Bone marrow progenitor cells contribute to repair and remodeling of ...

The FASEB Journal • Research Communication

Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension Jeffrey L. Spees,*,‡,1 Mandolin J. Whitney,* Deborah E. Sullivan,† Joseph A. Lasky,† Miguel Laboy,† Joni Ylostalo,* and Darwin J. Prockop* *Department of Medicine, Center for Gene Therapy and †Section of Pulmonary Diseases, Critical Care, and Environmental Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana, USA; and ‡Department of Medicine, Cardiovascular Research Institute, University of Vermont, Colchester, Vermont, USA Infusion of bone marrow stem or progenitor cells may provide powerful therapies for injured tissues such as the lung and heart. We examined the potential of bone marrow-derived (BMD) progenitor cells to contribute to repair and remodeling of lung and heart in a rat monocrotaline (MCT) model of pulmonary hypertension. Bone marrow from green fluorescent protein (GFP)-transgenic male rats was transplanted into GFP-negative female rats. The chimeric animals were injected with MCT to produce pulmonary hypertension. Significant numbers of male GFP-positive BMD cells engrafted in the lungs of MCT-treated rats. Microarray analyses and double-immunohistochemistry demonstrated that many of the cells were interstitial fibroblasts or myofibroblasts, some of the cells were hematopoietic cells, and some were pulmonary epithelial cells (Clara cells), vascular endothelial cells, and smooth muscle cells. A few BMD cells fused with pulmonary cells from the host, but the frequency was low. In the hypertrophied hearts of MCT-treated rats, we found a significant increase in the relative numbers of BMD cells in the right ventricle wall as compared with the left ventricle. Some of the BMD cells in the right ventricle were vascular cells and cardiomyocytes. We report BMD cardiomyocytes with a normal chromosome number, fusion of BMD cells with host cardiomyocytes, and, in some cases, nuclear fusion.—Spees, J. L., Whitney, M. J., Sullivan, D. E., Lasky, J. A., Laboy, M., Ylostalo, J., Prockop, D. J. Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension. FASEB J. 22, 1226 –1236 (2008)

suggests that most adult tissues harbor stem-like progenitor cells that are recruited initially during the repair process. In addition, data from many laboratories indicate that bone marrow progenitors participate in the repair of most if not all major organ systems (2). In some cases, the bone marrow-derived (BMD) cells have been shown to actively replace differentiated cells and also the stem-like cells of tissues (3). Whereas, in other instances, tissue repair by BMD progenitor cells occurs by virtue of their ability to secrete a wide variety of cytokines and growth factors that modulate the injury microenvironment and improve healing (4 –7). In addition, some reports indicate that stem-like cells can repair tissues by cell fusion (8, 9) and perhaps by transfer of mitochondria (10). Here we tested the potential of BMD progenitor cells to contribute to the repair and remodeling that occurs in the lung and heart during chronic and progressive pulmonary hypertension produced by monocrotaline (MCT). Rats exposed to the alkaloid plant toxin MCT develop several pathological symptoms of pulmonary hypertension, including vascular remodeling, increased pulmonary arterial pressures, increased breathing frequency, and cardiac right-ventricular hypertrophy (11). Defining the role of BMD stem or progenitor cells in the pathogenesis of pulmonary hypertension could lead to more effective therapies to alleviate both primary and secondary pulmonary hypertension.

Key Words: stem cell 䡠 monocrotaline 䡠 cardiac hypertrophy

Chimeric female rats with GFP-positive bone marrow were produced as described in the Supplemental Material. MCT

ABSTRACT

One of the unsolved questions regarding tissue repair and remodeling following injury is whether the participating cells are endogenous to the injured tissue or whether they originate from the bone marrow, migrating via the bloodstream (1). Current evidence 1226

MATERIALS AND METHODS MCT model of pulmonary hypertension

1 Correspondence: Department of Medicine, Cardiovascular Research Institute, University of Vermont, 208 South Park Dr., Ste. 2, Colchester, VT 05446, USA. E-mail: [email protected] doi: 10.1096/fj.07-8076com

0892-6638/08/0022-1226 © FASEB

(crotaline; Sigma, St. Louis, MO, USA) was suspended in 0.1 N HCl, adjusted to pH 7.0 with NaOH, and diluted in PBS. After 3 wk to allow hematopoietic reconstitution, the chimeric rats were anesthetized with isoflurane and tail veininjected with 0.5 ml of PBS containing 75 mg/kg MCT. Control bone marrow transplant (BMT) animals received PBS alone. Following another 3 wk for the development of pulmonary hypertension (6 wk post-transplant), rats were sacrificed by overdose with ketamine/xylazine and cardiac perfusion with 200 ml of cold PBS. The left lung and the heart were tied off, excised, and frozen for real-time polymerase chain reaction (PCR) assays. To preserve lung architecture, the right lung was inflated by intratracheal infusion with 10% formalin for 15 min and was postfixed overnight at 4°C by submersion in 10% formalin. The hearts of some animals were cut such that the atria were removed, and the right ventricle and the left ventricle plus the septum were weighed. The separated ventricles were then frozen for real-time PCR. The ventricular walls of some animals were cut in cross section for immunohistochemistry and in situ hybridization. The marrow from the tibiae and femurs of control BMT and MCT-treated animals was isolated as above for DNA extraction. Real-time quantitative PCR Genomic DNA was isolated from hearts, lungs, and bone marrow by extraction with sodium dodecyl sulfate (SDS)/ proteinase K and phenol/chloroform/isoamylalcohol (12). The DNA served as the template for Taqman real-time PCR assays for the rat Y chromosome. For comparison of cardiac right and left ventricular engraftment, the right ventricle was dissected away from the left ventricle. Following DNA isolation, real-time PCR assays were run in triplicate on 300 ng of each DNA sample. Microarray assays Total RNA was extracted from GFP⫹ cells isolated by FACS from the bone marrow, the blood, and the digested lungs of

two MCT-treated chimeric rats (High Pure RNA Isolation Kit, Roche Diagnostics, Indianapolis, IN, USA). As described in the Supplemental Material, the RNA was assayed by microarrays (RAE2305 and RAE230 2.0, Affymetrix, Santa Clara, CA, USA), and the data were analyzed by Microarray Suite 5.0 (Affymetrix), the dChip 1.3 program, and Gene Ontology terms. Additional detailed methods for immunohistochemistry, fluorescent in situ hybridization (FISH), and lectin binding and AcLDL uptake by live cells are available in the Supplemental Material.

RESULTS The BMT/MCT model In preliminary experiments, we determined that 60 or 120 mg/kg of MCT produced a dose-responsive increase in breathing frequency as well as a dose-responsive increase in cardiac right-ventricular hypertrophy in 1 wk (Fig. 1A, B). Following BMT, we observed that the rats were more sensitive to MCT; therefore, we elected to use 75 mg/kg. After injection of 75 mg/kg MCT, most rats died in 4 –5 wk; hence, we elected to examine them at 3 wk following administration of the toxin. The chimeric rats that received BMT and were exposed to 75 mg/kg MCT developed pulmonary hypertension, as demonstrated by increased right ventricular weight (Fig. 1C) and pulmonary vascular medial thickening (Fig. 1D–G). At 6 wk after BMT and 3 wk after MCT, Y chromosome real-time PCR assays demonstrated that the bone marrow chimerism of the rats that were exposed to MCT (96.11%⫾8.00) was not significantly different from that of control BMT animals that did not receive MCT (99.44%⫾1.36, P⫽0.3584).

Figure 1. BMT and MCT model of pulmonary hypertension. Female SpragueDawley rats were lethally irradiated (11 Gy) and transplanted with 5 ⫻ 106 bone marrow mononuclear cells from male transgenic GFP rats. After 3 wk, chimeric rats received tail-vein injections of 75 mg/kg MCT in PBS. Animals were sacrificed 3 wk later. A) Dose-dependent increase in breathing frequency 1 wk following 60 or 120 mg/kg MCT treatment of normal (non-BMT) rats. B) Dose-dependent effect of MCT on cardiac right-ventricular weight (due to right-ventricular hypertrophy) after 1 wk for normal (non-BMT) rats. RV/LV⫹S ⫽ right ventricle wet weight divided by the wet weight of the left ventricle. C) Development of pulmonary hypertension in BMT and MCT-treated rats as demonstrated by right-ventricular hypertrophy. Control, normal female rats; IRR, BMT rats following 6 wk recovery; IRR/MCT, BMT- and MCT-treated rats 3 wk after MCT exposure. D, F) Pulmonary blood vessels (arrows) in BMT rat (⫻10 and ⫻40, respectively). Sections were stained with hematoxylin and eosin. E, G) Pulmonary blood vessels (arrows) in BMT, MCT treated rat (⫻10 and ⫻40, respectively). *P ⬍ 0.05; **P ⬍ 0.01. BONE MARROW PROGENITORS AND PULMONARY HYPERTENSION

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DNA and protein assays of the lungs By immunostaining for GFP, we observed a wide distribution of bone marrow-derived cells throughout the lung parenchyma of chimeric rats and chimeric rats treated with MCT (Fig. 2A). To determine the number of male BMD cells in the lungs of control and MCT-treated rats, we used a real-time PCR assay for the rat Y chromosome. About 15% of the cells isolated from the lungs of control rats were GFP⫹ BMD cells, a result probably explained by the lung injury produced by the X-ray irradiation used to make the rats chimeric. After 3 wk of MCT-induced pulmonary hypertension, chimeric animals exhibited a significant (P⫽0.043) increase in the numbers of male BMD cells in the lungs as compared to control BMT rats (Fig. 2B). In addition, we digested the lungs from MCT-treated rats with collagenase and dispase and determined the percentage of GFP cells that had engrafted the lungs by FACS. The results demonstrated that 40 –59% of the lung cells were BMD in three animals assayed (Fig. 2C).

By sorting twice we obtained 97–99% pure GFP⫹/BMD cells from the lung (Fig. 2D). The FACS isolates from digested MCT-treated lung tissues were then compared to the whole digest prior to FACS by Western blotting. The GFP⫹ FACS isolate contained cells that expressed the epithelial proteins surfactant protein A (SPA), clara cell secretory protein (CC10/CCSP), and K17 (Fig. 2D). Assays for cell fusion and nuclear fusion To examine whether the isolated BMD cells were fused with pulmonary GFP-negative (host) cells, we cultured the isolated GFP cells for 3 d and then did double FISH for the Y chromosome and chromosome 4. We observed 4 categories of cells (Fig. 2E and Supplemental Table 1): 1) one nucleus with one Y and two 4s (normal BMD cell); 2) binucleated cells with two Ys and four 4s (multinucleated BMD cell such as a macrophage); 3) binucleated cells with one Y and four 4s (heterokaryon, cell fusion of BMD cell with host cell); and 4) cells containing one nucleus with one Y and three 4s (synkaryon, nuclear fusion product

Figure 2. A) Presence of BMD derived cells in section of lung from MCTtreated rat. Section was stained with antibody to GFP (⫻10). B) Significant increase in pulmonary BMD cells following MCT treatment (*P⫽0.043). BMD cells were quantified by real-time PCR assays for the Y chromosome. C) FACS of GFP⫹ (BMD) cells from PBS-perfused and collagenase/dispase-digested lung tissues of 3 individual MCT-treated rats. D) By sorting twice, GFP⫹ (BMD) cells can be enriched to 97–99%. Immunoblot shows levels of surfactant protein A (SPA), clara cell secretory protein (CC10, CCSP), keratin 17 (K17), and GFP in isolated (ISO) GFP⫹ cells compared with whole lung cell digest (L). E) FISH for the rat Y chromosome and chromosome 4 to assay for cell fusion. GFP⫹ cells isolated as in D were cultured for 48 h and fixed for double FISH. Green dots indicate Y chromosome signals. Red dots indicate chromosome 4 signals. From left to right: typical BMD cell (one Y, two 4s); a binucleated BMD cell, possibly a macrophage (two Y, four 4s); a binucleated cell derived from fusion of a donor cell with a host cell (heterokaryon, one Y, four 4s); a nuclear fusion (synkaryon, one Y, three 4s). One chromosome 4 was lost following nuclear fusion. 1228

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with loss of one chromosome 4). By epifluorescence microscopy we examined cells from double FISH of 2 sorts and found 0.52– 0.59% fusion (Supplemental Table 1). Of the fused cells, 0.3– 0.5% had undergone cell fusion of a BMD cell with a host lung cell followed by nuclear fusion during mitosis. Microarray assays of expressed genes To further characterize the BMD cells that had engrafted the MCT-injured lung, we performed microar-

ray assays on FACS-isolated GFP⫹ cells from the bone marrow, the blood, and the digested lungs of two MCT-treated chimeric rats. By cluster analysis, the mRNA profile of GFP⫹ cells isolated from the lung clustered separately from the profiles of GFP cells from the bone marrow and the blood for both rats (Fig. 3A and Supplemental Fig. 1A). Using the dCHIP 1.3 analysis program, we statistically analyzed the major patterns from the heat maps that were organized by gene ontologies (GO terms) (Fig. 3B and Supplemental Fig. 1B). Patterns 3 (Fig. 3B) and 8 (Supplemental Fig. 1B) from the

Figure 3. Microarray analysis of GFP⫹ cells from the bone marrow (BM), the blood (B), and the digested lung of an MCT-treated rat (L). GFP⫹ cells were isolated by FACS (double-sorted) from all tissues 3 wk after administration of the toxin. A) Cluster analysis from the dChip 1.3 software program indicating that bone marrow and blood GFP⫹ cell transcripts cluster together, separately from those of GFP⫹ cells isolated from the lung. B) Heat map of transcription profiles and profile patterns chosen for further analysis. GO terms and statistical analysis are provided in the Supplemental Material. C) Selected transcripts from B, primarily belonging to transcription pattern 3. Abbreviations: CCSP, clara cell secretory protein; SPA, surfactant protein A; SPC, surfactant protein C; SPD, surfactant protein D; K18, keratin 18; K19, keratin 19; SMA, smooth muscle ␣-actin; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; iNOS, inducible nitric oxide synthase; SOD2, superoxide dismutase isoform 2; ApoE, apolipoprotein E; CD133, prominin; CD45, protein tyrosine phosphatase, receptor type C (leukocyte common antigen). Several transcripts (CCSP, SPA, SPC, SPD, K18, K19, CD133) indicate engraftment of BMD cells as lung progenitor and epithelial phenotype. SMA and vimentin are indicative of engraftment as vascular cells or mesenchymal fibroblasts or myofibroblasts. Other transcripts demonstrate expression of cell adhesion molecules (ICAM and VCAM) and mRNAs for proteins known to modulate the vasculature (iNOS, SOD2, ApoE). Of special importance, the expression pattern of CD45 (a pan-hematopoietic marker) is highest in GFP⫹ cells assayed from the blood, with the lowest expression levels in the GFP⫹ cells obtained isolated from the lung. BONE MARROW PROGENITORS AND PULMONARY HYPERTENSION

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heat maps were of special interest because they contained genes that were expressed at low levels or absent in bone marrow and blood but highly expressed in lung. The most significant GO terms of patterns 3 and 8 included “response to wounding” and “inflammatory response” (Pⱕ0.000001; Supplemental Material). In addition, the microarray data using the newer Affymetrix chip registered significant GO terms for “blood vessel morphogenesis” (P⫽0.0002) and “blood vessel development” (P⫽0.000391). Some of the genes expressed in patterns 3 and 8 confirmed the immunohistochemistry results from lung tissue. The gene expressed for procollagen 1A2 was increased in the cells isolated from lung, an observation consistent with a large number of the cells becoming interstitial fibroblasts or myofibroblasts in the lung. In patterns 3 and 8, we observed that the epithelial transcripts for Clara cell secretory protein (CCSP) and surfactant proteins A and C were present in the GFP⫹ cells isolated from the lung but were absent in GFP⫹ cells from bone marrow and blood (Fig. 3C and Supplemental Fig. 1C), indicating that some BMD cells had contributed to the stem/ progenitor cell pool of the lung. The gene for surfactant protein D was expressed in the GFP⫹ cells from the lung in both rats examined. Interestingly, in one rat the SPD gene was expressed in the blood-derived GFP⫹ sample; in the other rat it was absent in both the bone marrow and blood samples (Fig. 3C and Supplemental Fig. 1C). The signal intensities for K18 and K19, smooth muscle ␣-actin (SMA), and vimentin were all higher in the lung cells relative to cells of either the bone marrow or the blood (Fig. 3C and Supplemental Fig. 1C). Of interest, in both samples examined, the GFP⫹ cells in the circulation expressed SMA, indicating that smooth muscle or myofibroblast progenitors may already express the mRNA for this differentiation marker prior to arriving in the lung. We also observed increased signal intensities for mRNAs of cellular adhesion proteins (VCAM, ICAM) in the lung cells as well as proteins known to modulate the vasculature (iNOS, SOD2, and ApoE) (Fig. 3C and Supplemental Fig. 1C). Notably, mRNAs for the pan-hematopoietic marker CD45 were expressed at much lower levels in GFP⫹ cells from the lung relative to those from blood, indicating that many of the BMD cells engrafting the lung were nonhematopoietic cells (Fig. 3C and Supplemental Fig. 1C). Assays for endothelial progenitor cells in cultured pulmonary cells In further experiments, we asked whether some of the BMD cells had engrafted in the MCT-injured lung as endothelial progenitor cells. Staining of 72 h live cell cultures derived from the digested lungs of MCT-treated rats demonstrated the specific uptake of Ulex europaeus lectin and AcLDL by many of the GFP⫹ cells, indicating an endothelial progenitor- or monocyte-like phenotype (Supplemental Fig. 2A–F, H, I). In culture, the putative 1230

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GFP⫹ endothelial progenitors were observed to adhere to GFP-negative smooth muscle cells (␣-smooth muscle actin-positive; Supplemental Fig. 2G) in a manner similar to that observed in primary bone marrow cultures where endothelial progenitors adhere to stromal cells (data not shown). A large number of GFP⫹ cells in the cultures were fibroblastic cells that did not take up Ulex lectin or AcLDL (Supplemental Fig. 2H, I). Engraftment and differentiation of BMD cells in the hypertensive lung As expected, double immunostaining demonstrated that a portion of the BMD cells in the lung were hematopoietic cells that stained with a monocyte/ macrophage-specific antibody (Fig. 5A–C). Many GFP⫹ cells were located interstitially in the lung parenchyma adjacent to epithelial cells (Fig. 4E). Isotype controls for the GFP immunostaining were diffuse and negative (Supplemental Fig. 2J). In addition, we observed a series of GFP⫹ cells that were nonhematopoietic cells. Some of the GFP⫹ cells differentiated into vascular smooth muscle cells that were ␣-smooth actin-positive cells and localized within the blood vessel walls (Fig. 4A–D). Deconvolution imaging of in situ hybridization for the Y chromosome combined with immunohistochemistry for ␣-smooth actin confirmed the engraftment of bone marrow-derived cells into blood vessel walls as smooth muscle cells (Fig. 4D). GFP⫹ cells that localized to the airway surface stained for Clara cell secretory protein, a marker for Clara cells that are progenitors of bronchial epithelium (CC10/CCSP, Fig. 4F–H). We also observed GFP⫹/CCSP⫹ cells in the lungs of control (BMT) animals that did not receive MCT (data not shown). To define further the phenotype of the BMD cells in the alveoli, we stained lung sections for GFP and T1␣, a protein specific to type I pneumocytes in the adult rat (Fig. 5D–F). GFP⫹ cells localized in close proximity with T1␣ staining, but because of the intricate structure of the alveoli, it was difficult to determine whether the GFP⫹ cells were type I cells or capillary endothelial cells. We believe that these cells were likely to be bone marrow-derived capillary endothelial cells. We observed that some BMD progenitors had engrafted and differentiated as endothelial cells in that they were vimentin-positive (data not shown) and von Willebrand factor-positive cells that lined the inner surfaces of blood vessel walls in the lung (Fig. 5G–I). The immunostaining was confirmed by FISH for the Y chromosome that demonstrated dramatic engraftment of male BMD cells surrounding and localized within blood vessel walls (Fig. 5J, K), including occluded vessels typical of MCT toxicity (Fig. 5K). Also in confirmation of the GFP staining, combined immunohistochemistry with FISH identified male cells in blood vessel walls that also stained for von Willebrand factor (Fig. 5L).

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Figure 4. BMD cell engraftment in the lungs of MCT-treated rats. Arrowheads: double-positive cells. Arrows: GFP⫹ cells that are negative for the relevant differentiation marker. A–C) GFP⫹ (red, ALEXA 594) smooth muscle cells (SMA; smooth muscle ␣-actin-positive, green, ALEXA 488) in a blood vessel wall. D) Deconvolution imaging of Y chromosome FISH (green dots) combined with immunohistochemistry for SMA (Vector Red) to confirm BMD blood vessel engraftment. E) Many GFP⫹ cells form interstitial tissue underlying bronchial epithelial cells (positive for Keratin 19). Note that the GFP⫹ cells do not colocalize with K19 in this image. F–H) GFP⫹ cells that localize to the airway epithelium colabel for CC10, a marker of Clara cells (bronchial epithelial cells).

The expression of von Willebrand factor in the BMD cells was confirmed in the cultures of GFP⫹ cell isolated from lung (for FACS gate see Fig. 2). After 2 wk of culture, we found GFP⫹ cells with an endothelial cell-like morphology that stained strongly for von Willebrand factor (Fig. 5M–O). Therefore, the results demonstrated that some of the cultured cells began as endothelial progenitor cells and differentiated into mature endothelial cells after 2 wk, as the initial cultures did not contain vWFpositive cells (data not shown). Engraftment and differentiation of BMD cells in the hypertrophied heart Real-time PCR assays for the Y chromosome demonstrated the presence of BMD cells in the perfused

hearts from both the control chimeric rats and the MCT-treated chimeric rats. However, no significant difference was found in the numbers of BMD cells that engrafted in the hearts of control BMT rats vs. those of MCT-treated rats (P⫽0.147; Fig. 6A). Unexpectedly, in control BMT animals that did not receive MCT, the left ventricle contained significantly greater numbers of BMD cells than did the right ventricle (Fig. 6B). Further real-time PCR assays for the Y chromosome demonstrated a significant increase in the homing of BMD cells to the right ventricle during repair and remodeling within the hearts of several MCT-treated animals (Fig. 6C). By dividing the right ventricular BMD cell engraftment by the left ventricular engraftment in the hearts of individual animals, we found a

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Figure 5. BMD cell engraftment in the lungs of MCT-treated rats. A–C) Some hematopoietic cells remain present in the lungs following perfusion. Arrowheads: GFP⫹ monocytes and macrophages. Arrows: GFP⫹ (BMD) cells that localize to epithelial or capillary endothelial positions in the lung architecure. D–F) T1␣ staining of type I epithelial cells in the lung. Arrowheads: engrafted BMD cells, which may be type I pneumocytes or capillary endothelial cells residing in between type I cells. Arrows: GFP⫹ cells that do not localize with T1␣ stain. G–I) von Willebrand factor staining of a GFP⫹ (BMD) cell in blood vessel endothelium (longitudinal section). J, K) Fluorescent in situ hybridization (FISH) for the rat Y chromosome (pink dots) in pulmonary vasculature of MCT-treated rats. Nuclei are stained with DAPI. L) FISH for Y chromosome (green dots) combined with immunostain for von Willebrand factor (red) identifies BMD endothelial cell. M–O) Cells isolated from the lungs of MCT-treated rats contain cells that mature to vWF-positive endothelial cells after 2 wk in culture. 1232

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significant difference between control BMT and MCTtreated rats (P⫽0.014; controls, n⫽8; MCT, n⫽6; Fig. 6D), indicating that the dramatic remodeling of the right heart that occurs during chronic pulmonary hypertension can influence the numbers of BMD cells that engraft the right ventricle wall. Double immunohistochemistry of heart sections from MCT-treated rats demonstrated that some of the GFP⫹ cells stained with the pan-hematopoietic marker CD45, and some GFP⫹ cells stained for a monocyte/ macrophage marker (data not shown). The majority of BMD cells in the heart stained for vimentin, a marker of mesodermal cells such as fibroblasts, myofibroblasts, and endothelial cells. Many of the GFP⫹/vimentin⫹ cells were associated with small blood vessels in the hypertrophied right ventricle (RV) wall, indicating engraftment as endothelial cells (Fig. 6E–G). FISH assays also identified BMD cells among cardiac myocytes and in the blood vessel walls (Fig. 6H). After double FISH assays for the Y chromosome and chromosome 4, we did not find any BMD cells in blood vessel walls that arose by cell fusion (Fig. 6I). Differentiation into cardiomyocytes and cell fusion in the hypertrophied heart To determine whether BMD cells differentiated into cardiomyocytes or fused with host-derived cardiomyocytes, we performed double FISH and, in some cases, double FISH combined with immunohistochemistry for cardiac-specific troponin. Double FISH for the Y chromosome and for chromosome 4 identified many BMD cells in the cardiac muscle layer that did not have extra copies of chromosome 4, indicating that they were not fused to host cells (Fig. 6H). By deconvolution microscopy of immunostains for GFP and cardiac alpha actinin, we identified GFP-positive myocytes in the right ventricle walls of MCT-treated rats (Fig. 7A). Numerous Y chromosome-positive cells in the hypertrophied RV wall also expressed cardiac troponin (Fig. 7B). To examine the possibility that some of the BMD cardiomyocytes arose from cell fusion of BMD cells with endogenous cardiac myocytes, we performed double FISH combined with immunostaining for cardiac troponin. A few of the BMD cardiomyocytes arose without cell fusion as demonstrated by one Y chromosome and two chromosome 4s or less because of loss during histological sectioning (Fig. 7C, D). Some BMD cardiomyocytes arose by cell fusion followed by nuclear fusion as demonstrated by the presence of one Y and three 4s (Fig. 7E). Rare BMD cardiomyocytes also appeared as binucleated cells (cardiomyocyte heterokaryon, Fig. 7F).

DISCUSSION The design of the experiments reported here had two critical features: 1) female rats were first transplanted with GFP⫹/male bone marrow, and 2) then they received a single dose of MCT that produced progres-

sively severe injury to the lungs and heart over a period of 3 wk. As a result, a continuous supply of fluorescently and genetically labeled BMD cells homed to the severely injured tissues over a prolonged period of time. The progressive MCT injury was severe enough that the MCT-treated rats would have died 1 to 2 wk after the date of sacrifice in our experimental design. We believe that the severity and progressive nature of the MCT injury combined with irradiation led to high numbers of BMD cells in the lungs of the MCT-treated chimeric rats. In control irradiated rats that received bone marrow transplants, ⬃15% of the cells in the lungs were GFP positive, whereas in the MCT-treated chimeric rats, on average 35– 45% of the cells in the lungs were GFP positive. The pulmonary GFP-positive cells in both cases were likely a mix of hematopoietic (inflammatory) and nonhematopoietic cells. Some hematopoietic cells were observed in both the injured lungs and the hypertrophied hearts of MCTtreated rats, even following extensive perfusion. However, numerous nonhematopoietic cells, including fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells, and epithelial cells were found in the injured lungs and were derived from the GFP⫹/male bone marrow. In addition, BMD cells were present in the lung as early progenitors of endothelial cells and epithelial cells. A large number of the BMD cells in lung were fibroblasts and myofibroblasts that may have contributed to fibrosis of the lung following irradiation or MCT treatment. Some of the differentiation of the BMD cells in lung may have occurred by cell fusion, but, as indicated by the FISH assays on isolated cells, the incidence of cell fusion in lung was very low. Aside from engrafted BMD vascular cells in the hearts of MCT-treated animals where we never observed cell fusion by our FISH assays, BMD cardiomyocyte engraftment in the heart appeared to involve a greater extent of cell fusion and some nuclear fusion of BMD cells with host-derived cardiomyocytes. The results did not establish which subpopulation of cells from bone marrow contributed to the repair and remodeling of the lung and heart, but the experiments provide a model system for defining such BMD subpopulations. The results are consistent with a series of earlier reports indicating engraftment of BMD cells into nonhematopoietic tissues (13–16). Kotton et al. (17) and Ortiz et al. (18) observed that BMD nonhematopoietic adult stem cells (MSCs) engrafted as pulmonary epithelial cells after intravenous infusion to mice in which lung injury was produced by bleomycin. Krause et al. (3) found that a highly purified bone marrow cell engrafted as epithelial cells in lung and other organs after infusion into marrowablated mice. Loi et al. (19) reported that bone marrowderived cells could contribute to CFTR-positive bronchial epithelium in the lungs of mice that were otherwise lacking the protein. Hayashida et al. (20) reported that bone marrow-derived cells contributed to pulmonary vascular remodeling in a hypoxia-based model of pulmonary hypertension in mice. Tissue-specific differentiation of

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Figure 6. Increased engraftment of cardiac BMD cells in response to right-ventricle hypertrophy. Y chromosome real-time PCR was used to quantify BMD cells that engrafted the heart. A) Y chromosome real-time PCR of DNA from ventricles and septum (no atria) of control (BMT) and MCT-treated rats. No significant difference was found in the overall numbers of BMD cells in the ventricles of control vs. MCT-treated rats (P⫽0.147). B) DNA isolated from the right ventricle compared with the left ventricle from hearts of control BMT animals demonstrates that significantly more BMD cells are typically found in the left ventricle as compared with the right. B–D) Cardiac right-ventricular hypertrophy in MCT-treated rats induces a significant increase in the number of BMD cells in the right ventricle. C1 to C8, control BMT rats (n⫽8); M1 to M6, BMT/MCT rats; 1234

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Figure 7. Cardiomyocyte engraftment of BMD cells in the hypertrophied RV wall. A) Deconvolution microscopy of immunohistochemistry for GFP and cardiac alpha actinin. Note that GFP is predominantly expressed in the nucleus of cells from the GFP transgenic rat. B) Combined immunohistochemistry for cardiac-specific troponin and Y FISH to demonstrate that some BMD cells become cardiomyocytes. C) Deconvolution microscopy to test for cell fusion. In some cases, we observed BMD cardiomyocytes with one Y chromosome and one 4 chromosome, apparently because of loss during histological sectioning. A binucleated host-derived cardiomyocyte is also shown (no Y chromosome and four 4 chromosomes). D) Deconvolution microscopy to test for cell fusion. One cardiomyocyte with normal chromosome number, and two with one Y and one 4 chromosome. A normal BMD cell is also shown in blood vessel. E) Deconvolution microscopy to test for cell fusion. Nuclear fusion of BMD cells with host cell (one Y and three 4s). As shown with pulmonary cells (Fig. 2), nuclear fusion may lead to loss of one chromosome 4. An apparently normal BMD cardiomyocyte is shown below. F) Deconvolution microscopy to test for cell fusion. A possible binucleated cardiomyocyte (heterokaryon, male nucleus with female nucleus in same cell). Both nuclei are normal. Arrowheads: BMD cells (Y chromosome, green dots). Arrows: chromosome 4 (pink dots). BV, blood vessel.

engrafted bone marrow cells in heart has been more difficult to demonstrate, but BMD cardiomyocytes were reported by several laboratories (21–23). Kajstura et al. (21) found that c-kit⫹ cells from bone marrow became cardiac myocytes and vascular cells without any evidence of cell fusion after the cells were directly injected into the myocardium. The observations in experimental animals were confirmed by observations with patients who received bone marrow or organ transplants. Engraftment of BMD cells was observed in liver (24), brain (25), lung (26), heart (27, 28), intestine (29), and kidney (30, 31). BMD cells have also been reported to contribute to both the smooth muscle (32) and endothelial (33) components of the vasculature in humans. In both the experiments in animals and the observations in patients, the highest levels of engraftment were generally seen in tissues that were severely injured. The observations as a whole are consistent with the conclusion that stem-like cells found in most or all tissues are the first source of repair following injury. As the tissue-endogenous stem cells are exhausted, they are likely to be complemented by additional stem or progenitor cells from the circulation

and the bone marrow (1, 34). The observations, however, do not exclude the possibility that the endogenous stem-like cells in tissues are continually being replenished at a slow rate by bone marrow cells and that the bone marrow is a major source of such cells over the lifetime of an individual. Recent reports suggest that naive or gene-modified endothelial progenitors from peripheral blood or bone marrow can ameliorate some of the symptoms of MCTinduced pulmonary hypertension (35, 36). To create effective and reliable cell-based therapies for pulmonary hypertension and many other diseases, it will now be important to clearly identify the population or subpopulations of adult BMD nonhematopoietic stem or progenitor cells that contribute to particular tissues and to develop culture conditions that will provide large numbers of reparative cells while maintaining their differentiation capacity and ability to durably engraft in vivo. This work was supported in part by U.S. National Institutes of Health grants HL077570 – 01 (to J.L.S.), AR47796 and AR48323 (to D.J.P.), The Oberkotter Foundation, HCA The Healthcare Company, and the Louisiana Gene Therapy Consortium.

RV/LV, ratio of right ventricle to left ventricle. *P⬍0.05, **P⬍0.01, ***P⬍0.001. E–G) Sections of RV doubly stained for vimentin and GFP⫹ cells. Vimentin⫹/GFP⫹ cells localize as endothelial cells of small blood vessels. H) Sections of RV examined for cell fusion by double FISH for the Y chromosome (red dots) and chromosome 4 (green dots) to look for cell fusion. Autofluorescence is turned up in the green (FITC) channel to show cardiac muscle. Arrowheads: BMD cells. Circles: BMD cardiomyotes with normal chromosome number (one Y, two 4s). Two BMD cells are engrafted into a large blood vessel (BV). I) Deconvolution microscopy of hypertrophied RV assayed for cell fusion by double FISH for Y (green)/4 (pink) chromosomes together with immunostaining for cardiac-specific troponin (red). The image is deconvolved from 18 visual sections of 0.5 ␮m each. Examination in x, y, and z planes ruled out overlay of the critical nuclei. Circles: BMD cells engrafted into blood vessel walls that possess the normal chromosome number. Arrowheads: BMD cells that have less than the normal number of chromosomes, apparently because of loss during sectioning. BONE MARROW PROGENITORS AND PULMONARY HYPERTENSION

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The FASEB Journal

Received for publication April 19, 2007. Accepted for publication October 25, 2007.

SPEES ET AL.