Toward Targeting GPR31 and GPR39 Signaling in Diabetic Retinopathy - Summary G-coupled protein receptor-31 and 39 (GPR31/GPR39) are specific receptors for 12/15-Lipoxygenase (12/15- LO) metabolites; 12- and 15-HETEs respectively. The role of GPR31/GPR39 in the pathogenesis of diabetic retinopathy (DR) has not yet been investigated. Our previous studies demonstrated that diabetes induces upregulation of retinal 12/15-LO and its metabolites, 12- and 15-HETEs, in human and experimental mice. Furthermore, 12/15-LO blockade preserved the blood-retinal barrier in diabetic mice and reduced retinal neovascularization in oxygen-induced retinopathy. Treatment of Müller cells (MCs) with 12/15-LO metabolites induced inflammatory cytokines and upregulated VEGF. However, there is still a critical gap in understanding the mechanism by which 12/15-LO metabolites activate retinal endothelial (RECs) and MCs. Our objective is to determine whether GPR31/GPR39 are involved in the pro-inflammatory and -angiogenic effects of 12/15-LO metabolites in RECs and MCs that lead to vascular dysfunction in DR. Our preliminary data demonstrated expression of GPR31/GPR39 in RECs and MCs and both receptors are upregulated in the retinas of diabetic mice. The transmembrane helices of the GPR31 and GPR39 proteins can be superimposed and 12- HETE bound to the cognate GPR31 receptor and GPR39 on the extracellular side of the proteins. Similarly, 15- HETE binds to GPR39 and GPR31. We will test the hypothesis that in RECs and MCs, GPR31 and GPR39 contribute to activation of signaling pathways that lead to vascular dysfunction in DR. We will test this hypothesis through two specific aims: Aim1: Determine the affinities and relative substrate specificities of GPR31 and GPR39 for 12- and 15-HETEs in RECs and MCs under normal and hyperglycemic conditions. Aim 2: Examine the effects of GPR31 or GPR39 gain-loss-of-function on RECS and MCs under normal or hyper-glycemic conditions. For this purpose, recombinant GPR31/GPR39 receptors will be expressed and purified and the interactions between the receptors and the HETEs will be examined by biolayer interferometry (BLI) and isothermal titration calorimetry (ITC). Affinity and selectivity of the GPR receptors will be tested against synthetic macrocyclic receptors for HETEs. We will test the formation of GPR-HETE complexes in human retinal endothelial cells (HRECs) and rat MCs under both normal glucose (NG), high glucose (HG) compared to osmotic control (OC). We predict increased GPR-HETE complexes by HG treatment. We will determine the effect of GPR31/GPR39 overexpression or inhibition on HRECs' barrier function, migration and tube formation under NG, HG, 12-HETE or 15-HETE conditions. Similarly, effect of GPR31/GPR39 gain-loss-of-function on MCs' viability, inflammatory response and levels of VEGF, and oxidative stress will be assessed. We predict that inhibition of GPR31 and/or GPR39 will improve HRECs barrier function and ameliorate inflammatory, oxidative, and VEGF pathways in MCs under HG or HETEs treatment. Successful completion of this R21 will establish GPR31/GPR39 as potential therapeutic targets to ameliorate vascular damage in DR.
