Lipopolysaccharide induces vascular dysfunction in vitro in vessels from nitric oxide synthase (NOSIII) knockout mice

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 in rodent models of sepsis is mediated by excessive production of nitric oxide by nitric oxide synthase (NOS) II, an inducible isoform of NOS. NOSIII, a constitutive form of NOS found in the endothelium, has also been implicated: NOSIII-/- mice show less haemodynamic instability during experimental endotoxaemia than wild-type (WT) mice (Connelly et al., 2005) . Vessels directly sense LPS through Toll-like receptor 4, which leads to the induction of NOSII and vascular dysfunction invitro (Cartwright et al., 2006) . We wish to determine the role of NOSIII in this pathway.

Male wild-type C57BK/6 or NOSIII-/- mice (29-33g) 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) or in DMEM containing LPS (10 μg/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. After constriction of vessels with phenylephrine (10-6 M), the responses to acetylcholine (10-5M) and SNP (10-5M) were tested. Results are expressed as mean ± SEM and analysed by 2-way ANOVA, unless otherwise stated. p<0.05 was considered significant.

After 24h culture, control vessels contracted to phenylephrine from NOSIII-/- (n=3, E max=2.56±0.18 mN) and C57BL/6 (n=5, E max=1.72±0.13). E max was significantly greater in vessels from NOSIII-/- mice (ANOVA, p<0.05). Culture with LPS significantly reduced contractility of aortae to phenylephrine in both C57BL/6 (n=5, p<0.001) and NOSIII-/- (n=3, p<0.001) mice, when compared to cultured controls. After exposure to LPS, the E max for phenylephrine was significantly less in vessels from NOSIII-/- mice (ANOVA, p<0.001). Acetylcholine significantly dilated control vessels from WT mice (ANOVA, p<0.001), whilst vessels cultured with LPS or vessels from NOSIII-/- mice showed no significant dilation with acetylcholine. Sodium nitroprusside (10-5M) fully dilated all vessels.

Theses data show that in vitro, vessels become hyporesponsive to phenylephrine after culture with LPS. Furthermore, the effect of LPS appears to be greater in vessels lacking NOSIII. Although in vivo studies have shown that NOSIII-/- mice are resistant to shock after administration of LPS, we show that the mechanism is unlikely to be due to a difference in vascular responses to pathogens. Interestingly, acetylcholine, a NOSIII dependent dilator, had no effect in vessels after culture with LPS, which may represent loss of NOSIII function.

Cartwright, N ., et al. (2006). Shock, in press.
Connelly, L ., et al. (2005). J Biol Chem, 280, 10040-6.

This work was funded by a grant from the British Heart Foundation.