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Capillary Defects and Exaggerated Inflammatory Response in the Airways of EphA2-Deficient Mice

2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês

10.2353/ajpath.2009.080949

1525-2191

Tatsuma Okazaki, Amy Ni, Peter Bałuk, Oluwasheyi A. Ayeni, Jennifer Kearley, Anthony J. Coyle, Alison A. Humbles, Donald M. McDonald,

Apelin-related biomedical research

Both Eph receptors and ephrin ligands have been implicated in blood vessel and neuronal development. Recent studies suggested that EphA2 inhibition reduces tumor angiogenesis, but its role in blood vessel development and inflammation is unclear. We examined these issues using either airways of pathogen-free, EphA2-deficient mice at various ages or EphA2-deficient mice whose airways were inflamed by either Mycoplasma pulmonis infection or ovalbumin sensitization and challenge. EphA2-deficient mice had fewer capillaries, a greater number of endothelial sprouts, and greater capillary diameters than age-matched, wild-type control mice. Moreover, capillaries in EphA2-deficient mice had significantly less pericyte coverage, suggesting abnormal interactions between endothelial cells and pericytes. These differences were apparent in early postnatal life but decreased during progression into adulthood. In inflamed airways, significantly more angiogenesis and lymphangiogenesis, a greater number of infiltrating leukocytes, and higher expression levels of inflammatory cytokine mRNA were present in EphA2-deficient mice after M. pulmonis infection. Additionally, in allergic airway inflammation with ovalbumin sensitization and challenge, a greater number of lymphatic sprouts and infiltrating leukocytes, higher mRNA expression levels of TH2 cytokines and chemokines related to allergic airway inflammation, and enhanced airway hyper-responsiveness were present in EphA2-deficient mice. We conclude that defective pericyte coverage causes capillary defects, abundant endothelial sprouts, and thick capillary diameters in EphA2-deficient mice, indicating that these animals have exaggerated responses to airway inflammation. Both Eph receptors and ephrin ligands have been implicated in blood vessel and neuronal development. Recent studies suggested that EphA2 inhibition reduces tumor angiogenesis, but its role in blood vessel development and inflammation is unclear. We examined these issues using either airways of pathogen-free, EphA2-deficient mice at various ages or EphA2-deficient mice whose airways were inflamed by either Mycoplasma pulmonis infection or ovalbumin sensitization and challenge. EphA2-deficient mice had fewer capillaries, a greater number of endothelial sprouts, and greater capillary diameters than age-matched, wild-type control mice. Moreover, capillaries in EphA2-deficient mice had significantly less pericyte coverage, suggesting abnormal interactions between endothelial cells and pericytes. These differences were apparent in early postnatal life but decreased during progression into adulthood. In inflamed airways, significantly more angiogenesis and lymphangiogenesis, a greater number of infiltrating leukocytes, and higher expression levels of inflammatory cytokine mRNA were present in EphA2-deficient mice after M. pulmonis infection. Additionally, in allergic airway inflammation with ovalbumin sensitization and challenge, a greater number of lymphatic sprouts and infiltrating leukocytes, higher mRNA expression levels of TH2 cytokines and chemokines related to allergic airway inflammation, and enhanced airway hyper-responsiveness were present in EphA2-deficient mice. We conclude that defective pericyte coverage causes capillary defects, abundant endothelial sprouts, and thick capillary diameters in EphA2-deficient mice, indicating that these animals have exaggerated responses to airway inflammation. Formation of blood vessel networks is regulated by coordinated attractive and repulsive guidance cues. 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A commonly used mouse model of allergic asthma is where animals are immunized with ovalbumin (OVA) and later exposed to inhaled OVA, this induces an influx of Th2 lymphocytes, eosinophils, and the development of airway hyper-responsiveness.43Humbles AA Lloyd CM McMillan SJ Friend DS Xanthou G McKenna EE Ghiran S Gerard NP Yu C Orkin SH Gerard C A critical role for eosinophils in allergic airways remodeling.Science. 2004; 305: 1776-1779Crossref PubMed Scopus (739) Google ScholarThe mouse trachea has a discrete and segmented network of blood vessels aligned with the framework of cartilage rings. This characteristic allows clear observation and quantification of blood vessel changes such as in inflammation and development.26Baluk P Tammela T Ator E Lyubynska N Achen MG Hicklin DJ Jeltsch M Petrova TV Pytowski B Stacker SA Yla-Herttuala S Jackson DG Alitalo K McDonald DM Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (502) Google Scholar, 44Baffert F Thurston G Rochon-Duck M Le T Brekken R McDonald DM Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1-induced plasticity of adult blood vessels.Circ Res. 2004; 94: 984-992Crossref PubMed Scopus (101) Google ScholarAccordingly, the present study sought to determine the distribution of immunoreactivity for EphA2 on airway blood vessels, the effect of EphA2 deficiency on neonatal blood vessel development from early neonatal life to adulthood, and the response to inflammation. Our approach was first to use immunohistochemistry to determine the distribution of immunoreactivity for EphA2 and its ligands in tracheas isolated from 2- to 25-week-old control wild-type and EphA2-deficient mice to compare the development of the tracheal blood vessels during the neonatal ages. Our study revealed defective capillary development and pericyte association with endothelial cells in EphA2-deficient mice. We used two models of airway inflammation induced by M. pulmonis infection or by sensitization and inhalation of allergen (ovalbumin). EphA2-deficient mice revealed an exaggerated response to both types of airway inflammation.Materials and MethodsMiceEphA2-deficient mice (strain 006028, Jackson Laboratories, Bar Harbor, ME) were a mixture of 129xC57BL/6 (strain B6129F2/J) background, backcrossed with C57BL/6 genetic background for two generations. These mice were originally described.45Brantley-Sieders DM Caughron J Hicks D Pozzi A Ruiz JC Chen J EphA2 receptor tyrosine kinase regulates endothelial cell migration and vascular assembly through phosphoinositide 3-kinase-mediated Rac1 GTPase activation.J Cell Sci. 2004; 117: 2037-2049Crossref PubMed Scopus (163) Google Scholar B6129F2/J mice (strain 101045, Jackson Laboratories) were used as control mice. Specific pathogen-free mice were housed under barrier conditions. Before being subjected to experimental procedures, mice were anesthetized by intramuscular injection of ketamine (83 mg/kg) and xylazine (13 mg/kg). All experimental procedures were approved by the Institutional Animal Care and Use Committees of the University of California, San Francisco, or MedImmune, Inc.M. pulmonis InfectionMice at 10 weeks of age were inoculated intranasally on day 0 with 50 μl of broth containing 3.3 × 105 colony-forming units of M. pulmonis organisms (strain CT7) as described previously.26Baluk P Tammela T Ator E Lyubynska N Achen MG Hicklin DJ Jeltsch M Petrova TV Pytowski B Stacker SA Yla-Herttuala S Jackson DG Alitalo K McDonald DM Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (502) Google Scholar Mice were anesthetized before inoculation and then allowed to recover. At 14 days after M. pulmonis infection, mice were anesthetized again for further studies. Body weights of the mice were measured daily, and relative body weights were shown in percentages by setting the body weights on day 0 as 100%.ImmunohistochemistryMice were perfused for 2 minutes with fixative (1% paraformaldehyde in phosphate-buffered saline; PBS, pH 7.4) from a cannula inserted through the left ventricle into the aorta.26Baluk P Tammela T Ator E Lyubynska N Achen MG Hicklin DJ Jeltsch M Petrova TV Pytowski B Stacker SA Yla-Herttuala S Jackson DG Alitalo K McDonald DM Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (502) Google Scholar Tracheas were removed and immersed in fixative for 1 hour. Tissues were washed and stained immunohistochemically by incubating whole mounts with one or more primary antibodies diluted in PBS containing 0.3% Triton X-100, 0.2% bovine serum albumin, 5% normal goat serum, and 0.1% sodium azide, as described previously.26Baluk P Tammela T Ator E Lyubynska N Achen MG Hicklin DJ Jeltsch M Petrova TV Pytowski B Stacker SA Yla-Herttuala S Jackson DG Alitalo K McDonald DM Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (502) Google Scholar The following antibodies were used at indicated concentrations: lymphatics: lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), 1:500 (rabbit polyclonal 07-538; Upstate Biotechnology, Billerica, MA or AngioBio, Del Mar, CA); CD31, 1:500 (hamster anti-mouse PECAM-1, clone 2H8; Thermo Scientific, Waltham, MA); pericytes: desmin, 1:2000 (rabbit polyclonal 8592; Abcam, Cambridge, MA); EphA2, 1:500 (goat polyclonal AF639; R&D Systems, Minneapolis, MN). Secondary antibodies were labeled with fluorescein isothiocyanate, cyanine 3 (Cy3), or Cy5, 1:500 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Specimens were viewed with a fluorescence microscope (Axiophot; Carl Zeiss MicroImaging, Inc.) with a 3-CCD low light RGB video camera (CoolCam, SciMeasure Analytical Systems, Atlanta, GA) or a confocal microscope (LSM-510; Carl Zeiss MicroImaging, Inc.) using AIM confocal software (version 3.2.2).The specificity of the EphA2 antibody (catalog number AF639, R&D Systems) has been documented. In direct enzyme-linked immunosorbent assay and Western blots, this antibody shows less than 1% cross-reactivity with rhEphA1, rmEphA3, rmEphA4, rmEphA6, rmEphA7, rmEphA8, and rrEphA5 R&D Systems product circular (anti-mouse EphA2 antibody, R&D Systems). In addition, the use of the EphA2 antibody AF639 has been documented in DMBA/TPA-induced skin tumors.46Guo H Miao H Gerber L Singh J Denning MF Gilliam AC Wang B Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin.Cancer Res. 2006; 66: 7050-7058Crossref PubMed Scopus (112) Google ScholarInduction of Airway Inflammation by OvalbuminMice were immunized with 10 μg of ovalbumin (OVA) (Sigma, St. Louis, MO) in alum (1 mg of aluminum hydroxide gel, Sigma) in 0.2 ml of sterile saline intraperitoneally on days 0 and 10. Sham-immunized mice received the same amount of saline in alum. Since induction of airway lymphangiogenesis required longer term OVA challenge, we performed two terms of OVA challenge. For airway hyper-responsiveness, cell counts in bronchoalveolar lavage fluid, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments, mice were exposed to aerosolized OVA (5%, 30 minutes) or saline (sham) on days 17 to 22. Airway hyper-responsiveness and airway inflammation were determined 24 hours later (day 23). For tracheal whole-mount staining, mice were intranasally inoculated with OVA as previously shown with some modifications.38Walsh ER Sahu N Kearley J Benjamin E Kang BH Humbles A August A Strain-specific requirement for eosinophils in the recruitment of T cells to the lung during the development of allergic asthma.J Exp Med. 2008; 205: 1285-1292Crossref PubMed Scopus (124) Google Scholar Mice were anesthetized and intranasally inoculated with 50 μl of OVA (0.667 mg/ml) in saline or saline alone (sham) on days 17 to 22, 24, 26, 28, 31, and 33. Tracheas were isolated 24 hours later (day 34).Assessment of Airway Hyper-ResponsivenessAirway hyper-responsiveness was determined using a modified version of previously described methods.47Wagers S Lundblad LK Ekman M Irvin CG Bates JH The allergic mouse model of asthma: normal smooth muscle in an abnormal lung?.J Appl Physiol. 2004; 96: 2019-2027Crossref PubMed Scopus (178) Google Scholar Briefly, mice were anesthetized with pentobarbital sodium and connected to a small animal ventilator (FlexiVent, Scireq, Montreal, Canada). Next baseline recordings of all parameters were obtained. The mice were then challenged with an aerosol of PBS and recordings of all parameters were made every 10 seconds for 3 minutes, alternating between signals (Snapshot and Quick-Prime, the latter measurements being made by interrupting the ventilation for a 1-second passive expiration followed by 2-second broadband (1–19.625 Hz) volume perturbation). Finally, two more deep lung inflations were given and the above protocol repeated three more times with aerosols containing methacholine (Sigma) at sequentially increasing concentrations of 3.125, 12.5, and 50 mg/ml.Morphometric MeasurementsFor morphometric measurements, the trachea was imaged using a Zeiss Axiophot fluorescence microscope equipped with a CoolCam fluorescence camera. Measurements were performed using digitizing software interfaced to the microscope and CoolCam camera. The number of capillaries per millimeter was measured by counting fluorescent images of CD31-positive blood vessels that crossed the center of cartilage rings.44Baffert F Thurston G Rochon-Duck M Le T Brekken R McDonald DM Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1-induced plasticity of adult blood vessels.Circ Res. 2004; 94: 984-992Crossref PubMed Scopus (101) Google Scholar Capillary diameters were assessed by measuring CD31-positive blood vessels at the center of cartilage rings.44Baffert F Thurston G Rochon-Duck M Le T Brekken R McDonald DM Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1-induced plasticity of adult blood vessels.Circ Res. 2004; 94: 984-992Crossref PubMed Scopus (101) Google Scholar Number of endothelial sprouts was measured by counting CD31-positive endothelial sprouts, defined as any thin, tapered CD31-positive projections. These CD31-positive vessel measurements were performed by observing 10 cartilage rings per trachea, each 0.2 mm2 in area. The number of lymphatic sprouts was measured viewing real-time fluorescent images of tracheal whole mount, we counted lymphatic sprouts, defined as tapered LYVE-1 positive projections visible at a screen magnification of x180, in 5 regions per trachea, each 1.5 mm2 in area.26Baluk P Tammela T Ator E Lyubynska N Achen MG Hicklin DJ Jeltsch M Petrova TV Pytowski B Stacker SA Yla-Herttuala S Jackson DG Alitalo K McDonald DM Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (502) Google Scholar Pericyte coverage was counted by measuring the length of capillaries covered by desmin-positive pericytes. Pericyte coverage percentage represents the length of capillaries covered by desmin-posit