Pulmonary emphysema is a respiratory condition characterized by alveolar destruction that leads to airflow limitation and reduced lung function. Although with extensive research, the pathophysiology of emphysema is poorly understood and effective treatments are still missing. Evidence suggests that mesenchymal stem cells (MSCs) possess the ability to engraft the injured tissues and induce repair via a paracrine effect. Thus, the aim of this study was to test the effects of the intratracheal administration of lung-derived mouse MSCs in a model of elastase-induced emphysema. Pulmonary function (static lung compliance) showed an increased stiffness induced by elastase, while morphometric findings (mean linear intercept and tissue/alveolar area) confirmed the severity of alveolar disruption. Contrarily, MSC administration partially restored lung elasticity and alveolar architecture. In the absence of evidence that MSCs acquired epithelial phenotype, we detected an increased proliferative activity of aquaporin 5- and surfactant protein C-positive lung cells, suggesting MSC-driven paracrine mechanisms. The data indicate the mediation of hepatocyte growth factor in amplifying MSC-driven tissue response after injury. Our study shed light on supportive properties of lung-derived MSCs, although the full identification of mechanisms orchestrated by MSCs and responsible for epithelial repair after injury is a critical aspect yet to be achieved.
Chronic obstructive pulmonary disease (COPD) and pulmonary emphysema, together with asthma, are highly prevalent lung diseases worldwide [
A significant body of evidence has demonstrated that mesenchymal stem cells (MSCs), harvested from adult organs such as bone marrow and adipose tissue and administered in the damaged tissue, may induce organ repair mainly through paracrine effects [
Most studies have focused attention on bone marrow- and adipose tissue-derived MSCs to assess their potential on lung diseases, and the orientation of scientific community for these sources is essentially dictated by the readiness to obtain MSCs from these sites. On the other hand, little is known about the biological significance of lung-derived MSCs also because of obvious difficulties to obtain lung biopsies that have limited the studies on these cells. Nonetheless, lung MSCs may be relevant in alveolar homeostasis and repair after injury and may need consideration as a potential tool or target for cell-based therapy that involves other pulmonary cell populations. Therefore, the aim of our study was to test the effects of intratracheal administration of pulmonary MSCs into elastase-injured emphysematous lungs. In contrast to the majority of studies that utilized the systemic administration of cells, in our work, the intratracheal delivery was used. This route provides benefits over a systemic infusion, such as the reduction of cell number and the low risk to engraft other organs.
Six/eight lungs were harvested from 2-month-old male C57BL/6J mice (Charles River Laboratories) for each isolation of murine lung-derived MSCs. Samples were collected in 100 mm diameter culture dishes and were quickly washed with DPBS w/o Ca2+ and Mg2+ (Euroclone) to wash out the blood. Large vascular and bronchial components were removed as well. In order to obtain a cell suspension, the lungs were tinily minced and enzymatically dissociated with a prewarmed collagenase solution [280 U/ml type II collagenase (Worthington), 100 U/ml penicillin, and 100
FACS analysis was performed for MSC phenotype characterization, and 10,000 cells were detected for each surface marker. PE-conjugated antibodies for CD14, CD34, CD44, CD45, CD73, CD90, and CD105 were used (BD Biosciences). Isotype control was utilized to define the threshold for each specific signal. Data were acquired by FACSAria (BD Biosciences) and analyzed by FCS Express 6 (De Novo Software).
MSCs were transduced with a Cignal Lentivirus carrying GFP and puromycin resistance genes at a MOI of 50 (Qiagen). After 24 h, cells were washed and transduction medium was replaced by fresh medium. At this point, Cignal reporter constructs were integrated into the genomic DNA of target cells. To select the cells that stably expressed the GFP reporter gene, puromycin (5
The experimental protocol was approved by the Animal Care and Use Committee of the University of Campania “Luigi Vanvitelli” (294/2016-PR 24.03.2016). Animal care complied with Italian regulations on the protection of animals used for experimental and other scientific purposes (116/1992) as well as with the EU guidelines for the use of experimental animals (2010/63/EU). Mice were housed in the Animal Facility of the University of Campania “Luigi Vanvitelli.” Food and water were supplied
Emphysema was induced in 2-month-old female C57BL/6J mice by intratracheal administration of porcine pancreatic elastase (PPE; 80 U/kg in 100
Prior to cell administration, mice were anesthetized with ketamine (40 mg/kg, i.p.) and medetomidine hydrochloride (0.15 mg/kg, i.p.). A 20-gauge custom-made catheter was inserted into the trachea via the mouth and connected to a mouse ventilator (Harvard Apparatus). After checking the correct placement of the catheter, the ventilator was disconnected and the delivery of the necessary vehicle (PPE, MSCs or medium) was carried out by using a syringe with a fine needle. Then, mice were mechanically ventilated for 3 min and placed in a warm chamber until they recovered consciousness (5–15 min).
After animal sacrifice, the body cavity was opened, an incision was made in the trachea, and a 20-gauge catheter was inserted and secured with a suture. Static lung compliance was measured with a 5 cc syringe connected to the trachea via a catheter and to a water manometer via a three-way stopcock. To get the inflation curves, 0.2 cc of air was manually injected, up to 3.0 cc. The resultant pressure from each incremental injection was read from the manometer approximately 1 s after the injection. Deflation was read in the same fashion, manually withdrawing 0.2 cc at a time, until reaching the maximum volume of 3.0 cc. The curves of inflation and deflation were measured twice for each animal. Volume was traced as a function of pressure. Static lung compliance was obtained through the average slope of each deflation curve at its midpoint [
For histology, the lungs were perfused and fixed as previously described [
Morphometric assessment included the determination of the average interalveolar distance (mean linear intercept) and the calculation of tissue and airspace areas, corrected for the alveolar number. The mean linear intercept was measured by superimposing a grid over each image and counting the number of times the alveolar walls intercepted the grid lines. The equation
Injected MSCs were detected by chicken polyclonal anti-GFP antibody (1 : 500, overnight at 4°C) (Abcam). Rat monoclonal CD45 (1 : 30, overnight at 4°C) (Novus Biological) was used to exclude the hematopoietic lineage in MSCs. Lung cells were identified by immunostaining for aquaporin 5 (AQP5; rabbit polyclonal, 1 : 100, overnight at 4°C) (Abcam) and surfactant protein C (SFTPC; rabbit polyclonal, 1 : 100, overnight at 4°C) (Santa Cruz Biotechnology). Cycling cells were visualized using mouse monoclonal anti-BrdU antibody (1 : 10, 1 h at 37°C) (Roche Diagnostics). The expression of hepatocyte growth factor (HGF; rabbit polyclonal, 1 : 100, overnight at 4°C) (Abcam) and its receptor c-Met (mouse monoclonal, 1 : 100, overnight at 4°C) (Cell Signaling) in the lung was also detected. Nuclei were stained with DAPI (Sigma-Aldrich). Secondary antibodies conjugated with FITC, TRITC, or Cy5 were used at the dilution of 1 : 100 for 1 h at 37°C (Jackson ImmunoResearch). The quantification of newly formed cells was performed by counting at least 200 AEC1 or AEC2 (
Tissue samples were homogenized in lysis buffer containing protease inhibitors (Sigma-Aldrich). Protein concentration was determined by Bradford assay (Bio-Rad Laboratories). 20
Results were reported as the mean ± SD. Statistics were performed by using GraphPad Prism (GraphPad Software). Significance among multiple comparisons was determined by the one-way ANOVA corrected with the Bonferroni’s posttest. A value of
The phenotype of MSCs isolated from the lung of healthy mice was addressed using flow cytometry. Lung-derived MSCs displayed the surface expression of CD44, CD73, CD90, and CD105, consistent with the profile of cells of mesenchymal origin. MSCs were also found to partially express the progenitor marker CD34 and to completely lack hematopoietic cell markers CD14 and CD45 (Figure
MSC characterization and engraftment. (a) Immunophenotypic profile by flow cytometry of MSCs isolated from adult mouse lungs. Grey-shaded peaks show CD markers; red histograms represent isotype control. (b) Representative image of MSCs after lentiviral transduction of GFP (green). (c, d)
Histological analysis revealed evident airspace enlargement and obliteration of the alveolar wall in the lungs injected with elastase. These changes were attenuated by the instillation of MSCs (Figure
Lung histology and function. (a) Hematoxylin/eosin staining on lung tissue at day 31. (b) Morphometric analysis of the mean linear intercept. (c) Quantification of tissue and alveolar area per alveolus. (d) Functional measurements of static lung compliance. Data are expressed as the mean ± SD (
To answer the question of how the presence of MSCs could participate to structural and functional changes observed in cell-treated mice, the
Biological effects mediated by MSCs. (a, b) Representative images of GFP-positive MSCs (green) lacking alveolar epithelial commitment in emphysematous pulmonary parenchyma. Alveolar type I (a) and type II (b) epithelial cells express aquaporin 5 (AQP5; magenta, pseudocolor) and surfactant protein C (SFTPC, red), respectively. (c, d) Protein expression of AQP5 (c) and SFTPC (d) in the lung by Western blotting. (e, f) Proliferative activity (BrdU; white, pseudocolor) in the PPE-MSC group; alveolar type I (e) and type II (f) epithelial cells expressed AQP5 (magenta, pseudocolor) and SFTPC (red), respectively. Scattered GFP-positive MSCs (green) are also present. Nuclei are counterstained with DAPI (blue). (g, h) Quantification of newly formed alveolar type I (g) and type II (h) epithelial cells. Data are expressed as the mean ± SD (
In the search for mechanistic insights that may drive repair and regenerative processes, we examined the expression of several growth factors such as EGF, VEGF, and HGF. The presence of these factors in the normal lung indicates their role in tissue homeostasis in physiological conditions. While Western blotting analysis revealed a nonsignificant modulation of EGF and VEGF, HGF expression, lower in PPE mice, was significantly boosted after the administration of MSCs (Figures
Growth factor profile and MSCs. (a–c) Protein expression of epidermal growth factor (EGF) (a), vascular endothelial growth factor (VEGF) (b), and hepatocyte growth factor (HGF) (c) in the lung by Western blotting. (d) Negative control for HGF staining. (e) Representative images displaying HGF (red) in the proximity of GFP-positive MSCs (green). (f) Intracellular content of HGF (red) in a GFP-positive MSC (green). (g–i) c-Met expression (red) in the lung parenchyma from the naïve (g), PPE (h), and PPE-MSC (i) groups; alveolar type II epithelial cells are shown by SFTPC (green). Nuclei are counterstained with DAPI (blue). Data are expressed as the mean ± SD (
We report that intratracheal administration of lung-derived MSCs ameliorated alveolar damage induced by elastase. This effect may have been mediated by the release of HGF as MSC-dependent paracrine mechanisms. Activation of HGF/c-Met system, by promoting survival and proliferation of alveolar epithelial cells, may be a major determinant to trigger a reparative response in emphysema lung.
The elastase model “translates” major pathogenic mechanisms accounting for COPD: the protease-antiprotease imbalance, characterized by elevated production of proteases by inflammatory cells that determines the interruption of alveolar integrity [
Phenotypic identity and plasticity of MSCs may depend on the tissue of origin and even vary within the same tissue. MSCs from multiple sources have the recurrent presence of mesenchymal markers and the concomitant absence of hematopoietic and endothelial markers. A number of different determinants, such as CD34, Sca-1, or CD117, may be expressed to different extents [
MSC-related therapeutic potential in lung diseases incorporates two main mechanisms: immunomodulation and multilineage differentiation [
The mechanisms through which MSCs may modulate the function of other cells involved in tissue homeostasis remain largely unexplored. There is a consensus that factors such as VEGF, EGF, and HGF are involved in the protective and reparative effects of bone marrow and adipose MSCs [
We report previously unrecognized properties of adult mouse lung-derived MSCs that after local administration boost and orchestrate a local response to damage. Although several aspects of cellular physiology and
Konrad Urbanek and Bruno D’Agostino are co-senior authors.
The authors declare no conflict of interest.
Donato Cappetta, Antonella De Angelis, Konrad Urbanek, and Bruno D’Agostino contributed equally to this work.
This work was supported by PRIN 2015 no. 201532AHAE_004 from the Italian Ministry of Education, University and Research (MIUR) and Scientific Publications Fund of the Department of Experimental Medicine, University of Campania “Luigi Vanvitelli,” no. 5 14.06.16.