Therapeutic accessibility of caspase-mediated cell death as a key pathomechanism in indirect acute lung injury

Abstract

Objective: Indirect acute lung injury is associated with high morbidity and mortality. However, the underlying pathophysiology is only marginally understood, and so far no pathophysiologic-based remedy exists. We hypothesized that apoptosis of lung epithelial cells is a pathophysiological relevant process in the development of indirect acute lung injury and that it should be accessible to a siRNA-based therapeutic intervention in vivo.

Design: Prospective, randomized, controlled animal study.

Setting: Basic science laboratory of a university affiliated level one trauma center.

Subjects: Male C3H/HeN mice, 8 wks old, n = 121.

Interventions: First, siRNA sequences to knock-down caspase-3 expression at a RNA and protein level were evaluated in vitro. Then, C3H/HeN mice were subjected to hemorrhagic shock, after which they received either a caspase-3 siRNA or a control/nonsense siRNA. Subsequently, they were then subjected to polymicrobial sepsis (induced by cecal ligation and puncture).

Measurements and main results: Twelve and 24 hrs after sepsis, increased lung epithelial apoptosis was observed, as evidenced by active caspase-3 Western blotting, caspase-3, TUNEL-, and M-30 immunohistochemistry. Hallmarks of acute lung injury, such as increased concentrations of pulmonary cytokines/chemokines, lung protein leakage, myeloperoxidase activity, and altered lung histology, were evident in response to these insults. The single intratracheal instillation of caspase-3 siRNA not only attenuated lung apoptosis and inflammation but also ameliorated the development of acute lung injury in treated mice. Most interestingly, this experimental therapeutic approach markedly improved 10-day survival of hemorrhaged septic mice.

Conclusions: Apoptosis of lung epithelial cells is a relevant pathomechanism in the development of hemorrhage-induced indirect septic acute lung injury, and caspase-3 appears to be a valuable therapeutic target accessible by siRNA treatment in vivo.

Figure 1

Pulmonary caspase-3 mRNA levels in mice having received control (CTRL) or caspase-3 (C-3) siRNA 12 hrs and 24 hrs following hemorrhagic shock and sepsis (HEM+Sepsis) compared to sham mice (dashed line = 100%) (Fig.1A). Integrated density (IDT) values of active caspase-3 relative to IDT values of β-actin of n=6 animals per group 12 hrs following HEM+Sepsis (Fig. 1B). Quantification via western blotting and densitometry. One Way ANOVA followed by Student Newman Keuls Test, *p<0.05 vs. corresponding sham, #p<0.05 vs. corresponding control-siRNA treatment (HEM+Sepsis).

Figure 2

Representative active caspase-3 (Fig.2A) and M30 (cleaved cytokeratin-18) (Fig.2B) immunohistochemistry of lung tissue from sham animals (Fig.2A1 and Fig.2B1, respectively), mice having received control-siRNA (Fig.2A2 and Fig.2B2 respectively) or caspase-3 siRNA (Fig.2A3 and Fig.2B3 respectively) 12 hrs after hemorrhagic shock and sepsis. n=4 animals/ group. Original magnifications, X 400.

Figure 3

TUNEL positive cells per high power field (HPF) in sham animals and at 12 and 24 hrs following hemorrhagic shock and sepsis after treatment with control (CTRL) or caspase-3 (C-3) siRNA. One Way ANOVA followed by Student Newman Keuls Test. n=4 animals/ group. * p< 0.05 vs. Sham, # p< 0.05 vs. corresponding control-siRNA treatment (HEM+Sepsis).

Figure 4

Lung TNF-α (Fig. 4A), IL-6 (Fig. 4B), KC (Fig. 4C), MCP-1 (Fig. 4D), MIP-2 (Fig. 4E) [pg/mg] in mice having received control (CTRL) or caspase-3 (C-3) siRNA 12 or 24 hrs following hemorrhagic shock and sepsis (HEM+Sepsis) compared to sham animals. Quantification via ELISA or CBA. N=7–8 /group. Two Way ANOVA followed by Student Newman Keuls Test, *p<0.05 vs. corresponding sham, #p<0.05 vs. corresponding control-siRNA treatment (HEM+Sepsis), +p<0.05 vs. corresponding 24 hrs time point (identical treatment and insult)

Figure 5

Plasma TNF-α ( Fig. 5A), IL-6 (Fig. 5B), KC (Fig. 5C), MCP-1 (Fig. 5D), MIP-2 (Fig. 5E) [pg/ml] concentrations in mice having received control (CTRL) or caspase-3 (C-3) siRNA 12 or 24 hrs following hemorrhagic shock and sepsis (HEM+Sepsis) compared to sham animals. Quantification via ELISA or CBA. N=7–8 /group. Two Way ANOVA followed by Student Newman Keuls Test. *p<0.05 vs. corresponding sham, #p<0.05 vs. corresponding control-siRNA treatment (HEM+Sepsis), +p<0.05 vs. corresponding 24 hrs time point (identical treatment and insult)

Figure 6

Ratio of fluorescently labeled albumin per µg lung tissue relative to fluorescently labeled albumin in the plasma (Fig.6A) in mice having received control (CTRL) or caspase-3 (C-3) siRNA 12 hrs following hemorrhagic shock and sepsis (HEM+Sepsis). Lung myeloperoxidase activity (Fig.6B) (U)Units/ mg lung tissue/ (min)minute in mice having received control (CTRL) or caspase-3 (C-3) siRNA 12 or 24 hrs following HEM+Sepsis compared to sham animals. N=5/ group for albumin leakage, n=7–8/group for myeloperoxidase activity. One (albumin) or Two Way ANOVA (myeloperoxidase activity) followed by Student Newman Keuls Test. *p<0.05 vs. corresponding sham, #p<0.05 vs. corresponding control-siRNA treatment (HEM+Sepsis).

Figure 7

Representative H&E preparation of lung tissue slides from sham animals (Fig.7A) and mice 12 hrs after hemorrhagic shock and sepsis after having received control-siRNA (Fig.7B) or caspase-3 siRNA (Fig.7C). N=4 animals/ group. Original magnifications, X 400.

Figure 8

Ten day survival in animals after having received control- (CTRL) or caspase-3 (C-3) siRNA following hemorrhagic shock (HEM) and subsequent sepsis 24 hrs thereafter. Kaplan-Meier, LogRank Test, n=15 animals per group, p< 0.05.