Inhibition of GPR39 restores defects in endothelial cell–mediated neovascularization under the duress of chronic hyperglycemia: evidence for regulatory roles of the sonic hedgehog signaling axis

Impaired endothelial cell (EC)–mediated angiogenesis contributes to critical limb ischemia in diabetic patients. The sonic hedgehog (SHH) pathway participates in angiogenesis but is repressed in hyperglycemia by obscure mechanisms. We investigated the orphan G protein–coupled receptor GPR39 on SHH pathway activation in ECs and 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 significantly elevated in T2D. The EC proliferation, migration, and tube formation were attenuated by adenovirus-mediated GPR39 overexpression (Ad-GPR39) or GPR39 agonist TC-G-1008 in vitro. The production of proangiogenic factors was reduced by Ad-GPR39. Conversely, human ECs transfected with GPR39 siRNA or the mouse aortic ECs isolated from GPR39 global knockout (GPR39KO) mice displayed enhanced migration and proliferation compared with their respective controls. GPR39 suppressed the basal and ligand-dependent activation of the SHH effector GLI1, leading to attenuated EC migration. Coimmunoprecipitation revealed that the GPR39 direct binding of the suppressor of fused (SUFU), the SHH pathway endogenous inhibitor, may achieve this. Furthermore, in ECs with GPR39 knockdown, the robust GLI1 activation and EC migration were abolished by SUFU overexpression. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, the GPR39KO mice demonstrated a faster pace of revascularization from hind limb ischemia and lower incidence of tissue necrosis than GPR39 wild-type (GPR39WT) counterparts. These findings have provided a conceptual framework for developing therapeutic tools that ablate or inhibit GPR39 for ischemic tissue repair under metabolic stress

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

MicroRNA-466 and microRNA-200 increase endothelial permeability in hyperglycemia by targeting Claudin-5

Endothelial cell (EC) permeability is essential to vascular homeostasis in diabetes. MicroRNAs are critical gene regulators whose roles in the EC permeability have yet to be characterized. This study aims to examine the change in cell permeability induced by miR-200 and miR-466 in ECs. Human aortic ECs and dermal microvascular ECs from healthy subjects and type 2 diabetic patients were used. Our in vitro experiments unveiled higher expressions of miR-200 family members and miR-466 in diabetic ECs and in healthy ECs when exposed to high glucose. Overexpression of both miR-200 and miR-466 significantly increased EC permeability through transcriptional suppression of Claudin-5, the cell tight junction protein, by directly binding to its 3′ untranslated region. In a mouse model of chronic hyperglycemia mimicking type 2 diabetes in humans (db/db mice), the delayed closure rate of a full-thickness excisional wound was partly rescued by topical application of the miR-200 inhibitor. The topical application of both miR-200 and miR-466 inhibitors exhibited improved efficacy in accelerating wound closure compared with the topical application of miR-200 inhibitor alone. Our study demonstrated the potentially effective approach of miR-200/miR-466 cocktail inhibition to restore vascular integrity and tissue repair in hyperglycemia