Lung apopotosis (A) and macrophage infiltration (C) are not improved by DEX in GR ^{EC KO} mice.

<p>Both GR ^{EC KO} mice and controls show an improvement in liver apoptosis (B) and macrophage infiltration (D) following DEX. *p<0.05 compared to similarly treated controls.</p

Heightened inflammation in GR ^{EC KO} mice following DEX pre-treatment.

<p>(A) No differences in corticosterone level were observed between GR ^{EC KO} mice and controls for any of the conditions tested. (B) Total nitric oxide levels in GR ^{EC KO} mice are increased following DEX+LPS. (C) TNF-α and (D) IL-6 levels are significantly increased in GR ^{EC KO} mice following DEX+LPS treatment while they are nearly unchanged from baseline in controls. All blood samples were collected 8 hours after LPS treatment (and 10 hours after DEX pre-treatment, if applicable). *p<0.05 compared to similarly treated controls.</p

Loss of the Endothelial Glucocorticoid Receptor Prevents the Therapeutic Protection Afforded by Dexamethasone after LPS

<div><p>Glucocorticoids are normally regarded as anti-inflammatory therapy for a wide variety of conditions and have been used with some success in treating sepsis and sepsis-like syndromes. We previously demonstrated that mice lacking the glucocorticoid receptor in the endothelium (GR ^{EC KO} mice) are extremely sensitive to low-dose LPS and demonstrate prolonged activation and up regulation of NF-κB. In this study we pre-treated these GR ^{EC KO} mice with dexamethasone and assessed their response to an identical dose of LPS. Surprisingly, the GR ^{EC KO} mice fared even worse than when given LPS alone demonstrating increased mortality, increased levels of the inflammatory cytokines TNF-α and IL-6 and increased nitric oxide release after the dexamethasone pre-treatment. As expected, control animals pre-treated with dexamethasone showed improvement in all parameters assayed. Mechanistically we demonstrate that GR ^{EC KO} mice show increased iNOS production and NF-κB activation despite treatment with dexamethasone.</p></div

Expression profile of GRα and GRβ mRNA in endothelial cells.

<p>Real time PCR analysis was performed on mouse lung endothelial cells treated as described. Values were normalized to untreated control siRNA GRα levels and represent mean ± SEM for 3 independent samples. Cells were isolated from C57/BL6 mice. *p <0.05 compared to untreated control siRNA GRα levels.</p

Impaired survival in GR ^{EC KO} mice after DEX.

<p>(A) GR ^{EC KO} mice show increased mortality after LPS treatment. (B) Mortality in GR ^{EC KO} mice is further increased in GR ^{EC KO} mice following DEX pre-treatment while controls are fully rescused following DEX+LPS. (C) Continuous blood pressure monitoring demonstrates hemodynamic instability in GR ^{EC KO} mice following pre-treatement with DEX while blood pressure is completely stabilized in control mice. *p<0.05</p

Increased iNOS expression and NF-κB activation in GR ^{EC KO} mice.

<p>Western blot of aortic homogenates from controls and GR ^{EC KO} mice treated with LPS alone or DEX+LPS and harvested at the indicated timepoints. Densitometry values are indicated below each lane. Activation of NF-κB was assayed in the same homogenates. (A) Control mice show decreased expression of iNOS when given DEX+LPS as compared to LPS alone and while (B) GR ^{EC KO} mice show increased iNOS levels following DEX+LPS as compared to LPS alone. (C) Activation of NF-κB is suppressed following DEX pre-treatement in control animals while in (D) GR ^{EC KO} mice increased activation of NF-κB is shown at every time point. *p<0.05 compared to similarly treated control. U = untreated, D = dexamethasone.</p