Radiation-Induced Lung Injury Is Mitigated by Blockade of Gastrin-Releasing Peptide
2013; Elsevier BV; Volume: 182; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2012.12.024
ISSN1525-2191
AutoresShutang Zhou, Esther Nissao, Isabel L. Jackson, Wei Leong, Lindsay Dancy, Frank Cuttitta, Željko Vujašković, Mary E. Sunday,
Tópico(s)Cancer, Stress, Anesthesia, and Immune Response
ResumoGastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RiLI). Using C57BL/6 mouse model of pulmonary fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68+ macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. Ten weeks later radiation plus PBS controls had significantly increased pSmad2/3+ nuclei/cm2. GRP blockade with 77427 treatment diminished CD68+, GRP+, and pSmad2/3+ cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel radiation fibrosis mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally. Gastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RiLI). Using C57BL/6 mouse model of pulmonary fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68+ macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. Ten weeks later radiation plus PBS controls had significantly increased pSmad2/3+ nuclei/cm2. GRP blockade with 77427 treatment diminished CD68+, GRP+, and pSmad2/3+ cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel radiation fibrosis mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally. Radiation fibrosis is a serious complication that affects normal lung following unintentional exposure or due to therapeutic ionizing radiation of thoracic tumors. Despite advances in radiobiology, precise mechanisms by which radiation induces lung injury remain controversial.1Vujaskovic Z. Marks L.B. Anscher M.S. The physical parameters and molecular events associated with radiation-induced lung toxicity.Semin Radiat Oncol. 2000; 10: 296-307Abstract Full Text PDF PubMed Scopus (108) Google Scholar Classically, radiation-induced lung injury (RiLI) is characterized by a latent period that can last for weeks to months after radiation exposure, followed by 2 stages of overt lung injury that can lead to life-threatening and debilitating pulmonary toxic effects.2Dorr W. Baumann M. Herrmann T. Radiation-induced lung damage: a challenge for radiation biology, experimental and clinical radiotherapy.Int J Radiat Biol. 2000; 76: 443-446Crossref PubMed Scopus (24) Google Scholar, 3Morgan G.W. Breit S.N. Radiation and the lung: a reevaluation of the mechanisms mediating pulmonary injury.Int J Radiat Oncol Biol Phys. 1995; 31: 361-369Abstract Full Text PDF PubMed Scopus (250) Google Scholar Acute inflammatory lung injury arises 1 to 6 months after radiation exposure, with diffuse alveolar damage, similar to acute respiratory distress syndrome. Later, chronic interstitial and intra-alveolar fibrosis develops, predominantly in irradiated segments, with myofibroblast proliferation and collagen deposition. It is unclear why only approximately 15% of radiation-exposed patients develop RiLI.1Vujaskovic Z. Marks L.B. Anscher M.S. The physical parameters and molecular events associated with radiation-induced lung toxicity.Semin Radiat Oncol. 2000; 10: 296-307Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 4Marks L.B. Yu X. Vujaskovic Z. Small Jr., W. Folz R. Anscher M.S. Radiation-induced lung injury.Semin Radiat Oncol. 2003; 13: 333-345Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar General cytoprotective agents, such as a catalytic antioxidant metalloporphyrin (AEOL10113), can reduce the severity of RiLI by decreasing free radical injury after radiation.5Vujaskovic Z. Batinic-Haberle I. Rabbani Z.N. Feng Q.F. Kang S.K. Spasojevic I. Samulski T.V. Fridovich I. Dewhirst M.W. Anscher M.S. A small molecular weight catalytic metalloporphyrin antioxidant with superoxide dismutase (SOD) mimetic properties protects lungs from radiation-induced injury.Free Radic Biol Med. 2002; 33: 857-863Crossref PubMed Scopus (162) Google Scholar Our novel paradigm links gastrin-releasing peptide (GRP) to radiation lung injury. We hypothesized that GRP is a mediator of RiLI: promoting both macrophage accumulation and fibrosis. We propose that ionizing radiation triggers pulmonary neuroendocrine cell (PNEC) hyperplasia, leading to GRP secretion, which then mediates chronic lung injury. GRP receptor (GRPR) gene expression is detected and functional in pulmonary epithelial cells, fibroblasts, endothelial cells, and macrophages.6Siegfried J.M. DeMichele M.A.A. Hunt J.D. Davis A.G. Vohra K.P. Pilewski J.M. Expression of mRNA for gastrin-releasing peptide receptor by human bronchial epithelial cells: association with prolonged tobacco exposure and responsiveness to bombesin-like peptides.Am J Respir Crit Care Med. 1997; 156: 358-366Crossref PubMed Scopus (45) Google Scholar, 7Subramaniam M. Bausch C. Twomey A. Andreeva S. Yoder B.A. Chang L.Y. Crapo J.D. Pierce R.A. Cuttitta F. Sunday M.E. Bombesin-like peptides modulate alveolarization and angiogenesis in bronchopulmonary dysplasia.Am J Respir Crit Care Med. 2007; 176: 902-912Crossref PubMed Scopus (43) Google Scholar, 8Jensen R.T. Battey J.F. Spindel E.R. Benya R.V. International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states.Pharmacol Rev. 2008; 60: 1-42Crossref PubMed Scopus (418) Google Scholar, 9De la Fuente M. Del Rio M. Ferrandez M.D. Hernanz A. Modulation of phagocytic function in murine peritoneal macrophages by bombesin, gastrin-releasing peptide and neuromedin C.Immunology. 1991; 73: 205-211PubMed Google Scholar, 10Zhou S. Potts E.N. Cuttitta F. Foster W.M. Sunday M.E. Gastrin-releasing peptide blockade as a broad-spectrum anti-inflammatory therapy for asthma.Proc Natl Acad Sci U S A. 2011; 108: 2100-2105Crossref PubMed Scopus (27) Google Scholar GRP is a proinflammatory neuropeptide that functions as an inflammatory cell activator, mitogen, and cell differentiation factor.8Jensen R.T. Battey J.F. Spindel E.R. Benya R.V. International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states.Pharmacol Rev. 2008; 60: 1-42Crossref PubMed Scopus (418) Google Scholar, 10Zhou S. Potts E.N. Cuttitta F. Foster W.M. Sunday M.E. Gastrin-releasing peptide blockade as a broad-spectrum anti-inflammatory therapy for asthma.Proc Natl Acad Sci U S A. 2011; 108: 2100-2105Crossref PubMed Scopus (27) Google Scholar, 11Majumdar I.D. Weber H.C. Biology of mammalian bombesin-like peptides and their receptors.Curr Opin Endocrinol Diabetes Obes. 2010; 18: 68-74Crossref Scopus (43) Google Scholar GRP is expressed at the highest levels in PNEC in fetal lung,12Wharton J. Polak J.M. Bloom S.R. Ghatei M.A. Solcia E. Brown M.R. Pearse A.G.E. Bombesin-like immunoreactivity in the lung.Nature. 1978; 273: 769-770Crossref PubMed Scopus (329) Google Scholar where it can promote lung development.13Sunday M.E. Cutz E. Role of neuroendocrine cells in fetal and postnatal lung.in: Mendelson C.R. Humana Press, Totowa, NJ2000: 299-336Google Scholar After birth, GRP production normally decreases, but elevated levels are associated with many inflammatory lung conditions, including chronic lung disease of newborns (bronchopulmonary dysplasia).14Johnson D.E. Lock J.E. Elde R.P. Thompson T.R. Pulmonary neuroendocrine cells in hyaline membrane disease and bronchopulmonary dysplasia.Pediatr Res. 1982; 16: 446-454Crossref PubMed Scopus (119) Google Scholar, 15Meloni F. Ballabio P. Pistorio A. Todarello C. Montoli C. Berrayah L. Meloni C. Grassi C. Aguayo S.M. Urinary levels of bombesin-related peptides in a population sample from northern Italy: potential role in the pathogenesis of chronic obstructive pulmonary disease.Am J Med Sci. 1998; 315: 258-265Crossref PubMed Scopus (5) Google Scholar, 16Sunday M.E. Yoder B.A. Cuttitta F. Haley K.J. Emanuel R.L. Bombesin-like peptide mediates lung injury in a baboon model of bronchopulmonary dysplasia.J Clin Invest. 1998; 102: 584-594Crossref PubMed Scopus (64) Google Scholar, 17Cullen A. Van Marter L.J. Moore M. Parad R. Sunday M.E. Urine bombesin-like peptide elevation precedes clinical evidence of bronchopulmonary dysplasia.Am J Respir Crit Care Med. 2002; 165: 1093-1097Crossref PubMed Scopus (54) Google Scholar PNEC hyperplasia can be triggered by inflammation or exposure to oxygen or ozone10Zhou S. Potts E.N. Cuttitta F. Foster W.M. Sunday M.E. Gastrin-releasing peptide blockade as a broad-spectrum anti-inflammatory therapy for asthma.Proc Natl Acad Sci U S A. 2011; 108: 2100-2105Crossref PubMed Scopus (27) Google Scholar, 16Sunday M.E. Yoder B.A. Cuttitta F. Haley K.J. Emanuel R.L. Bombesin-like peptide mediates lung injury in a baboon model of bronchopulmonary dysplasia.J Clin Invest. 1998; 102: 584-594Crossref PubMed Scopus (64) Google Scholar, 18Sunday M.E. Shan L. Subramaniam M. Immunomodulatory functions of the diffuse neuroendocrine system: implications for bronchopulmonary dysplasia.Endocr Pathol. 2004; 15: 91-106Crossref PubMed Google Scholar and can take weeks to reach peak levels.19Sunday M.E. Willett C.G. Induction and spontaneous regression of intense pulmonary neuroendocrine cell differentiation in a model of preneoplastic lung injury.Cancer Res. 1992; 52: 2677s-2686sPubMed Google Scholar The present investigation tests the hypothesis that GRP contributes to radiation-induced pulmonary fibrosis in C57BL/6 mice. One hour post exposure to thoracic radiation (15 Gy), we treated mice i.p. with either PBS or GRP blockade by using small molecule 77427. We have quantified results of immunohistochemistry (IHC) by using image analysis with ImageJ version 1.46e (NIH, Bethesda, MD) to determine whether GRP contributes to radiation-induced inflammatory responses and/or fibrosis, specifically including assessment of active transforming growth factor (TGF)-β signaling. C57BL/6 mice at 8 weeks of age (Charles River Laboratories, Morrisville, NC) were selected for these experiments because they are susceptible to radiation fibrosis.20Johnston C.J. Williams J.P. Elder A. Hernady E. Finkelstein J.N. Inflammatory cell recruitment following thoracic irradiation.Exp Lung Res. 2004; 30: 369-382Crossref PubMed Scopus (75) Google Scholar, 21Adawi A. Zhang Y. Baggs R. Rubin P. Williams J. Finkelstein J. Phipps R.P. Blockade of CD40-CD40 ligand interactions protects against radiation-induced pulmonary inflammation and fibrosis.Clin Immunol Immunopathol. 1998; 89: 222-230Crossref PubMed Scopus (75) Google Scholar, 22Epperly M.W. Sikora C.A. DeFilippi S.J. Gretton J.E. Bar-Sagi D. Archer H. Carlos T. Guo H. Greenberger J.S. Pulmonary irradiation-induced expression of VCAM-I and ICAM-I is decreased by manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) gene therapy.Biol Blood Marrow Transplant. 2002; 8: 175-187Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Females were used because GRPR is X-linked and females have twice the level of GRPR gene expression as males.23Shriver S.P. Bourdeau H.A. Gubish C.T. Tirpak D.L. Davis A.L. Luketich J.D. Siegfried J.M. Sex-specific expression of gastrin-releasing peptide receptor: relationship to smoking history and risk of lung cancer.J Natl Cancer Inst. 2000; 92: 24-33Crossref PubMed Scopus (183) Google Scholar Radiation dose and uniformity of distribution were determined before initiating the study as described.24Dileto C.L. Travis E.L. Fibroblast radiosensitivity in vitro and lung fibrosis in vivo: comparison between a fibrosis-prone and fibrosis-resistant mouse strain.Radiat Res. 1996; 146: 61-67Crossref PubMed Scopus (74) Google Scholar In brief, X-ray dosimetry was performed using a calibrated ionization chamber,25Beddar A.S. Salehpour M. Briere T.M. Hamidian H. Gillin M.T. Preliminary evaluation of implantable MOSFET radiation dosimeters.Phys Med Biol. 2005; 50: 141-149Crossref PubMed Scopus (34) Google Scholar, 26Briere T.M. Lii J. Prado K. Gillin M.T. Sam Beddar A. Single-use MOSFET radiation dosimeters for the quality assurance of megavoltage photon beams.Phys Med Biol. 2006; 51: 1139-1144Crossref PubMed Scopus (12) Google Scholar with dose rate variation across the field being 2 and <20 μm by using ImageJ version 1.46e. The total area of tissue per photomicrograph was determined by thresholding all of the tissue, followed by binary conversion and measurement of the tissue area. For GRP and PGP9.5, all positive cells per slide were counted manually because they most often occur in clusters that are not recognized as separate cells by ImageJ. This was achieved by counting numbers of nuclei present in cells with immunopositive cytoplasm. The numbers of foci (clusters) and total number of PNECs were normalized for the total lung tissue area on the slide because many PNECs are present in alveolar ducts. The total lung area was determined on nonoverlapping photomicrographs of the whole section at ×2 magnification, which then underwent thresholding, binary conversion, and area measurement. SMA immunostaining (without any counterstain) was quantified in photomicrographs within 1 mm of the pleural surface (but excluding the pleura) by thresholding in the red visibility range (224 to 255), followed by binary conversion and measurement of area. These values were normalized for the total area of the alveolar tissue in each photomicrograph (excluding conducting airways and their associated blood vessels). Quantification of Trichrome staining was performed using blue collagen staining without any hematoxylin counterstain by thresholding in the blue visibility range (134 to 211), followed by binary conversion and area measurement, again normalized for total tissue in each photomicrograph. Groups were compared using the unpaired Student's t-test, with P < 0.05 defined as the level of significance. To assess possible roles for GRP in tissue injury responses after radiation, mice were given 500 nmol/L final concentration of 77427, twice weekly, beginning 1 hour after radiation. Lung tissues were harvested for inflation fixation and staining at 2, 4, 6, 8, 10, and 20 weeks later. Bright field microscopy and ImageJ screening demonstrated altered numbers of immunopositive cells as follows: macrophages (CD68) only at 4 weeks, neuroendocrine (NE) cells (GRP, PGP9.5) mainly at 6 weeks, activated TGF-β signaling (pSmad2/3) only at 10 weeks, and myofibroblasts (SMA) at 20 weeks (Figure 1, Figure 2, Figure 3, Figure 4). Trichrome staining was performed only at the 20-week time point.Figure 2GRP blockade reduces peak numbers of PNECs at 6 weeks after radiation. A: Sham plus PBS controls have rare GRP+ cells (black arrow) most often at branch points in the airways (GRP immunostaining). B: Six weeks after radiation, there are numerous GRP+ cells in distal bronchiolar epithelium (many indicated by arrows). Most GRP+ cells are also PGP9.5+ (D–F), consistent with a neuroendocrine phenotype (GRP immunostaining). Many of these cells in irradiated mice given PBS are also CC10+ (unpublished data), consistent with a multipotent epithelial phenotype emerging after radiation. C: Mice exposed to radiation and then injected with 77427 twice a week have fewer GRP+ cells in bronchiolar epithelium at 6 weeks (GRP immunostaining). D: Sham plus PBS controls have rare PGP9.5+ cells (none in this field) despite strong positive control staining for PGP9.5 in nerve fibers (white arrows in right lower corner) (PGP9.5 immunostaining). E: Six weeks after radiation, there are numerous PGP9.5+ cells in distal bronchiolar epithelium (arrows) (PGP9.5 immunostaining). PGP9.5+ cells are also positive for GRP (A–C) and CC10 (not shown), consistent with a multipotent epithelial phenotype after radiation. F: Mice exposed to radiation then injected with 77427 twice a week have fewer PGP9.5+ cells in bronchiolar epithelium at 6 weeks (PGP9.5 immunostaining). G: Quantitative image analysis shows a large increase in GRP+ and PGP9.5+ cells in lungs of mice exposed 6 weeks earlier to radiation, which is significantly decreased by treatment with 77427. Compared with sham controls, ∗P < 0.001 and ‡P < 0.01 for either #GRP+ foci per cm2 or #PGP9.5+ cells/cm2 in radiation-treated lung tissue area. Compared with radiation-treated mice; †P < 0.01 for either §P < 0.04 (PGP9.5) for mice exposed to radiation, then further injected with 77427 twice a week. Scale bars: 25 μm (A–F). L, airway lumen; V, blood vessel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3GRP blockade diminishes pSmad2/3+ cells in mouse lung at 10 weeks after radiation. A: Sham plus PBS group. B: Radiation plus PBS group. C: Radiation plus 77427 group. D: Image analysis. A–C: Nuclear pSmad2/3 immunostaining (black arrows) was significantly elevated at 10 weeks after radiation (compared with negative nuclei counterstained with methyl green, white arrows). Sham controls had relatively few pSmad2/3+ cells; then the relative numbers of these cells increased more than fourfold after radiation. ∗P < 0.001 for radiation plus PBS versus sham plus PBS (fourfold increase) or radiation plus 77427; †P < 0.01 for radiation plus 77427 was only 30% elevated over sham plus 77427; ‡P < 0.001 for sham controls given only 77427 had a doubling of pSmad2/3+ cells versus the sham plus PBS group. Scale bars: 25 μm (A–C). V, blood vessel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Increased SMA-immunostaining and collagen deposition 20 weeks after radiation is abrogated by GRP blockade. A: Normal SMA immunostaining (red) in sham plus PBS lung occurs in airway and vascular smooth muscle. In subpleural lung (photomicrographs for ImageJ), a few small blood vessels are present. B: Mice exposed to radiation plus PBS have widespread, patchy SMA+ cells throughout the alveolar interstitium (white arrows). C: In radiation plus 77427 mice, SMA immunostaining is mostly restricted to normal small blood vessels. D: Normal trichrome staining (blue to purplish blue) in sham plus PBS lung occurs around airways and blood vessels. In the subpleural region shown here and used for ImageJ analysis, blue outlines of small blood vessels are visible (black arrows). E: Mice exposed to radiation plus PBS have collagen deposition in the alveolar interstitium (some blue to purplish blue interstitium, white arrows), as well as blue outlines of small blood vessels (black arrows). F: In radiation plus 77427 mice, collagen is mostly restricted to small blood vessels (black arrows). G: Quantitative image analysis was performed as detailed in Materials and Methods. For both SMA and trichrome, ∗P < 0.0001 for radiation plus PBS versus sham plus PBS; †P < 0.001 for radiation plus PBS versus radiation plus 77427. Asterisks in A and C indicate pleural surface. Scale bars: 25 μm (A–F). V, blood vessel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Increased numbers of alveolar macrophages have been reported to occur in mouse lung between 1 and 4 months after radiation as part of an inflammatory phase.29Zhang H. Han G. Liu H. Chen J. Ji X. Zhou F. Zhou Y. Xie C. The development of classically and alternatively activated macrophages has different effects on the varied stages of radiation-induced pulmonary injury in mice.J Radiat Res (Tokyo). 2011; 52: 717-726Crossref PubMed Scopus (40) Google Scholar, 30Chiang C.S. Liu W.C. Jung S.M. Chen F.H. Wu C.R. McBride W.H. Lee C.C. Hong J.H. Compartmental responses after thoracic irradiation of mice: strain differences.Int J Radiat Oncol Biol Phys. 2005; 62: 862-871Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar In sham controls, we demonstrated scattered CD68+ cells both in alveolar spaces and in the distal lung interstitium (Figure 1A). At 4 weeks after radiation, CD68+ cells were increased, almost exclusively localized to the alveolar interstitium (Figure 1B). GRP blockade by 77427 reduced this macrophage accumulation in the pulmonary interstitium (Figure 1C). By quantitative image analysis using ImageJ, the CD68+ cells in radiation plus PBS mice were nearly threefold increased compared with sham plus PBS mice (Figure 1D). We did not observe any significant differences in numbers of macrophages at the other time points. Rare GRP+ and/or PGP9.5+ cells were detected in sham controls (Figure 2, A and D), typically in bronchiolar epithelium near branch points, which is a niche for epithelial progenitor or stem cells in lung.31Reynolds S.D. Giangreco A. Power J.H. Stripp B.R. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar At 4 weeks after radiation, three of the eight mice had increased numbers of GRP+ and PGP9.5+ PNECs (data not shown). By 6 weeks after radiation, all animals had elevated numbers of GRP+ (Figure 2B) and PGP9.5+ (Figure 2E) cells, increased up to 20-fold per square centimeter of lung (Figure 2G). This PNEC hyperplasia was linear, manifested primarily as ≥10-fold increase of small GRP+ foci with a mean of two cells per focus. There were significantly fewer PNECs when mice were treated with 77427 (Figure 2, C, F, and G). Both the GRP+ and PGP9.5+ cells were localized almost entirely to small bronchioles (Figure 2, B and E) and yielded similar results of quantitative image analysis (Figure 2G). There was no difference in PCNA labeling of these cells. Interestingly, many GRP+ cells also co-stained for CC10, a Clara cell–specific marker (not shown), indicating that these may fall into the category of regenerative multipotent cells after injury.31Reynolds S.D. Giangreco A. Power J.H. Stripp B.R. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration.Am J Pathol. 2000; 156: 269-278Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar There was no apparent difference in total numbers of CC10+ cells at any time point (data not shown). Phospho-Smad2/3 (pSmad2/3) is a recognized marker of active TGF-β signaling, as occurs in bleomycin-induced pulmonary fibrosis,32Higashiyama H. Yoshimoto D. Okamoto Y. Kikkawa H. Asano S. Kinoshita M. Receptor-activated Smad localisation in bleomycin-induced pulmonary fibrosis.J Clin Pathol. 2007; 60: 283-289Crossref PubMed Scopus (29) Google Scholar carbon tetrachloride–induced hepatic fibrosis, and glomerular fibrosis.33Flanders K.C. Smad3 as a mediator of the fibrotic response.Int J Exp Pathol. 2004; 85: 47-64Crossref PubMed Scopus (523) Google Scholar, 34Kaimori A. Potter J. Kaimori J.Y. Wang C. Mezey E. Koteish A. Transformi
Referência(s)