Hyperoxia causes angiopoietin 2–mediated acute lung injury and necrotic cell death

Abstract

The angiogenic growth factor angiopoietin 2 (Ang2) destabilizes blood vessels, enhances vascular leak and induces vascular regression and endothelial cell apoptosis. We considered that Ang2 might be important in hyperoxic acute lung injury (ALI). Here we have characterized the responses in lungs induced by hyperoxia in wild-type and Ang2−/− mice or those given either recombinant Ang2 or short interfering RNA (siRNA) targeted to Ang2. During hyperoxia Ang2 expression is induced in lung epithelial cells, while hyperoxia-induced oxidant injury, cell death, inflammation, permeability alterations and mortality are ameliorated in Ang2−/− and siRNA-treated mice. Hyperoxia induces and activates the extrinsic and mitochondrial cell death pathways and activates initiator and effector caspases through Ang2-dependent pathways in vivo. Ang2 increases inflammation and cell death during hyperoxia in vivo and stimulates epithelial necrosis in hyperoxia in vitro. Ang2 in plasma and alveolar edema fluid is increased in adults with ALI and pulmonary edema. Tracheal Ang2 is also increased in neonates that develop bronchopulmonary dysplasia. Ang2 is thus a mediator of epithelial necrosis with an important role in hyperoxic ALI and pulmonary edema.

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Figure 1: Effect of hyperoxia on Ang-1, Ang-2 and Tie 2.
Figure 2: Effect of hyperoxia on survival, inflammation, permeability and tissue injury.
Figure 3: Role of Ang2 in hyperoxia-induced oxidant and DNA injury.
Figure 4: Effects of rAng2 and Ang2 siRNA on hyperoxia-induced responses.
Figure 5: Role of Ang2 in hyperoxia-induced alterations in apoptosis and angiogenic regulators and the effect of rAng2 on MLE-12 cell survival.
Figure 6: Ang2 in biological fluids from human adults and neonates.

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Acknowledgements

We thank P. Tan for siRNA design; P. Hadwiger, I. Röhl and K. Charisse for siRNA synthesis; P. Deuerling and S. Krause for help with Ang2 siRNA activity testing; M. Manoharan, H.P. Vornlocher and V. Kotelianski for siRNA discussions; and K. Bertier for administrative assistance. This work was supported in part by grants HL-74195 (to V.B.), HL-64242, HL-61904 and HL-56389 (to J.A.E.) from the National Heart, Lung, and Blood Institute of the US National Institutes of Health.

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Correspondence to Jack A Elias.

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Supplementary information

Supplementary Fig. 1

Levels of Ang2 mRNA expression in mice exposed to hyperoxia and given Bcl-2 or control siRNA. (PDF 29 kb)

Supplementary Fig. 2

Detection of Ang2 protein in mice exposed to hyperoxia and given low dose (LD) Ang-2 or control siRNA. Immunohistochemistry was used to localize Ang2 protein from mice exposed to room air or 100% O2 for 72h. The single labeling experiments highlight prominent staining in alveolar epithelial cells (S2a). The arrows highlight representative Type II alveolar cells that label with the anti-Ang2 antibody. Double labeling experiments were also undertaken using antibodies against Ang2 (green) and SP-C (red) (S2b). The arrows highlight Type II alveolar cells that label with both antibodies (yellow) in lungs from mice exposed to hyperoxia and treated with siRNA controls. Similar double positive cells were seen at a much lower frequency in lungs from mice exposed to 100%O2 and the Ang2 siRNA. (PDF 176 kb)

Supplementary Fig. 3

Role of Ang2 in hyperoxia-induced alterations in VEGF. BAL levels of VEGF were assessed from Ang2+/+, Ang2+/− and Ang2−/− mice breathing RA and exposed to hyperoxia for 72h. The values are the mean ± SEM of evaluations in a minimum of 6 mice. *P<0.0001, #P=0.02. (PDF 22 kb)

Supplementary Fig. 4

Dose-dependent specific inhibition of Ang2 by siRNA in vitro. a. Mean Ang2 mRNA silencing at 100 nM: B-16V cells were transfected with 100 nM Ang2 siRNA or irrelevant control siRNA, respectively. Mean of 3 separate experiments ± SD. b. Dose response curve for Ang2 siRNA: B-16V cells were transfected with different Ang2 siRNA concentrations. IC50 value was 3.0 nM; one of two representative experiments ± SD. In both experiments, residual Ang2 mRNA levels were measured by bDNA and normalized to GAPDH; Ang2 mRNA values are expressed as percent relative to control siRNA-treated. (PDF 92 kb)

Supplementary Fig. 5

Levels of Ang2 mRNA in mice exposed to hyperoxia and given low dose (LD) Ang-2 or control siRNA, as assessed by real-time RT-PCR. The values are the mean ± SEM of evaluations in a minimum of 3 mice. *P<0.0001, **P<0.01. (PDF 22 kb)

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Bhandari, V., Choo-Wing, R., Lee, C. et al. Hyperoxia causes angiopoietin 2–mediated acute lung injury and necrotic cell death. Nat Med 12, 1286–1293 (2006). https://doi.org/10.1038/nm1494

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