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Maria B Grant, Vaishnavi Jadhav, Jude Al-Sabah, Brahim Chaqour, Ashay D Bhatwadekar; Vascular Dysfunction in Per2m/m mice is Mediated via the Upregulation Of Connective Tissue Growth Factor. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2716.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that Per2m/m mice that are lacking circadian rhythms recapitulate the retinal vascular alterations associated with diabetic retinopathy (DR). The vascular dysfunction in Per2m/m mice was largely attributed to dysregulated expression of connective tissue growth factor (CTGF/CCN2), a downstream target and core component of the TGF-β pathway. At the molecular levels, CTGF gene expression is dependent on the canonical Wnt/β-catenin pathway. Interestingly, signaling through PER2 leads to increased casein kinase 1 (CK1)-ε activity, a signaling events also associated with β-catenin phosphorylation and degradation. Therefore, we hypothesized that silencing of Per2 increases β-catenin stability and nuclear translocation and subsequent transactivation of the CTGF gene.
Eyes from wild type and Per2m/m were enucleated, sectioned and stained for CTGF. Human retinal endothelial cells (HRECs) were transfected with siRNA for Per2 and the protein expression of CTGF and β-catenin was determined using qRT-PCR and western blot analysis. The nuclear localization of β-catenin was examined by immuonofluorescence. ATCF/LEF luciferase reporter assay (TOPFLASH) assay was used to validate the involvement of β-catenin in the activation of CTGF.
Per2m/m retinas showed increased CTGF immunostaining in the ganglion cell layer and retinal endothelium. Loss of Per2 function resulted in increased expression of CTGF and β-catenin. siRNA-mediated silencing of Per2 in HRECs increased nuclear accumulation of β-catenin as compared to the control siRNA. The TOPFALSH assay showed an increase in luminescence for HRECs transfected with Per2 siRNA.
Our studies show that loss of Per2 results in activation of CTGF via nuclear entry of β-catenin. The future therapies aimed at targeting the β-catenin-CTGF pathway may provide a novel tool for correction of vascular dysfunction in DR.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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