides, which cause translational arrest or degradation of mRNAs through base pairing with 3’UTR of the target transcript. miRNAs regulate several cellular functions and are of importance in physiologic and pathologic processes. We investigated miRNA alterations in diabetic retinopathy and other diabetic vascular complications from a potential therapeutic standpoint.

We used microRNA array analyses of endothelial cells exposed to various levels of glucose and retinas from the nondiabetic and diabetic rats. Based on the information generated by microRNA array and bioinformatics analysis, we selected two miRNAs for detailed study; they are miR146a (target fibronectin 1, FN) and miR 200b (target vascular endothelial growth factor, VEGF). Cell culture studies in the endothelial cells demonstrated that both these miRNAs are downregulated when the cells were exposed to 25mM glucose, compared to 5mM glucose. Simultaneously, histone acetylator p300, VEGF and FN mRNAs and proteins were upregulated along with histone acetylation. Transfection of of miR146a and 200b mimics, although were able to correct FN and VEGF upregulation respectively. In addition p300 eaxpression was regulated by miR200b, whereas it regulated miR146a levels. We established, using luciferase assay, that miR200b and miR146a binds to 3’UTR of VEGF and FN gene respectively. We then used intravitreal injections of specific miR200b mimic or miR146a mimic (once a week for 4 weeks) in the left eye, whereas the right eye received scrambled control. Analysis of retinal tissues after one week of the last injection showed increased retinal miR200b or miR146a expression, indicating efficiency of this delivery method. Analysis of retinal tissues after one week of the last injection showed normalization of retinal miR200b and miR146a expression. Such treatments further led to the reduction of diabetes induced VEGF and FN mRNA and protein upregulation respectively. We further carried out investigation of the retinal tissues of diabetic rats with respect to increased vascular permeability using an immunocytochemical stain with albumin. The retinas from the diabetic rats showed increased vascular permeability, which was prevented by intravitreal miR200b injection. Human retinal tissue from autopsy showed similar changes. In addition, cardiac, renal and retinal tissues from both type 1 and type 2 diabetic animals showed similar changes.

Hence a complex web of pathways including intracellular signaling, epigenetics and miRNA alterations are involved in the pathogenesis of functional and structural changes in chronic diabetic complications. Specific miRNAs, such as miR200b or miR146a may act as potential targets for the treatment of diabetic retinopathy and other diabetic vascular complications.