Abstract 172: Toll-like Receptor-4 Mediates Augmented Contractility In Diabetic Resistance Vessels

Abstract

Hyperglycemia, the hallmark of diabetes, plays a central role in the pathogenesis of diabetic vascular dysfunction; however the underlying mechanisms are not entirely understood. We previously reported that inhibition of Toll-Like Receptor- 4 (TLR4), a key receptor of the innate immune system, improves vascular function in spontaneously hypertensive rats (SHR) revealing a novel role of TLR4 in the vascular system. In our current study, we hypothesized that upregulation of TLR4 in the vasculature following hyperglycemia leads to augmented vascular contractility. Third order mesenteric arteries from streptozotocin (STZ)-induced diabetic Sprague-Dawley rats (glucose 279 ± 11.6mg/dL) and their respective controls (98 ± 8.03mg/dL ), were incubated with either neutralizing antibody to TLR4 (1ug/mL) or IgG (1ug/mL) for 30 min and contractile responses to phenylephrine (PE) were evaluated ex vivo. Vessels from STZ rats exhibited greater maximal contractile force to PE compared to control (Emax 38.99 ± 1.45 vs. 23.98 ± 0.73 (mN), p<0.05, respectively). Anti-TLR4 incubation attenuated this response to PE in vessels from STZ rats (29.71 ± 1.69mN) but had no effect in control vessels (21.03 ± 1.02mN). To test whether hyperglycemia modulates vascular TLR4, mesenteric vascular smooth muscle cell (MVSM) cultures were stimulated with high glucose (HG 25mM) or normal glucose (NG 5mM) for 48 hours. HG increased protein expression of TLR4 in MVSM compared to MVSM stimulated with NG (2.74±0.34 vs 1.38±0.20, p<0.05). Concurrently, HG caused activation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) (5.04±0.83 vs 3.80±0.90, p<0.05), increased levels of PCNA, a marker of proliferation, (1.34±0.11 vs 0.35±0.04, p<0.05), and decreased levels of calponin, a marker of contractile phenotype, (1.45±0.14 vs 3.62±0.40, p<0.05). Levels of total intracellular reactive oxygen species (ROS) were robustly augmented under HG condition (2.2 fold of the normal glucose). Our results suggest that hyperglycemia upregulates vascular TLR4 leading to augmented vascular contractility, potentially due to activation of ERK1/2, increased ROS and MSMCs proliferation.