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Pamela M Martin, Deeksha Gambhir, Wanwisa Promsote, Rajalakshmi Veeranan-Karmegam, ; Absence of the anti-inflammatory receptor GPR109A is associated with compromised outer blood-retinal barrier integrity. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2956.
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Diabetic retinopathy (DR) is a leading cause of blindness worldwide. Alterations of blood-retinal barrier (BRB) integrity contribute principally to cellular dysfunction and vision loss in the disease. Much attention has been given to factors and mechanisms regulating the maintenance of inner-BRB integrity however, the outer-BRB, of which RPE is a major component, has received considerably less attention. We demonstrated previously, the expression of the Gi-linked protein coupled receptor, GPR109A, in retina, a tissue in which the receptor localizes largely to the RPE basolateral membrane. Subsequent in vitro and in vivo studies of GPR109A function in this cell type revealed a role for the receptor in the regulation of inflammatory signaling. Increased inflammation and BRB breakdown go hand-in-hand. Therefore, here we examined the relevance of GPR109A expression to factors relevant to the preservation of outer-BRB integrity in Gpr109a+/+ and Gpr109a-/- mouse retina.
Micron III technology was used to obtain fundoscopic images of Gpr109a+/+ (wild type, WT) and Gpr109a-/- (knockout, KO) mouse eyes at various ages. ZO-1 and occludin expression was examined in WT and KO RPE flatmount preparations by immunofluorescence. qPCR and Western blot analyses of these proteins in RNA and protein samples obtained from WT and KO RPE/eyecup tissues were also performed. Primary RPE cell cultures were established from additional WT and KO mouse eyes and cultured on permeable supports for a period of 6 weeks. These polarized cells were then used for in vitro permeability assays.
Disruptions in RPE morphology were readily apparent upon fundoscopic imaging of KO mouse eyes and immunofluorescence analysis of ZO-1 and occludin expression in corresponding RPE flatmount preparations. Additionally, levels of ZO-1 and occludin mRNA and protein were significantly reduced. Altered RPE junctional protein expression/compromised barrier function was confirmed by in vitro permeability assays which revealed increased apical to basolateral leakage of FITC-Dextran dye in KO RPE.
GPR109A expression is important to the maintenance of outer-BRB integrity. Strategies to augment GPR109A expression in pathologic conditions such as diabetes may be useful to the prevention and treatment of outer-BRB breakdown and in turn to the prevention of vision loss and blindness.
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