α5β1 Integrin Blockade Inhibits Lymphangiogenesis in Airway Inflammation
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080942
ISSN1525-2191
AutoresTatsuma Okazaki, Amy Ni, Oluwasheyi A. Ayeni, Peter Bałuk, Li-Chin Yao, Doerte Vossmeyer, Gunther Zischinsky, Grit Zahn, Jochen Knolle, Claudia Christner, Donald M. McDonald,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoThe integrin α5β1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of α5β1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, α5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have α5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the α5β1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth. The integrin α5β1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of α5β1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, α5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have α5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the α5β1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth. 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Springer, New York2008: 15-31Google Scholar Blood capillaries span the rings in a ladder-like pattern, and arterioles, venules, and lymphatics are restricted to the regions between the rings.31Baluk 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 (510) Google Scholar This stereotype architecture makes it straightforward to detect and quantify the growth and remodeling of blood vessels and lymphatics in inflammatory conditions.31Baluk 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 (510) Google Scholar Respiratory tract infection caused by Mycoplasma pulmonis results in conspicuous growth and remodeling of blood vessels and lymphatics in the airways.31Baluk 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 (510) Google Scholar, 39Thurston G Murphy TJ Baluk P Lindsey JR McDonald DM Angiogenesis in mice with chronic airway inflammation: strain-dependent differences.Am J Pathol. 1998; 153: 1099-1112Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 40Thurston G Maas K Labarbara A McLean JW McDonald DM Microvascular remodelling in chronic airway inflammation in mice.Clin Exp Pharmacol Physiol. 2000; 27: 836-841Crossref PubMed Scopus (30) Google Scholar, 41McDonald DM Angiogenesis and remodeling of airway vasculature in chronic inflammation.Am J Respir Crit Care Med. 2001; 164: S39-S45Crossref PubMed Scopus (348) Google Scholar We used the vascular changes accompanying M. pulmonis infection to determine whether α5β1 integrin plays an essential role in blood vessel remodeling and lymphangiogenesis in airway inflammation. The role of α5β1 integrin was tested by using 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), a novel selective small molecule inhibitor of α5β1 integrin function. JSM8757 is an orally available analogue of JSM6427, which was previously shown to inhibit choroidal neovascularization and inflammatory lymphangiogenesis in the cornea.11Dietrich T Onderka J Bock F Kruse FE Vossmeyer D Stragies R Zahn G Cursiefen C Inhibition of inflammatory lymphangiogenesis by integrin alpha5 blockade.Am J Pathol. 2007; 171: 361-372Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 22Umeda N Kachi S Akiyama H Zahn G Vossmeyer D Stragies R Campochiaro PA Suppression and regression of choroidal neovascularization by systemic administration of an alpha5beta1 integrin antagonist.Mol Pharmacol. 2006; 69: 1820-1828Crossref PubMed Scopus (78) Google Scholar, 42Stragies R Osterkamp F Zischinsky G Vossmeyer D Kalkhof H Reimer U Zahn G Design and synthesis of a new class of selective integrin alpha5beta1 antagonists.J Med Chem. 2007; 50: 3786-3794Crossref PubMed Scopus (58) Google Scholar Our approach was first to determine the distribution of α5β1 integrin in the trachea by using α5 integrin subunit immunoreactivity as a readout. Next, we examined the efficacy of JSM8757 in inhibiting proliferation of cultured lymphatic endothelial cells and then on vascular remodeling and lymphangiogenesis after M. pulmonis infection. We found that the α5β1 integrin blockade reduced lymphatic sprouting and growth in airway inflammation but did not reduce blood vessel remodeling or macrophage recruitment. Specific pathogen-free 8-week-old female C57BL/6 mice (Charles River, Hollister, CA) were housed under barrier conditions. 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 at San Francisco. All reagents were purchased from Sigma unless indicated otherwise. JSM8757, which was synthesized at Jerini AG (Berlin, Germany) with a purity of >99%, is a small-molecule antagonist of integrin α5β1. JSM8757 mimics the RGD (Arg-Gly-Asp) recognition motif in fibronectin, where it interacts with α5 and β1 integrin subunits with subnanomolar IC50 values. The agent inhibits α5β1/ligand binding with an IC50 value of 0.9 nmol/L assessed by enzyme-linked immunosorbent assay and an IC50 value of 30 nmol/L in blocking the adhesion of α5β1-positive HEK293 cells to fibronectin. JSM8757 has much higher affinity for α5β1 integrin than for other RGD-binding integrins, with IC50 values >670-fold greater for αvβ3 integrin, >13,300-fold greater for αvβ5 integrin, and >34,000-fold greater for αIIbβ3 integrin. The enzyme-linked immunosorbent assay and IC50 determination was performed as described previously.42Stragies R Osterkamp F Zischinsky G Vossmeyer D Kalkhof H Reimer U Zahn G Design and synthesis of a new class of selective integrin alpha5beta1 antagonists.J Med Chem. 2007; 50: 3786-3794Crossref PubMed Scopus (58) Google Scholar Mice at 8 weeks of age were anesthetized and inoculated intranasally on day 0 with 50 μl of broth containing 106 colony-forming units of M. pulmonis organisms (strain CT7) as described previously.31Baluk 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 (510) Google ScholarM. pulmonis organisms activate an immune response with a time course similar to other airway infections.43Aurora AB Baluk P Zhang D Sidhu SS Dolganov GM Basbaum C McDonald DM Killeen N Immune complex-dependent remodeling of the airway vasculature in response to a chronic bacterial infection.J Immunol. 2005; 175: 6319-6326PubMed Google Scholar Mice were concurrently injected intraperitoneally with vehicle or JSM8757 (100 mg/kg) twice a day for 14 days. Previous pharmacokinetic studies show that this dose of JSM8757 results in plasma concentrations (Cmax) of approximately 40 μmol/L. At 14 days after M. pulmonis infection, mice were anesthetized again and tissues were harvested for further studies. Mice 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.31Baluk 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 (510) Google Scholar Tracheas were removed and immersed in fixative for 1 hour at 4°C. Tissues were washed and stained immunohistochemically by incubating whole mounts or 60-μm cryostat sections 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.31Baluk 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 (510) Google Scholar The following antibodies were used at the indicated concentrations: lymphatics: lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), 1:500 (rabbit polyclonal 07–538; AngioBio, Del Mar, CA); endothelial cells: CD31, 1:500 (hamster anti-mouse PECAM-1, clone 2H8; Thermo, Waltham, MA); monocyte/macrophages: CD11b, 1:500 (rat anti-mouse CD11b, clone M1/70; eBioscience, San Diego, CA), ionized calcium-binding adapter molecule 1 (Iba1), 1:500 (rabbit polyclonal 019-19741; Wako Pure Chemical Industries, Osaka, Japan); α5 integrin/CD49e: 1:500 (rat anti-mouse CD49e, clone 5H10-27; BD Biosciences PharMingen, San Diego, CA). Because α5 integrin subunits (CD49e) pair solely with β1 subunits, anti-α5 integrin antibody was regarded as binding exclusively to α5β1 integrin.6Mahabeleshwar GH Chen J Feng W Somanath PR Razorenova OV Byzova TV Integrin affinity modulation in angiogenesis.Cell Cycle. 2008; 7: 335-347Crossref PubMed Scopus (63) Google Scholar, 16Humphries JD Byron A Humphries MJ Integrin ligands at a glance.J Cell Sci. 2006; 119: 3901-3903Crossref PubMed Scopus (1181) Google Scholar, 17Kumar CC Signaling by integrin receptors.Oncogene. 1998; 17: 1365-1373Crossref PubMed Scopus (240) Google Scholar Secondary antibodies were labeled with fluorescein isothiocyanate, Cyanine3 (Cy3), or Cy5, 1:500 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Specimens were viewed with an Axiophot fluorescence microscope (Carl Zeiss) with a 3-CCD low light RGB video camera (CoolCam; SciMeasure Analytical Systems, Atlanta, GA) or a LSM510 confocal microscope (Carl Zeiss) using AIM confocal software (version 3.2.2). Measurements were performed using digitizing software interfaced to the Axiophot microscope and CoolCam camera. The number of lymphatic sprouts was measured in projected real-time fluorescent images of tracheal whole mounts. Lymphatic sprouts were counted and defined as tapered LYVE-1-positive projections visible at a screen magnification of 180×, in five regions per trachea, each 1.5 mm2 in area.31Baluk 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 (510) Google Scholar Area densities (percentage of total tissue area) of LYVE-1-positive lymphatics and CD31-positive blood vessels viewed in real-time fluorescent images of tracheal whole mounts were measured by stereological point counting of 10 regions per trachea, each 1.7 mm2 in area.31Baluk 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 (510) Google Scholar The fluorescence intensity was measured as an estimate of the expression of α5 integrin in the mucosa overlying cartilage rings. The fluorescence intensity was measured on digital images stained for α5 integrin immunoreactivity using ImageJ software.44Inai T Mancuso M Hashizume H Baffert F Haskell A Baluk P Hu-Lowe DD Shalinsky DR Thurston G Yancopoulos GD McDonald DM Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts.Am J Pathol. 2004; 165: 35-52Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 45Kamba T Tam BY Hashizume H Haskell A Sennino B Mancuso MR Norberg SM O'Brien SM Davis RB Gowen LC Anderson KD Thurston G Joho S Springer ML Kuo CJ McDonald DM VE
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