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Ashay D. Bhatwadekar, Jeffrey S. Thinschmidt, Yuanqing Yan, James M. Dominguez, Choogon Lee, Julia V. Busik, Maria B. Grant; Retinal Vascular Dysfunction Induced By Mutations In Per2 Clock Gene Is Mediated By Upregulation Of TGF-β1 Pathway. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4117.
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Diabetic retinopathy is associated with upregulation of transforming growth factor β1 (TGF-β1) and its downstream effectors. Mutations of clock gene Per2 induce endothelial dysfunction and inadequate vascular repair. In this study, we hypothesized that retinal vascular dysfunction in Per2 mutant mice is associated with modulation of TGF-β1 and an imbalance of nitric oxide (NO) which could mimic the retinal vascular dysfunction seen in diabetes.
Per2 (Per2tm1Brd/J) or wild type (WT) mice of 4 and 12 months of age were utilized for the study. Retinas were processed for mRNA expression of TGF-β1, DNA-binding protein inhibitor (ID-1), connective tissue growth factor (CTGF), fibronectin (FN) and plasminogen activator inhibitor (PAI-1), eNOS and iNOS by RT-PCR. Retinal vascularity was assessed on flat mounted retinas following the injection of rhodamine conjugated BS-1 isolectin. Acellular capillaries were evaluated using trypsin digestion. In parallel studies bone marrow progenitor cells (BMPCs) were evaluated for NO levels.
Per2 mutant retinas showed a 2 fold increase in TGFβ1 mRNA, accompanied by upregulation in PAI-1 (2 fold; p<0.05) and Id-1 expression (2 fold p<0.05). CTGF, an important downstream mediator of pro-fibrotic effect of TGFβ1 was upregulated by 2.5-3 folds (p<0.05) while FN expression was increased 2.5 fold (p<0.05) in Per2 mutant mice. There was no change in number of branch points of retinal vasculature of Per2 mutant mice, however there was a 40 % increase (p<0.05) in number of acellular capillaries in Per2 mutant mice when compared to WT. BMPCs obtained from Per2 mutant mice showed reduced proliferative response with 1.5 fold (p<0.05) decrease in NO levels.
In conclusion, retinal vascular dysfunction in Per2 mutant mice is mediated through upregulation of TGF-β1 and enhanced oxidative stress, this together with reduced contribution by BMPCs and increased acellular capillaries results in pathologic alterations of retina similar to diabetic retinopathy.
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