Circulating Vascular Progenitor Cells Do Not Contribute to Compensatory Lung Growth

Circulating Vascular Progenitor Cells Do Not Contribute to Compensatory Lung Growth Robert Voswinckel, Tibor Ziegelhoeffer, Matthias Heil, Sawa Kostin, Georg Breier, Tanja Mehling, Rainer Haberberger, Matthias Clauss, Andreas Gaumann, Wolfgang Schaper, Werner Seeger Abstract—The biological principles that underlie the induction and process of alveolization in the lung as well as the maintenance of the complex lung tissue structure are one of the major obstacles in pulmonary medicine today. Bone marrow– derived cells have been shown to participate in angiogenesis, vascular repair, and remodeling of various organs. We addressed this phenomenon in the lung vasculature of mice in a model of regenerative lung growth. C57BL/6 mice were transplanted with bone marrow from one of three different reporter gene–transgenic strains. flk-1⫹/lacZ mice, tie-2/lacZ transgenic mice (both exhibiting endothelial cell–specific reporter gene expression), and ubiquitously enhanced green fluorescent protein (eGFP)-expressing mice served as marrow donors. After hematopoietic recovery, compensatory lung growth was induced by unilateral pneumonectomy and led to complete restoration of initial lung volume and surface area. The lungs were consecutively investigated for bone marrow– derived vascular cells by lacZ staining and immunohistochemistry for phenotype identification of vascular cells. lacZ- or eGFP-expressing bone marrow– derived endothelial cells could not be found in microvascular regions of alveolar septa. Single eGFP-positive endothelial cells were detected in pulmonary arteries at very low frequencies, whereas no eGFP-positive vascular smooth muscle cells were observed. In conclusion, we demonstrate in a model of lung growth and alveolization in adult mice the absence of significant bone marrow– derived progenitor cell contribution to the concomitant vascular growth and remodeling processes. (Circ Res. 2003;93:372-379.) Key Words: stem cells 䡲 plasticity 䡲 vascular endothelium 䡲 vascular smooth muscle 䡲 alveolization

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any recent reports present evidence for unexpected plasticity of bone marrow– derived stem or progenitor cells.1–11 The transplantation of whole bone marrow or enriched hematopoietic or mesenchymal stem cells was shown to lead to the integration of progeny into blood vessels and to their differentiation into endothelial cells and perivascular cells in different organs under physiological or pathological conditions.3,12–14 Bone marrow– derived endothelial cells were found to contribute to vascular growth and repair in adult mammals, a phenomenon that was termed postnatal vasculogenesis.12,15 Significant integration of bone marrow– derived vascular cells was shown in animal models of cardiac infarction16 and stroke.17,18 Furthermore, endothelial cells from circulating progenitors have been shown to significantly contribute to tumor angiogenesis.19 The derivation of endothelial cells and hematopoietic cells from one proximal stem cell, the so-called hemangioblast, has been observed in embryonic tissues20 and recently at sites of vascular growth in adult animals.21 The integration of bone marrow– derived vascular cells into growing or aging blood vessels is not only of biological interest per se but would give rise to new therapeutic concepts of vascular diseases.

Loss of functional lung tissue attributable to a variety of lung diseases is already one of the main causes of mortality in industrial societies.22,23 Chronic obstructive pulmonary disease and emphysema are now the twelfth leading cause of disability and will become the third leading cause of death in industrial countries in 2025. It is therefore of utmost importance to gain better insight into the mechanisms and regulation of maintenance, repair, and regeneration of the pulmonary gas exchange surface. The mechanisms that control the onset, rate, and cessation of alveolus formation as well as the confounding epithelial and pulmonary vascular growth and differentiation are minimally understood.24 Pulmonary angiogenesis and endothelial survival are critical for alveolus formation and homeostasis.25,26 Compensatory lung growth is an ideal model to investigate processes and programs of alveolization in adult mammals.27–31 The contribution of bone marrow– derived cells to growth, repair, and maintenance of the lung vascular system is unknown. In this study, we specifically addressed this question for the first time by using animal models of bone marrow transplantation with marrow derived from one of three different transgenic donors that

Original received March 17, 2003; revision received July 10, 2003; accepted July 10, 2003. From the Department of Internal Medicine (R.V., T.M., W.S.), University Clinic Giessen, Giessen, Germany; Departments of Molecular and Cellular Biology & Experimental Cardiology (R.V., T.Z., M.H., S.K., G.B., M.C., A.G., W.S.), Max-Planck-Institute for Physiological and Clinical Research, Bad Nauheim, Germany; and Department of Anatomy and Cellular Biology (R.H.), University of Giessen, Giessen, Germany. Correspondence to Robert Voswinckel, MD, Medical Clinic 2, University Clinic Giessen, Klinikstrasse 36, 35392 Giessen, Germany. E-mail [email protected] © 2003 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org

DOI: 10.1161/01.RES.0000087643.60150.C2

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express reporter genes under endothelial cell–specific or ubiquitously active promoters to investigate whether pulmonary endothelial and perivascular cells derive from circulating progenitors. C57BL/6 wild-type mice were transplanted with bone marrow of mice that express the lacZ reporter gene under the endothelial cell–specific flk-132 (vascular endothelial growth factor receptor 2) or tie-233 (angiopoietin receptor) promoters. Bone marrow from ubiquitary enhanced green fluorescent protein (eGFP)-expressing mice34 was used as a control to identify all bone marrow– derived cells after bone marrow transplantation into wild-type mice. The transplanted cells and their progeny could later be detected in the lung tissue because of their reporter gene expression. In wild-type mice, which were bone marrow–transplanted with the endothelial cell–specific lacZ reporter gene– expressing cells, only bone marrow– derived differentiated endothelial cells will express lacZ, whereas transplantation of the eGFP-transgenic marrow allows for the detection of progeny by GFP expression in all tissues independently of the cellular differentiation.

Materials and Methods Bone Marrow Transplantation and Transgenic Mice C57BL/6J wild-type mice 12 to 16 weeks of age were lethally irradiated with 11.0 Gy, and 2 to 5⫻106 transgenic bone marrow cells were transplanted. Bone marrow was harvested by flushing tibias and femurs of 8- to 12-week old mice with RPMI 1640 containing 1% FCS, 100 U/mL penicillin, and 1000 U/mL streptomycin. The first group of transgenic donor mice was C57BL/6TgN(ACTbEGFP)1Osb (Jackson Laboratory, Bar Harbor, Maine). These mice ubiquitously express eGFP, which leads to green fluorescence of all cells except erythrocytes and hair follicle cells.34 Success of bone marrow transplantation (BMT) was monitored by flow cytometry of peripheral blood (FACScan, Becton Dickinson). At 6 weeks after BMT, ⱖ80% of cells expressed eGFP, and after 6 months, ⱖ90% expressed eGFP. The second group of transgenic donor mice was heterozygous flk-1⫹/lacZ knock-in mice, which express the lacZ reporter gene from the endogenous flk-1-locus, resulting in reporter gene expression in all endothelial cells during embryogenesis and in the pulmonary vasculature during adulthood.35 The third group of transgenic donor mice was tie-2/lacZ mice expressing lacZ under control of the tie-2 promoter/intronic enhancer, which target reporter gene expression to virtually all endothelial cells during embryogenesis and adulthood.33

Animal Surgery Mice underwent left-sided pneumonectomy 6 to 8 weeks after BMT. The mice were anesthetized with an intraperitoneal injection of 60 mg/kg ketamine and 2 mg/kg xylazine, orally intubated, and mechanically ventilated with a mouse ventilator (Hugo Sachs Elektronik, March-Hugstetten). The left lung was carefully lifted through an incision in the 6th intercostal space, tied at the hilus, and resected. The animals recovered in a warmed cage with chow and water provided ad libitum. The animal handling and study protocol conformed with the guidelines for animal experiments of the University of Giessen and were approved by the local authorities for animal ethics and animal experiments.

Histological Analysis of Mice Transplanted With eGFP Transgenic Marrow The animals were euthanized with halothane. The lung was inflated with a pressure of 10 cm H2O and perfusion fixed with 1% buffered paraformaldehyde (Sigma-Aldrich) with a pressure of 25 cm H2O. The lungs were postfixed overnight in 1% PFA at 4°C, dehydrated over a graded series of alcohol, and paraffin embedded. Sections of 4 to 10 ␮m were cut on a microtome (Leica). Antigen retrieval was

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performed by incubation with trypsin solution (Digest All 2, Zymed) for 10 minutes at 37°C. Antibody staining was performed following standard procedures. All incubations and washes were done with PBS ⫹2.5% calf serum plus 0.1% Triton X-100. Unspecific binding sites were blocked over 30 minutes with PBS containing 2.5% calf serum and 10% goat serum (Sigma-Aldrich). Incubation times for primary and secondary antibodies were 60 and 30 minutes, respectively. The following were used as primary antibodies: CD31 (clone MEC13.3; dilution 1:100), CD34 (clone MEC14.7; 1:100; both Pharmingen), CD45 (1:50; Cymbus Biotechnologies), ␣-smooth muscle actin (1:400) and vimentin (1:200; both Sigma-Aldrich), von Willebrand factor (1:400; Dako), anti-␤-galactosidase (1:1000; Cortex), and anti-GFP (1:400; Abcam, Cambridge, UK). Tissue staining with the biotinylated griffonia simplicifolia isolectin B4 (5 ␮g/mL; Sigma-Aldrich) was performed according to Hellstrom et al.36 Anti–flk-1 antibody was provided by Dr S. Nishikawa (Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Japan). As secondary antibodies, goat anti–rat-Cy3, streptavidin-Cy3 (1:1000), goat anti–rabbit-FITC (1:100; all Pharmingen) or goat anti-rat-alexa488, and goat anti–rabbit-alexa555 (1:2000; Molecular Probes) were used. The sections were examined with a Leica TCS confocal microscope (Leica) using the 488-nm line of the Argon laser. Fluorescent signals from eGFP/FITC and Cy3 were viewed simultaneously in separate detector channels. True color overlays of single and serial sections were generated with the Leica confocal software.

Histological Analysis of Mice Transplanted With lacZ Transgenic Marrow OCT compound was instilled intratracheally, and the lungs were embedded in OCT and shock frozen. Cryosections were obtained on a cryostat (Leica), air dried, and fixed with 2% PFA for 20 minutes at room temperature. X-gal staining was done as described previously.33 Each staining was controlled by parallel staining of a lung section from a wild-type mouse (negative control) and the flk-1⫹/lacZ or tie-2/lacZ donor mouse (positive control). The slides were viewed with a Zeiss Axiovert scope (Zeiss).

Whole-Mount lacZ Staining Perfusion-fixed lungs were placed in X-gal staining solution at 30°C overnight.33 Afterward, the lungs were washed three times in PBS, dehydrated over a graded series of ethanol, and cleared in a 1:2 solution of benzyl alcohol and benzyl benzoate (Sigma) to translucence for macroscopic assessment under a stereomicroscope (Zeiss Stemi SV11).

Determination of Lung Volume Lungs were instillation-fixed with 4% paraformaldehyde plus 0.1% glutaraldehyde in PBS for 1 hour with a pressure of 20-cm water column. Lung volume was measured by fluid displacement according to the method of Scherle.37

Results Compensatory Lung Growth We partially characterized compensatory lung growth in C57BL/6 mice by quantification of lung volume and alveolar surface area after left-sided pneumonectomy. Volumes of right lungs 21 days after pneumonectomy were measured and compared with the total volumes of left and right lungs of nonoperated control mice. Lung volumes were normalized to individual body mass (mass-specific volume). Mass-specific volumes of right lungs 21 days after pneumonectomy (30.6⫾0.9 cm2/g) did not significantly differ from massspecific volumes of controls (29.7⫾0.9 cm2/g, Figure 1). Furthermore, mass-specific alveolar surface of both groups did not differ as well, which proves substantial alveolization

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Figure 1. Compensatory lung growth leads to rapid and total restoration of lung volume. Lungs were inflated and fixed with a pressure of 20-cm water column, and the lung volume was determined by fluid displacement. Mass specific total lung volumes of control mice (open bar) were not significantly different (NS) from mass-specific right lung volumes of mice 21 days after left-sided pneumonectomy (black bar, n⫽5 in each group). Data are shown as mean⫾SEM.

and septum formation (R. Voswinckel, H. Fehrenbach, unpublished data, 2003).

Transplantation With flk-1ⴙ/lacZ or tie-2/lacZ Transgenic Marrow and Consecutive Compensatory Lung Growth To investigate bone marrow– derived endothelial cells in pulmonary vessels after regenerative lung growth, wild-type mice were transplanted with bone marrow of either flk-1⫹/lacZ or tie-2/lacZ transgenic mice, which express the lacZ reporter gene specifically in endothelial cells (Figures 2b and 2d through 2f). Endothelial expression of ␤-gal in flk-1⫹/lacZ mice as well as flk-1 and tie-2 in adult postpneumonectomy lungs was shown by specific antibody staining (Figures 2a through 2d). After the reconstitution of the bone marrow, the mice were unilaterally pneumonectomized to evoke rapid growth of lung tissue and pulmonary vessels. Four weeks after the pneumonectomy, when compensatory lung growth was completed, histological analysis of the lungs failed to reveal integrated, lacZ-positive bone marrow– derived endothelial cells in the lungs of all animals of either the flk-1⫹/lacZ or the tie-2/lacZ transplanted group (Figures 2g and 2h). At least 10 50-␮m thick sections of each lung were examined without counterstain to ensure even the detection of single, weakly lacZ-positive cells (pictures not shown). lacZ expression of integrated endothelial cells could neither be found in alveolar septa nor in larger vessels. Whole-mount lacZ staining of the lungs of two flk-1⫹/lacZ and two tie-2/lacZ bone marrow– transplanted wild-type mice 21 days after pneumonectomy, performed to view the whole lungs for the identification of regional clusters of bone marrow– derived endothelial cells, confirmed the absence of lacZ-positive cells (Figure 3).

Figure 2. Endothelial cell–specific lacZ expression is detectable in pulmonary vessels of transgenic bone marrow donor mice but not in lungs of bone marrow–transplanted mice. Double staining of adult wild-type (a) and flk-1⫹/lacZ (b) lung for CD31 (green) and ␤-gal (red) shows constitutive endothelial ␤-gal expression in the flk-1⫹/lacZ lung (nuclear stain with Hoechst 33342). Immunohistochemistry of lungs 4 weeks after pneumonectomy shows flk-1 expression (brown staining) in alveolar endothelial and bronchial epithelial cells (c) as well as tie-2 expression in endothelial cells (d). After X-gal staining of 50-␮m sections for lacZ expression, adult flk-1⫹/lacZ mice present with predominant blue staining of pulmonary capillaries and, interestingly, of bronchial epithelium (e), whereas tie-2/lacZ transgenic mice show strong lacZ expression also in larger pulmonary vessels (f). In wild-type C57BL/6 mice bone marrow–transplanted with either flk-1⫹/lacZ (g) or tie-2/lacZ (h) transgenic bone marrow and consecutive induction of compensatory lung growth attributable to unilateral pneumonectomy, no lacZ-positive bone marrow– derived endothelial cells could be detected in the lungs (n⫽5 mice in each group; scale bar⫽50 ␮m).

To control successful bone marrow engraftment, polymerase chain reaction (PCR) for ␤-galactosidase was performed on the bone marrows of the transplanted animals and was positive in all cases (Figures 4a and 4b). Furthermore, lacZ staining of bone marrow showed lacZ-positive cells, demonstrating that flk-1– driven and tie-2– driven reporter gene expression was detectable in transplanted animals (Figures 4c and 4d).

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Figure 3. Absence of endothelial lacZ expression in lungs of mice after transplantation with flk-1⫹/lacZ or tie-2/lacZ bone marrow and unilateral pneumonectomy. To detect focal areas of bone marrow– derived endothelial cell engraftment, lungs of mice transplanted with either flk-1⫹/lacZ or tie-2/lacZ marrow were perfusion-fixed and whole mount–stained for lacZ expression and the tissue was cleared to translucence. A representative picture from an flk-1⫹/lacZ bone marrow–transplanted mouse is shown. Blue lacZ staining was absent in lung tissue viewed macroscopically and under a stereomicroscope as shown in panel a. The partially air-filled bronchial tree is visible through the cleared tissue (arrows). For comparison, whole-mount lacZ stained lung of an flk-1⫹/lacZ bone marrow donor mouse is shown in panel b, where high expression of lacZ (blue staining) can be found in bronchial epithelium (arrows) and lower levels of expression in endothelial cells delicately delineating the vessels (arrowheads). Scale bars⫽50 ␮m.

Transplantation With eGFP Transgenic Marrow Followed by Consecutive Compensatory Lung Growth To additionally substantiate the lacZ-transgenic marrow experiments, we used eGFP-transgenic marrow transplantation that allowed for the identification of all progeny irrespective of the cellular differentiation. The success of the transplantation with eGFP transgenic bone marrow was assessed by flow cytometric analysis for eGFP fluorescence of the mononuclear cells from peripheral blood. Six weeks after transplantation, ⬎80% of peripheral blood of the transplanted animal was GFP-positive (Figures 5a through 5d), a number that increased to ⬎90% after 6 months. Flow cytometric analysis for differentiation markers of lymphocytes, monocytes, and granulocytes revealed a normal hematopoietic system at 6 weeks. Single GFP-positive cardiomyocytes were detected at time of tissue sampling, possibly representing engraftment of bone marrow– derived cells in this tissue (Figures 5e and 5f). Paraffin sections of 4-␮m thickness were immunostained for endothelial antigens and examined for colocalization of GFP-positive bone marrow– derived cells and the endothelial markers by use of conventional fluorescence microscopy. Because of the utmost proximity of endothelial cells and intravascular leukocytes in alveolar septa, a definitive distinction of both cell types in these regions without accepting many false-positive events was in our hands not possible with this standard technique (Figure 6a). To gain maximal resolution, we investigated 10-␮m sections with a confocal laser scan microscope. For each antigen investigated, at least three sections of each lung were stained. Histological analysis of the eGFP-transplanted mice revealed abundant bone marrow– derived cells in alveolar regions as well as in peribronchial and perivascular regions. The shapes of the bone marrow– derived cells in alveolar regions varied from small rounded morphology to large, delicately elongated cells with thin protrusions, which

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stretched sometimes over more than one alveolar septum. Most of the alveolar macrophages were GFP-positive at this time point (10 to 12 weeks after bone marrow transplantation). Endothelial cell immunostaining with anti-CD31, antiCD34, anti–von Willebrand factor, and GSI-B4 lectin revealed absence of colocalization of these markers and GFPfluorescence in alveolar regions (Figures 6b through 6f). Rarely, single bone marrow– derived cells with either morphologically or immunologically determined endothelial phenotype were found to be integrated in the intimal layer of pulmonary arteries (⬇1 cell in every 10th section, Figures 7a through 7d). Virtually all GFP-positive cells in alveolar, peribronchial, and perivascular regions stained positive for the pan-leukocyte marker CD45. Even elongated cells in the alveolar septa, which could have been judged by morphology to be endothelial or epithelial cells, all expressed CD45 and were consequently considered leukocytes, most probably resembling dendritic cells (Figures 7e and 7f). Furthermore, groups of bone marrow– derived leukocytes were detected peribronchially at branching points of the airways, the location of the bronchus-associated lymphatic tissue. Staining for ␣-smooth muscle actin and vimentin, performed to detect putative differentiation of bone marrow– derived cells into smooth muscle cells or fibroblasts, revealed complete absence of GFP-positive smooth muscle or fibroblastic cells in all slides investigated (pictures not shown).

Figure 4. Positive PCR for genomic lacZ from bone marrow samples and lacZ expression in bone marrow cells of flk-1⫹/lacZ and tie-2/lacZ transplanted mice prove successful for marrow engraftment. To assess the success of bone marrow engraftment, PCR for the genomic lacZ transgene was performed on bone marrow samples of wild-type mice transplanted either with flk-1⫹/lacZ knock-in bone marrow (a) or tie-2/lacZ transgenic bone marrow (b) at the time of lung tissue sampling. lacZ PCR was strongly positive for all bone marrows, indicating substantial engraftment of transplanted cells. Positive (⫹) and negative (⫺) lacZ PCR controls are shown. As an additional internal control of bone marrow engraftment, some bone marrow cells stained positive for lacZ (arrows) in all transplanted mice. Representative sections of bone marrow from flk-1⫹/lacZ (c) and tie-2/lacZ (d) transplanted mice are shown. Scale bar⫽100 ␮m.

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Figure 5. Positive controls for successful eGFP-transgenic bone marrow transplantation. Representative flow cytometric data of peripheral blood cells derived from wild-type control mice (a) and wildtype mice 6 weeks after bone marrow transplantation with eGFP transgenic marrow (b through d). Top, Dot plots of forward scatter (FSC) and green eGFPfluorescence intensity (eGFP). Bottom, Respective histograms, where the transplanted mice present with at least 80% eGFP-positive cells in the circulation. Some eGFP-positive cardiomyocytes were detected in hearts of GFP bone marrow–transplanted mice after pneumonectomy (e and f). These may serve as internal positive controls of bone marrow cell engraftment into structural organ tissues. Confocal laser scan microscopy: panel e is a reconstruction of 20 sections through a 7-␮m slice; panel f is a single section. Scale bars⫽25 ␮m.

Discussion We investigated the contribution of bone marrow– derived precursor cells to pulmonary vascular growth and maintenance in adult mice. To induce substantial lung growth in adult animals, we applied a model of compensatory lung growth that follows left-sided pneumonectomy and leads to total restoration of lung volume, alveolar number, alveolar surface, and cellular number in small rodents.27,38 Compensatory lung growth in adult mice restored initial lung volume (Figure 1) and gas exchange area over 21 days and therefore represents an excellent model for the investigation of alveolization and pulmonary vascular growth. In this study, adult wild-type C57BL/6 mice underwent bone marrow transplantation with transgenic marrows containing endothelial cell– specific32,33 (flk-1⫹/lacZ or tie-2/lacZ) or ubiquitous reporter gene expression (cac/eGFP).34 After hematopoietic recovery, the left lung was resected. The right lungs were removed 3 weeks later and examined for bone marrow– derived vascular cells. In mice transplanted with flk-1⫹/lacZ or tie-2/lacZ transgenic bone marrow, lacZ expression was completely absent in large vessels as well as in pulmonary capillaries of the alveolar septa after pneumonectomy, which suggests that bone marrow– derived endothelial cells do not participate in compensatory lung growth. We can exclude false-negative results attributable to unsuccessful bone marrow transplanta-

tion, because PCR for the lacZ transgene was strongly positive in bone marrow samples of all transplanted animals at the time of tissue sampling (10 to 12 weeks after transplantation), indicating that a significant amount of donor cells engrafted. Furthermore, cells staining positive for lacZ were detected in bone marrow of the transplanted animals, which was not the case in controls, and served as an internal control of transplantation success. In addition, exactly the same protocol was applied to the eGFP-transgenic marrowtransplanted mice, where bone marrow engraftment and function could be monitored and quantified by FACS analysis for eGFP expression in peripheral blood mononuclear cells, which reproducibly showed efficient engraftment and hematopoietic reconstitution. Single cardiomyocytes in GFPtransplanted mice expressed the reporter gene and may resemble engraftment of progenitor cells into heart muscle. No mouse died after the lethal irradiation and consecutive lacZ-transgenic bone marrow transplantation, which indicates proper marrow engraftment. We showed, by X-gal staining of flk-1⫹/lacZ and tie-2/lacZ donor animal lungs and by specific flk-1 and tie-2 antibody staining of postpneumonectomy lungs, that these receptors are constitutively expressed in pulmonary vessels in adulthood and 4 weeks after pneumonectomy. The constitutive expression of flk-1 in adult lung capillaries, which is an exception from the rule of downregu-

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Figure 6. Phenotypic characterization of GFP-expressing bone marrow– derived cells in lungs of mice by staining for endothelial cell markers. Conventional fluorescence microscopy for coexpression of GFP (green fluorescence) and endothelial markers (red) derived colocalization of both colors for many cells in alveolar septa as shown for CD34 (a, 4-␮m paraffin section). These colocalization signals could not be confirmed using confocal laser scan microscopy for the readout of CD34 (b), CD31 (c and d), von Willebrand factor (e), and GSI-B4 lectin expression (f). All eGFP-expressing cells in alveolar septa were intravascular or perivascular cells that did not express endothelial markers. d and f, Reconstructions of 20 confocal sections through 7 ␮m to show more clearly the polymorphic shape or intravascular localization of these cells, respectively. Scale bar⫽25 ␮m.

lation of flk-1 expression in mature vessels, is specific for this organ and seems to be critical for pulmonary endothelial cell survival.25,26 This argues against the concern that the absence of lacZ-positive, bone marrow– derived endothelial cells could be attributable to downregulation of the flk-1– driven or tie-2– driven lacZ reporter gene. In addition, the same negative results were obtained by investigating mice that had been transplanted with eGFP transgenic bone marrow. In these animals, eGFP-expressing cells were abundantly present in alveolar, perivascular, and peribronchial regions. However, applying high-resolution confocal microscopy, we could confirm that virtually all GFP-expressing cells in alveolar septa were of leukocytic origin. The utmost proximity of intravascular leukocytes and capillary endothelial cells in our hands required confocal microscopy to exclude false-positive colocalization of GFP and immunofluorescent stain for endothelial markers. Very rarely, single bone marrow– derived endothelial cells were found to be integrated in the intimal layer of pulmonary arteries. Smooth muscle cells, pericytes, and fibroblasts, phenotyped by staining for ␣-smooth muscle actin and vimentin, in addition proved not to be bone marrow– derived in our model. We consider it highly unlikely that the absence of bone marrow– derived vascular cells, observed with all three donor strains, could be attributable to an immune response against the reporter gene expressing cells in the lung. After myeloablative irradiation and bone marrow transplantation, the newly formed hematopoietic system, and with it the immune system, would be completely donor derived. An immune reaction against bone marrow– derived structural cells of the lung would mean an autoimmune reaction. To our knowledge, no data exist so far concerning the contribution of bone marrow– derived cells to vascular

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growth or regeneration of the lung. The positive findings of other groups, which investigated vessel growth under pathological conditions in heart,16 liver,39 and brain,17 do not necessarily imply the same biologic phenomenon to be a cause of lung regeneration as well. Many of these investigations applied bone marrow or stem cell injections as a therapy for experimental tissue damage. Our experiments were not designed as a way of applying stem cells therapeutically at the time of an organ damage like ischemia or inflammation but rather to investigate the proportion of bone marrow contribution to a process of regulated regenerative growth without interfering with endogenous circulating stem cell concentrations. Recent studies suggested differences in stem cell recruitment attributable to different organ lesions.39 Based on these findings it is still possible that in varying models of lung disease, bone marrow– derived cells may play a significant role. In comparable models, bone marrow– derived cells were investigated in organ maintenance over 12 months10 and after a bleomycin-induced lung lesion.40 Both groups reported significant integration of progeny and differentiation into pulmonary epithelial type 2 or type 1 cells, respectively. The comparably high bone marrow– derived epithelial cell type 2 number in the lung was discussed to be attributable to the high pulmonary irradiation sensitivity that could in return provoke substantial epithelial regeneration. Additionally, the continuous growth of mice during their lifetime could contribute to the ongoing generation of lung tissue. Endothelial cells, on the other hand, are very sensitive to irradiation, and

Figure 7. Bone marrow– derived endothelial cells in pulmonary vessels after compensatory lung growth represent a very rare event. Rarely, bone marrow– derived endothelial cells (green fluorescence) were detected in the intimal layer of pulmonary arteries. Confocal microscopy images of a GFP-expressing cell stained for CD31 is shown in panel a (arrow). A rather small cell expressing GFP and binding GSI-B4 lectin is shown in panel b. Cells of endothelial cell appearance based on morphology (elongated cell and nuclear shape, direct contact to lamina elastica interna [arrowheads]) are shown in sections stained for ␣-smooth muscle actin (c) and lycopersicon esculentum lectin (d). Phenotypic characterization for the pan-leukocyte antigen CD45 (e and f) showed that virtually all bone marrow– derived cells in the lung represented hematopoietic cells despite their sometimes very intriguing cellular shapes that might have suggested structural parenchymal cell types. c, e, and f, Reconstructions of 20 confocal sections through 7-␮m tissue depth. Scale bar⫽25 ␮m.

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endothelial damage contributes to pneumonitis after irradiation of the lung.41,42 We therefore assumed that endothelial repair after irradiation as well as the vascular growth concomitant to the compensatory lung growth would be a stimulus for the integration of endothelial precursor cells. The fact that we could not show this phenomenon in our experiments may be attributable to the comparably shorter time course of our experiments, where the tissues were investigated 10 to 12 weeks after bone marrow transplantation. However, the repair and regenerative growth of the lungs are completed by then, and it is unlikely that a substantial contribution of donor cells to the endothelial or perivascular cell pool will be found later on. Recent investigations by Wagers et al43 questioned earlier results of abundant stem cell contribution to lung tissue maintenance and the integration of bone marrow– derived pulmonary epithelium. Nevertheless, it may be of great but yet unknown importance which kinds of stem cells are used, if they went through purification or cell culture steps, or if they are of hematopoietic or mesenchymal phenotype. In conclusion, we investigated the contribution of bone marrow– derived cells to pulmonary angiogenesis and vascular remodeling by applying a model of lung growth and alveolization in adult mice. Using three different transgenic mice strains as bone marrow donors, we provide evidence for the failure of bone marrow– derived vascular precursor cells to significantly contribute to the generation of endothelial cells, pericytes, vascular smooth muscle cells, and fibroblasts in postpneumonectomy lung growth in the adult mouse, which implies that the proliferative capacity of endogenous cell compartments of the lung would be sufficient for this process.

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Acknowledgments This work was funded in part by the Max-Planck-Society. The authors thank Dr Urban Deutsch from the Max-Planck-Institute for Vascular Biology in Muenster for kindly providing the Tie-2/lacZ mice.

References 1. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science. 1999;284:1168 –1170. 2. Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, Mulligan RC. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature. 1999;401:390 –394. 3. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, Hammond WP. Evidence for circulating bone marrow-derived endothelial cells. Blood. 1998;92:362–367. 4. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science. 1998;279:1528 –1530. 5. Eglitis MA, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci U S A. 1997;94:4080 – 4085. 6. Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science. 2000;290:1779 –1782. 7. Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, NadalGinard B, Bodine DM, Leri A, Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001;98:10344 –10349. 8. Lagasse E, Connors H, Al Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M. Purified hematopoietic

21.

22.

23.

24.

25.

26.

27. 28.

29.

stem cells can differentiate into hepatocytes in vivo. Nat Med. 2000;6: 1229 –1234. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK, Goodell MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest. 2001;107:1395–1402. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell. 2001;105:369 –377. Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002;8:403– 409. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, Kearne M, Magner M, Isner JM. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999;85:221–228. Carmeliet P, Luttun A. The emerging role of the bone marrow-derived stem cells in (therapeutic) angiogenesis. Thromb Haemost. 2001;86: 289 –297. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest. 2002;109:337–346. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249 –257. Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Transplanted adult bone marrow cells repair myocardial infarcts in mice. Ann N Y Acad Sci. 2001;938:221–229. Zhang ZG, Zhang L, Jiang Q, Chopp M. Bone marrow-derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res. 2002;90:284 –288. Hess DC, Hill WD, Martin-Studdard A, Carroll J, Brailer J, Carothers J. Bone marrow as a source of endothelial cells and NeuN-expressing cells after stroke. Stroke. 2002;33:1362–1368. Lyden D, Hattori K, Dias S, Costa C, Blaikie P, Butros L, Chadburn A, Heissig B, Marks W, Witte L, Wu Y, Hicklin D, Zhu Z, Hackett NR, Crystal RG, Moore MA, Hajjar KA, Manova K, Benezra R, Rafii S. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med. 2001;7:1194 –1201. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998; 125:725–732. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med. 2002;8:607– 612. National Center for Health Statistics, Division of Data Services, Hyattsville, Md. Report of Final Mortality Statistics; 2002:1– 86. Accessible at: http://www.cdc.gov/nchs/releases/02facts/final2000.htm. Chung F, Barnes N, Allen M, Angus R, Corris P, Knox A, Miles J, Morice A, O’Reilly J, Richardson M. Assessing the burden of respiratory disease in the UK. Respir Med. 2002;96:963–975. Massaro G, Radaeva S, Clerch LB, Massaro D. Lung alveoli: endogenous programmed destruction and regeneration. Am J Physiol Lung Cell Mol Physiol. 2002;283:L305–L309. Jakkula M, le Cras TD, Gebb S, Hirth KP, Tuder RM, Voelkel NF, Abman SH. Inhibition of angiogenesis decreases alveolarization in the developing rat lung. Am J Physiol Lung Cell Mol Physiol. 2000;279: L600 –L607. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, Voelkel NF. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest. 2000;106: 1311–1319. Rannels DE, Rannels SR. Compensatory growth of the lung following partial pneumonectomy. Exp Lung Res. 1988;14:157–182. Yuan S, Hannam V, Belcastro R, Cartel N, Cabacungan J, Wang J, Diambomba Y, Johnstone L, Post M, Tanswell AK. A role for plateletderived growth factor-BB in rat postpneumonectomy compensatory lung growth. Pediatr Res. 2002;52:25–33. Kaza AK, Kron IL, Leuwerke SM, Tribble CG, Laubach VE. Keratinocyte growth factor enhances post-pneumonectomy lung growth by alveolar proliferation. Circulation. 2002;106(suppl I):I-120 –I-124.

Downloaded from http://circres.ahajournals.org/ by guest on August 4, 2015

Voswinckel et al

Vascular Stem Cells in Compensatory Lung Growth

30. Leuwerke SM, Kaza AK, Tribble CG, Kron IL, Laubach VE. Inhibition of compensatory lung growth in endothelial nitric oxide synthasedeficient mice. Am J Physiol Lung Cell Mol Physiol. 2002;282: L1272–L1278. 31. Brown LM, Rannels SR, Rannels DE. Implications of postpneumonectomy compensatory lung growth in pulmonary physiology and disease. Respir Res. 2001;2:340 –347. 32. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature. 1995;376:62– 66. 33. Schlaeger TM, Bartunkova S, Lawitts JA, Teichmann G, Risau W, Deutsch U, Sato TN. Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc Natl Acad Sci U S A. 1997;94:3058 –3063. 34. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. Green mice as a source of ubiquitous green cells. FEBS Lett. 1997;407:313–319. 35. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature. 1995;376:62– 66. 36. Hellstrom M, Kaln M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-␤ in recruitment of vascular smooth muscle cells

37. 38.

39. 40.

41.

42.

43.

379

and pericytes during embryonic blood vessel formation in the mouse. Development. 1999;126:3047–3055. Scherle W. A simple method for volumetry of organs in quantitative stereology. Mikroskopie. 1970;26:57– 60. Brown LM, Malkinson AM, Rannels DE, Rannels SR. Compensatory lung growth after partial pneumonectomy enhances lung tumorigenesis induced by 3-methylcholanthrene. Cancer Res. 1999;59:5089 –5092. Forbes S, Vig P, Poulsom R, Thomas H, Alison M. Hepatic stem cells. J Pathol. 2002;197:510 –518. Kotton DN, Ma BY, Cardoso WV, Sanderson EA, Summer RS, Williams MC, Fine A. Bone marrow-derived cells as progenitors of lung alveolar epithelium. Development. 2001;128:5181–5188. Tee PG, Travis EL. Basic fibroblast growth factor does not protect against classical radiation pneumonitis in two strains of mice. Cancer Res. 1995; 55:298 –302. Langley RE, Bump EA, Quartuccio SG, Medeiros D, Braunhut SJ. Radiation-induced apoptosis in microvascular endothelial cells. Br J Cancer. 1997;75:666 – 672. Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002;297:2256 –2259.

Downloaded from http://circres.ahajournals.org/ by guest on August 4, 2015

Circulating Vascular Progenitor Cells Do Not Contribute to Compensatory Lung Growth Robert Voswinckel, Tibor Ziegelhoeffer, Matthias Heil, Sawa Kostin, Georg Breier, Tanja Mehling, Rainer Haberberger, Matthias Clauss, Andreas Gaumann, Wolfgang Schaper and Werner Seeger Circ Res. 2003;93:372-379; originally published online July 24, 2003; doi: 10.1161/01.RES.0000087643.60150.C2 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2003 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

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