Endothelial glucocorticoid receptor is required for protection against sepsis

Abstract

The glucocorticoid receptor (GR) is ubiquitously expressed on nearly all cell types, but tissue-specific deletion of this receptor can produce dramatic whole organism phenotypes. In this study we investigated the role of the endothelial GR in sepsis in vivo and in vitro. Mice with an endothelial-specific GR deletion and controls were treated with 12.5 mg/kg LPS and phenotyped. Mice lacking GR showed significantly increased mortality, more hemodynamic instability, higher nitric oxide levels, and higher levels of the inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) compared with controls. There were no differences in rates of apoptosis or macrophage recruitment between the two groups. Both endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression were increased after LPS challenge in mice with endothelial GR deficiency, and aminoguanidine, a specific iNOS inhibitor in mice was able to rescue hemodynamic collapse in these animals. In vitro, human umbilical vein cells (HUVECs) subjected to GR knockdown by siRNA showed increased expression of eNOS at baseline that persisted after treatment with LPS. Both eNOS and iNOS mRNA was increased by qPCR. In HUVECs lacking GR, NF-κB levels and NF-κB-dependent genes tissue factor and IL-6 were increased compared with controls. Thus, endothelial GR is a critical regulator of NF-κB activation and nitric oxide synthesis in sepsis.

Fig. 1.

Sensitivity and specificity of LPS-induced mortality in GR^{EC KO} mice with associated hemodynamic collapse. (A) GR^{EC KO} (n = 12), GR fl/fl (n = 13), and Tie-1 Cre+ mice (n = 7) were treated with LPS 12.5 mg/kg i.p. and monitored for the time shown. GR^{EC KO} mice had poorer survival compared with the control groups. (B) GR^{VSM KO} mice (n = 5) treated with the same dose of LPS did not demonstrate increased mortality, suggesting the effect was specific to the endothelium. (C) GR^{EC KO} mice show significantly lower BP 4 h after treatment with 5 mg/kg i.v. LPS compared with controls. (D) Heart rate in GR^{EC KO} mice is also significantly lower than controls 4 h after i.v. LPS. *P < 0.05.

Fig. 2.

Phenotyping of GR^{EC KO} and control animals. No differences were seen in (A) baseline cholesterol or HDL levels, (B) corticosterone levels, (C) ALT or AST levels. (D) TNF-α and IL-6 levels were similar at baseline and increased as expected after LPS. However, GR^{EC KO} mice had significantly greater levels of inflammatory cytokines after LPS. (E) NO levels after LPS increased in both groups but were significantly higher in GR^{EC KO} mice. All parameters were measured 8 h after LPS challenge.

Fig. 3.

Increased iNOS expression in GR^{EC KO} mice following LPS challenge. GR^{EC KO} and GR fl/fl mice (n = 3 per group) were treated with either vehicle or LPS 12.5 mg/kg. After 4 and 8 h, the animals were killed and their aortas harvested and processed. (A) GR^{EC KO} mice show significantly higher iNOS mRNA 8 h after LPS treatment compared with controls. (B) Western blot of aortic homogenates shows increased iNOS expression in the GR^{EC KO} mice at both time points studied. Three separate experiments yielded similar results. (C) Aminoguanidine, a specific iNOS inhibitor, can rescue GR^{EC KO} mice after LPS. GR^{EC KO} mice were treated with LPS only (n = 11) or aminoguanidine at +2 and +6 h after LPS (n = 7) or aminoguanidine at +2, +6, +24, and +30 h after LPS (n = 7). Survival was monitored for the time shown. Mice treated with four doses of aminoguanidine had a statistically significant improvement in their survival compared with the other two groups (P = 0.04).

Fig. 4.

GR knockdown increases eNOS expression. (A) Controls and GR^{EC KO} mice (n = 3 per group) were treated with vehicle or LPS 12.5 mg/kg and aortas were processed 8 h later. eNOS mRNA was significantly higher in GR^{EC KO} mice. (B) At baseline GR knockdown in HUVECs resulted in increased eNOS mRNA. After 24 h of treatment with LPS 10 μg/mL control cells showed a decrease in eNOS mRNA, as expected, whereas GR knockout cells continued to show increased mRNA levels. (C) Representative Western blot showing increased eNOS expression in GR knockdown cells both at baseline and after LPS treatment. (D) eNOS densitometry for three separate experiments. *P < 0.05 compared with nontreated control, **P < 0.05 compared with similarly treated control.

Fig. 5.

GR knockdown in HUVECs prolongs NF-κB activation. (A) HUVECs were treated with TNF 10 μg/mL. Both control and GR siRNA-treated cells showed similar peak NF-κB activation but GR siRNA-treated cells showed prolonged activation. (B) Densitometry of three separate experiments. (C and D) HUVECs were treated with LPS 20 μg/mL. After 2 and 6 h, GR siRNA-treated cells showed significantly increased tissue factor and IL-6 mRNA. This effect was abolished at 12 and 24 h. *P < 0.05 compared with nontreated control, **P < 0.05 compared with similarly treated control.