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TNF-[alpha] is a proinflammatory cytokine and is an important mediator of maternal endothelial dysfunction leading to preeclampsia. In this study, we tested whether IL-10 protects against TNF-[alpha]-induced endothelial dysfunction in murine aorta. In in vitro experiments, aortic rings of C57BL/6 female mice were incubated in Dulbecco's modified Eagle's medium in the presence of either vehicle (distilled [H.sub.2]O), TNF-[alpha] (4 nmol/1), or recombinant mouse IL-10 (300 ng/ml) or in the presence of both TNF-[alpha] and IL-10 for 22 h at 37[degrees]C. In in vivo experiments C57BL6/IL-10 knockout female mice were treated with saline or TNF-[alpha] (220 ng x [kg.sup.-1] x [day.sup.-1]) for 14 days. Aortic rings were isolated from in vitro and in vivo experiments and mounted in a wire myograph (Danish Myotech) and stretched to a tension of 5 mN. Endothelium-dependent relaxation was assessed by constructing cumulative concentration-response curves to acetylcholine (ACh, 0.001-10 [micro]mol/l) during phenylephrine (10 [micro]mol/l)-induced contraction. As a result, overnight exposure of aortic rings to TNF-[alpha] resulted in significant blunted maximal relaxing responses ([E.sub.max]) to ACh compared with untreated rings (22 [+ or -] 4 vs. 82 [+ or -] 3%, respectively). IL-10 knockout mice treated with TNF-[alpha] showed significant impairment in ACh responses ([E.sub.max]) compared with C57BL/6 mice treated with TNF-[alpha] (51 [+ or -] 3 vs. 72 [+ or -] 3%, respectively). Western blot analysis showed that endothelial nitric oxide synthase (eNOS) expression was reduced by TNF-[alpha] in in vitro and in vivo experiments, whereas IL-10 restored the eNOS expression. In conclusion, the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation caused by TNF-[alpha] by protecting eNOS expression. tumor necrosis factor-[alpha]; nuclear factor-[kappa]B doi: 10.1152/ajpheart.00763.2009.

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1beta into mature IL-1beta, which is then secreted. Because both LPS (in vivo) and IL-1beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco's modified Eagle's medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-l-arginine methyl ester or N-([3-(aminomethyl)phenyl]methyl)ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1beta induced iNOS protein expression. Thus, activation of P2X(7) receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1beta-mediated release of nitric oxide by iNOS.