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 (GPR39 ^KO ) 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 GPR39 ^KO mice demonstrated a faster pace of revascularization from hind limb ischemia and lower incidence of tissue necrosis than GPR39 wild-type (GPR39 ^WT ) counterparts. These findings have provided a conceptual framework for developing therapeutic tools that ablate or inhibit GPR39 for ischemic tissue repair under metabolic stress.

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.

GPR35 (G-protein–coupled receptor 35) is a poorly characterized receptor that has garnered increased interest as a therapeutic target through its implications in a range of inflammatory and cardiovascular diseases, but its biological functions stay largely unknown. The current study evaluated the effect of GPR35 on endothelial cell (EC) functions and hemodynamic homeostasis. In primary human aortic ECs, the expression of GPR35 was manipulated by transfections of adenovirus carrying either GPR35 cDNA or shRNA against GPR35, using adenovirus carrying β-gal as control. Mouse aortic ECs were isolated and cultured from GPR35 knockout and wild-type control mice. Our results indicated that genetic inhibition of GPR35 in human and mouse ECs significantly promoted cell proliferation, migration, and tube formation in vitro. The GCH1 (guanosine triphosphate cyclohydrolase I)-mediated biosynthesis of tetrahydrobiopterin was enhanced, reducing intracellular superoxide. Knocking down GCH1 or eNOS (endothelial nitric oxide synthase) significantly blunted the robust angiogenesis induced by GPR35 suppression. Male GPR35 knockout mice demonstrated reduced basal arterial blood pressure and an attenuated onset of hypertension in deoxycorticosterone acetate-salt induced hypertensive model compared with male GPR35 wild-type control mice in vivo, with concomitant improved endothelium-dependent vasodilation and decreased superoxide in isolated aortas. The difference in arterial blood pressure was absent between female GPR35 wild-type control and female GPR35 knockout mice. Our study provides novel insights into the roles of GPR35 in endothelial function and vascular tone modulation that critically contribute to the pathophysiology of blood pressure elevation. Antagonizing GPR35 activity might represent a potentially effective therapeutic approach to restore EC function and hemodynamic homeostasis.

Animal models of metabolic disorders are essential to studying pathogenic mechanisms and developing therapies for diabetes, but the induction protocols vary, and sexual dimorphism often exists. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, blood glucose and lipid profiles were measured. The high-fat (HF) diet damaged insulin sensitivity and increased triglycerides, total cholesterol, LDL-cholesterol, HDL-cholesterol, and liver lipid deposition. STZ increased blood glucose and liver fibrosis with less effects on blood lipids or liver lipid deposition. The combination of DIO and STZ treatments led to significant liver lipid deposition and fibrosis. Female mice showed delayed body weight gain on HF diet and resisted STZ-induced hyperglycemia. However, once they developed DIO, which occurs around 26 weeks of HF diet, the female mice were prone to STZ-induced hyperglycemia. In hindlimb ischemia, male mice in the DIO-STZ group showed significantly worse neovascularization compared with DIO or STZ groups. The DIO-STZ females showed significantly worse recovery than the DIO-STZ males. Our observations suggest that DIO-STZ is a plausible model for studying metabolic and cardiovascular disorders in obesity and diabetes. Moreover, the findings in female animals stress the need to assess sexual dimorphism and investigate the underlying mechanisms that contribute to the worse vasculopathy manifestations in females in metabolic models.

Highly homologous members of the Gα _i family, Gα _i1-3 , have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gα _i1 effector, however, it is poorly activated by Gα _i2 . Here, in a proteomic proximity labeling screen we observed a strong preference for Gα _i1 relative to Gα _i2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gα _i1 into Gα _i2 or substitution of a single amino acid, A230 in Gα _i2 to the corresponding D in Gα _i1 , largely rescues PRG activation and interactions with other Gα _i targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gα _i2 relative to Gα _i1 and that mutation of A230 ^s4h3.3 to D in Gα _i2 stabilizes HD-RLD interactions through formation of an ionic interaction with R145 ^HD.11 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229 ^s4h3.3 in Gα _i1 interacts with R144 ^HD.11 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gα _i2 is unable to form the “ionic lock” to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gα _i1 or Gα _i2 by GPCRs.