title: Uncoupling of eNOS contributes to redox-sensitive leukocyte recruitment and microvascular leakage elicited by methylglyoxal.
abstract: Elevated levels of the glycolysis metabolite methylglyoxal (MG) have been implicated in impaired leukocyte-endothelial interactions and vascular complications in diabetes, putative mechanisms of which remain elusive. Uncoupling of endothelial nitric oxide synthase (eNOS) was shown to be involved in endothelial dysfunction in diabetes. Whether MG contributes to these effects has not been elucidated. By using intravital microscopy in vivo, we demonstrate that MG-triggered reduction in leukocyte rolling velocity and increases in rolling flux, adhesion, emigration and microvascular permeability were significantly abated by scavenging reactive oxygen species (ROS). In murine cremaster muscle, MG treatment reduced tetrahydrobiopterin (BH4)/total biopterin ratio, increased arginase expression and stimulated ROS and superoxide production. The latter was significantly blunted by ROS scavengers Tempol (300μM) or MnTBAP (300μM), by BH4 supplementation (100μM) or by NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 20μM). In these tissues and cultured murine and human primary endothelial cells, MG increased eNOS monomerization and decreased BH4/total biopterin ratio, effects that were significantly mitigated by supplementation of BH4 or its precursor sepiapterin but not by L-NAME or tetrahydroneopterin, indicative of MG-triggered eNOS uncoupling. MG treatment further decreased the expression of guanosine triphosphate cyclohydrolase I in murine primary endothelial cells. MG-induced leukocyte recruitment was significantly attenuated by supplementation of BH4 or sepiapterin or suppression of superoxide by L-NAME confirming the role of eNOS uncoupling in MG-elicited leukocyte recruitment. Together, our study uncovers eNOS uncoupling as a pivotal mechanism in MG-induced oxidative stress, microvascular hyperpermeability and leukocyte recruitment in vivo.
authors.affiliation: Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5.
authors.forename: Yang
authors.initials: Y
authors.lastname: Su
authors.forename: Syed M
authors.initials: SM
authors.lastname: Qadri
authors.forename: Mokarram
authors.initials: M
authors.lastname: Hossain
authors.forename: Lingyun
authors.initials: L
authors.lastname: Wu
authors.forename: Lixin
authors.initials: L
authors.lastname: Liu
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects*; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010084:Oxidation-Reduction; D011765:Pyruvaldehyde / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D001708:Biopterins; D011765:Pyruvaldehyde; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin
keywords: Leukocyte recruitment; Methylglyoxal; Microvascular permeability; Superoxide; eNOS uncoupling