Vascular Expression of Toll‐Like Receptor 4 and Myeloid Differentiation Factor‐88 Mediate Endothelial Dysfunction in Response to Lipopolysaccharide Independent of Circulating Immune Cells

Abstract

The goal of this study was to test the hypothesis that vascular expression of toll‐like receptor 4 (TLR4) and its primary downstream signaling pathway, myeloid‐differentiation factor‐88 (MyD88), mediate endothelial dysfunction in response to lipopolysaccharide (LPS). Carotid arteries from wild‐type (C57Bl/6), Tlr4‐Lps‐d (Tlr4 mutant mice), and Myd88‐deficient (Myd88−/−) mice were incubated with either vehicle (saline) or LPS (0.05–0.50 μg/ml) for 22 hrs. LPS, but not vehicle, treatment produced concentration‐dependent impairment (P<0.05) of endothelium‐dependent relaxation to acetylcholine (ACh) in wild‐type mice. In contrast, in carotid arteries from Tlr4‐Lps‐d or Myd88−/− mice endothelial responses to ACh following LPS incubation were found to be similar (P>0.05) to responses in wild‐type mice incubated with vehicle. LPS has no effect (P>0.05) on vascular response to sodium nitroprusside in any of the six experimental groups. Both basal and NADPH‐stimulated superoxide levels were selectively increased (P<0.05) in vessels from wild‐type, but not Tlr4‐Lps‐d or Myd88−/−, mice treated with LPS. RNA expression revealed that both TLR4 and MyD88 are indeed expressed in the vascular wall under basal conditions and that LPS induces differential expression of a number of inflammatory gene products, including Tlr4, Myd88, and Nfkb1. Pathway analysis revealed a linkage between TLR4 and MyD88 in the vessels wall with IL‐6 and downstream targets such as Nox2 and Nox4. These findings serve to link activation of TLR4 and downstream signaling through MyD88 and NFkB1 with increases in vascular superoxide and functional impairment of endothelial function in response to LPS. Taken together, these findings demonstrate that both Tlr4 and Myd88 expression and activity within the vascular wall contributes to LPS‐induced impairment of endothelial function. The fact that experiments were performed ex vivo, demonstrate that the effects of LPS on endothelial function occur directly within the vascular wall independently of circulating immune cell activation.Support or Funding InformationSupported in part by NIH/NIBIB R01‐EB2006.