Endothelial dysfunction is a key risk factor in diabetes-related multiorgan damage. Methylglyoxal (MGO), a highly reactive dicarbonyl generated primarily as a by-product of glycolysis, is increased in both type 1 and type 2 diabetic patients. MGO can rapidly bind with proteins, nucleic acids, and lipids, resulting in structural and functional changes. MGO can also form advanced glycation end products (AGEs). How MGO causes endothelial cell dysfunction, however, is not clear. Human aortic endothelial cells (HAECs) from healthy (H-HAECs) and type 2 diabetic (D-HAECs) donors were cultured in endothelial growth medium (EGM-2). D-HAECs demonstrated impaired network formation (on Matrigel) and proliferation (MTT assay), as well as increased apoptosis (caspase-3/7 activity and TUNEL staining), compared with H-HAECs. High glucose (25 mM) or AGEs (200 ng/ml) did not induce such immediate, detrimental effects as MGO (10 µM). H-HAECs were treated with MGO (10 µM) for 24 h with or without the ATP-sensitive potassium (KATP) channel antagonist glibenclamide (1 µM). MGO significantly impaired H-HAEC network formation and proliferation and induced cell apoptosis, which was reversed by glibenclamide. Furthermore, siRNA against the KATP channel protein Kir6.1 significantly inhibited endothelial cell function at basal status but rescued impaired endothelial cell function upon MGO exposure. Meanwhile, activation of MAPK pathways p38 kinase, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) (determined by Western blot analyses of their phosphorylated forms, p-JNK, p-p38, and p-ERK) in D-HAECs were significantly enhanced compared with those in H-HAECs. MGO exposure enhanced the activation of all three MAPK pathways in H-HAECs, whereas glibenclamide reversed the activation of p-stress-activated protein kinase/JNK induced by MGO. Glyoxalase-1 (GLO1) is the endogenous MGO-detoxifying enzyme. In healthy mice that received an inhibitor of GLO1, MGO deposition in aortic wall was enhanced and endothelial cell sprouting from isolated aortic segment was significantly inhibited. Our data suggest that MGO triggers endothelial cell dysfunction by activating the JNK/p38 MAPK pathway. This effect arises partly through activation of KATP channels. By understanding how MGO induces endothelial dysfunction, our study may provide useful information for developing MGO-targeted interventions to treat vascular disorders in diabetes.

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Introduction: Endothelial cell (EC) permeability is essential to vascular homeostasis in diabetes. MicroRNAs (miRs) are critical gene regulators whose roles in the EC permeability have yet to be elucidated. It remains unclear whether miR-200 and miR-466 regulate EC permeability. We hypothesized that the inhibition of miR-466 and miR-200 can restore vascular integrity and accelerate wound healing via targeting Claudin-5. Methods and Results: Human dermal microvascular ECs from healthy subjects and type 2 diabetic patients at passages 5-7 were cultured in vitro. The EC permeability was significantly increased by high glucose (25mM)or silencing Claudin-5 as measured by FITC-Dextran Transwell assay (n = 5, p<0.05). Claudin-5 protein levels were significantly reduced in diabetic ECs and healthy ECs exposed to high glucose. The reduction of Claudin-5 protein was restored by the inhibition of miR-466 (Western Blot, n = 5, p<0.05), though both miR-200 and miR-466 are directly bound with the 3’ UTR of Claudin-5 mRNA (luciferase 3’UTR reporter assay). Furthermore, silencing Claudin-5 diminished the protection of miR-200/miR-466 inhibition on EC permeability damaged by high glucose (n = 5, p<0.05). In the in vivo study, the topical application of miR-200 and miR-466 inhibitors showed enhanced efficacy in accelerating the closure of a 7-mm full-thickness excisional wound in a type 2 diabetic (db/db) mouse model, with concomitant robust wound capillary formation (CD31 immunochemistry), compared with the topical application of miR-200 inhibitor alone (n = 5, p<0.05). Claudin-5 levels were increased in the wounds receiving miR-466 inhibitor alone or both miR-200 and miR-466 inhibitors (n = 5, p<0.05). Conclusions: Our study demonstrated the potentially effective approach of miR-200/miR-466 cocktail inhibition to restore vascular integrity and tissue repair in hyperglycemia. This may provide a novel therapeutic strategy targeting miRs for the treatment of wound healing in diabetes.