Endothelial nitric oxide synthase activation is critical for vascular leakage during acute inflammation in vivo

Abstract

The role of endothelium-derived nitric oxide (NO) in acute inflammation is not known. Here, we examine acute inflammation in congenic endothelial NO synthase-deficient (eNOS-/-) mice. Intraplantar injection of carrageenan induces a biphasic inflammatory response. The early phase (0-6 h) is largely eliminated, and the secondary phase (24-96 h) is markedly reduced in eNOS-/- but not WT mice. Inhibition of phosphatidylinositol 3-kinase or hsp90, pathways upstream of eNOS activation, also reduces carrageenan-stimulated edema formation. To separate the ability of eNOS to regulate leukocyte trafficking vs. vascular permeability, zymosan-stimulated leukocyte infiltration and protein extravasation were assessed in WT and eNOS-/- mice. Zymosan increases inflammatory cell extravasation to the same extent in WT and eNOS-/- mice, whereas the extravasation of plasma protein is lower in eNOS-/- mice. Inhibition of phosphatidylinositol 3-kinase and hsp90 also blocks protein leakage, but not leukocyte influx. These data collectively support the critical role for eNOS in regulating the magnitude of the acute inflammatory response and show that eNOS is critical for regulating microcirculatory endothelial barrier function in vivo.

Fig. 1.

Reduced paw edema in eNOS^-/- mice. (A) eNOS^-/- mice display a reduced edema. Both the first and second phases of the carrageenan-induced edema (n = 6 per group) are influenced. (B) eNOS^-/- mice display a reduction in tissue MPO levels at the 2-h time point (n = 6 per group; C). Data are expressed as mean ± SEM. ^**, P < 0.01; ^***, P < 0.001. (C) Hematoxylin and eosin staining of saline- (Left) and carrageenan-injected (Right) paws. Paws from eNOS^-/- mice show a marked reduction in interstitial edema formation after carrageenan injection compared with paws from injected WT (C57BL/6J) mice.

Fig. 2.

Carrageenan acutely increases eNOS and COX-1 protein levels and chronically induces both iNOS and COX-2. (A) After carrageenan injections into mouse paws, there is a significant increase in eNOS expression as compared with saline injection at 2 and 4 h. iNOS and COX-2 are not expressed until the 24-h time point (n = 8 per group). (B) Densitometric analysis of semiquantitative Western data from three separate experiments. Data are expressed as mean ± SEM. ^**, P < 0.01.

Fig. 3.

Inhibition of PI3-K with LY294002 reduces edema. Local intraplantar administration of LY294002 (2 and 20 μg per paw with 0.01% DMSO as a vehicle control) significantly inhibits paw edema (A) but not of tissue MPO levels (B). Data are expressed as mean ± SEM; n = 8 per group. ^*, P < 0.05; ^**, P < 0.001.

Fig. 4.

Inhibition of hsp90 function with GA reduces edema formation. Local intraplantar administration of GA (2 and 20 μg per paw with 0.01% DMSO as a vehicle control) significantly inhibits both edema (A) and MPO levels (B). Data are expressed as mean ± SEM; n = 8 per group; ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001.

Fig. 5.

Zymosan-induced protein extravasation, but not cellular infiltrates, are reduced in eNOS^-/- mice and inhibitors of eNOS activation. Zymosan treatment increased white cell infiltration (A) and MPO activity (B) to the same extent in WT and eNOS^-/- mice; however, the proteinaceous content (C) of the exudates was markedly reduced in eNOS^-/- mice. In WT mice, administration of LY294002 (20 μg per pouch) or GA (20 μg per pouch) did not modify zymosan-induced cellular infiltration (D) but inhibited protein extravasation (E). DMSO is used as a control as above. Data are expressed as mean ± SEM. ^***, P < 0.001; n = 5 mice per group for A-C and n = 8 per group for D and E.