Nature of the research:
This is a fundamental research project investigating the influence of microRNAs on the initiation and progression of Diabetic Retinopathy. In the project, a variety of in vitro and in vivo models is utilized.
Diabetic retinopathy is the main cause of blindness in adults in the Industrialized World. Diabetic Retinopathy is a chronic microvascular complication affecting virtually all patients with diabetes. Diabetic Retinopathy is characterized by progressive alterations in the retinal microvasculature, i.e. endothelial dysfunction, vascular hyperpermeability, hyperglycemia-induced ROS production, pericyte dropout.
MicroRNAs are small non-coding nucleotides that affect cell function by translational repression of their target genes. MicroRNA dysregulation is associated with diabetic retinopathy and affects endothelial function and angiogenesis. We have recently identified several specific microRNAs that augment endothelial dysfunction by modulating crucial endothelial signaling transduction pathways and hypothesize that dysregulation of microRNAs occurs when diabetic complications develop.
Research question / problem definition
The main aim of this project is to clarify how novel diabetes-related microRNA (diamiR) ‘master switches’ can be manipulated to restore endothelial phenotype and function in diabetic retinopathy. Hereto we aim to (1) identify diamiRs which are associated with diabetic retinopathy and their gene targets, (2) to investigate the influence of diamiRs on the development of endothelial dysfunction and diabetic retinopathy, and (3) to therapeutically modulate diamiR expression to alleviate diabetic retinopathy in a mouse model.
Workplan
To identify differentially expressed diamiRs during hyperglycemic injury (in vitro) and diabetic retinal microangiopathy (in vivo), rEC are stimulated with high glucose (30mM D-glucose) medium for 5 days and miR expression is analyzed by conventional array technology. In the Ins2Akita mouse model, retinas will be isolated from 3 month-old individuals and their miR expression profiles compared to non-diabetic wild type controls. DiamiRs, which are differentially expressed miRs in both systems, will be further validated by qRTPCR analysis and used in further assays. This analysis identifies the ‘acutely’ and ‘chronically dysregulated’ hyperglycemia-induced diamiRs.
To identify the gene targets of identified diamiRs, TARGETome analysis will be used. In short, diamiRs are lentivirally transduced in rEC and subjected to hyperglycemia. Cells are lysed and AGO2 (part of the miR Induced Silencing Complex; RISC) is precipitated with all its bound mRNAs. These mRNAs are analyzed on whole genome expression arrays in a treatment-to-no-treatment fashion. To validate the causal interaction of identified diamiRs with their target genes. Reporter constructs are generated that express the Luciferase reporter gene under control of specific 3’ UTR sequences derived from above identified diamiR targets. Co-transfection of these reporter constructs with the identified diamiRs into COS cells allows for analysis of interaction between the miR and its predicted target mRNA by conventional Luciferase activity measurements.
To address the upstream mediators that cause diamiR deregulation in rEC, we analyze the expression of identified diamiRs after hyperglycemic treatment in the presence of pharmacological pathway inhibitors. Subsequently, conventional promoter analysis is used to validate a role for specific signal transduction cascades in the regulation of diamiR expression.
To address the effects of a specific diamiR on rEC function, the expression of diamiRs is modulated in vitro, either by lentivirally transducing diamiRs (increasing the expression of a specific miR) or antimiRs (miR-inhibitory-RNAs that abolish its expression) into rEC. Next, hyperglycemic injury is induced and the effect of diamiR expression on target gene availability, endothelial activation state (cytokine production, adhesion molecule expression) and function (NO/ROS production, sprouting ability) is analyzed by qRTPCR, immunohistochemistry, western blotting and functional measurements (i.e. ROS accumulation and Nitrate production).
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