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Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

2015; Elsevier BV; Volume: 185; Issue: 11 Linguagem: Inglês

10.1016/j.ajpath.2015.07.010

1525-2191

Catherine T. Le, Grace Laidlaw, Christopher Morehouse, Brian Naiman, Philip Z. Brohawn, Tomas Mustelin, Jane R. Connor, Donald M. McDonald,

Lymphatic Disorders and Treatments

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response. Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response. Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.1Wilson J.W. Hii S. The importance of the airway microvasculature in asthma.Curr Opin Allergy Clin Immunol. 2006; 6: 51-55Crossref PubMed Scopus (38) Google Scholar, 2Kunstfeld R. Hirakawa S. Hong Y.K. Schacht V. Lange-Asschenfeldt B. Velasco P. Lin C. Fiebiger E. Wei X. Wu Y. Hicklin D. Bohlen P. Detmar M. 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Zaid H. Adams A. Baluk P. Lashnits E. Morisada T. Le T. O'Brien S. Epstein D.M. Koh G.Y. McDonald D.M. Pericyte requirement for anti-leak action of angiopoietin-1 and vascular remodeling in sustained inflammation.Am J Pathol. 2011; 178: 2897-2909Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.6Baluk P. Tammela T. Ator E. Lyubynska N. Achen M.G. Hicklin D.J. Jeltsch M. Petrova T.V. Pytowski B. Stacker S.A. Yla-Herttuala S. Jackson D.G. Alitalo K. McDonald D.M. Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (510) Google Scholar, 8Yao L.C. Baluk P. Feng J. McDonald D.M. Steroid-resistant lymphatic remodeling in chronically inflamed mouse airways.Am J Pathol. 2010; 176: 1525-1541Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 9Baluk P. 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Angiopoietin/Tie2 signaling transforms capillaries into venules primed for leukocyte trafficking in airway inflammation.Am J Pathol. 2010; 176: 2009-2018Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 5Tabruyn S.P. Colton K. Morisada T. Fuxe J. Wiegand S.J. Thurston G. Coyle A.J. Connor J. McDonald D.M. Angiopoietin-2-driven vascular remodeling in airway inflammation.Am J Pathol. 2010; 177: 3233-3243Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 6Baluk P. Tammela T. Ator E. Lyubynska N. Achen M.G. Hicklin D.J. Jeltsch M. Petrova T.V. Pytowski B. Stacker S.A. Yla-Herttuala S. Jackson D.G. Alitalo K. McDonald D.M. Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (510) Google Scholar, 7Fuxe J. Tabruyn S. Colton K. Zaid H. Adams A. Baluk P. Lashnits E. Morisada T. Le T. O'Brien S. Epstein D.M. Koh G.Y. McDonald D.M. 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The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)33McCall M.N. Bolstad B.M. Irizarry R.A. Frozen robust multiarray analysis (fRMA).Biostatistics. 2010; 11: 242-253Crossref PubMed Scopus (429) Google Scholar, 34Kramer A. Green J. Pollard Jr., J. Tugendreich S. Causal analysis approaches in Ingenuity Pathway Analysis.Bioinformatics. 2014; 30: 523-530Crossref PubMed Scopus (2771) Google Scholar and the Database for Annotation, Visualization and Integrated Discovery (DAVID).35Huang D.W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25346) Google Scholar The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response. Female 8-week-old C57BL/6 mice (Charles River, Hollister, CA; or Jackson Laboratory, Bar Harbor, MN), housed under barrier conditions, were used for all experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. Mice were anesthetized with an i.m. injection of 10 mg/kg ketamine and 20 mg/kg xylazine and inoculated intranasally with 50 μL culture media containing 1 × 106 colony-forming units of M. pulmonis (strain CT8), divided as 25 μL into each nostril.4Fuxe J. Lashnits E. O'Brien S. Baluk P. Tabruyn S.P. Kuhnert F. Kuo C. Thurston G. McDonald D.M. Angiopoietin/Tie2 signaling transforms capillaries into venules primed for leukocyte trafficking in airway inflammation.Am J Pathol. 2010; 176: 2009-2018Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 9Baluk P. Adams A. Phillips K. Feng J. Hong Y.K. Brown M.B. McDonald D.M. Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation.Am J Pathol. 2014; 184: 1577-1592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Mice were studied 7 days after infection. Function-blocking monoclonal human anti-Ang2 antibody (anti-Ang2; clone 3.19.3; AZD5180; 10 mg/kg; MedImmune LLC, Gaithersburg, MD)5Tabruyn S.P. Colton K. Morisada T. Fuxe J. Wiegand S.J. Thurston G. Coyle A.J. Connor J. McDonald D.M. Angiopoietin-2-driven vascular remodeling in airway inflammation.Am J Pathol. 2010; 177: 3233-3243Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 36Brown J.L. Cao Z.A. Pinzon-Ortiz M. Kendrew J. Reimer C. Wen S. Zhou J.Q. Tabrizi M. Emery S. McDermott B. Pablo L. McCoon P. Bedian V. Blakey D.C. A human monoclonal anti-ANG2 antibody leads to broad antitumor activity in combination with VEGF inhibitors and chemotherapy agents in preclinical models.Mol Cancer Ther. 2010; 9: 145-156Crossref PubMed Scopus (131) Google Scholar and/or monoclonal rat anti-murine TNF antibody (anti-TNF; MP6-XT22; 3 mg/kg; BioLegend, San Diego, CA)37Deepe Jr., G.S. Gibbons R.S. 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Angiopoietin/Tie2 signaling transforms capillaries into venules primed for leukocyte trafficking in airway inflammation.Am J Pathol. 2010; 176: 2009-2018Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 9Baluk P. Adams A. Phillips K. Feng J. Hong Y.K. Brown M.B. McDonald D.M. Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation.Am J Pathol. 2014; 184: 1577-1592Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Tracheas were removed, immersed in 1% paraformaldehyde for 1 hour, and then incubated overnight in combinations of two or three of the following primary antibodies, diluted in a mixture of 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO), 0.2% bovine serum albumin, and 0.1% sodium azide in PBS containing 10% goat serum: platelet endothelial cell adhesion molecule (PECAM-1; hamster anti-mouse CD31, clone 2H8, 1:500; Thermo Fisher Scientific, Waltham, MA), lymphatic vessel endothelial hyaluronan receptor-1 (rabbit anti-mouse lymphatic vessel endothelial hyaluronan receptor-1, catalog number 11-034, 1:500; AngioBio, Del Mar, CA), desmin (rabbit anti-mouse desmin, clone Y66, 1:500; Thermo Fisher Scientific), P-selectin (rat anti-mouse CD62P, RB40.34; 1 mg/mL; BD Biosciences, San Diego, CA), or S100a9 (rat anti-mouse S100a9, 2B10; 1:500; Abcam, Cambridge, England). Tracheas were incubated overnight in secondary antibodies conjugated to Alexa Fluor-488, Cy3, or Alexa Fluor-647 (all used at 1:500; Jackson ImmunoResearch, West Grove, PA), fixed for 5 minutes in 1% paraformaldehyde, and mounted as whole mounts. Digital images were acquired with a Zeiss Axiophot fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with single- and dual-fluorescence filters and a low-light, three-chip, charge-coupled device camera (480 × 640-pixel RGB-color images; CoolCam; SciMeasure Analytical Systems, Atlanta, GA) and linked to a digitizing tablet (Digi-Pad; GTCO CalComp, Columbia, MD). Confocal digital images were obtained with a Zeiss LSM-510 confocal microscope equipped (Carl Zeiss Microscopy GmbH) with argon and helium-neon lasers, using AIM software version 3.2.2. The diameter of blood vessels was measured in tracheal whole mounts stained for PECAM-1. Measurements were made of mucosal vessels specifically overlying the rostrocaudal midpoint of cartilage rings, which in normal tracheas consisted only of capillaries that were located approximately halfway between the arteriole and venule.5Tabruyn S.P. Colton K. Morisada T. Fuxe J. Wiegand S.J. Thurston G. Coyle A.J. Connor J. McDonald D.M. Angiopoietin-2-driven vascular remodeling in airway inflammation.Am J Pathol. 2010; 177: 3233-3243Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 7Fuxe J. Tabruyn S. Colton K. Zaid H. Adams A. Baluk P. Lashnits E. Morisada T. Le T. O'Brien S. Epstein D.M. Koh G.Y. McDonald D.M. Pericyte requirement for anti-leak action of angiopoietin-1 and vascular remodeling in sustained inflammation.Am J Pathol. 2011; 178: 2897-2909Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 8Yao L.C. Baluk P. Feng J. McDonald D.M. Steroid-resistant lymphatic remodeling in chronically inflamed mouse airways.Am J Pathol. 2010; 176: 1525-1541Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar The diameter of 15 vessels was measured over each of five cartilage rings per trachea. To assess the status of pericytes, the number of cytoplasmic processes of pericytes was determined in the same regions as the vessels in tracheal whole mounts stained for desmin and PECAM-1. Pericyte processes were defined as desmin-positive extensions of the cell body. Processes on 20 pericytes were counted on vessels over each of five to eight cartilage rings per trachea. Remodeling of lymphatics for lymphatic vessel endothelial hyaluronan receptor-1 was evaluated by counting sprouts (tapered projections) from lymphatic vessels (10× objective, 1× Optovar; Carl Zeiss Microscopy GmbH). Sprouts were counted in five regions, each having an area of 0.53 mm2, in each trachea. Values were expressed per square millimeter of tracheal mucosa. Measurements were made with a digitizing tablet on real-time images viewe