Abstract

Overproduction of nitric oxide (NO) is thought to be a key mediator of the vascular dysfunction and severe hypotension in patients with endotoxaemia and septic shock. The contribution of NO produced directly in the vasculature by endothelial cells to the hypotension seen in these conditions, vs. the broader systemic increase in NO, is unclear. To determine the specific role of endothelium derived NO in lipopolysaccharide (LPS)-induced vascular dysfunction we administered LPS to mice deficient in endothelial cell tetrahydrobiopterin (BH4), the essential co-factor for NO production by NOS enzymes. Mice deficient in endothelial BH4 production, through loss of the essential biosynthesis enzyme Gch1 (Gch1fl/flTie2cre mice) received a 24hour challenge with LPS or saline control. In vivo LPS treatment increased vascular GTP cyclohydrolase and BH4 levels in aortas, lungs and hearts, but this increase was significantly attenuated in Gch1fl/flTie2cre mice, which were also partially protected from the LPS-induced hypotension. In isometric tension studies, in vivo LPS treatment reduced the vasoconstriction response and impaired endothelium-dependent and independent vasodilatations in mesenteric arteries from wild-type mice, but not in Gch1fl/flTie2cre mesenteric arteries. Ex vivo LPS treatment decreased vasoconstriction response to phenylephrine in aortic rings from wild-type and not in Gch1fl/flTie2cre mice, even in the context of significant eNOS and iNOS upregulation. These data provide direct evidence that endothelial cell NO has a significant contribution to LPS-induced vascular dysfunction and hypotension and may provide a novel therapeutic target for the treatment of systemic inflammation and patients with septic shock.

Highlights

  • Endotoxaemia is a leading cause of morbidity and mortality, characterised by systemic inflammation, decreased peripheral vascular resistance, microvascular leak and decreased cardiac output leading to refractory hypotension [24]

  • We have demonstrated the specific role of endothelial cell BH4-dependent NOS regulation in the pathogenesis of LPSinduced vascular dysfunction and hypotension

  • The lack of endothelial cell BH4 results in an attenuation of LPS-induced hypotension. Together these findings demonstrate for the first time that deficiency in endothelial cell GTP cyclohydrolase 1 (Gch1) and BH4 biosynthesis is alone sufficient to protect against LPS-induced vascular dysfunction and hypotension induced by the dose of LPS used here, indicating a novel role of endothelial cell Gch1 and BH4-dependent NOS regulation in the pathogenesis of LPS-induced vascular dysfunction and hypotension

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Summary

Introduction

Endotoxaemia is a leading cause of morbidity and mortality, characterised by systemic inflammation, decreased peripheral vascular resistance, microvascular leak and decreased cardiac output leading to refractory hypotension [24]. High levels of nitric oxide (NO) production by inducible nitric oxide synthase (iNOS, encoded by NOS2), which can be induced by lipopolysaccharide (LPS), are believed to be a key mediator of these phenomena [21]. The synthesis of NO by all NOS isoforms requires the cofactor tetrahydrobiopterin, BH4. Biosynthesis of BH4 is Abbreviations: Gch, GTP cyclohydrolase 1; BH4, tetrahydrobiopterin; LPS, lipopolysaccharide. ☆ Author contribution: S.C. and E.M. performed the experiments, analysed and interpreted the data. S.C., E.M., and K.M.C. designed the experiments. S.C., E.M., and K.M.C. wrote the manuscript. All authors read and approved the final manuscript

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