Abstract 20372: High-Mobility Group Box-1 Activates Toll-Like Receptor 4: A Potential Novel Mechanism in Endothelial Dysfunction in Diabetes Mellitus
Background: Macro and microvascular complications are the main problem of all forms of diabetes mellitus. Endothelial dysfunction plays a key role in the pathogenesis of vascular complications, and most anti-diabetic drugs still do not avoid the development of endothelial dysfunction. Recent evidence shows that diabetic patients have increased serum high-mobility group box-1 (HMGB1) levels and upregulation of Toll-like receptor 4 (TLR4), a key component of innate immunity; however whether HMGB-1 is linked with TLR4 and it has impact in diabetic endothelial dysfunction is unknown. We hypothesize that HMGB1 activates TLR4 in endothelial cells (EC), which in turn increases generation of reactive oxygen species (ROS) and reduces NO bioavailability thereby causing endothelial dysfunction.
Methods: Human EC were treated with recombinant 200ng/mL HMGB1 for 24 hours and intracellular ROS were detected by H2DCF-DA fluorescence and protein expression of MyD88, a downstream adaptor of TLR4 signaling, was measured by immunoblotting. Diabetes was induced in wild-type (WT) and knockout (KO) TLR4 -/- mice by streptozotocin (STZ) (50mg/kg; 5 weeks) treatment and ROS levels were determined in blood vessel segments. Endothelial function was assessed in mesenteric arteries from STZ-induced diabetic rats treated with 50ug/day CLI095, an inhibitor of TLR4, over 14 days using a wire myograph.
Results: HMGB1 reduced phosphorylated eNOS levels (0.8 fold, p<0.001), increased ROS generation in EC (2.2 fold, p<0.05) and increased MyD88 expression (2 fold, p<0.05), compared with unstimulated cells. Diabetic mice displayed exaggerated ROS levels in blood vessel segments (4.1 fold vs. control, p<0.05) which was attenuated in KO TLR4-/- mice (1.8 fold vs.diabetic WT, p<0.001). Co-Immunoprecipitation showed increased TLR4-MyD88 interaction in mesenteric arteries from STZ rats and impaired acethylcoline-induced relaxation (30.41±5.24% vs. 85.70±3.50% control, p<0.05), which was ameliorated with CLI95 treatment (65.84±4.25%, p<0.05).
Conclusions: Our data suggest that TLR4 signaling plays role in the diabetic endothelial dysfunction via increased ROS levels and implies HMGB1 as a novel trigger of endothelial dysfunction in diabetes through TLR4 signaling.
Author Disclosures: M. Carrillo-Sepulveda: None. R. Webb: None.
This research has received full or partial funding support from the American Heart Association
- © 2014 by American Heart Association, Inc.