Intratracheal Cell Transfer Demonstrates the Profibrotic Potential of Resident Fibroblasts in Pulmonary Fibrosis
2015; Elsevier BV; Volume: 185; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2015.07.022
ISSN1525-2191
AutoresTatsuya Tsukui, Satoshi Ueha, Shigeyuki Shichino, Yutaka Inagaki, Kouji Matsushima,
Tópico(s)Medical Imaging and Pathology Studies
ResumoPulmonary fibrosis is a devastating disease for which there are few effective therapies. Activated fibroblasts form subepithelial clusters known as fibroblastic foci, which are characterized by excessive collagen deposition. The origin of activated fibroblasts is controversial and needs to be clarified to understand their pathogenicity. Here, using an intratracheal adoptive cell transfer method, we show that resident fibroblasts in alveolar walls have the highest profibrotic potential. By using collagen I(α)2-green fluorescent protein and neural/glial antigen 2-DsRed fluorescent reporter mice, we identified resident fibroblasts and pericytes in the alveolar walls based on surface marker expression and ultrastructural characteristics. In the early phase of bleomycin-induced pulmonary fibrosis, activated fibroblasts migrated into epithelium-denuded alveolar airspaces. Purified resident fibroblasts delivered into injured alveoli by an intratracheal route showed similar activated signatures as activated fibroblasts and formed fibroblastic foci. Neither pericytes nor epithelial cells had the same profibrotic potential. Transferred resident fibroblasts highly up-regulated profibrotic genes including α-smooth muscle actin and were a significant source of collagen deposition. These data provide insights into the cellular mechanisms of fibrogenesis and show intratracheal cell transfer to be a useful tool for exploring novel therapeutic targets against pulmonary fibrosis. Pulmonary fibrosis is a devastating disease for which there are few effective therapies. Activated fibroblasts form subepithelial clusters known as fibroblastic foci, which are characterized by excessive collagen deposition. The origin of activated fibroblasts is controversial and needs to be clarified to understand their pathogenicity. Here, using an intratracheal adoptive cell transfer method, we show that resident fibroblasts in alveolar walls have the highest profibrotic potential. By using collagen I(α)2-green fluorescent protein and neural/glial antigen 2-DsRed fluorescent reporter mice, we identified resident fibroblasts and pericytes in the alveolar walls based on surface marker expression and ultrastructural characteristics. In the early phase of bleomycin-induced pulmonary fibrosis, activated fibroblasts migrated into epithelium-denuded alveolar airspaces. Purified resident fibroblasts delivered into injured alveoli by an intratracheal route showed similar activated signatures as activated fibroblasts and formed fibroblastic foci. Neither pericytes nor epithelial cells had the same profibrotic potential. Transferred resident fibroblasts highly up-regulated profibrotic genes including α-smooth muscle actin and were a significant source of collagen deposition. These data provide insights into the cellular mechanisms of fibrogenesis and show intratracheal cell transfer to be a useful tool for exploring novel therapeutic targets against pulmonary fibrosis. Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease of unknown etiology that is characterized by architectural destruction of alveoli and the formation of fibroblastic foci.1Noble P.W. Barkauskas C.E. Jiang D. Pulmonary fibrosis: patterns and perpetrators.J Clin Invest. 2012; 122: 2756-2762Crossref PubMed Scopus (338) Google Scholar, 2Barkauskas C.E. Noble P.W. Cellular mechanisms of tissue fibrosis. 7. New insights into the cellular mechanisms of pulmonary fibrosis.Am J Physiol Cell Physiol. 2014; 306: C987-C996Crossref PubMed Scopus (108) Google Scholar, 3King Jr., T.E. Pardo A. Selman M. Idiopathic pulmonary fibrosis.Lancet. 2011; 378: 1949-1961Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar Activated fibroblasts, such as myofibroblasts that express α-smooth muscle actin (α-SMA), accumulate in fibroblastic foci and deposit excessive amounts of extracellular matrix components such as type 1 collagen, leading to loss of organ function. Characterization of the pathogenic effects of activated fibroblasts will help to clarify the underlying mechanisms in fibrotic diseases and support the development of specific treatment strategies against fibrosis. The origin of activated fibroblasts in fibrotic diseases remains controversial.4Kramann R. DiRocco D.P. Humphreys B.D. Understanding the origin, activation and regulation of matrix-producing myofibroblasts for treatment of fibrotic disease.J Pathol. 2013; 231: 273-289Crossref PubMed Scopus (164) Google Scholar Although previous reports have proposed a contribution of circulating fibrocytes or epithelial cells through epithelial-to-mesenchymal transition, recent studies using parabiosis or epithelial cell–specific Cre-reporter mice in a model of bleomycin-induced pulmonary fibrosis have challenged these views.5Rock J.R. Barkauskas C.E. Cronce M.J. Xue Y. Harris J.R. Liang J. Noble P.W. Hogan B.L.M. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition.Proc Natl Acad Sci U S A. 2011; 108: E1475-E1483Crossref PubMed Scopus (687) Google Scholar, 6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar A recent lineage tracing study showed a major contribution of Foxd1-Cre–labeled lung pericytes to the myofibroblast population.7Hung C. Linn G. Chow Y.H. Kobayashi A. Mittelsteadt K. Altemeier W.A. Gharib S.A. Schnapp L.M. Duffield J.S. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis.Am J Respir Crit Care Med. 2013; 188: 820-830Crossref PubMed Scopus (250) Google Scholar There is mounting evidence that perivascular cells including pericytes play significant roles in fibrosis in multiple organs.8Goritz C. Dias D.O. Tomilin N. Barbacid M. Shupliakov O. Frisen J. A pericyte origin of spinal cord scar tissue.Science. 2011; 333: 238-242Crossref PubMed Scopus (581) Google Scholar, 9Dulauroy S. Di Carlo S.E. Langa F. Eberl G. Peduto L. Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury.Nat Med. 2012; 18: 1262-1270Crossref PubMed Scopus (289) Google Scholar, 10Kramann R. Schneider R.K. DiRocco D.P. Machado F. Fleig S. Bondzie P.A. Henderson J.M. Ebert B.L. Humphreys B.D. Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis.Cell Stem Cell. 2015; 16: 51-66Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar However, it is not clear whether the cell populations described in these studies play equivalent roles in all organs and diseases. Moreover, there is a lack of studies that have performed rigorous lineage tracing of resident fibroblasts, which classically have been suggested to be major progenitors of myofibroblasts. In the present study, we performed lineage tracing of resident fibroblasts using intratracheal adoptive cell transfer. By this approach, we show that resident fibroblasts have the highest profibrotic potential compared with other lung cell populations. Transferred resident fibroblasts formed fibroblastic foci similar to those observed in IPF patients, suggesting a profibrotic role of resident fibroblasts in pulmonary fibrosis. Collagen I(α)2-green fluorescent protein (GFP) reporter mice (Col-GFP mice; C57BL/6 background) were generated in a previous study.11Higashiyama R. Moro T. Nakao S. Mikami K. Fukumitsu H. Ueda Y. Ikeda K. Adachi E. Bou–Gharios G. Okazaki I. Inagaki Y. Negligible contribution of bone marrow-derived cells to collagen production during hepatic fibrogenesis in mice.Gastroenterology. 2009; 137: 1459-1466.e1Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar Neural/Glial antigen 2 (NG2)-DsRed mice were purchased from the Jackson Laboratory (stock number 008241; Bar Harbor, ME). NG2-DsRed mice were crossed with either Col-GFP or C57BL/6 mice and the F1 offspring were used for experiments after genotyping. ROSA-CAG-SCAT3.1 mice were generated from BDF1-derived embryonic stem cells and back-crossed to C57BL/6 mice for at least two generations.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Tomura M. Mori Y.S. Watanabe R. Tanaka M. Miyawaki A. Kanagawa O. Time-lapse observation of cellular function with fluorescent probe reveals novel CTL-target cell interactions.Int Immunol. 2009; 21: 1145-1150Crossref PubMed Scopus (11) Google Scholar Host C57BL/6 mice used for intratracheal transfer experiments were purchased from CLEA Japan (Tokyo, Japan). Experiments were performed on 8- to 12-week-old mice. Mice were maintained in specific pathogen–free facilities at the University of Tokyo. All animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Tokyo. Intratracheal instillation for bleomycin (BLM) treatment or cell transfer experiments was performed as described previously.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Briefly, mice were anesthetized before instillation by oropharyngeal aspiration with 50 μL of either 2.0 to 2.5 mg/kg BLM sulfate (Toronto Research Chemical, Toronto, Canada) or saline as a negative control. For intratracheal transfer experiments, 50 μL of either cell suspension or phosphate-buffered saline (PBS) vehicle were instilled in the same way. For bromodeoxyuridine (BrdU) incorporation analysis, 0.8 mg/mL BrdU (Sigma-Aldrich, St. Louis, MO) was included in the drinking water of host mice commencing 1 day before intratracheal transfer and continuing until the day of analysis. Lung cell suspensions were prepared as described previously, with minor modifications.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Briefly, lungs were harvested after perfusion with 5 mL of PBS via the right ventricle. The lobes were minced and digested in protease solution [0.2% collagenase (Wako Pure Chemical Industries, Osaka, Japan), 0.1 mg/mL Dispase II (Roche, Basel, Switzerland), and 2000 U/mL DNase I (Merck, Darmstadt, Germany) in RPMI medium (Sigma-Aldrich) supplemented with 10 mmol/L HEPES (Nacalai Tesque, Kyoto, Japan)] for 60 minutes at 37°C with trituration by micropipette every 20 minutes. After digestion, cells were passed through a 70-μm strainer (BD Biosciences, San Jose, CA), washed with PBS, and suspended in RPMI medium with 0.5% bovine serum albumin (Nacalai Tesque). The antibodies used were as follows: anti-CD31 (clone 390; BV421, PerCP/Cy5.5, and biotin; BioLegend, San Diego, CA), anti-CD31 (AF3628; R&D Systems, Minneapolis, MN), anti-CD45 [clone 30-F11; allophycocyanin (APC)/Cy7 and biotin; BD Biosciences], anti-CD45.2 (clone 104; APC; BioLegend), anti–platelet-derived growth factor receptor (PDGFR)α (clone APA5; APC and biotin; BioLegend), anti-PDGFRβ (AF1042; R&D Systems), anti-PDGFRβ (clone C82A3; Cell Signaling Technology, Danvers, MA), anti-CD146 (clone ME-9F1; PerCP/Cy5.5, APC, and unlabeled; BioLegend), anti-epithelial cell adhesion molecule (EpCAM) [clone G8.8; phycoerythrin (PE)/Cy7, APC/Cy7, and biotin; BioLegend], anti-Ter119 (clone Ter119; APC/Cy7 and biotin; BD Biosciences), anti–α-SMA (clone 1A4; APC; R&D Systems), anti–collagen I (LSL-LB-1102; LSL, Tokyo, Japan), anti-collagen IV (LSL-LB-1403; LSL), anti-BrdU (clone Bu20a; APC; BioLegend), anti-BrdU (clone MoBu-1; Alexa 647; Life Technologies, Grand Island, NY), anti-APC (130-090-855; microbeads; Miltenyi Biotec, San Diego, CA), anti-goat IgG (A-21447; Alexa 647; Life Technologies), anti-rabbit IgG (A-31572 and A-31573; Alexa 555 and 647; Life Technologies), and anti-rat IgG (A-21247; Alexa 647; Life Technologies). Cell numbers in whole-lung cell suspensions were counted using Flow-Count Fluorospheres (Beckman Coulter, Brea, CA). Subsequently, cells were incubated with an anti-CD16/32 antibody to block nonspecific binding, then stained for lineage markers. For α-SMA staining, cells were fixed with Cytofix/Cytoperm buffer (BD Biosciences) for 20 minutes before staining with the anti–α-SMA antibody. BrdU incorporation was analyzed using a BrdU flow kit (BD Biosciences). Antibody-labeled cells were washed with PBS and analyzed using a Gallios flow cytometer (Beckman Coulter). Flow cytometry data were analyzed using FlowJo software version 7.6.5 (Tree Star, Ashland, OR). For enrichment of Col-GFP+ PDGFRα+ resident fibroblasts, whole-lung cell suspensions were stained for CD31, CD45, EpCAM, and Ter119, followed by negative selection using an AutoMACS cell separator (Miltenyi Biotec). Cells in negative fractions were checked for purity and cell number. After calculating the cell concentration, cells were washed and suspended in PBS for intratracheal transfer. For fluorescence-activated cell sorting purification of Col-GFP+, Col-GFP+ NG2-DsRed+, NG2-DsRed+, and EpCAM+ cells, whole-lung cell suspensions were divided into two fractions. One fraction was stained for CD31, CD45, CD146, and Ter119 before negative selection by AutoMACS, after which the negative fractions were stained for EpCAM and then sorted with a MoFlo Astrios flow cytometer (Beckman Coulter) for Col-GFP+ autofluorescence+ cells or EpCAM+ cells. The other fraction was stained for CD31, CD45, PDGFRα, EpCAM, and Ter119 before negative selection by AutoMACS, after which the negative fractions were sorted with the MoFlo Astrios flow cytometer for Col-GFP+ NG2-DsRed+ cells or NG2-DsRed+ cells. Sorted cells were checked for purity and cell number, and suspended in PBS ready for intratracheal transfer. Immunohistochemistry was performed as described previously.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Z-stack images were reconstructed in 3D by surface rendering using Velocity 3D image analysis software version 6.2.1 (PerkinElmer, Waltham, MA). Col-GFP+ cells (1 × 107) were transferred into host mice on day 7 after BLM treatment. At an additional 7 days after intratracheal transfer, whole lungs were harvested and whole-lung cell suspensions were stained for CD31, CD45, CD146, EpCAM, and Ter119 before negative selection by AutoMACS. Whole-lung cells also were prepared from untreated Col-GFP mice as a negative control. Col-GFP+ cells were sorted from the AutoMACS negative fraction using a MoFlo Astrios flow cytometer. After confirming cell purity, cells were lyzed in TRIzol reagent (Life Technologies) and total RNA was isolated according to the manufacturer's protocol. RNA was reverse-transcribed to cDNA using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed using Thunderbird SYBR qPCR Mix (Toyobo) with an ABI 7500 real-time PCR system (Life Technologies). The primers used were as follows: Rps3 forward 5′-CGGTGCAGATTTCCAAGAAG-3′ and reverse 5′-GGACTTCAACTCCAGAGTAGCC-3′; Col1a1 forward 5′-AGACATGTTCAGCTTTGTGGAC-3′ and reverse 5′-GCAGCTGACTTCAGGGATG-3′; Acta2 forward 5′-GTCCCAGACATCAGGGAGTAA-3′ and reverse 5′-TCGGATACTTCAGCGTCAGGA-3′; Spp1 forward 5′-GGAGGAAACCAGCCAAGG-3′ and reverse 5′-TGCCAGAATCAGTCACTTTCAC-3′; S100a4 forward 5′-GGAGCTGCCTAGCTTCCTG-3′ and reverse 5′-TCCTGGAAGTCAACTTCATTGTC-3′; Fn1 forward 5′-CGGAGAGAGTGCCCCTACTA-3′ and reverse 5′-CGATATTGGTGAATCGCAGA-3′; Tnc forward 5′-GGGCTATAGAACACCGATGC-3′ and reverse 5′-CATTTAAGTTTCCAATTTCAGGTTC-3′; Il1b forward 5′-AGGCAGGCAGTATCACTCATTGT-3′ and reverse 5′-CGTCACACACCAGCAGGTTATC-3′; Il5 forward 5′-AAGGATGCTTCTGCACTTGAGT-3′ and reverse 5′-TCTCCAATGCATAGCTGGTGAT-3′; Il13 forward 5′-CTTGCCTTGGTGGTCTCG-3′ and reverse 5′-CGTTGCACAGGGGAGTCT-3′; Il17a forward 5′-AAGCTCAGCGTGTCCAAACA-3′ and reverse 5′-GGCACTGAGCTTCCCAGATC-3′; and Tgfb1 forward 5′-CCCGAAGCGGACTACTATGC-3′ and reverse 5′-CCCGAATGTCTGACGTATTGAA-3′. Relative gene expression levels were calculated after normalization to expression of the reference gene Rps3. The migratory capacity of Col-GFP+ cells was evaluated using an Oris cell migration assay kit (fibronectin-coated) (Platypus Technologies, Madison, WI). After purifying Col-GFP+ cells, 1 × 105 cells in 100 μL of Dulbecco’s modified Eagle’s medium (DMEM) medium (Sigma-Aldrich) containing 2% fetal bovine serum (Sigma-Aldrich) were dispensed into each well of a 96-well plate containing a cell-seeding stopper. After a 24-hour incubation in a CO2 incubator, the stopper was removed and the wells were washed with PBS to remove nonadherent cells. The wells were refilled with 100 μL DMEM containing 2% fetal bovine serum; images of the Col-GFP+ cells were acquired using an SP-5 confocal microscope (Leica Microsystems, Wetzlar, Germany) after a further 0, 12, or 24 hours of incubation. The area of the cell-free zone on the center of each well was quantified using ImageJ version 1.47t (NIH, Bethesda, MD; http://imagej.nih.gov/ij). The migration index was calculated as a ratio relative to the cell-free area at time 0 hour. Invasion assays were performed using the Cultrex 96-well basement membrane extract cell invasion assay kit (Trevigen, Gaithersburg, MD). Purified Col-GFP+ cells on plastic dishes were incubated in DMEM containing 2% fetal bovine serum for 24 hours. After trypsinizing the cells, 5 × 104 cells in serum-free DMEM were dispensed into the upper chambers of the assay kit, which were coated with 0.5 × basement membrane extract. The lower chambers were filled with DMEM containing 2% fetal bovine serum. After a 36-hour incubation, all cells were fixed with 4% paraformaldehyde (Nacalai Tesque), and the cells on the upper side of the membrane were removed by wiping with a cotton swab. The cells on the lower side of the membrane were stained with Diff-Quik (Sysmex, Kobe, Japan) and counted. The invasion index was calculated by normalizing the cell number for treated Col-GFP+ cells to the average cell number for control Col-GFP+ cells. Hydroxyproline assays were performed as described previously.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Data are expressed as means ± SEM where applicable. Statistical comparisons were performed by unpaired t-tests (two-tailed) or by one-way analysis of variance with the Tukey-Kramer post-test for multiple groups. P < 0.05 was considered statistically significant. Statistical analysis was performed using Prism software version 5.01 (GraphPad Software, La Jolla, CA). The lungs are composed of numerous types of cells that contribute to the complex structure of the organ and enable efficient gas exchange.13Hogan B.L. Barkauskas C.E. Chapman H.A. Epstein J.A. Jain R. Hsia C.C. Niklason L. Calle E. Le A. Randell S.H. Rock J. Snitow M. Krummel M. Stripp B.R. Vu T. White E.S. Whitsett J.A. Morrisey E.E. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.Cell Stem Cell. 2014; 15: 123-138Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar A clear classification of stromal cell subsets in the healthy state is necessary to understand cellular origin in pathologic conditions. We previously showed that resident fibroblasts in the alveolar walls are labeled with GFP in Col-GFP mice.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar To further characterize the resident fibroblast population and to determine the overlap between the Col-GFP+ population and other stromal cell populations, we cross-bred Col-GFP mice with NG2-DsRed mice. NG2 is expressed on some, but not all, pericytes.7Hung C. Linn G. Chow Y.H. Kobayashi A. Mittelsteadt K. Altemeier W.A. Gharib S.A. Schnapp L.M. Duffield J.S. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis.Am J Respir Crit Care Med. 2013; 188: 820-830Crossref PubMed Scopus (250) Google Scholar, 14Song S. Ewald A.J. Stallcup W. Werb Z. Bergers G. PDGFRbeta+ perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival.Nat Cell Biol. 2005; 7: 870-879Crossref PubMed Scopus (460) Google Scholar Flow cytometric analysis of whole-lung cells from Col-GFP+/- NG2-DsRed+/- mice showed three distinct transgene-expressing populations in lineage marker–negative (ie, CD45- Ter119- EpCAM- CD31-) cells: Col-GFP+ (population R1), Col-GFP+ NG2-DsRed+ (population R2), and NG2-DsRed+ (population R3) (Figure 1A). It has been reported previously that PDGFRα is expressed on resident fibroblasts and that CD146 is expressed on smooth muscle cells or pericytes.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 7Hung C. Linn G. Chow Y.H. Kobayashi A. Mittelsteadt K. Altemeier W.A. Gharib S.A. Schnapp L.M. Duffield J.S. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis.Am J Respir Crit Care Med. 2013; 188: 820-830Crossref PubMed Scopus (250) Google Scholar, 15Middleton J. Americh L. Gayon R. Julien D. Mansat M. Mansat P. Anract P. Cantagrel A. Cattan P. Reimund J.M. Aguilar L. Amalric F. Girard J.P. A comparative study of endothelial cell markers expressed in chronically inflamed human tissues: MECA-79, Duffy antigen receptor for chemokines, von Willebrand factor, CD31, CD34, CD105 and CD146.J Pathol. 2005; 206: 260-268Crossref PubMed Scopus (103) Google Scholar The R1 population contained 86.7% ± 1.29% PDGFRα+ cells and 5.26% ± 0.37% CD146+ cells (Figure 1A), whereas the R2 and R3 populations were composed mostly of CD146+ cells (Figure 1A). Histologically, peribronchiolar and perivascular Col-GFP+ cells were CD146+ (Figure 1B). Considering that perivascular but not peribronchiolar smooth muscle cells had NG2-DsRed expression (Figure 1B), CD146+ cells in the R1 population are likely to represent peribronchiolar smooth muscle cells, whereas the CD146+ cells in the R2 population are likely to represent perivascular smooth muscle cells. In the alveolar area, we observed NG2-DsRed+ cells, which also expressed PDGFRβ (a marker of pericytes), but not all PDGFRβ+ cells expressed NG2-DsRed (Figure 1C). In addition, we occasionally detected complete overlap of NG2-DsRed signals with Col-GFP signals in the alveolar walls (8.5% ± 1.4% of NG2-DsRed+ cells) (Supplemental Figure S1A). Although some of these NG2-DsRed+ Col-GFP+ regions could represent the overlap of two distinct cells, it also is possible that some pericytes have a fibroblast-like phenotype, as reported previously.7Hung C. Linn G. Chow Y.H. Kobayashi A. Mittelsteadt K. Altemeier W.A. Gharib S.A. Schnapp L.M. Duffield J.S. Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis.Am J Respir Crit Care Med. 2013; 188: 820-830Crossref PubMed Scopus (250) Google Scholar Ultrastructural analysis of NG2-DsRed+ cells in the alveolar walls confirmed characteristics typical of pericytes, which were located in close proximity to CD31+ endothelial cells and were enveloped together by the endothelial basement membrane (Figure 1D and Supplemental Movie S1).16Armulik A. Genové G. Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.Dev Cell. 2011; 21: 193-215Abstract Full Text Full Text PDF PubMed Scopus (1714) Google Scholar Thus, the R3 population represented a proportion of the pericytes in the alveolar walls, whereas the R2 population represented a mix of perivascular smooth muscle cells and, to a lesser extent, Col-GFP+ DsRed+ cells in the alveolar walls. Col-GFP+ cells in alveolar walls expressed PDGFRα (Figure 1E), and Col-GFP+ PDGFRα+ cells showed autofluorescence when excited by a violet laser, possibly owing to their storage of lipid droplets (Supplemental Figure S1B).17Barkauskas C.E. Cronce M.J. Rackley C.R. Bowie E.J. Keene D.R. Stripp B.R. Randell S.H. Noble P.W. Hogan B.L. Type 2 alveolar cells are stem cells in adult lung.J Clin Invest. 2013; 123: 3025-3036Crossref PubMed Scopus (953) Google Scholar, 18Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. Iwaisako K. Moore-Morris T. Scott B. Tsukamoto H. Evans S.M. Dillmann W. Glass C.K. Brenner D.A. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.Proc Natl Acad Sci U S A. 2012; 109: 9448-9453Crossref PubMed Scopus (542) Google Scholar An endothelial basement membrane separated Col-GFP+ cells from the majority of the endothelium, a characteristic that distinguished these cells from PDGFRβ+ pericytes, which were convoluted within the endothelial basement membrane (Figure 1, F and G). These features of Col-GFP+ cells were consistent with the ultrastructural definition of resident fibroblasts in the alveolar walls.19Sirianni F.E. Chu F.S. Walker D.C. Human alveolar wall fibroblasts directly link epithelial type 2 cells to capillary endothelium.Am J Respir Crit Care Med. 2003; 168: 1532-1537Crossref PubMed Scopus (92) Google Scholar Col-GFP+ cells form clusters after BLM-induced injury, whereas they exist only sparsely in alveolar walls in the normal state.6Tsukui T. Ueha S. Abe J. Hashimoto S. Shichino S. Shimaoka T. Shand F.H. Arakawa Y. Oshima K. Hattori M. Inagaki Y. Tomura M. Matsushima K. Qualitative rather than quantitative changes are hallmarks of fibroblasts in bleomycin-induced pulmonary fibrosis.Am J Pathol. 2013; 183: 758-773Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Col-GFP+ cells in the alveolar walls were surrounded by a basement membrane and epithelial cells in the normal state (Figure 2A). In the early phase of BLM-induced injury, some Col-GFP+ cells migrated out of the interstitium into alveolar airspaces, with one end of the cell remaining attached to the denuded alveolar walls (Figure 2B). Consistent with this observation, Col-GFP+ cells isolated at day 7 after bleomycin treatment showed increased migratory capacity as assessed by a gap migration assay and increased invasive capacity (Supplemental Figure S2, A–C). The migration of fibroblasts into alveolar airspaces also has been described in electron microscopy studies of experimental lung fibrosis or IPF.20Fukuda Y. Ferrans V.J. Schoenberger C.I. Rennard S.I. Crystal R.G. Patterns of pulmonary structural remodeling after experimental paraquat toxicity. The morphogenesis of intraalveolar fibrosis.Am J Pathol. 1985; 118: 452-475PubMed Google Scholar, 21Fukuda Y. Basset F. Ferrans V.J. Yamanaka N. Significance of early intra-alveolar fibrotic lesions and integrin expression in lung biopsy specimens from patients with idiopathic pulmonary fibrosis.Hum Pathol. 1995; 26: 53-61Abstract Full Text PDF PubMed Scopus (74) Google Scholar In those studies, the investigators reported that fibroblasts with well-developed endoplasmic reticula are exposed to alveolar airspaces after epithelial injury, and that these fibroblasts attach to the luminal surface of epithelial basement membranes.20Fukuda Y. Ferrans V.J. Schoenberger C.I. Rennard S.I. Crystal R.G. Patterns of pulmonary structural remodeling after experimental paraquat toxicity. The morphogenesis of intraalveolar fibrosis.Am J Pathol. 1985; 118: 452-475PubMed Google Scholar Fibroblastic foci in IPF patients are considered to be formed by the exudation of fibroblasts into alveolar airspaces.22Noble P.W. Back to the future: historical perspective on the pathogenesis of idiopathic pulmonary fibrosis.Am J Respir Cell Mol Biol. 2005; 33: 113-120Crossref PubMed Scopus (76) Google Scholar To investigate whether fibroblasts in alveolar airspaces develop into fibroblast clusters in the interstitium, we isolated resident fibroblasts from the lungs of untreated Col-GFP mice and transferred this cell suspension into BLM-treated, wild-type mice via an intratracheal route (intratracheal transfer) (Figure 2C). The purity of Col-GFP+ PDGFRα+ cells was greater than 80% in each experiment (Supplemental Figure S2D). On day 21 after BLM treatment, we detected Col-GFP+ cells by both flow cyt
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