Mechanisms of Methylglyoxal-elicited Leukocyte Recruitment

Methylglyoxal (MG) is a reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. In hyperglycemic conditions, an increased MG level has been linked to the development of diabetes and the accompanying vascular inflammation encountered at both macro- and microvascular levels. The present study explores the mechanisms of MG-induced leukocyte recruitment in mouse cremasteric microvasculature. Biochemical and intravital microscopy studies performed suggest that administration of MG (25 and 50 mg/kg) to mouse cremaster muscle tissue induces dose-dependent leukocyte recruitment in cremasteric vasculature with 84-92% recruited cells being neutrophils. MG treatment up-regulated the expression of endothelial cell (EC) adhesion molecules P-selectin, E-selectin and intercellular adhesion molecule-1 (ICAM-1) via the activation of nuclear factor-κB (NF-κB) signalling pathway and contributed to the increased leukocyte rolling flux, reduced leukocyte rolling velocity, and increased leukocyte adhesion, respectively. The inhibition of NF-κB blunted MG-induced endothelial adhesion molecule expression and thus attenuated leukocyte recruitment. Further study of signalling pathways revealed that MG induced Akt-regulated transient glycogen synthase kinase 3 (GSK3) activation in ECs, which was responsible for NF-κB activation at early time-points (< 1 h). After MG activation for 1 h, the endothelial GSK3 activity was decreased due to the up-regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1), which was responsible for maintaining NF-κB activity at later time-points. Silencing GSK3 or SGK1 attenuated P-selectin, E-selectin and ICAM-1 expression in ECs, and abated MG-induced leukocyte recruitment. SGK1 also promoted cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activity which was partially involved in ICAM-1 expression. Silencing CREB blunted ICAM-1 expression while P-selectin and E-selectin levels remained unaffected. MG also induced GSK3 activation in isolated neutrophils after 30 min treatment, an effect that was not responsible for MG-elicited Mac-1 expression. These data suggest the sequential activation of GSK3 and SGK1 in ECs as the pivotal signalling mechanism in MG-elicited leukocyte recruitment. Additionally, MG-treatment led to uncoupling of endothelial nitric oxide synthase (eNOS) following MG-induced superoxide generation in ECs. MG triggered eNOS uncoupling and hypophosphorylation associated with superoxide generation and biopterin depletion in EA.hy926 ECs. In cremaster muscle, as well as in cultured murine and human primary ECs, MG increased eNOS monomerization and decreased 5,6,7,8-tetrahydroboipterin (BH4)/total biopterin ratio, effects that were significantly mitigated by supplementation of BH4 or its precursor sepiapterin but not by NG-nitro-L-arginine methyl ester (L-NAME) or 5,6,7,8-tetrahydroneopterin (NH4). These observations confirm that MG administration triggers eNOS uncoupling. In murine cremaster muscle, MG triggered the reduction of leukocyte rolling velocity and the increases in rolling flux, adhesion, emigration and microvascular permeability. 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. MG treatment further decreased the expression of guanosine triphosphate cyclohydrolase I in murine primary ECs, suggesting the impaired BH4 biosynthesis caused by MG. Taken together, these data suggest that vascular inflammation and endothelial dysfunction occurring in diabetes may be linked to GSK3/SGK1 regulated adhesion molecule expression, as well as the uncoupling of eNOS evoked by elevated levels of MG. These findings not only provide a better understanding of the role of MG in the development of diabetic vascular inflammation, but also suggest the potential therapeutic targets for MG-sensitive endothelial dysfunction in diabetes