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

EMMPRIN, GOLPH3 and eNOS are co-localized at the Golgi apparatus in endothelial cells.

<p>(<b>A</b>) Immunolabeling of EMMPRIN, eNOS, GOLPH3 and GM130 (a Golgi marker) in bovine aortic endothelial cells (BAECs). The upper panel shows EMMPRIN (red, left) and eNOS (green, center) and their merged image with DNA in blue (right). Arrows indicate where EMMPRIN and eNOS are located. The lower panel shows GOLPH3 (red, left), GM130 (green, center), and their merged image with nucleus in blue (right). Arrows indicate where GOLPH3 and GM130 are located. Scale bar; 10 µm. Images were taken using a Nikon E800 Microscope with a Plan-Fluorchromat 40×/0.75 objective (Nikon, Melville, NY). Shown are representative images from at least 3 independent experiments. (<b>B</b>) Immunolabeling of COS cells that were transfected with wild-type eNOS (blue, upper left panel), YFP-EMMPRIN (green, upper right panel) and HA-tagged GOLPH3 (red, lower left panel). The lower right panel shows their merged image. Scale bar; 10 µm. Arrows indicate eNOS-, EMMPRIN- and GOLPH3-rich areas. Scale bar; 10 µm. Shown are representative images from at least 3 independent transfection experiments. (<b>C</b>) Immunoprecipitation (IP) using anti-EMMPRIN (EMP) and total goat IgG from BAEC lysates. IP samples were blotted with eNOS and GOLPH3 antibodies. Input refers to eNOS levels in lysates before IP, indicating an equal amount of proteins in lysates used for IP. The blots are representative images from three independent experiments.</p

EMMPRIN and GOLPH3 are S-nitrosylated in endothelial cells.

<p>(<b>A</b>) S-nitrosylation of EMMPRIN was detected by the biotin-switch assay in bovine aortic endothelial cells (BAECs). Input refers to EMMPRIN levels in lysates before performing the biotin-switch assay. Specificity of biotinylation was confirmed by incubating samples in the presence or absence of 2 mM ascorbic acid (AA) during the biotin-switch assay. Arrows indicate S-nitrosylated EMMPRIN (EMP). Shown are representative results from 3 independent experiments. (<b>B</b>) S-nitrosylated EMMPRIN levels in BAECs in the basal (non-stimulated, left panel) and after eNOS stimulation with 10 µM of a calcium ionophore (A23187) for 30 min to promote NO production (middle panel). Arrow indicates S-nitrosylated EMMPRIN. BAEC lysates immunoprecipitated (IP) with EMMPRIN (EMP, left panel) or S-nitroso-cysteine (SNO-cys, middle panel) antibodies were blotted with SNO-cys or EMMPRIN antibodies, respectively. The bar graph on the right panel shows band intensities of S-nitrosylated EMMPRIN (arrow in the middle panel) in BAECs stimulated with or without A23187. The blots are representative images from 3 independent experiments. (<b>C</b>) S-nitrosylation of GOLPH3 was detected by the biotin-switch assay in BAECs. Input refers to GOLPH3 levels in lysates before performing the biotin-switch assay. Specificity of biotinylation was confirmed by incubating samples in the presence or absence of 2 mM ascorbic acid (AA) during the biotin-switch assay. Arrows indicate S-nitrosylated GOLPH3. The blots are representative images from 3 independent experiments. (<b>D</b>) S-nitrosylated GOLPH3 levels in BAECs after eNOS stimulation with 10 µM of A23187 for 30 min to promote NO production (left panel). BAEC lysates immunoprecipitated with S-nitrosocysteine (SNO-cys) antibodies were blotted for GOLPH3. The graph on the right panel shows band intensities of S-nitrosylated GOLPH3 (arrow in the left panel) in BAECs stimulated with or without A23187. Shown are representative results from 3 independent experiments.</p

PDGF-AA, PDGF-BB and survivin levels are increased in eNOS (−/−) mice in response to remodeling stimulus.

<p>(A), Immunohistochemistry of PDGF-BB protein shows that it is present in adventitia of RC but is increased in all layers of LC post-ligation. Scale bar, 10 µm. (B), Quantitative RT-PCR of PDGF-AA and BB shows there was no change of PDGF mRNA level in wild type mice after LEC ligation. However, PDGF-AA and BB mRNA are all elevated in ligated LC compare to contralateral RC in eNOS (−/−) mice, 18 s was used as internal control. (C), Survivin immunohistochemistry shows strong staining throughout whole vessel wall of LC, and there is little or no staining in RC. Scale bar equals to 10 µm. (D), RT-PCR shows equal expression of survivin mRNA of contralateral RC and ligated LC in wild type mice. However, survivin was upregulated in ligated LC of eNOS (−/−) mice. 18 s was used as internal control.</p

S-nitrosylation of EMMPRIN is increased in the aorta isolated from cirrhotic rats.

<p>Aorta samples were lysed in a lysis buffer. Three aorta samples were combined per group to obtain a sufficient amount of proteins for the biotin-switch assay. Lysates before the biotin-switch assay were blotted for EMMPRIN and a loading control, heat shock protein 90 (Hsp90) (input, left panel). Equal amounts of proteins (500 µg) in the lysates were used for the biotin-switch assay. Biotinylated proteins were captured by streptavidin agarose beads and blotted with an EMMPRIN antibody (right panel). The aorta from cirrhotic rats showed a higher level of S-nitrosylated EMMPRIN than that of normal rats. Interestingly, only those glycosylated EMMPRIN were S-nitrosylated in the aorta.</p