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Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair
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Mei Wang, Guoru Zhang, Yaling Wang, Tao Liu, Yang Zhang, Yu An, Yongjun Li ∗ Department of Cardiology, the Second Hospital of Hebei Medical University, 215 Hepingxin Road, Shijiazhuang, China
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Article history: Received 28 August 2014 Received in revised form 15 October 2014 Accepted 3 November 2014 Available online xxx
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Keywords: Mesenchymal stem cells Cardiac muscle repair Macrophages Transforming growth factor  1 BMP7
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1. Introduction
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Transplantation of bone-marrow derived mesenchymal stem cells (MSCs) has potential therapeutic effects on cardiac muscle repair. However, the underlying mechanism remains not completely clarified. Here we show that transplantation of MSCs significantly increased local recruitment of macrophages to facilitate cardiac muscle repair. MSCs-induced recovery of cardiac function and attenuation of fibrosis after injury were all abolished by either impaired macrophage infiltration, or by MSCs depletion after macrophage recruitment. However, angiogenesis seemed to be only affected by depletion of macrophages, but not by depletion of MSCs, suggesting that macrophages are responsible for the augmented angiogenesis after MSCs transplantation, while MSCs do not directly contribute to angiogenesis in the functional cardiac repair. Moreover, high level of transforming growth factor  1 (TGF1) was detected in macrophages and high level of BMP7 was detected in MSCs, suggesting that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGF1 by macrophages. Our study thus sheds new insight on the interaction of MSCs and macrophages in a functional cardiac repair triggered by MSCs transplantation. © 2014 Published by Elsevier Ltd.
Bone marrow-derived stem/progenitor cells can promote neovascularization in the ischemic myocardium for a functional improvement (Losordo and Dimmeler, 2004a,b). However, the underlying mechanism is not completely understood (Balsam et al., 2004; Fukata et al., 2013; Murry et al., 2004). Moreover, the outcome of transplantation of bone marrow-derived stem/progenitor cells into an injured heart has been extremely variable, which may largely result from the difference in the grafted population of the bone marrow-derived stem/progenitor cells (Balsam et al., 2004, Fukata et al., 2013; Murry et al., 2004; Wu et al., 2010). Mesenchymal stem cells (MSCs) have first been isolated from the bone marrow, and are the most important bone marrow-derived stem/progenitor cells. MSCs have now been identified in all postnatal tissues, and have been shown to be critical for tissue renewal
Abbreviations: MSCs, mesenchymal stem cells; BMP7, bone morphogenetic protein 7; TGF1, transforming growth factor  1; DMSO, dexamethasone; AMI, acute myocardial infarction; DTR, diphtheria toxin receptor; DTA, diphtheria toxin A; LVDd, LV diastolic dimensions; LVFS, percentage of LV fractional shortening. ∗ Corresponding author. Tel.: +86 31187046901; fax: +86 31187046901. E-mail address: yongjun [email protected] (Y. Li).
and repair (Cao et al., 2014; Fawzy et al., 2013; Monsefi et al., 2013; Song et al., 2014; Ye et al., 2013; Zhao et al., 2013). In the current study, we addressed the question about the exact role of MSCs in the cardiac repair, and especially their crosstalk with macrophages. We found that MSCs transplantation significantly increased local infiltration of macrophages, which are necessary for the improved cardiac function, increased angiogenesis and reduction in fibrosis. MSCs themselves, although not required for angiogenesis, appeared to be needed for inhibition of fibrosis and improvement of cardiac function, possibly through their production of bone morphogenetic protein 7 (BMP7) to contradict the fibrogenic effect of transforming growth factor  1 (TGF1) by macrophages.
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2. Materials and methods
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2.1. Mouse handling
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All mouse experiments were approved by the Institutional Animal Care and Use Committee at the Second Hospital of Hebei Medical University (Animal Welfare Assurance). Surgeries were performed under ketamine/xylazine anesthesia, according to the Principles of Laboratory Care. Male 10 week-old C57BL/6 mice were
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Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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used in the current study. Five mice were analyzed in each experimental condition.
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2.2. MSCs isolation, culturing and differentiation
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The MSCs were isolated and grown in culture as has been described previously (Cao et al., 2014; Tropel et al., 2004; Zhou et al., 2007). Briefly, plugs of marrow from five 10-week-old male C57/6BL mice (about 27 g body weight) were dispersed in DMEM and then centrifuged at 900g for 5 min. The pellets were re-suspended and plated at 105 cells/cm2 in DMEM containing 10% FBS. After 10 passages’ selection of attached cells, the cells were sorted for Stro-1 (BD) by flow cytometry to get rid of contaminating cells. A positive clone was selected after subjected to chondrogenetic, osteogenic, and adipogenic differentiation assays for confirming phenotype. For chondrogenetic induction, 2.5 × 105 MSC were induced with 5 mL chondrogenetic induction medium containing 10 g TGF1 (R&D), 50 g insulin growth factor 1 (R&D), and 2 mg/mL dexamethasone (DMSO, Sigma) followed by centrifugation at 500g for 5 min. The cell pellets were maintained in the chondrogenetic induction medium for 14 days and subjected to Alcian blue staining. For osteogenic induction, cells were digested and seeded onto a 24-well plate at a density of 104 cells/well, and then maintained in osteogenic induction medium containing 10 nM Vitamin D3 (Sigma) and 10 mM -phosphoglycerol and 0.1 M DMSO for 14 days and were subjected to Von kossa staining. For adipogenic induction, cells were digested and seeded onto a
24-well plate at a density of 104 cells/well, and then maintained in the adipogenic induction medium containing 0.5 mM 3isobutyl-1-methylxanthine (IBMX), 200 M indomethacin, 10 M insulin and 1 M DMSO for 14 days and subjected to Oil red O staining.
2.3. Transduction of MSCs A recombinant lentivirus expressing diphtheria toxin receptor (DTR) and green fluorescence protein (GFP) under the control of a CMV promoter (Invivogen: Itsint) efficiently transduced MSCs at MOI 100 in high efficiency. The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. Successfully transduced MSCs were selected by flow cytometry based on GFP.
2.4. Acute myocardial infarction (AMI) model and MSCs transplantation A standardized mouse AMI model was applied as has been described before (Kusano et al., 2005). For MSCs transplantation, 106 MSCs were intravenously injected via tail vein to the mice immediately after surgical induction of AMI.
Fig. 1. Isolation, culturing, differentiation and labeling of primary mouse MSCs. (A) Schematic of the viral construct. (B) Transduced green fluorescent MSCs in culture. (C) Purification of transduced MSCs by flow cytometry based on GFP. (D–F) Differentiation assays were performed, including oil red O staining (D), alcian blue staining (E) and von kossa staining (F). (G) The expression level of adipogenic marker GOS2 increased by 88.5 ± 6.2 fold in adipocytes differentiated from MSCs (Adi), compared to undifferentiated MSCs. The expression level of chondrogenic marker BGN increased by 10.5 ± 1.2 fold in chondrocytes differentiated from MSCs (Cho), compared to undifferentiated MSCs. The expression level of osteogenic marker DKK1 increased by 12.5 ± 1.5 fold in osteocytes differentiated from MSCs (Ost), compared to undifferentiated MSCs (G). (H) In vitro administration of DTA was found to efficiently kill cultured DTR-expressing MSCs, by Propidium iodide (PI) staining of dead cells. Scale bars are 50 m. N = 5.
Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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2.5. Clodronate and diphtheria toxin A injection
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For macrophage depletion, 200 l clodronate-liposomes (Clodronateliposome) were i.v. injected to the mice every other day, from the first day of surgical induction of AMI and MSCs transplantation until analysis. Control mice were injected with liposomes containing PBS, showing same results for all parameters that were analyzed in the current study, compared to the mice treated with AMI and MSCs. For MSCs depletion, 100 ng diphtheria toxin A (DTA) was i.p. injected to the mice that had received DTR-transduced MSCs, for 3 consecutive days since 7 days after MSCs transplantation.
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2.6. Tomato lectin labeling of the vessels
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When the mice were sacrificed, adequate perfusion with saline is performed to remove lectin in the circulation, which avoids its interference with quantification. For evaluating functional vessel density in the heart, DyLight594-labeled Lycopersicon esculentum (tomato lectin, Vector labs) was i.v. injected from the tail vein to the mice 5 min before sacrifice.
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2.7. Heart function evaluation
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Mice underwent echocardiography just before MI (0 week) and at 1, 2, and 4 weeks after AMI as has been described before (Kusano et al., 2005). Transthoracic echocardiography was performed with
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a 6- to 15-MHz transducer (SONOS 5500, Hewlett Packard). Twodimensional images were obtained in the parasternal long and short axis and apical four-chamber views. M-mode images of the left ventricular (LV) short axis were taken just below the level of mid-papillary muscles. LV end-diastolic and end-systolic dimensions were measured and functional shortening was determined according to modified recommendations of American Society of Echocardiography. A mean value of three measurements was determined for each time point. 2.8. Flow cytometry The mouse hearts were digested into single cells as has been described before (Li et al., 2014). The digests were then incubated with M-cadherin antibody (M-Cad, Millipore, USA) followed by labeling with PE-conjugated anti-mouse second antibody (BD, USA). Afterwards, flow cytometry analysis was performed in a FACSAria (Becton Dickinson) flow cytometer. MSCs were recognized by direct fluorescence of GFP. 2.9. Immunohistochemistry Mouse hearts were dissected out and fixed with 4% paraformaldehyde for 6 h, and then cyro-protected in 20% sucrose overnight. Samples were then sectioned in 6 M. For immunostaining, primary antibody is rabbit anti-M-cadherin (M-Cad) and rat anti-F4/80 (Invitrogen, USA). Secondary antibody
Fig. 2. MSCs improved heart function after cardiac muscle injury. MSCs were transplanted immediately after induction of AMI. (A, B) LV end-diastolic and end-systolic dimensions were measured and functional shortening was determined before AMI (week 0) and on 1, 2, 3 and 4 weeks post-AMI. The echocardiography 2, 3, 4 weeks post-AMI showed significantly improved LV diastolic dimensions (LVDd) (A) and percentage of LV fractional shortening (LVFS) (B) in mice transplanted with MSCs compared to saline control. (C, D) Masson’s Trichrome-stained sections showed markedly reduced LV fibrosis, by representative images (C), and by quantification (D). (E, F) Functional cardiac vessel density was quantified by lectin, shown by quantification (E), and by representative images (F). Scale bars are 50 m. * : p < 0.05. N = 5.
Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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is Cy3-conjugated anti-rabbit antibody (Jackson Labs, USA). GFP and tomato lectin were visualized by direct green and red fluorescence, respectively. DAPI was used to stain nucleus. Vessel density was determined with ImageJ (NIH) software by measuring the percentage of lectin-positive area to the total heart area. For quantification of heart fibrosis, heart sections were stained with Masson trichrome as has been described before (Shen et al., 2014). Evaluation of heart fibrosis in each sample was performed based on 20 randomly selected fields per section, which were examined under ×400 magnification for assessment of the degree of heart fibrosis by visualizing blue-stained areas, exclusive of staining that co-localized with perivascular or intramural vascular structures, the endocardium, or LV trabeculae. ImageJ software was used to determine blue-stained areas and non-stained myocyte areas from each section using color-based threshold. The percentage of total fibrosis area was calculated as the summed blue-stained areas divided by total ventricular area, as has been described previously (Wolf et al., 2005).
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2.10. Quantitative real-time PCR (RT-qPCR)
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RNA was extracted from cultured cells with RNeasy (Qiagen, Hilden, Germany) for cDNA synthesis. RT-qPCR was performed in duplicates with QuantiTect SYBR Green PCR Kit (Qiagen). All primers were purchased from Qiagen. Values of genes were normalized against ␣-tubulin and then compared to the control (=1).
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2.11. Statistics
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All values are depicted as mean ± standard deviation from five individuals and are considered significant if p < 0.05. All data were statistically analyzed using one-way ANOVA with a Bonferoni correction.
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3. Results
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3.1. Isolation, culturing, differentiation and labeling of primary mouse MSCs Primary mouse MSCs were isolated from male C57BL/6 mice, expanded in culture, and then transduced with lentivirus carrying DTR and GFP under the control of a CMV promoter (Fig. 1A). Successfully transduced MSCs were selected by flow cytometry based on GFP (Fig. 1B and C). Differentiation assays were then applied to confirm the MSC-phenotype of the transduced cells, including Von kossa staining to evaluate osteogenic induction, Oil red O staining to evaluate adipogenic induction and Alcian blue staining to evaluate chondrogenetic induction (Fig. 1D–F). Differentiation of MSCs was assured by specific lineage gene expression (Fig. 1G). The expression level of adipogenic marker GOS2 increased by 88.5 ± 6.2 fold in adipocytes differentiated from MSCs, compared to undifferentiated MSCs. The expression level of chondrogenic marker BGN increased by 10.5 ± 1.2 fold in chondrocytes differentiated from MSCs, compared to undifferentiated MSCs. The expression level of osteogenic marker DKK1 increased by 12.5 ± 1.5 fold in osteocytes differentiated from MSCs, compared to undifferentiated MSCs (Fig. 1G). Moreover, in vitro administration of DTA was found to efficiently kill cultured DTR-expressing MSCs (Fig. 1H). 3.2. Therapeutic effects of isogenic MSCs transplantation after cardiac muscle injury In order to evaluate the effect of transplantation of MSCs on injured heart, we transplanted the labeled MSCs immediately after
Fig. 3. Grafted MSCs did not differentiate into cardiac muscle cells. (A) A representative immunostaining image in MSCs-grafted AMI-heart. M-cad: M-cadherin. DAPI: nucleus staining. (B–D) Flow cytometry was applied to M-cad-stained heart digests from mice that received MSCs and AMI, shown by representative flow chart (B). Less than 0.1% GFP-positive cells were M-cad-positive (C), and in all M-cad-positive cells, the percentage of GFP-positive cells was less than 0.01% (D), quantified by immunohistochemistry (IHC) or by FACS. Scale bar is 50 m. * : p < 0.05. NS: non-significant. N = 5.
induction of AMI, as has been described before (Kusano et al., 2005). LV end-diastolic and end-systolic dimensions were measured. Functional shortening was determined before AMI (week 0) and on 1, 2, 3 and 4 weeks post-AMI. The echocardiography 2, 3, 4 weeks post-AMI showed significantly improved LV diastolic dimensions (LVDd) (Fig. 2A) and percentage of LV fractional shortening (LVFS) (Fig. 2B) in mice transplanted with MSCs, compared to the control mice that received saline. Masson’s Trichrome-stained sections showed markedly reduced LV fibrosis, by representative images (Fig. 2C), and by quantification (Fig. 2D). Moreover, functional cardiac vessel density was quantified by perfused lectin, showing significant increase in the mice that received MSCs after AMI (Fig. 2E and F). Lectin labels functional vessels, and appears to be a better marker for evaluating vessel function, compared to CD31, CD144 and CD106, etc. These data suggest that grafted isogenic MSCs improve heart function, reduce fibrosis, and increase angiogenesis after heart injury.
3.3. Grafted MSCs very rarely differentiated into cardiac muscle cells We next examined whether grafted MSCs may differentiate into cardiac muscle cells. We detected many GFP-positive MSCs in the injured mouse heart after MSCs transplantation, but very few of them appeared to express a specific cardiac muscle cell marker, M-cadherin (M-Cad), by immunostaining (Fig. 3A), and by flow cytometry analyses on heart digests (Fig. 3B). Quantification show that less than 0.1% GFP-positive MSCs were M-cad-positive (Fig. 3C), and in all M-cad-positive cells, the percentage of GFPpositive cells was less than 0.01% (Fig. 3D). These data thus suggest that a direct contribution of MSCs to the regenerated cardiac muscle cells is very limited.
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Fig. 4. MSCs transplantation recruited macrophages. (A) RT-qPCR for F4/80 in mouse heart hat received AMI and control saline, or mice that received AMI and MSCs with or without clodronate. (B, C) Analyses of F4/80+ cells in the heart digests by flow cytometry, shown by quantification (B), and by representative flow charts (C). (D) The population of M1 and M2 macrophages in the injured heart appeared to be similar by analysis with a M2 macrophage-specific marker CD163. (E) These macrophages can be readily visualized in the AMI-treated MSCs-grafted heart. Scale bars are 50 m. Clod: clodronate. * : p < 0.05. NS: non-significant. N = 5.
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3.4. Recruited macrophages were necessary for cardiac repair Macrophages are well-known for their essential roles in the tissue regeneration and repair, which prompted us to examine the involvement of macrophages in this model. F4/80 is a specific marker for macrophages. We detected significantly increase in F4/80 transcript level in the AMI-heart grafted with MSCs, compared to the AMI-heart treated with saline, 7 days after AMI and transplantation of MSCs (Fig. 4A). Moreover, significantly higher percentage of F4/80+ cells were detected in the AMI-heart grafted with MSCs, compared to the AMI-heart treated with saline (Fig. 4B and C), suggesting that MSCs recruited macrophages in the injured heart. Moreover, the population of M1 and M2 macrophages in the injured heart appeared to be similar by analysis with a M2 macrophage-specific marker CD163 (Fig. 4D). These macrophages can be readily visualized in the AMI-treated MSCs-grafted heart (Fig. 4E). In order to find out whether macrophages are necessary for the MSCs-induced therapeutic effects on an injured heart, we chemically depleted macrophage infiltration with clodronate, as has been previously described (Cao et al., 2014; Shen et al., 2014; Song et al., 2014; van Rooijen et al., 1997; Xiao et al., 2014). Our data showed that clodronate administration reduced the recruited macrophages
by 70% in the AMI-heart after MSC transplantation (Fig. 4A–C), which significantly abolished the improved LVDd (Fig. 5A) and LVFS (Fig. 5B), the reduction in fibrosis (Fig. 5C), and the augmented angiogenesis (Fig. 5D and E) by MSCs transplantation. These data suggest that the recruited macrophages are necessary for the functional cardiac repair induced by MSCs. 3.5. MSCs had effects other than recruiting macrophages during cardiac repair Then we aimed to figure out whether the effects of MSCs on cardiac repair are limited as to recruit macrophages. We gave the MSCs-grafted AMI-mice with DTA, to eliminate DTR-carrying MSCs, at 7 days after AMI and transplantation of MSCs, when macrophages had been already recruited. We found that MSCs were efficiently killed by DTA, by analyzing GFP transcripts in the heart by RT-qPCR (Fig. 6A), and GFP+ cells in the heart digests by flow cytometry (Fig. 6B). Of note, elimination of MSCs after macrophage recruitment resulted in significant abolishment of the improved LVDd (Fig. 6C) and LVFS (Fig. 6D) and the reduced fibrosis (Fig. 6E), but not of the augmented angiogenesis (Fig. 6F and G) by MSCs transplantation. These data suggest that the recruited macrophages appear to be responsible for the augmented angiogenesis, while MSCs play a
Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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Fig. 5. Macrophages are necessary for the functional cardiac repair induced by MSCs. (A–E) Clodronate (Clod) was given to deplete macrophages, which significantly abolished the improved LVDd (A) and LVFS (B), the reduction in fibrosis (C), and the increased functional vessel density (D, E) by MSCs transplantation. (D, E) Functional cardiac vessel density was quantified by perfused lectin, shown by quantification (D), and by representative images (E). Scale bars are 50 m. * : p < 0.05. N = 5.
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substantial role in the cardiac functional repair, other than a direct effect on angiogenesis, besides recruitment of macrophages.
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3.6. MSCs seem to contradict the fibrogenic effect of macrophages
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Previous studies have highlighted an important counteraction between TGF1 and BMP7 in the epithelial-to-mesenchymal transition for fibrosis (Buijs et al., 2007; Khan et al., 2011; Liu et al., 2014; Shen et al., 2014; Wang and Hirschberg, 2003). Since TGF1 has been reported to be highly expressed by macrophages (Ding et al., 1993; Lazdins et al., 1991; Standiford et al., 2011; Vignola et al., 1996; Xiao et al., 2014), we thus examined the expression of TGF1 and BMP7 in the isolated macrophages and MSCs from the recipient heart (Fig. 7A). We detected high TGF1 level in macrophages and high BMP7 level in MSCs (Fig. 7B). These data may imply that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGF1 by macrophages (Fig. 7C). 4. Discussion Transplantation of bone-marrow derived MSCs has an established therapeutic effect on cardiac muscle injury. However, previous studies failed to reach consistent results on the underlying mechanism (Balsam et al., 2004; Fukata et al., 2013; Murry et al., 2004; Wu et al., 2010), which prompted us to address this question in the current study. We chose a strict inbred strain, C57BL/6 mice, for isogenic transplantation in our study. We first isolated and cultured primary
mouse MSCs from male C57BL/6 mice, and then labeled these cells with a GFP reporter and a DTR, for in vivo lineage tracing and lineage ablation, respectively. The GFP labeling of the MSCs allow us to analyze their possible contribution to other lineages in vivo, by double immunostaining, and by flow cytometry. Indeed, we found little of differentiation of MSCs into cardiac muscle cells. The toxicity of DTA is extremely high that one molecule of DTA in the cytosol may be enough to kill the cell. However, mouse cells do not have the DTR to allow DTA to enter the cells. Thus, administration of DTA to the mice did not kill any cells from the recipient mice. On the other hand, since MSCs were transduced with DTR, they became very susceptible to DTA toxicity. The power of this system was assured in our experiment using DTA to eliminate the transplanted MSCs. The phenotypes of the transduced MSCs were further confirmed by three differentiation assays. In order to evaluate the effect of transplantation of MSCs on injured heart, we transplanted the MSCs immediately after induction of AMI. Significant improvement in LVDd and LVFS, reduced fibrosis, and increased vascularization in MSCs-grafted mice demonstrate a substantial role of MSCs in cardiac muscle repair. Macrophages are well-known for their importance in the tissue regeneration and repair, and in the organ remodeling and fibrosis. We found five times more macrophages in the AMI-heart grafted with MSCs, compared to the AMI-hearted treated with saline, suggesting that MSCs recruited macrophages in the injured heart. We then used clodronate to deplete macrophages to see whether the effects of MSCs on cardiac repair may be affected by macrophage depletion. Clodronate is a hydrophilic molecule that is packaged in a
Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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Fig. 6. MSCs have an angiogenesis-unrelated role in the functional cardiac repair. (A–G) DTA was given to the MSCs-grafted AMI mice to eliminate DTR-carrying MSCs, at 7 days after AMI and MSCs transplantation, when macrophages were already recruited. (A) MSCs were efficiently killed by DTA, by analyzing GFP transcripts in the heart (A), and GFP+ cells in the heart digests (B). (C–G) Removal of MSCs significantly abolished the improved LVDd (C) and LVFS (D), the reduction in fibrosis (E), but had no effects on the increased functional vessel density (F, G). (F, G) Functional cardiac vessel density was quantified by perfused lectin, shown by quantification (F), and by representative images (G). Scale bars are 50 m. * : p < 0.05. N = 5.
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liposome to mediate internalization clodronate into macrophages. This molecule has a short half-life when released in the circulation, but does not easily cross phospholipid bilayers of liposomes or cell membranes. As a consequence, once ingested by a macrophage in a liposome encapsulated form, it will be accumulated within the cell as soon as the liposomes are digested with the help of its lysosomal phospholipases. At a certain intracellular clodronate concentration, the macrophage is eliminated by apoptosis. This method has proven its efficacy and specificity for depletion of macrophage subsets in various organs (van Rooijen et al., 1997). We found that depletion of macrophage significantly abolished the improved heart function, the reduced fibrosis, and the augmented angiogenesis by MSCs transplantation, suggesting that the recruited macrophages are necessary for the functional cardiac repair induced by MSCs.
Interestingly, depletion of MSCs after macrophage recruitment similarly abolished the improved heart function and the reduced fibrosis, but not the augmented angiogenesis, suggesting that MSCs transplantation may induce angiogenesis through macrophages, rather than directly by themselves. In line with it, macrophages may secrete angiogenic factors to promote angiogenesis. These data also suggest that besides recruiting macrophages, MSCs themselves are also necessary for the functional cardiac repair. Since previous studies have highlighted an important counteraction between TGF1 and BMP7 in the epithelial-to-mesenchymal transition for fibrosis (Buijs et al., 2007; Khan et al., 2011; Liu et al., 2014; Shen et al., 2014; Wang and Hirschberg, 2003), and macrophages express high TGF1 (Ding et al., 1993; Lazdins et al., 1991; Standiford et al., 2011; Vignola et al., 1996; Xiao
Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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Fig. 7. MSCs contradicted the fibrogenic effect of macrophages. (A) Macrophages (blue rectangle) and MSCs (green rectangle) were isolated from the recipient mouse heart digests by flow cytometry. (B) Expression of TGF1 and BMP7 was analyzed by RT-qPCR in purified macrophages and MSCs, compared to those in the unsorted heart digests Q3 (=1). High TGF1 level was detected in macrophages and high BMP7 level was detected in MSCs. (C) Schematic of the model. * : p < 0.05. N = 5. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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et al., 2014), we thus hypothesize that macrophages may promote fibrosis, and thus have adverse effects on the functional cardiac recovery. Since the effects of MSCs appeared to be positive, it seemed that the fibrogenic effect of macrophages might have been inhibited. Interestingly, our data support our hypothesis, showing that MSCs expressed high BMP7, a known contradictor of TGF1, through which macrophages may promote fibrosis. Our data thus suggest that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGF1 by macrophages. To summarize, our study sheds new insight on the interaction between MSCs and macrophages after MSCs transplantation in a functional cardiac repair. Future studies may address combination of BMP7 and adoptive transfer of macrophages as a novel therapeutic method to facilitate heart recovery from injury.
Conflict of interest None disclosed.
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Wu Y, Zhao RC, Tredget EE. Concise review: bone marrow-derived stem/progenitor cells in cutaneous repair and regeneration. Stem Cells 2010;28:905–15. Xiao X, Gaffar I, Guo P, Wiersch J, Fischbach S, Peirish L, et al. M2 macrophages promote beta-cell proliferation by up-regulation of SMAD7. Proc Natl Acad Sci USA 2014;111:E1211–20. Ye L, Zhang P, Duval S, Su L, Xiong Q, Zhang J. Thymosin beta4 increases the potency of transplanted mesenchymal stem cells for myocardial repair. Circulation 2013;128:S32–41. Zhao QS, Xia N, Zhao N, Li M, Bi CL, Zhu Q, et al. Localization of human mesenchymal stem cells from umbilical cord blood and their role in repair of diabetic foot ulcers in rats. Int J Biol Sci 2013;10:80–9. Zhou B, Cao XC, Fang ZH, Zheng CL, Han ZB, Ren H, et al. Prevention of diabetic microangiopathy by prophylactic transplant of mobilized peripheral blood mononuclear cells. Acta Pharmacol Sin 2007;28:89–97.
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair
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Mei Wang, Guoru Zhang, Yaling Wang, Tao Liu, Yang Zhang, Yu An, Yongjun Li ∗ Department of Cardiology, the Second Hospital of Hebei Medical University, 215 Hepingxin Road, Shijiazhuang, China
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Article history: Received 28 August 2014 Received in revised form 15 October 2014 Accepted 3 November 2014 Available online xxx
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Keywords: Mesenchymal stem cells Cardiac muscle repair Macrophages Transforming growth factor  1 BMP7
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1. Introduction
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Transplantation of bone-marrow derived mesenchymal stem cells (MSCs) has potential therapeutic effects on cardiac muscle repair. However, the underlying mechanism remains not completely clarified. Here we show that transplantation of MSCs significantly increased local recruitment of macrophages to facilitate cardiac muscle repair. MSCs-induced recovery of cardiac function and attenuation of fibrosis after injury were all abolished by either impaired macrophage infiltration, or by MSCs depletion after macrophage recruitment. However, angiogenesis seemed to be only affected by depletion of macrophages, but not by depletion of MSCs, suggesting that macrophages are responsible for the augmented angiogenesis after MSCs transplantation, while MSCs do not directly contribute to angiogenesis in the functional cardiac repair. Moreover, high level of transforming growth factor  1 (TGF1) was detected in macrophages and high level of BMP7 was detected in MSCs, suggesting that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGF1 by macrophages. Our study thus sheds new insight on the interaction of MSCs and macrophages in a functional cardiac repair triggered by MSCs transplantation. © 2014 Published by Elsevier Ltd.
Bone marrow-derived stem/progenitor cells can promote neovascularization in the ischemic myocardium for a functional improvement (Losordo and Dimmeler, 2004a,b). However, the underlying mechanism is not completely understood (Balsam et al., 2004; Fukata et al., 2013; Murry et al., 2004). Moreover, the outcome of transplantation of bone marrow-derived stem/progenitor cells into an injured heart has been extremely variable, which may largely result from the difference in the grafted population of the bone marrow-derived stem/progenitor cells (Balsam et al., 2004, Fukata et al., 2013; Murry et al., 2004; Wu et al., 2010). Mesenchymal stem cells (MSCs) have first been isolated from the bone marrow, and are the most important bone marrow-derived stem/progenitor cells. MSCs have now been identified in all postnatal tissues, and have been shown to be critical for tissue renewal
Abbreviations: MSCs, mesenchymal stem cells; BMP7, bone morphogenetic protein 7; TGF1, transforming growth factor  1; DMSO, dexamethasone; AMI, acute myocardial infarction; DTR, diphtheria toxin receptor; DTA, diphtheria toxin A; LVDd, LV diastolic dimensions; LVFS, percentage of LV fractional shortening. ∗ Corresponding author. Tel.: +86 31187046901; fax: +86 31187046901. E-mail address: yongjun [email protected] (Y. Li).
and repair (Cao et al., 2014; Fawzy et al., 2013; Monsefi et al., 2013; Song et al., 2014; Ye et al., 2013; Zhao et al., 2013). In the current study, we addressed the question about the exact role of MSCs in the cardiac repair, and especially their crosstalk with macrophages. We found that MSCs transplantation significantly increased local infiltration of macrophages, which are necessary for the improved cardiac function, increased angiogenesis and reduction in fibrosis. MSCs themselves, although not required for angiogenesis, appeared to be needed for inhibition of fibrosis and improvement of cardiac function, possibly through their production of bone morphogenetic protein 7 (BMP7) to contradict the fibrogenic effect of transforming growth factor  1 (TGF1) by macrophages.
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2. Materials and methods
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2.1. Mouse handling
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All mouse experiments were approved by the Institutional Animal Care and Use Committee at the Second Hospital of Hebei Medical University (Animal Welfare Assurance). Surgeries were performed under ketamine/xylazine anesthesia, according to the Principles of Laboratory Care. Male 10 week-old C57BL/6 mice were
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used in the current study. Five mice were analyzed in each experimental condition.
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2.2. MSCs isolation, culturing and differentiation
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The MSCs were isolated and grown in culture as has been described previously (Cao et al., 2014; Tropel et al., 2004; Zhou et al., 2007). Briefly, plugs of marrow from five 10-week-old male C57/6BL mice (about 27 g body weight) were dispersed in DMEM and then centrifuged at 900g for 5 min. The pellets were re-suspended and plated at 105 cells/cm2 in DMEM containing 10% FBS. After 10 passages’ selection of attached cells, the cells were sorted for Stro-1 (BD) by flow cytometry to get rid of contaminating cells. A positive clone was selected after subjected to chondrogenetic, osteogenic, and adipogenic differentiation assays for confirming phenotype. For chondrogenetic induction, 2.5 × 105 MSC were induced with 5 mL chondrogenetic induction medium containing 10 g TGF1 (R&D), 50 g insulin growth factor 1 (R&D), and 2 mg/mL dexamethasone (DMSO, Sigma) followed by centrifugation at 500g for 5 min. The cell pellets were maintained in the chondrogenetic induction medium for 14 days and subjected to Alcian blue staining. For osteogenic induction, cells were digested and seeded onto a 24-well plate at a density of 104 cells/well, and then maintained in osteogenic induction medium containing 10 nM Vitamin D3 (Sigma) and 10 mM -phosphoglycerol and 0.1 M DMSO for 14 days and were subjected to Von kossa staining. For adipogenic induction, cells were digested and seeded onto a
24-well plate at a density of 104 cells/well, and then maintained in the adipogenic induction medium containing 0.5 mM 3isobutyl-1-methylxanthine (IBMX), 200 M indomethacin, 10 M insulin and 1 M DMSO for 14 days and subjected to Oil red O staining.
2.3. Transduction of MSCs A recombinant lentivirus expressing diphtheria toxin receptor (DTR) and green fluorescence protein (GFP) under the control of a CMV promoter (Invivogen: Itsint) efficiently transduced MSCs at MOI 100 in high efficiency. The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. Successfully transduced MSCs were selected by flow cytometry based on GFP.
2.4. Acute myocardial infarction (AMI) model and MSCs transplantation A standardized mouse AMI model was applied as has been described before (Kusano et al., 2005). For MSCs transplantation, 106 MSCs were intravenously injected via tail vein to the mice immediately after surgical induction of AMI.
Fig. 1. Isolation, culturing, differentiation and labeling of primary mouse MSCs. (A) Schematic of the viral construct. (B) Transduced green fluorescent MSCs in culture. (C) Purification of transduced MSCs by flow cytometry based on GFP. (D–F) Differentiation assays were performed, including oil red O staining (D), alcian blue staining (E) and von kossa staining (F). (G) The expression level of adipogenic marker GOS2 increased by 88.5 ± 6.2 fold in adipocytes differentiated from MSCs (Adi), compared to undifferentiated MSCs. The expression level of chondrogenic marker BGN increased by 10.5 ± 1.2 fold in chondrocytes differentiated from MSCs (Cho), compared to undifferentiated MSCs. The expression level of osteogenic marker DKK1 increased by 12.5 ± 1.5 fold in osteocytes differentiated from MSCs (Ost), compared to undifferentiated MSCs (G). (H) In vitro administration of DTA was found to efficiently kill cultured DTR-expressing MSCs, by Propidium iodide (PI) staining of dead cells. Scale bars are 50 m. N = 5.
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2.5. Clodronate and diphtheria toxin A injection
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For macrophage depletion, 200 l clodronate-liposomes (Clodronateliposome) were i.v. injected to the mice every other day, from the first day of surgical induction of AMI and MSCs transplantation until analysis. Control mice were injected with liposomes containing PBS, showing same results for all parameters that were analyzed in the current study, compared to the mice treated with AMI and MSCs. For MSCs depletion, 100 ng diphtheria toxin A (DTA) was i.p. injected to the mice that had received DTR-transduced MSCs, for 3 consecutive days since 7 days after MSCs transplantation.
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2.6. Tomato lectin labeling of the vessels
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When the mice were sacrificed, adequate perfusion with saline is performed to remove lectin in the circulation, which avoids its interference with quantification. For evaluating functional vessel density in the heart, DyLight594-labeled Lycopersicon esculentum (tomato lectin, Vector labs) was i.v. injected from the tail vein to the mice 5 min before sacrifice.
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2.7. Heart function evaluation
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Mice underwent echocardiography just before MI (0 week) and at 1, 2, and 4 weeks after AMI as has been described before (Kusano et al., 2005). Transthoracic echocardiography was performed with
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a 6- to 15-MHz transducer (SONOS 5500, Hewlett Packard). Twodimensional images were obtained in the parasternal long and short axis and apical four-chamber views. M-mode images of the left ventricular (LV) short axis were taken just below the level of mid-papillary muscles. LV end-diastolic and end-systolic dimensions were measured and functional shortening was determined according to modified recommendations of American Society of Echocardiography. A mean value of three measurements was determined for each time point. 2.8. Flow cytometry The mouse hearts were digested into single cells as has been described before (Li et al., 2014). The digests were then incubated with M-cadherin antibody (M-Cad, Millipore, USA) followed by labeling with PE-conjugated anti-mouse second antibody (BD, USA). Afterwards, flow cytometry analysis was performed in a FACSAria (Becton Dickinson) flow cytometer. MSCs were recognized by direct fluorescence of GFP. 2.9. Immunohistochemistry Mouse hearts were dissected out and fixed with 4% paraformaldehyde for 6 h, and then cyro-protected in 20% sucrose overnight. Samples were then sectioned in 6 M. For immunostaining, primary antibody is rabbit anti-M-cadherin (M-Cad) and rat anti-F4/80 (Invitrogen, USA). Secondary antibody
Fig. 2. MSCs improved heart function after cardiac muscle injury. MSCs were transplanted immediately after induction of AMI. (A, B) LV end-diastolic and end-systolic dimensions were measured and functional shortening was determined before AMI (week 0) and on 1, 2, 3 and 4 weeks post-AMI. The echocardiography 2, 3, 4 weeks post-AMI showed significantly improved LV diastolic dimensions (LVDd) (A) and percentage of LV fractional shortening (LVFS) (B) in mice transplanted with MSCs compared to saline control. (C, D) Masson’s Trichrome-stained sections showed markedly reduced LV fibrosis, by representative images (C), and by quantification (D). (E, F) Functional cardiac vessel density was quantified by lectin, shown by quantification (E), and by representative images (F). Scale bars are 50 m. * : p < 0.05. N = 5.
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is Cy3-conjugated anti-rabbit antibody (Jackson Labs, USA). GFP and tomato lectin were visualized by direct green and red fluorescence, respectively. DAPI was used to stain nucleus. Vessel density was determined with ImageJ (NIH) software by measuring the percentage of lectin-positive area to the total heart area. For quantification of heart fibrosis, heart sections were stained with Masson trichrome as has been described before (Shen et al., 2014). Evaluation of heart fibrosis in each sample was performed based on 20 randomly selected fields per section, which were examined under ×400 magnification for assessment of the degree of heart fibrosis by visualizing blue-stained areas, exclusive of staining that co-localized with perivascular or intramural vascular structures, the endocardium, or LV trabeculae. ImageJ software was used to determine blue-stained areas and non-stained myocyte areas from each section using color-based threshold. The percentage of total fibrosis area was calculated as the summed blue-stained areas divided by total ventricular area, as has been described previously (Wolf et al., 2005).
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2.10. Quantitative real-time PCR (RT-qPCR)
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RNA was extracted from cultured cells with RNeasy (Qiagen, Hilden, Germany) for cDNA synthesis. RT-qPCR was performed in duplicates with QuantiTect SYBR Green PCR Kit (Qiagen). All primers were purchased from Qiagen. Values of genes were normalized against ␣-tubulin and then compared to the control (=1).
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2.11. Statistics
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All values are depicted as mean ± standard deviation from five individuals and are considered significant if p < 0.05. All data were statistically analyzed using one-way ANOVA with a Bonferoni correction.
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3. Results
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3.1. Isolation, culturing, differentiation and labeling of primary mouse MSCs Primary mouse MSCs were isolated from male C57BL/6 mice, expanded in culture, and then transduced with lentivirus carrying DTR and GFP under the control of a CMV promoter (Fig. 1A). Successfully transduced MSCs were selected by flow cytometry based on GFP (Fig. 1B and C). Differentiation assays were then applied to confirm the MSC-phenotype of the transduced cells, including Von kossa staining to evaluate osteogenic induction, Oil red O staining to evaluate adipogenic induction and Alcian blue staining to evaluate chondrogenetic induction (Fig. 1D–F). Differentiation of MSCs was assured by specific lineage gene expression (Fig. 1G). The expression level of adipogenic marker GOS2 increased by 88.5 ± 6.2 fold in adipocytes differentiated from MSCs, compared to undifferentiated MSCs. The expression level of chondrogenic marker BGN increased by 10.5 ± 1.2 fold in chondrocytes differentiated from MSCs, compared to undifferentiated MSCs. The expression level of osteogenic marker DKK1 increased by 12.5 ± 1.5 fold in osteocytes differentiated from MSCs, compared to undifferentiated MSCs (Fig. 1G). Moreover, in vitro administration of DTA was found to efficiently kill cultured DTR-expressing MSCs (Fig. 1H). 3.2. Therapeutic effects of isogenic MSCs transplantation after cardiac muscle injury In order to evaluate the effect of transplantation of MSCs on injured heart, we transplanted the labeled MSCs immediately after
Fig. 3. Grafted MSCs did not differentiate into cardiac muscle cells. (A) A representative immunostaining image in MSCs-grafted AMI-heart. M-cad: M-cadherin. DAPI: nucleus staining. (B–D) Flow cytometry was applied to M-cad-stained heart digests from mice that received MSCs and AMI, shown by representative flow chart (B). Less than 0.1% GFP-positive cells were M-cad-positive (C), and in all M-cad-positive cells, the percentage of GFP-positive cells was less than 0.01% (D), quantified by immunohistochemistry (IHC) or by FACS. Scale bar is 50 m. * : p < 0.05. NS: non-significant. N = 5.
induction of AMI, as has been described before (Kusano et al., 2005). LV end-diastolic and end-systolic dimensions were measured. Functional shortening was determined before AMI (week 0) and on 1, 2, 3 and 4 weeks post-AMI. The echocardiography 2, 3, 4 weeks post-AMI showed significantly improved LV diastolic dimensions (LVDd) (Fig. 2A) and percentage of LV fractional shortening (LVFS) (Fig. 2B) in mice transplanted with MSCs, compared to the control mice that received saline. Masson’s Trichrome-stained sections showed markedly reduced LV fibrosis, by representative images (Fig. 2C), and by quantification (Fig. 2D). Moreover, functional cardiac vessel density was quantified by perfused lectin, showing significant increase in the mice that received MSCs after AMI (Fig. 2E and F). Lectin labels functional vessels, and appears to be a better marker for evaluating vessel function, compared to CD31, CD144 and CD106, etc. These data suggest that grafted isogenic MSCs improve heart function, reduce fibrosis, and increase angiogenesis after heart injury.
3.3. Grafted MSCs very rarely differentiated into cardiac muscle cells We next examined whether grafted MSCs may differentiate into cardiac muscle cells. We detected many GFP-positive MSCs in the injured mouse heart after MSCs transplantation, but very few of them appeared to express a specific cardiac muscle cell marker, M-cadherin (M-Cad), by immunostaining (Fig. 3A), and by flow cytometry analyses on heart digests (Fig. 3B). Quantification show that less than 0.1% GFP-positive MSCs were M-cad-positive (Fig. 3C), and in all M-cad-positive cells, the percentage of GFPpositive cells was less than 0.01% (Fig. 3D). These data thus suggest that a direct contribution of MSCs to the regenerated cardiac muscle cells is very limited.
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Fig. 4. MSCs transplantation recruited macrophages. (A) RT-qPCR for F4/80 in mouse heart hat received AMI and control saline, or mice that received AMI and MSCs with or without clodronate. (B, C) Analyses of F4/80+ cells in the heart digests by flow cytometry, shown by quantification (B), and by representative flow charts (C). (D) The population of M1 and M2 macrophages in the injured heart appeared to be similar by analysis with a M2 macrophage-specific marker CD163. (E) These macrophages can be readily visualized in the AMI-treated MSCs-grafted heart. Scale bars are 50 m. Clod: clodronate. * : p < 0.05. NS: non-significant. N = 5.
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3.4. Recruited macrophages were necessary for cardiac repair Macrophages are well-known for their essential roles in the tissue regeneration and repair, which prompted us to examine the involvement of macrophages in this model. F4/80 is a specific marker for macrophages. We detected significantly increase in F4/80 transcript level in the AMI-heart grafted with MSCs, compared to the AMI-heart treated with saline, 7 days after AMI and transplantation of MSCs (Fig. 4A). Moreover, significantly higher percentage of F4/80+ cells were detected in the AMI-heart grafted with MSCs, compared to the AMI-heart treated with saline (Fig. 4B and C), suggesting that MSCs recruited macrophages in the injured heart. Moreover, the population of M1 and M2 macrophages in the injured heart appeared to be similar by analysis with a M2 macrophage-specific marker CD163 (Fig. 4D). These macrophages can be readily visualized in the AMI-treated MSCs-grafted heart (Fig. 4E). In order to find out whether macrophages are necessary for the MSCs-induced therapeutic effects on an injured heart, we chemically depleted macrophage infiltration with clodronate, as has been previously described (Cao et al., 2014; Shen et al., 2014; Song et al., 2014; van Rooijen et al., 1997; Xiao et al., 2014). Our data showed that clodronate administration reduced the recruited macrophages
by 70% in the AMI-heart after MSC transplantation (Fig. 4A–C), which significantly abolished the improved LVDd (Fig. 5A) and LVFS (Fig. 5B), the reduction in fibrosis (Fig. 5C), and the augmented angiogenesis (Fig. 5D and E) by MSCs transplantation. These data suggest that the recruited macrophages are necessary for the functional cardiac repair induced by MSCs. 3.5. MSCs had effects other than recruiting macrophages during cardiac repair Then we aimed to figure out whether the effects of MSCs on cardiac repair are limited as to recruit macrophages. We gave the MSCs-grafted AMI-mice with DTA, to eliminate DTR-carrying MSCs, at 7 days after AMI and transplantation of MSCs, when macrophages had been already recruited. We found that MSCs were efficiently killed by DTA, by analyzing GFP transcripts in the heart by RT-qPCR (Fig. 6A), and GFP+ cells in the heart digests by flow cytometry (Fig. 6B). Of note, elimination of MSCs after macrophage recruitment resulted in significant abolishment of the improved LVDd (Fig. 6C) and LVFS (Fig. 6D) and the reduced fibrosis (Fig. 6E), but not of the augmented angiogenesis (Fig. 6F and G) by MSCs transplantation. These data suggest that the recruited macrophages appear to be responsible for the augmented angiogenesis, while MSCs play a
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Fig. 5. Macrophages are necessary for the functional cardiac repair induced by MSCs. (A–E) Clodronate (Clod) was given to deplete macrophages, which significantly abolished the improved LVDd (A) and LVFS (B), the reduction in fibrosis (C), and the increased functional vessel density (D, E) by MSCs transplantation. (D, E) Functional cardiac vessel density was quantified by perfused lectin, shown by quantification (D), and by representative images (E). Scale bars are 50 m. * : p < 0.05. N = 5.
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substantial role in the cardiac functional repair, other than a direct effect on angiogenesis, besides recruitment of macrophages.
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3.6. MSCs seem to contradict the fibrogenic effect of macrophages
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Previous studies have highlighted an important counteraction between TGF1 and BMP7 in the epithelial-to-mesenchymal transition for fibrosis (Buijs et al., 2007; Khan et al., 2011; Liu et al., 2014; Shen et al., 2014; Wang and Hirschberg, 2003). Since TGF1 has been reported to be highly expressed by macrophages (Ding et al., 1993; Lazdins et al., 1991; Standiford et al., 2011; Vignola et al., 1996; Xiao et al., 2014), we thus examined the expression of TGF1 and BMP7 in the isolated macrophages and MSCs from the recipient heart (Fig. 7A). We detected high TGF1 level in macrophages and high BMP7 level in MSCs (Fig. 7B). These data may imply that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGF1 by macrophages (Fig. 7C). 4. Discussion Transplantation of bone-marrow derived MSCs has an established therapeutic effect on cardiac muscle injury. However, previous studies failed to reach consistent results on the underlying mechanism (Balsam et al., 2004; Fukata et al., 2013; Murry et al., 2004; Wu et al., 2010), which prompted us to address this question in the current study. We chose a strict inbred strain, C57BL/6 mice, for isogenic transplantation in our study. We first isolated and cultured primary
mouse MSCs from male C57BL/6 mice, and then labeled these cells with a GFP reporter and a DTR, for in vivo lineage tracing and lineage ablation, respectively. The GFP labeling of the MSCs allow us to analyze their possible contribution to other lineages in vivo, by double immunostaining, and by flow cytometry. Indeed, we found little of differentiation of MSCs into cardiac muscle cells. The toxicity of DTA is extremely high that one molecule of DTA in the cytosol may be enough to kill the cell. However, mouse cells do not have the DTR to allow DTA to enter the cells. Thus, administration of DTA to the mice did not kill any cells from the recipient mice. On the other hand, since MSCs were transduced with DTR, they became very susceptible to DTA toxicity. The power of this system was assured in our experiment using DTA to eliminate the transplanted MSCs. The phenotypes of the transduced MSCs were further confirmed by three differentiation assays. In order to evaluate the effect of transplantation of MSCs on injured heart, we transplanted the MSCs immediately after induction of AMI. Significant improvement in LVDd and LVFS, reduced fibrosis, and increased vascularization in MSCs-grafted mice demonstrate a substantial role of MSCs in cardiac muscle repair. Macrophages are well-known for their importance in the tissue regeneration and repair, and in the organ remodeling and fibrosis. We found five times more macrophages in the AMI-heart grafted with MSCs, compared to the AMI-hearted treated with saline, suggesting that MSCs recruited macrophages in the injured heart. We then used clodronate to deplete macrophages to see whether the effects of MSCs on cardiac repair may be affected by macrophage depletion. Clodronate is a hydrophilic molecule that is packaged in a
Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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Fig. 6. MSCs have an angiogenesis-unrelated role in the functional cardiac repair. (A–G) DTA was given to the MSCs-grafted AMI mice to eliminate DTR-carrying MSCs, at 7 days after AMI and MSCs transplantation, when macrophages were already recruited. (A) MSCs were efficiently killed by DTA, by analyzing GFP transcripts in the heart (A), and GFP+ cells in the heart digests (B). (C–G) Removal of MSCs significantly abolished the improved LVDd (C) and LVFS (D), the reduction in fibrosis (E), but had no effects on the increased functional vessel density (F, G). (F, G) Functional cardiac vessel density was quantified by perfused lectin, shown by quantification (F), and by representative images (G). Scale bars are 50 m. * : p < 0.05. N = 5.
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liposome to mediate internalization clodronate into macrophages. This molecule has a short half-life when released in the circulation, but does not easily cross phospholipid bilayers of liposomes or cell membranes. As a consequence, once ingested by a macrophage in a liposome encapsulated form, it will be accumulated within the cell as soon as the liposomes are digested with the help of its lysosomal phospholipases. At a certain intracellular clodronate concentration, the macrophage is eliminated by apoptosis. This method has proven its efficacy and specificity for depletion of macrophage subsets in various organs (van Rooijen et al., 1997). We found that depletion of macrophage significantly abolished the improved heart function, the reduced fibrosis, and the augmented angiogenesis by MSCs transplantation, suggesting that the recruited macrophages are necessary for the functional cardiac repair induced by MSCs.
Interestingly, depletion of MSCs after macrophage recruitment similarly abolished the improved heart function and the reduced fibrosis, but not the augmented angiogenesis, suggesting that MSCs transplantation may induce angiogenesis through macrophages, rather than directly by themselves. In line with it, macrophages may secrete angiogenic factors to promote angiogenesis. These data also suggest that besides recruiting macrophages, MSCs themselves are also necessary for the functional cardiac repair. Since previous studies have highlighted an important counteraction between TGF1 and BMP7 in the epithelial-to-mesenchymal transition for fibrosis (Buijs et al., 2007; Khan et al., 2011; Liu et al., 2014; Shen et al., 2014; Wang and Hirschberg, 2003), and macrophages express high TGF1 (Ding et al., 1993; Lazdins et al., 1991; Standiford et al., 2011; Vignola et al., 1996; Xiao
Please cite this article in press as: Wang M, et al. Crosstalk of mesenchymal stem cells and macrophages promotes cardiac muscle repair. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.11.003
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Fig. 7. MSCs contradicted the fibrogenic effect of macrophages. (A) Macrophages (blue rectangle) and MSCs (green rectangle) were isolated from the recipient mouse heart digests by flow cytometry. (B) Expression of TGF1 and BMP7 was analyzed by RT-qPCR in purified macrophages and MSCs, compared to those in the unsorted heart digests Q3 (=1). High TGF1 level was detected in macrophages and high BMP7 level was detected in MSCs. (C) Schematic of the model. * : p < 0.05. N = 5. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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et al., 2014), we thus hypothesize that macrophages may promote fibrosis, and thus have adverse effects on the functional cardiac recovery. Since the effects of MSCs appeared to be positive, it seemed that the fibrogenic effect of macrophages might have been inhibited. Interestingly, our data support our hypothesis, showing that MSCs expressed high BMP7, a known contradictor of TGF1, through which macrophages may promote fibrosis. Our data thus suggest that MSCs not only may recruit macrophages to enhance angiogenesis to promote regeneration, but also may secrete BMP7 to contradict the fibrogenic effect of TGF1 by macrophages. To summarize, our study sheds new insight on the interaction between MSCs and macrophages after MSCs transplantation in a functional cardiac repair. Future studies may address combination of BMP7 and adoptive transfer of macrophages as a novel therapeutic method to facilitate heart recovery from injury.
Conflict of interest None disclosed.
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