Plasminogen Regulates Cardiac Repair After Myocardial ... - JACC

Journal of the American College of Cardiology 2014 by the American College of Cardiology Foundation Published by Elsevier Inc.

Vol. 63, No. 25, 2014 ISSN 0735-1097/$36.00 http://dx.doi.org/10.1016/j.jacc.2013.11.070

PRE-CLINICAL RESEARCH

Plasminogen Regulates Cardiac Repair After Myocardial Infarction Through its Noncanonical Function in Stem Cell Homing to the Infarcted Heart Yanqing Gong, PHD,*y Yujing Zhao, BS,* Ying Li, BS,* Yi Fan, MD, PHD,z Jane Hoover-Plow, PHD* Cleveland, Ohio; and Philadelphia, Pennsylvania Objectives

The purpose of this study was to investigate the role of plasminogen (Plg) in stem cell–mediated cardiac repair and regeneration after myocardial infarction (MI).

Background

An MI induces irreversible tissue damage, eventually leading to heart failure. Bone marrow (BM)–derived stem cells promote tissue repair and regeneration after MI. Thrombolytic treatment with Plg activators significantly improves the clinical outcome in MI by restoring cardiac perfusion. However, the role of Plg in stem cell–mediated cardiac repair remains unclear.

Methods

An MI was induced in Plg-deficient (Plg/) and wild-type (Plgþ/þ) mice by ligation of the left anterior descending coronary artery. Stem cells were visualized by in vivo tracking of green fluorescent protein (GFP)-expressing BM cells after BM transplantation. Cardiac function, stem cell homing, and signaling pathways downstream of Plg were examined.

Results

Granulocyte colony-stimulating factor, a stem cell mobilizer, significantly promoted BM-derived stem cell (GFPþc-kitþ cell) recruitment into the infarcted heart and stem cell–mediated cardiac repair in Plgþ/þ mice. However, Plg deficiency markedly inhibited stem cell homing and cardiac repair, suggesting that Plg is critical for stem cell–mediated cardiac repair. Moreover, Plg regulated C-X-C chemokine receptor type 4 (CXCR4) expression in stem cells in vivo and in vitro through matrix metalloproteinase-9. Lentiviral reconstitution of CXCR4 expression in BM cells successfully rescued stem cell homing to the infarcted heart in Plg-deficient mice, indicating that CXCR4 has a critical role in Plg-mediated stem cell homing after MI.

Conclusions

These findings have identified a novel role for Plg in stem cell–mediated cardiac repair after MI. Thus, targeting Plg may offer a new therapeutic strategy for stem cell–mediated cardiac repair after MI. (J Am Coll Cardiol 2014;63:2862–72) ª 2014 by the American College of Cardiology Foundation

Ischemic heart disease, including myocardial infarction (MI), is a major cause of death and disability worldwide. Obstruction of coronary arteries leads to MI, with the associated death of cardiomyocytes. Treatments aimed at

From the *Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Departments of Cardiovascular Medicine and Molecular Cardiology, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio; yDivision of Translational Medicine and Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and the zDepartment of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. This study was funded in part by grants from the American Heart Association (09BGIA2050157 and 12SDG9050018) and the National Institutes of Health, National Heart, Lung, and Blood Institute (R01HL078701 and R01HL17964 to Dr. Hoover-Plow and K99HL103792 to Dr. Fan). The authors have reported that they have no relationships relevant to the contents of this paper to disclose. Manuscript received August 7, 2013; revised manuscript received November 5, 2013, accepted November 26, 2013.

restoring blood supply rapidly in the infarct heart, thrombolytic therapy and primary angioplasty, have significantly decreased early mortality of patients with MI. However, continuous overloads in the surviving myocardium eventually leads to heart failure. Epidemiological data have shown that about 60% of heart failure results from ischemic heart disease (1). Standard therapy for heart failure that addresses See page 2873

the fundamental problem of cardiomyocytes loss is cardiac transplantation, but this treatment has limited application because of insufficient donor hearts and the need for longterm immunosuppressive therapy. New discoveries on the regenerative potential of stem cells for preventing heart

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failure have transformed experimental research and led to an explosion in clinical investigation (2–6). Stem cell homing to the heart to promote cardiac repair and regeneration after MI is a naturally occurring process. However, it occurs too slowly, and recruited stem cells are not sufficient to be meaningful for the recovery of heart function (7). To reach the efficient regeneration of damaged myocardium, therapeutic treatments have been developed by transplantation of a large number of stem cells into the bloodstream or infarcted heart or enhanced mobilization of stem cells from bone marrow by treatments with cytokines, such as granulocyte colony-stimulating factor (G-CSF) (8,9). However, insufficient stem cell engraftment and survival during these treatments have limited the application of stem cell therapy for treating MI (10,11). Therefore, a better understanding of the underlying mechanisms regulating stem cell function during cardiac repair may lead to the development of novel approaches for stem cell therapies. Plasminogen (Plg) is the main enzyme responsible for fibrinolysis. The Plg system contains a proenzyme, Plg, which is converted to the active enzyme plasmin by tissue Plg activator or urokinase Plg activator (12). As a thrombolytic agent, tissue Plg activator (tPA) has been utilized as the first line of treatment of acute MI for almost 2 decades. In addition to its canonical function, Plg is critical for cardiac repair after MI, wound healing, and liver injury (13–15). However, the mechanism for Plg-regulated cardiac repair remains largely unknown. In this study, using a knockout mouse model, stem cell tracking and genetic lentiviral approaches, and an experimental MI model, we have elucidated a novel mechanism by which Plg induces recruitment of bone marrow (BM)– derived stem cells to the infarcted heart, promoting cardiac repair after MI. Moreover, our data reveal that Plg induces C-X-C chemokine receptor type 4 (CXCR4) expression in migrating BM stem cells and may contribute to stem cell recruitment to the infarcted heart. Methods All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee. The BM transplantation and induction of MI, flow cytometric analysis, histochemistry and immunohistology of heart sections, Western blot of CXCR4 expression, zymography of matrix metalloproteinase-9 (MMP-9) activity, in vitro chemotaxis assay, and data analysis are described in the Online Appendix. Results Plg is critical for G-CSF–stimulated cardiac function recovery and tissue repair after infarction. G-CSF promotes the recruitment of BM-derived stem cell to injured tissue, leading to improvement of tissue repair and heart function recovery in MI models (9,16–18). To investigate

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the role of Plg in stem cell– Abbreviations and Acronyms mediated tissue repair and heart function recovery, MI was inBM = bone marrow duced in Plgþ/þ and Plg/ mice BMNC = bone marrow by ligation of the left anterior mononuclear cell descending coronary artery. Mice CXCR4 = C-X-C chemokine were treated with G-CSF to receptor type 4 stimulate stem cell mobilization EC = endothelial cells from BM and recruitment to the FACS = fluorescence infarct heart, and cardiac funcactivated cell sorting tion was analyzed by echocardiG-CSF = granulocyte ography. Plg deficiency did not colony-stimulating factor alter normal heart function, as GFP = green fluorescent indicated by the similar ejection protein fraction of the left ventricle LV = left ventricle/ (LV) observed in Plgþ/þ (86.7 ventricular 2.4%, n ¼ 6) and Plg/ MMP = matrix (87.6 0.8%, n ¼ 8) mice metalloproteinase (Fig. 1A). The ligation surPlg = plasminogen gery induced inadequate cardiac SDF = stromal cell-derived function, reducing LV ejection factor fraction by about 50% in Plgþ/þ SMC = vascular smooth and Plg/ mice (Fig. 1A). No muscle cells spontaneous recovery of heart function was observed in the 28 days after MI surgery, as shown by decreased ejection faction detected in Plgþ/þ and Plg/ mice. Importantly, G-CSF significantly increased ejection fraction by one-half in Plgþ/þ, but not in Plg/ mice (Fig. 1A). Consistently, G-CSF reduced LV internal diameter by approximately 30% to 40% in Plgþ/þ, but not in Plg/ mice (Fig. 1B). These data indicate that Plg is critical for G-CSF–stimulated heart function recovery after MI. We investigated the role of Plg in G-CSF–mediated tissue repair after MI. G-CSF treatment significantly improved post-MI tissue repair in Plgþ/þ mice 4 weeks after MI surgery, as evidenced by decreased infarct size by 30% (Figs. 1C and 1D). In contrast, Plg/ mice had even slightly larger infarct size, indicating that Plg is required for G-CSF–stimulated tissue repair. Formation of new blood vessels (i.e., neovascularization) is a fundamental process during cardiac repair after MI. G-CSF induced a significant, 2-fold increase in microvascular density in the infarcted area in Plgþ/þ mice (Figs. 1E and 1F). Plg deficiency slightly decreased basal neovascularization (without G-CSF treatment), and completely abolished G-CSF–induced neovascularization. Together, these data establish a critical role of Plg in G-CSF–stimulated tissue repair and cardiac function recovery after MI, implicating that Plg may promote stem cell recruitment to improve cardiac repair and regeneration. Plg is required for BM-derived stem cell recruitment in the infarcted heart. G-CSF treatment is widely used to enhance stem cell mobilization and recruitment to improve cardiac repair after MI (9,18,19). Our previous work has shown that Plg is required for G-CSF–induced stem cell mobilization from BM to the circulation (20). To evaluate

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Figure 1


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Plg Is Required for G-CSF-Stimulated Heart Function Recovery After MI

(A to F) Myocardial infarction (MI) was induced in mice by ligation of the left anterior descending coronary artery, followed by treatment with or without granulocyte colonystimulating factor (G-CSF) for 5 days. (A and B) Heart function was evaluated by echocardiography before MI and 3 and 28 days after MI, and ejection fraction and left ventricular (LV) interval diameter were measured. Data expressed as mean SEM (n ¼ 5 to 8). (C to F) At 28 days after MI, heart tissue was harvested. (C and D) Heart sections were stained with Masson’s trichrome dye. (C) Representative whole-heart images for serial sections. (D) Quantified data for infarct size (mean SEM, n ¼ 5 to 6). (E and F) Heart sections were subjected to immunofluorescence analysis with anti-CD31 antibody. (E) Representative images. (F) Quantified data for vessel density (mean SEM, n ¼ 4). *p < 0.05. Plg ¼ plasminogen.

the role of Plg in stem cell recruitment to the infarct heart and cardiac repair after MI, we initially analyzed stem cell mobilization after MI. An MI itself induced a moderate stem cell mobilization, as indicated by a 2-fold increase in cell number of stem cells (Linc-kitþ cells) detected by fluorescence-activated cell sorting (FACS) in circulation in Plgþ/þ mice (Fig. 2A). G-CSF treatment robustly promoted stem cell mobilization, with an 8-fold increase in the number of stem cells in the blood of Plgþ/þ MI mice. Plg deficiency almost completely inhibited MIinduced stem cell mobilization and significantly inhibited

G-CSF–stimulated mobilization, suggesting a necessary role of Plg in stem cell mobilization in response to cardiac infarction with or without G-CSF administration. To track BM-derived stem cell recruitment in vivo after MI, we performed BM transplantation to allow a visualization of BM-derived green fluorescent protein (GFPþ) cells in the infarcted cardiac tissue (Fig. 2B). Immunofluorescence analysis showed that MI induced a mild recruitment of BM-derived GFPþ cells and c-kitþ stem cells in the infarct site, whereas G-CSF treatment induced marked cell recruitment in Plgþ/þ mice (Fig. 2C). Importantly,

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Figure 2


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Plg Is Critical for Stem Cell Mobilization and Recruitment From BM to Infarcted Heart After MI

(A) At 5 days after MI surgery or sham operation with or without G-CSF injection, Lin–c-kitþ cells in blood were counted by fluorescence-activated cell sorting (FACS) (mean SEM, n ¼ 6). *p < 0.05. (B) Scheme of experimental protocol. Bone marrow (BM) cells isolated from green fluorescent protein (GFP) mice were transplanted into lethally irradiated mice. An MI was induced, followed by different analyses. (C and D) At 10 days after MI surgery, heart tissue was harvested and subjected to immunofluorescence analysis with anti-GFP and anti–c-kit antibodies. Nuclei were stained with 40 ,6-diamidino-2-phenylindole. (C) Representative images. GFPþc-kitþ cells (yellow in merged pictures) indicate BM-derived stem cells. (D) Graph represents the percentage of GFPþc-kitþ cells in infarct zone (mean SEM, n ¼ 4 to 5). **p < 0.01. CXCR ¼ C-X-C chemokine receptor type 4; other abbreviations as in Figure 1.

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Figure 3


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Plg is Required for Neovascularization Mediated by BM Cell-Derived Cells

(A to D) GFPþ BM cells were transplanted to recipient mice, and MI was induced, followed by treatment with or without G-CSF. At 10 days after MI surgery, heart tissue was harvested and immunohistochemically stained. (A) Heart sections were stained with anti-GFP and anti-CD31 antibodies. GFPþCD31þ cells indicate BM-derived endothelial cells (yellow in merged pictures). (B) Heart sections were stained with anti-GFP and anti–a-vascular smooth muscle cell (SMC) actin antibodies. GFPþa-SMC actinþ cells indicate BM-derived endothelial cells (yellow in merged pictures). (C and D) Graph represents the percentage of GFPþCD31þ and GFPþa-SMC actinþ cells in infarct zone (mean SEM, n ¼ 4 to 5). **p < 0.01. Abbreviations as in Figures 1 and 2.

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Figure 4


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Plg Deficiency Inhibits CXCR4 Expression in Infarcted Cardiac Tissue and BM Cells

(A to D) An MI was induced in mice, followed by treatment with or without G-CSF. At 10 days after MI surgery, heart tissue was collected and immunohistochemically stained with anti–SDF-1 (A and B) and anti-CXCR4 (C and D) antibodies. (A) Representative images for SDF-1 expression in infarct heart. (B) Graph represents SDF-1 expression level in infarct heart (mean SEM, n ¼ 4 to 5). (C) Representative images for CXCR4 expression in infarct heart. (D) Graph represents CXCR4 expression level in infarct heart (mean SEM, n ¼ 4 to 5). (E and F) An MI was induced, followed by treatment with or without G-CSF. At 5 days after MI surgery, BM cells were collected. (E) Cell lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblot with anti-CXCR4 antibody. (F) The percentage of CXCR4þc-kitþ cells was measured by FACS analysis (mean SEM, n ¼ 7 to 9). *p < 0.05. **p < 0.01. NS ¼ no significance; other abbreviations as in Figures 1 and 2.

Plg deficiency significantly reduced both MI-induced and G-CSF–stimulated stem cell recruitment. Quantitative data showed that Plg deficiency almost completely abolished BM-derived GFPþc-kitþ stem cells after MI with or without G-CSF treatment (Fig. 2D). Interestingly, Plg deficiency had a much more profound effect on stem cell recruitment (an 11-fold reduction, almost a reduction to basal level) than cell mobilization (a 2-fold reduction), suggesting a new, direct role of Plg in stem cell recruitment

and engraftment in addition to its known function in stem cell mobilization. Plg induces stem cell–mediated neovascularization after MI. Neovascularization is critical for cardiac repair and function recovery after MI. Stem cells promote post-MI neovascularization and tissue regeneration by differentiation to new vascular cells, including endothelial cells (EC) and vascular smooth muscle cells (SMC) (21). We investigated the role of Plg in stem cell–mediated neovascularization.

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Figure 5


Plg Induces CXCR4 Expression and Cell Migration in BM Cells

(A and B) BMNCs were isolated from BM and treated with Plg for 40 hours. (A) Cell lysate was analyzed by immunoblot with anti-CXCR4 antibody. (B) Cells treated with or without Plg (10 mg/ml) were immunostained with anti-CXCR4 antibody, followed by FACS analysis. Inset: quantitative data for percentage of CXCR4þ cells (C) Cell migration of BMNCs was induced in response to SDF-1 with or without Plg (10 mg/ml) and AMD3100. Migrated cells were collected and counted by hemocytometry, and subjected to FACS analysis. Data expressed as mean SEM (n ¼ 3). **p < 0.01.

After BM transplantation with GFPþ BM cells and MI induction, BM-derived cells, EC, and SMC were visualized by immunofluorescence with anti-GFP, -CD31, and -a-SMC actin antibodies, respectively. Colocalization analysis showed that over one-half of CD31þ and a-SMC actinþ cells expressed GFP, confirming their BM origin and suggesting that BM-derived stem cells significantly

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contribute to newly generated EC and SMC (Figs. 3A and 3B). G-CSF substantially increased the cells number of BM-derived EC and SMC, detected as GFPþCD31þ and GFPþa-SMC actinþ cells, respectively (Figs. 3C and 3D), likely due to the stimulation in stem cell recruitment. Plg deficiency inhibited post-MI regeneration of EC and SMC by BM-derived cells with or without G-CSF treatment, as indicated by the remarkably diminished co-localization of GFPþCD31þ and GFPþa-SMC actinþ cells in the infarct site of Plg/ mice. These data suggest that Plg is critical for stem cell–mediated neovascularization, contributing to cardiac repair after MI. Plg promotes CXCR4 expression during stem cell recruitment. Stromal cell-derived factor-1 (SDF-1)/ CXCR4 is the major chemoattractant ligand/receptor for stem cell recruitment after MI (22–24). Immunohistochemistry analysis showed that G-CSF induced robust expression of both SDF-1 and CXCR4 in infarcted cardiac tissue (Figs. 4A to 4D), consistent with their function in enhancing stem cell mobilization and recruitment. To explore the molecular mechanism of Plg in stem cell recruitment, we investigated whether Plg regulates SDF-1/ CXCR4 expression. Plg did not affect SDF-1 expression in infarcted heart tissue, as evidenced by the lack of a difference in SDF-1 expression detected in Plgþ/þ and Plg/ mice treated with or without G-CSF (Figs. 4A and 4B). However, Plg deficiency significantly inhibited CXCR4 expression in heart infarcts and abolished G-CSF–enhanced CXCR4 expression (Figs. 4C and 4D), but did not affect basal expression of CXCR4 in normal cardiac tissue without MI (data not shown), suggesting that Plg is critical for CXCR4 expression in infarcted heart and particularly important for GCSF–stimulated CXCR4 expression. The reduced CXCR4 expression in heart infarcts in Plg/ mice may be due to a decrease in the recruitment of CXCR4þ stem cells and/or a decrease in the expression of CXCR4 in BM-derived stem cells. To test the latter hypothesis, CXCR4 expression was analyzed in the BM cells of Plgþ/þ and Plg/ mice who received MI surgery and were treated with or without G-CSF. Immunoblot analysis showed that MI surgery induced CXCR4 expression in BM cells and that G-CSF further increased its expression in Plgþ/þ mice (Fig. 4E), consistent with their functions in promotion of stem cell mobilization to circulation and recruitment in heart infarcts (Fig. 2A), whereas basal expression of CXCR4 without MI stress or G-CSF challenge was barely detectable in BM cells of Plgþ/þ and Plg/ control mice (Fig. 4E). Plg deficiency completely abolished the CXCR4 expression in BM cells, suggesting that Plg is necessary for MI-inducible, G-CSF–stimulated CXCR4 expression. Similarly, FACS analysis of BM c-kitþ stem cells showed that MI and G-CSF up-regulated CXCR4 expression in BM stem cells of Plgþ/þ mice, and Plg deficiency significantly inhibited MI-inducible, G-CSF–stimulated CXCR4 expression in BM stem cells (Fig. 4F). These data suggest that MI stress and G-CSF challenge induce Plg-dependent CXCR4 expression in

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Figure 6


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CXCR4 Expression Is Required for Plg-Mediated Stem Cell Recruitment

(A to F) The BM cells isolated from GFP mice were transduced with lentivirus that expressed CXCR4 and were transplanted into lethally irradiated recipient mice. At 4 weeks after transplantation, MI was induced, followed with G-CSF injection treatment (n ¼ 6). (A) Scheme of experimental protocol. (B) The BM cells were lentivirally transduced, and cell lysate was immunoblotted with anti-CXCR4 and anti–b-actin antibodies. (C) At 10 days after MI, BM cells were isolated from BM transplantation recipient mice and subjected to immunoblot analysis with anti-CXCR4 antibody. (D to F) At 10 days after MI, cardiac tissue was harvested and subjected to immunofluorescence analysis with anti-GFP and anti–c-kit antibodies. Nuclei were stained blue by 40 ,6-diamidino-2-phenylindole. (D) Representative whole-heart images for GFP. White dashed lines indicate infarct area. (E) Representative images for BM-derived stem cells (GFPþc-kitþ, yellow in merged pictures) in infarcted area. (F) Graph represents the percentage of GFPþc-kitþ cells in infarct zone (mean SEM, n ¼ 4 to 5). **p < 0.01. Abbreviations as in Figures 1 and 2.

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Figure 7


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Plg-Mediated CXCR4 Expression and Stem Cell Migration Depends on MMP-9 Activation

(A and B) An MI was induced in mice, followed by treatment with or without G-CSF for 5 days. Protease activity in blood plasma was analyzed by gelatin zymography, and the proenzyme and active forms of MMP-9 were identified by molecular weight relative to markers. (A) Representative images. (B) Quantitative data of the intensity of MMP-9 bands are shown (mean SEM, n ¼ 3). (C) Bone marrow mononuclear cells (BMNCs) were treated with Plg (10 mg/ml) for 40 h. Cell culture medium was collected and subjected to gelatin zymography analysis. (D) Cell migration in BMNCs was induced by SDF-1 in the presence or absence of Plg with control immunoglobulin (Ig)G and MMP-9 antibody. Cell number of migrated BMNCs was counted (mean SEM, n ¼ 3). (E) The BMNCs were treated with Plg in the presence of control IgG or MMP-9 neutralizing antibody and were subjected to FACS analysis with anti-CXCR4 antibody. Inset: quantitative data for percentage of CXCR4þ cells. *p < 0.05. **p < 0.01. Abbreviations as in Figures 1 and 2.

BM-derived stem cells, leading to stem cell recruitment to infarct site and cardiac repair after MI. Plg induces cell migration by up-regulating CXCR4 expression in BM-derived cells. To test whether Plg directly regulates CXCR4 expression in BM cells, bone marrow mononuclear cells (BMNCs) were isolated and treated with Plg in vitro. Immunoblot analysis showed that Plg induced CXCR4 expression in BMNCs in a dosedependent manner (Fig. 5A). FACS analysis confirmed a 2-fold up-regulation in CXCR4 expression induced by 2 mg/ml Plg (Fig. 5B).We further investigated whether CXCR4 is critical for Plg-mediated stem cell migration. BMNC migration was induced by SDF-1 chemotaxis in the presence or absence of AMD3100, a CXCR4 antagonist that inhibits CXCR4 binding to SDF-1 and blocks CXCR4 function. Plg significantly increased cell migration of total BM cells and Linc-kitþ stem cell migration in response to SDF-1 (Fig. 5C), as determined by counting migrated BMNCs by hemocytometry and Linckitþ stem cells by FACS (Fig. 5C). Importantly, AMD3100 significantly inhibited Plg-stimulated BM cell migration and completed abolished Plg-mediated Linckitþ stem cell migration, indicating that CXCR4 is required for Plg-stimulated cell

migration in BM cells, particularly in BM-derived stem cells. CXCR4 is required for Plg-mediated BM-derived stem cell recruitment to the infarct site after MI. To investigate the role of CXCR4 in Plg-mediated stem cell recruitment after MI, we tested whether CXCR4 overexpression rescues the diminished stem cell recruitment induced by Plg deficiency (Fig. 6A). GFPþ BM cells were lentivirally transduced to overexpress CXCR4, as indicated by the 2-fold higher expression at the protein level (Fig. 6B). The transduced BM cells were transplanted to lethally radiated Plgþ/þ and Plg/ recipient mice, which efficiently restored CXCR4 expression in BM cells in Plg/ mice (Fig. 6C). Mice were subjected to MI induction and G-CSF challenge. Immunofluorescence analysis showed that restoration of CXCR4 expression efficiently rescued the recruitment of BM-derived cells (GFPþ cells) in infarcted heart tissue of Plg/ mice (Fig. 6D). Similarly, recruitment of BM-derived stem cells, detected as GFPþc-kitþ cells, was significantly enhanced by the CXCR4 overexpression in Plg/ mice (Figs. 6E and 6F), suggesting that CXCR4 is critical for Plg-mediated stem cell recruitment after MI.

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Plg-mediated CXCR4 expression depends on MMP-9 activation. Matrix metalloproteinases, including MMP-3, -9, and -13, serve as downstream targets of plasmin activity (25,26), which may be involved in Plg-mediated stem cell recruitment. Zymography assay showed that no detectable MMP-9 activation was observed in the BM of Plgþ/þ or Plg/ mice before MI surgery (data not shown); however, MI induced remarkable MMP-9 activation in the BM of Plg/ mice, which was further increased by G-CSF treatment (Figs. 7A and 7B). Importantly, Plg deficiency significantly attenuated the MMP-9 activation induced by MI with or without G-CSF challenge, suggesting that Plg induces MMP-9 activation in BM cells after MI. Consistently, Plg treatment in vitro with BMNCs isolated from BM cells induced MMP-9 activation (Fig. 7C). To test whether MMP-9 activation is required for Plg-mediated cell migration, BMNCs were induced to migrate in response to SDF-1 in the presence of Plg. MMP-9 activation was blocked by adding a neutralizing antibody, which efficiently abolished MMP-9 activity, as previously shown (25). MMP-9 neutralization inhibited the Plg-induced cell migration in response to SDF-1 in BMNCs (data not shown) and Linc-kitþ stem cells (Fig. 7D), indicating that MMP-9 is essential for Plgmediated stem cell migration. We also tested the role of MMP-9 in Plg-mediated CXCR4 expression. MMP-9 neutralizing antibody significantly inhibited Plg-regulated CXCR4 expression (Fig. 7D). These results suggest that MMP-9 may serve as a mediator for Plg-mediated CXCR4 expression and stem cell recruitment after MI. Discussion Despite many breakthroughs in cardiovascular medicine, MI remains a major cause of mortality in the United States. Stem cell recruitment and differentiation to mature cells, including cardiomyocytes and vascular cells, to replace injured cardiac tissue is a key repair process after MI. Our study elucidates a Plg-mediated molecular mechanism regulating stem cell recruitment and cardiac repair after MI. CXCR4 is a major chemotactic receptor for stem cell migration, which is critical for stem cell mobilization and recruitment. Here, we reveal an important mechanism for up-regulation of CXCR4 expression, leading to stem cell recruitment and tissue repair. These findings will contribute to the development of new therapeutic strategies (e.g., targeting Plg/MMP-9) for strengthening stem cell therapy for sole or G-CSF–combined MI treatment. In addition to its canonical, antithrombosis function, Plg is critical for tissue repair after MI, wound healing, and liver injury (13–15,27,28). Notably, deficiency of urokinase Plg activator completely protected against rupture but impaired scar formation and infarct revascularization, whereas Plg deficiency abolished inflammatory cell infiltration and necrotic cardiomyocyte removal, suggesting that the Plg system is required for the cardiac repair after MI (13,29). Recent studies have also shown that Plg is critical for stem


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cell mobilization after 5-fluoruracil–induced myelosuppression and G-CSF challenge (30–33). Here, for the first time to our knowledge, we demonstrate that Plg regulates stem cell homing to the infarcted heart and contributes to cardiac repair after MI. SDF-1/CXCR4 is a critical chemotaxis pathway for stem cell mobilization and recruitment after MI (22–24,34). Upon injury, CXCR4 expression is induced in the stem cells, and CXCR4þ stem cells are recruited from BM to the lesion in response to SDF-1 that is overexpressed in the lesion, which is critical for cardiac repair after MI. Based on this function, several therapeutic strategies have been developed to enhance stem cell–mediated tissue repair, including local SDF-1 delivery and inhibition of SDF-1 cleavage, pharmacological mobilization of CXCR4þ cells from BM, and genetic up-regulation of CXCR4 expression in BM-derived stem cells (35,36). The mechanism underlying regulation of SDF-1/CXCR4 signals during stem cell recruitment has not been fully understood. Here, we show that Plg contributes importantly to regulation of CXCR4 expression in recruiting stem cells after MI. CXCR4 expression is subject to transcriptional regulation as well as receptor desensitization and degradation by multiple signals including calcium, cAMP, PKA, and PKC (37). Whether and how Plg regulates these signals remain unclear. MMP-9 serves as a common downstream signal for Plg to regulate inflammatory cell migration in many pathological processes, including aneurysm formation and atherosclerosis development (25,38,39). Here, we reveal that MMP-9 is required for Plg-mediated CXCR4 expression in stem cells during cardiac repair after MI. Consistently, a recent study has shown that CXCR4 is critical for MMP-9–mediated stem cell migration, as evidenced by G-CSF stimulation of MMP9 activity in BM cells, but no stem cell mobilization in CXCR4/ mice, and by the fact that transplantation of CXCR4þVEGFR2þ cells into MMP-9/ mice rescues the impaired ischemic revascularization (40,41). Our findings provide the direct evidence that Plg-dependent MMP-9 activation regulates CXCR4 expression for stem cell recruitment. Several possible mechanisms may contribute to this process, including Plg/MMP-9–mediated up-regulation of CXCR4 messenger ribonucleic acid expression by activation of intracellular signals or autocrine/paracrine effects by stimulated growth factors and cytokines, and protease-inducible inhibition of CXCR4 desensitization and degradation. Conclusions We elucidate a distinct mechanism underlying stem cell– mediated cardiac repair after MI. In this newly identified pathway, Plg regulates CXCR4 expression in BM-derived stem cells by activating MMP-9, particularly in the setting of G-CSF challenge, to induce cell homing to the lesion, promoting cardiac repair after MI. Thus, targeting Plg may offer a therapeutic opportunity to enhance stem cell– mediated cardiac repair after MI.

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Acknowledgements

The authors thank Drs. Marc Penn and Edward Plow for their helpful discussions. Reprint requests and correspondence: Dr. Yanqing Gong, Division of Translational Medicine and Human Genetics, University of Pennsylvania School of Medicine, 3400 Civic Center Boulevard, Philadelphia, Pennsylvania 19104. E-mail: gongy@ mail.med.upenn.edu.

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Key Words: BM-derived stem cells plasminogen.

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CXCR4

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myocardial infarction

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APPENDIX

For a supplemental Methods section, please see the online version of this article.