Trauma-hemorrhagic shock-induced pulmonary epithelial and endothelial cell injury utilizes different programmed cell death signaling pathways

Abstract

Intestinal ischemia after trauma-hemorrhagic shock (T/HS) results in gut barrier dysfunction and the production/release of biologically active and tissue injurious factors in the mesenteric lymph, which, in turn, causes acute lung injury and a systemic inflammatory state. Since T/HS-induced lung injury is associated with pulmonary endothelial and epithelial cell programmed cell death (PCD) and was abrogated by mesenteric lymph duct ligation, we sought to investigate the cellular pathways involved. Compared with trauma-sham shock (T/SS) rats, a significant increase in caspase-3 and M30 expression was detected in the pulmonary epithelial cells undergoing PCD, whereas apoptosis-inducing factor (AIF), but not caspase-3, was detected in endothelial cells undergoing PCD. This AIF-mediated pulmonary endothelial PCD response was validated in an in situ femoral vein assay where endothelial cells were found to express AIF but not caspase-3. To complement these studies, human umbilical vein endothelial cell (HUVEC), human lung microvascular endothelial cell (HLMEC), and human alveolar type II epithelial cell (A549) lines were used as in vitro models. T/HS lymph induced the nuclear translocation of AIF in HUVEC and HLMEC, and caspase inhibition in these cells did not afford any cytoprotection. For proof of principle, AIF silencing in HUVEC reversed the cytotoxic effects of T/HS on cell viability and DNA fragmentation. In A549 cells, T/HS lymph activated caspase-3-mediated apoptosis, which was partially abrogated by N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). Additionally, T/HS lymph did not cause the nuclear translocation of AIF in A549 cells. Collectively, T/HS-induced pulmonary endothelial PCD occurs via an AIF-dependent caspase-independent pathway, whereas epithelial cells undergo apoptosis by a caspase-dependent pathway.

Fig. 1.

Trauma-hemorrhagic shock (T/HS)-induced pulmonary cell death. A: TdT-mediated dUTP nick end labeling (TUNEL) staining of lung tissue from rat subjected to T/HS. Arrows indicate endothelial (Endo) and nonendothelial apoptotic cells that stained blue. Magnification, ×100, and inset, ×40. B: number of TUNEL-positive endothelial and nonendothelial cells per 100 high-power fields (hpf) in rats subjected to trauma-sham shock (T/SS) or T/HS. Data are shown as mean values ± SD (n = 4 per group). *P < 0.01 vs. T/SS.

Fig. 2.

T/HS induces alveolar epithelial cell apoptosis via caspase-3-dependent pathway. A: caspase-3 immunohistochemical staining of lung tissue from rat subjected to T/HS. Arrows indicate cleaved caspase-3-positive alveolar epithelial cells. Magnification, ×100. B: number of cleaved caspase-3-positive epithelial cells per 100 hpf. Data are shown as mean values ± SD (n = 4–6 per group). *P < 0.01 vs. T/SS. C: M30 immunohistochemical staining of lung tissue from rats subjected to T/SS or T/HS. Magnification, ×40. D: number of M30-positive alveolar epithelial cells per 40 hpf. Data are shown as mean values ± SD (n = 4–6 per group). *P < 0.001 vs. T/SS.

Fig. 3.

T/HS induces apoptosis-inducing factor (AIF) expression in pulmonary endothelial cells. A: AIF and von Willebrand factor (vWf) immunofluorescent staining in lung tissue of rats subjected to T/SS or T/HS. Magnifications shown are ×20 and ×63. B: number of AIF-positive endothelial and nonendothelial cells per 100 hpf from rats subjected to T/HS or T/SS. Data are shown as mean values ± SD. *P < 0.01 vs. T/SS (n = 4 per group).

Fig. 4.

AIF activation is associated with T/HS lymph-induced rat femoral endothelial cell death. A: TUNEL staining of normal rat femoral vein incubated with 10% T/HS mesenteric lymph for 3 h. Arrows depict TUNEL-positive endothelial cells. Magnification, ×40. B: number of TUNEL-positive endothelial per vein ring section incubated with 10% T/SS or T/HS lymph. Data are shown as mean values ± SD (n = 4). *P < 0.01 vs. T/SS lymph. C: AIF immunofluorescent staining in normal rat femoral veins incubated with 10% T/SS or T/HS lymph for 3 h. Magnification, ×40. Inset: magnification image of an endothelial cell (×100). D: number of AIF-positive endothelial cells per vein ring section incubated with 10% T/SS or T/HS lymph. Data are shown as mean values ± SD (n = 4 per group). *P < 0.01 vs. T/SS lymph.

Fig. 5.

Inhibition of caspases by N-benzyloxycarbonyl-Val-Ala-Asp (zVAD) does not protect against the cytotoxic effects of T/HS lymph in human lung microvascular endothelial cells (HLMEC). HLMEC were pretreated with zVAD (100 μM) or DMSO vehicle control for 1 h and then exposed to medium, staurosporine (STS; 1 μM), and 5% T/HS and 5% T/SS lymph for 3 h. Cell death was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. In the bar graph, the data are expressed as mean values ± SD. *P < 0.001 vs. STS plus zVAD (n = 5 per condition).

Fig. 6.

T/HS lymph induces the nuclear translocation of AIF in human umbilical vein endothelial cells (HUVEC). A: HUVEC were exposed to medium (Med), STS (200 nM), and 5% T/HS and 5% T/SS lymph for 3 h, immunostained for AIF (green), and examined by immunofluorescent microscopy. The nuclei were stained with propidium iodide (PI; red). In HUVEC exposed to medium or T/SS lymph, AIF expression was excluded from the nucleus and appeared diffuse in the cytoplasm and perinuclear regions, whereas in HUVEC exposed to T/HS or STS, AIF was redistributed into the nucleus (green-red merge). Magnification, ×63. B: the mean values ± SD shown in the bar graphs for the number of AIF-positive stained cells per 100 cells (*P < 0.001 vs. T/SS and medium) were from 4 independent experiments.

Fig. 7.

T/HS lymph induces the nuclear translocation of AIF in HLMEC. A: HLMEC were exposed to medium, STS (200 nM), and 5% T/HS and 5% T/SS lymph for 3 h, immunostained for AIF (green), and examined by immunofluorescent microscopy. The nuclei were stained with PI (red). In HLMEC exposed to medium or T/SS lymph, AIF expression was excluded from the nucleus and appeared diffuse in the cytoplasm and perinuclear regions, whereas in HLMEC exposed to T/HS or STS, AIF was redistributed into the nucleus (green-red merge). Magnification, ×63. B: the mean values ± SD shown in the bar graphs for the number of AIF-positive stained cells per 100 cells (*P < 0.001 vs. T/SS or medium) were from 4 independent experiments.

Fig. 8.

Knockdown of AIF expression inhibits T/HS-induced endothelial cell apoptosis. A: whole cell extracts (WCEs) were prepared from HUVEC that were mock transfected or transfected with small interfering RNA (siRNA) against AIF for 72 h. AIF and actin protein levels were determined by Western blotting. B and C: after 48–72 h of transfection, HUVEC were exposed to medium and 5% T/HS and 5% T/SS lymph for 3 h, and cell viability and DNA fragmentation were assessed by MTT cell viability assay (B) and nucleosomal release ELISA (C). The optical density (OD) correlates with the number of free nucleosomes. In the bar graphs, the data are expressed as mean values ± SD for percent cell viability (B) and the number of free nucleosomes (C). *P < 0.001 vs. mock-transfected HUVEC incubated with medium or T/SS lymph, and #P < 0.001 vs. mock-transfected HUVEC incubated with T/HS lymph (n = 6–18 per condition).

Fig. 9.

Endonuclease G (EndoG) is activated in HUVEC exposed to T/HS lymph. A: WCEs from HUVEC were analyzed for EndoG and actin expression by Western blotting. B: densitometry was performed to quantify EndoG and actin expression. In the bar graph, the data are expressed as mean values ± SD for EndoG/actin. *P < 0.001 vs. T/SS lymph (n = 6–10 per condition).

Fig. 10.

Inhibition of caspases by zVAD protects against T/HS lymph-induced A549 cell apoptosis. A: A549 cells were exposed to 5% T/HS and 5% T/SS lymph for 3 h, immunostained with M30 (green), and examined by immunofluorescence. The nuclei were stained with PI (red). B: the mean values ± SD shown in the bar graph for the number of M30 apoptotic A549 cells counted per 100 cells. *P < 0.001 vs. T/SS lymph. C: A549 cells were pretreated with zVAD (100 μM) or DMSO vehicle control for 1 h and then exposed to medium, STS (1 μM), and 5% T/HS and 5% T/SS lymph for 3 h. Cell death was assessed by MTT cell viability assay. In the bar graph, the data are expressed as mean values ± SD for percent cell viability. *P < 0.01 STS vs. STS, and **P < 0.01 T/HS vs. T/HS and zVAD (n = 6–8 per condition).

Fig. 11.

T/HS lymph induces caspase-3 activation in A549 cells. WCEs prepared from A549 (A) and HUVEC (B) exposed to medium and 5% T/SS lymph for 3 h were examined for cleaved caspase-3 protein levels by Western blotting. STS was used as a positive control for apoptosis in HUVEC (200 nM) and A549 (1 μM) for 3 h. Data are representative of 3–4 independent lymph or medium samples per condition. The numbers represent individual samples per condition.

Fig. 12.

T/HS lymph does not induce nuclear translocation of AIF in A549 cells. A: A549 cells were exposed to STS (1 μM) and 5% T/HS and 5% T/SS lymph for 3 h, immunostained for AIF (green), and examined by immunofluorescence. The nuclei were stained with PI (red). In A549 cells exposed to T/SS or T/HS, AIF expression was excluded from the nucleus, whereas in A549 cells exposed to STS, AIF was redistributed into the nucleus (green-red merge). Original magnification, ×63. B: the mean values ± SD shown in the bar graphs for the number of AIF-positive stained cells per 100 cells (*P < 0.001 vs. all groups) were from 3 independent experiments.