Neither flagellin nor unmethylated CpG-containing synthetic oligonucleotides lead to vascular dysfunction in vitro

Neil Cartwright, Timothy W Evans and Jane A Mitchell. Department of Critical Care, National Heart and Lung Institute, Imperial College, London SW3 6LY.

Vascular dysfunction, characterised by hyporesponsiveness to vasopressor agents such as phenylephrine, is pivotal to the onset of septic shock. Blood vessels directly sense pathogen associated molecular patterns (PAMPS) through Toll-like receptors (TLRs) leading to the induction of nitric oxide synthase II, excessive production of nitric oxide and vascular dysfunction (Cartwright et al., 2006) . Eschericia coli (E.coli) is a common cause of septic shock and contains several PAMPs, including lipopolysaccharide (LPS), flagellin and unmethylated CpG-containing bacterial DNA. LPS activates TLR4, leading to vascular dysfunction in vitro. However, the effects of flagellin, a TLR5 agonist, or bacterial CpG-containing DNA, a TLR9 agonist, are not known. We wish to assess the role of TLR5 and TLR9 in vascular function.

Male wild-type C57BK/6 mice (29-31g) were killed by overdose of CO2, and the descending thoracic aortae removed. Rings of aortae, approximately 2mm long, were cultured for 24h in DMEM alone (control), in DMEM containing flagellin (50μg/ml) or CpG ODN (2μg/ml). Following culture, aortic rings were mounted on a DMT wire myograph in physiological saline solution containing L-arginine (10-3 M) at 37°C bubbled with 5%CO2, 95%O2 (PSS). Phenylephrine (PE) was added cumulatively (10-8–3x10-6 M) and isometric force was measured. Tension was calculated from vessel length. Results were analysed by 2-way ANOVA, p<0.05 was considered significant.

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Figure 1. Culture of murine aortae with flagellin (A) or CpG ODN (B) does not lead to hyporesponsiveness to phenylephrine

The contractility of vessels was not significantly changed following culture with flagellin when compared to cultured controls (n=4, p=0.7443). Similarly, culture with CpG ODN did not significantly change the contractility of vessels when compared to cultured controls (n=6, p0.4885) (figure 1).

We conclude that neither TLR5 nor TLR9 agonists lead to vascular dysfunction in vitro.

Cartwright N ., et al. (2006). Shock, in press.

This work was funded by the British Heart Foundation