Abstract 91: Inhibition of Toll-like Receptor 4 Suppresses Reactive Oxygen Species Generation in Diabetic Vasculature
Reactive oxygen species (ROS) are augmented in diabetes and play a central role in vascular dysfunction. TLR4, a key component of the innate immune system, is elevated during diabetes; however, the mechanistic link between TLR4, ROS, and vascular dysfunction remains elusive. Our previous results show that under high glucose (HG), TLR4 is upregulated in vascular smooth muscle cells (VSMCs) and mediates HG-induced ROS production. We hypothesized that TLR4 augments ROS in diabetic resistance vessels, which contributes to vascular dysfunction. To test our hypothesis, streptozotocin (STZ)-induced diabetic Sprague-Dawley rats (65mg/kg; 5weeks) were treated with 50ug/day of CLI-095, an inhibitor of TLR4, over 14 days. Glucose levels were increased (390±13 vs. 97±8.2mg/dL control) and body weight decreased (310±38.4 vs. 385±21g control) in the STZ group. These effects were unchanged by CLI-095 treatment. Mesenteric resistance arteries (MRA) from STZ rats exhibited impaired acetylcholine-induced relaxation (35.26±4.38% vs. 81.83±2.61% control, p<0.05), and CLI-095 treatment ameliorated this effect (71.99±3.48%, p<0.05). Moreover, dihydroethidium staining showed that the increase in ROS due to STZ (1.8 fold vs. control, p<0.05) was attenuated after CLI-095 treatment (1.6 fold vs. STZ, p<0.05). To test if TLR4 signaling is activated in MRA from the STZ rats, MyD88 protein levels, a downstream adaptor molecule, were measured. Arteries from the STZ rats showed increased expression of MyD88 (1.6 fold vs. control, p<0.05) which was reduced after CLI-095 treatment (1.2 fold vs. STZ, p<0.05). Finally, we assessed if TLR4 signaling in VSMCs following HG treatment is altered. Immunoprecipitation showed that HG did not alter the interaction between TLR4 and MyD88 in endothelial cells. In contrast, HG conditions increased TLR4 and MyD88 interaction in VSMCs. Concurrently, HG-induced activation of NFKB in VSMCs was diminished in cells pre-treated with CLI-095. Together our data suggest that activation of TLR4 signaling in VSMCs leads to ROS generation thereby contributing to a reduction in nitric oxide bioavailabitity contributing to endothelial dysfunction in diabetes. Thus, TLR4 is a putative target for the treatment of diabetic vascular complications.
- © 2013 by American Heart Association, Inc.