Deletion Of G Protein-Coupled Receptor 39 In Endothelial Cells Protects Angiogenesis In Hyperglycemia

Aberrant endothelial cell (EC)-mediated angiogenesis is a crucial feature contributing to critical limb ischemia in patients with diabetes. The sonic hedgehog (SHH) pathway robustly participates in angiogenesis but is repressed in hyperglycemia by not fully understood mechanisms. This study investigated the impact of an orphan G protein-coupled receptor (GPR), GPR39, on SHH pathway activation in ECs and on ischemia-induced angiogenesis in animals with chronic hyperglycemia. Human aortic ECs from healthy and type 2 diabetic (T2D) donors were cultured in vitro. GPR39 mRNA expression was highly elevated in T2D ECs. EC proliferation, migration, tube formation, and production of multiple proangiogenic factors in healthy ECs were significantly attenuated by adenovirus-mediated GPR39 gene transfer in vitro. The GPR39 agonist TC-G-1008 diminished EC proliferation, migration, and tube formation in healthy and T2D ECs. On the contrary, mouse aortic ECs isolated from GPR39 global knockout (GPR39KO) mice displayed enhanced migration and proliferation than the wildtype (GPR39WT) mouse ECs. GPR39 suppressed the constitutive (basal) and ligand-dependent activation of the SHH effector GLI-1. Co-immunoprecipitation experiments revealed that this is potentially achieved by directly activating the Suppressor of Fused (SUFU), the inhibitory signaling molecule on the SHH pathway. Furthermore, augmented migration induced by GPR39 gene knockdown or deletion was blunted by simultaneously knocking down GLI1. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, male GPR39KO mice demonstrated a much faster pace of revascularization from ischemia in their hind limb and lower incidence of tissue necrosis than GPR39WT counterparts. However, these benefits were abolished when the SHH pathway inhibitor cyclopamine suppressed the activation of the SHH pathway. Intramuscular injection of adeno-associated virus (AAV)-mediated shRNA against GPR39 significantly accelerated revascularization and prevented tissue necrosis compared to AAV-mediated scramble shRNA treatment. Our results reveal that inhibition of GPR39 promotes angiogenesis by enhancing SHH signaling in ECs and restores revascularization to ischemic injury in animals with chronic hyperglycemia. These novel findings have provided state-of-the-art evidence and a conceptional framework for developing molecules that genetic ablate or pharmacologically inhibit GPR39 as a potential therapeutic tool in ischemic tissue repair under chronic metabolic stress.Keywords: G Protein-Coupled Receptor 39, endothelial function, limb ischemia, angiogenesis, hyperglycemi