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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References
Barazzone, C., Horowitz, S., Donati, Y.R., Rodriguez, I. & Piguet, P.F. Oxygen toxicity in mouse lung: pathways to cell death. Am. J. Respir. Cell Mol. Biol. 19, 573–581 (1998).
Barazzone, C. & White, C.W. Mechanisms of cell injury and death in hyperoxia: role of cytokines and Bcl-2 family proteins. Am. J. Respir. Cell Mol. Biol. 22, 517–519 (2000).
Crapo, J.D. Morphologic changes in pulmonary oxygen toxicity. Annu. Rev. Physiol. 48, 721–731 (1986).
O'Reilly, M.A. et al. Bcl-2 family gene expression during severe hyperoxia induced lung injury. Lab. Invest. 80, 1845–1854 (2000).
Ward, N.S. et al. Interleukin-6-induced protection in hyperoxic acute lung injury. Am. J. Respir. Cell Mol. Biol. 22, 535–542 (2000).
Waxman, A.B. et al. Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation. J. Clin. Invest. 101, 1970–1982 (1998).
Mantell, L.L., Horowitz, S., Davis, J.M. & Kazzaz, J.A. Hyperoxia-induced cell death in the lung—the correlation of apoptosis, necrosis, and inflammation. Ann. NY Acad. Sci. 887, 171–180 (1999).
Carmeliet, P. Angiogenesis in health and disease. Nat. Med. 9, 653–660 (2003).
Ferrara, N., Gerber, H.P. & LeCouter, J. The biology of VEGF and its receptors. Nat. Med. 9, 669–676 (2003).
Neufeld, G., Cohen, T., Gengrinovitch, S. & Poltorak, Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 13, 9–22 (1999).
McDonald, D.M. Angiogenesis and remodeling of airway vasculature in chronic inflammation. Am. J. Respir. Crit. Care Med. 164, S39–S45 (2001).
Thurston, G. et al. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat. Med. 6, 460–463 (2000).
Thurston, G. et al. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286, 2511–2514 (1999).
Yancopoulos, G.D. et al. Vascular-specific growth factors and blood vessel formation. Nature 407, 242–248 (2000).
Jain, R.K. Molecular regulation of vessel maturation. Nat. Med. 9, 685–693 (2003).
Sandhu, R. et al. Reciprocal regulation of angiopoietin-1 and angiopoietin-2 following myocardial infarction in the rat. Cardiovasc. Res. 64, 115–124 (2004).
Tait, C.R. & Jones, P.F. Angiopoietins in tumours: the angiogenic switch. J. Pathol. 204, 1–10 (2004).
Bloch, W. et al. The angiogenesis inhibitor endostatin impairs blood vessel maturation during wound healing. FASEB J. 14, 2373–2376 (2000).
He, C.H. et al. Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury. J. Clin. Invest. 115, 1039–1048 (2005).
Gale, N.W. et al. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by angiopoietin-1. Dev. Cell 3, 411–423 (2002).
Horowitz, S. Pathways to cell death in hyperoxia. Chest 116, 64S–67S (1999).
O'Reilly, M.A. DNA damage and cell cycle checkpoints in hyperoxic lung injury: braking to facilitate repair. Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L291–L305 (2001).
Raffray, M. & Cohen, G.M. Apoptosis and necrosis in toxicology: a continuum or distinct modes of cell death? Pharmacol. Ther. 75, 153–177 (1997).
Richard, C. et al. Androgens modulate the balance between VEGF and angiopoietin expression in prostate epithelial and smooth muscle cells. Prostate 50, 83–91 (2002).
Oshima, Y. et al. Angiopoietin-2 enhances retinal vessel sensitivity to vascular endothelial growth factor. J. Cell. Physiol. 199, 412–417 (2004).
Tsigkos, S., Koutsilieris, M. & Papapetropoulos, A. Angiopoietins in angiogenesis and beyond. Expert Opin. Investig. Drugs 12, 933–941 (2003).
Karmpaliotis, D. et al. Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 283, L585–L595 (2002).
Joza, N., Kroemer, G. & Penninger, J.M. Genetic analysis of the mammalian cell death machinery. Trends Genet. 18, 142–149 (2002).
Lin, E.Y., Orlofsky, A., Berger, M.S. & Prystowsky, M.B. Characterization of A1, a novel hemopoietic-specific early-response gene with sequence similarity to bcl-2. J. Immunol. 151, 1979–1988 (1993).
Werner, A.B., de Vries, E., Tait, S.W., Bontjer, I. & Borst, J. Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax. J. Biol. Chem. 277, 22781–22788 (2002).
Wang, X. et al. Necrotic cell death in response to oxidant stress involves the activation of the apoptogenic caspase-8/bid pathway. J. Biol. Chem. 278, 29184–29191 (2003).
O'Reilly, M.A., Staversky, R.J., Watkins, R.H. & Maniscalco, W.M. Accumulation of p21Cip1/WAF1 during hyperoxic lung injury in mice. Am. J. Respir. Cell Mol. Biol. 19, 777–785 (1998).
Mitsutake, N. et al. Tie-2 and angiopoietin-1 expression in human thyroid tumors. Thyroid 12, 95–99 (2002).
Nakayama, T. et al. Expression of Tie-1 and 2 receptors, and angiopoietin-1, 2 and 4 in gastric carcinoma; immunohistochemical analyses and correlation with clinicopathological factors. Histopathology 44, 232–239 (2004).
Wurmbach, J.H., Hammerer, P., Sevinc, S., Huland, H. & Ergun, S. The expression of angiopoietins and their receptor Tie-2 in human prostate carcinoma. Anticancer Res. 20, 5217–5220 (2000).
Kim, I. et al. The angiopoietin-tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis. Cardiovasc. Res. 49, 872–881 (2001).
Lobov, I.B., Brooks, P.C. & Lang, R.A. Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc. Natl. Acad. Sci. USA 99, 11205–11210 (2002).
Beck, H., Acker, T., Wiessner, C., Allegrini, P.R. & Plate, K.H. Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat. Am. J. Pathol. 157, 1473–1483 (2000).
Zagzag, D. et al. Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis. Lab. Invest. 80, 837–849 (2000).
Ware, L.B. & Matthay, M.A. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 163, 1376–1383 (2001).
Anandarajah, A.P. & Ritchlin, C.T. Pathogenesis of psoriatic arthritis. Curr. Opin. Rheumatol. 16, 338–343 (2004).
Fearon, U. et al. Angiopoietins, growth factors, and vascular morphology in early arthritis. J. Rheumatol. 30, 260–268 (2003).
Kimura, H., Mochida, S., Inao, M., Matsui, A. & Fujiwara, K. Angiopoietin/tie receptors system may play a role during reconstruction and capillarization of the hepatic sinusoids after partial hepatectomy and liver necrosis in rats. Hepatol. Res. 29, 51–59 (2004).
Brat, D.J. & Van Meir, E.G. Vaso-occlusive and prothrombotic mechanisms associated with tumor hypoxia, necrosis, and accelerated growth in glioblastoma. Lab. Invest. 84, 397–405 (2004).
Zhu, S., Ware, L.B., Geiser, T., Matthay, M.A. & Matalon, S. Increased levels of nitrate and surfactant protein a nitration in the pulmonary edema fluid of patients with acute lung injury. Am. J. Respir. Crit. Care Med. 163, 166–172 (2001).
Pugin, J., Verghese, G., Widmer, M.C. & Matthay, M.A. The alveolar space is the site of intense inflammatory and profibrotic reactions in the early phase of acute respiratory distress syndrome. Crit. Care Med. 27, 304–312 (1999).
Koga, K. et al. Expression of angiopoietin-2 in human glioma cells and its role for angiogenesis. Cancer Res. 61, 6248–6254 (2001).
Lip, P.L. et al. Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy: effects of laser photocoagulation and angiotensin receptor blockade. Br. J. Ophthalmol. 88, 1543–1546 (2004).
Ohashi, H. et al. Alterations in expression of angiopoietins and the Tie-2 receptor in the retina of streptozotocin induced diabetic rats. Mol. Vis. 10, 608–617 (2004).
Bhandari, A. & Bhandari, V. Pathogenesis, pathology and pathophysiology of pulmonary sequelae of bronchopulmonary dysplasia in premature infants. Front. Biosci. 8, e370–e380 (2003).
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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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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DOI: https://doi.org/10.1038/nm1494
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