Author + information
- Received August 7, 2013
- Revision received November 5, 2013
- Accepted November 26, 2013
- Published online July 1, 2014.
- Yanqing Gong, PhD∗,†∗ (, )
- Yujing Zhao, BS∗,
- Ying Li, BS∗,
- Yi Fan, MD, PhD‡ and
- Jane Hoover-Plow, PhD∗
- ∗Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Departments of Cardiovascular Medicine and Molecular Cardiology, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio
- †Division of Translational Medicine and Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
- ‡Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
- ↵∗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.
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 (Plg+/+) 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 (GFP+c-kit+ cell) recruitment into the infarcted heart and stem cell–mediated cardiac repair in Plg+/+ 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.
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 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 the fundamental problem of cardiomyocytes loss is cardiac transplantation, but this treatment has limited application because of insufficient donor hearts and the need for long-term immunosuppressive therapy. New discoveries on the regenerative potential of stem cells for preventing heart 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.
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.
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 the role of Plg in stem cell–mediated tissue repair and heart function recovery, MI was induced in Plg+/+ and Plg−/− mice by ligation of the left anterior descending coronary artery. Mice were treated with G-CSF to stimulate stem cell mobilization from BM and recruitment to the infarct heart, and cardiac function was analyzed by echocardiography. Plg deficiency did not alter normal heart function, as indicated by the similar ejection fraction of the left ventricle (LV) observed in Plg+/+ (86.7 ± 2.4%, n = 6) and Plg−/− (87.6 ± 0.8%, n = 8) mice (Fig. 1A). The ligation surgery induced inadequate cardiac function, reducing LV ejection fraction by about 50% in Plg+/+ and Plg−/− mice (Fig. 1A). No spontaneous recovery of heart function was observed in the 28 days after MI surgery, as shown by decreased ejection faction detected in Plg+/+ and Plg−/− mice. Importantly, G-CSF significantly increased ejection fraction by one-half in Plg+/+, but not in Plg−/− mice (Fig. 1A). Consistently, G-CSF reduced LV internal diameter by approximately 30% to 40% in Plg+/+, 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 Plg+/+ 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 Plg+/+ 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 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 (Lin−c-kit+ cells) detected by fluorescence-activated cell sorting (FACS) in circulation in Plg+/+ 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 Plg+/+ MI mice. Plg deficiency almost completely inhibited MI-induced 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 (GFP+) cells in the infarcted cardiac tissue (Fig. 2B). Immunofluorescence analysis showed that MI induced a mild recruitment of BM-derived GFP+ cells and c-kit+ stem cells in the infarct site, whereas G-CSF treatment induced marked cell recruitment in Plg+/+ mice (Fig. 2C). Importantly, 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 GFP+c-kit+ 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. After BM transplantation with GFP+ BM cells and MI induction, BM-derived cells, EC, and SMC were visualized by immunofluorescence with anti-GFP, -CD31, and -α-SMC actin antibodies, respectively. Colocalization analysis showed that over one-half of CD31+ and α-SMC actin+ cells expressed GFP, confirming their BM origin and suggesting that BM-derived stem cells significantly 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 GFP+CD31+ and GFP+α-SMC actin+ 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 GFP+CD31+ and GFP+α-SMC actin+ 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 Plg+/+ 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 G-CSF–stimulated CXCR4 expression. The reduced CXCR4 expression in heart infarcts in Plg−/− mice may be due to a decrease in the recruitment of CXCR4+ 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 Plg+/+ 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 Plg+/+ 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 Plg+/+ 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-kit+ stem cells showed that MI and G-CSF up-regulated CXCR4 expression in BM stem cells of Plg+/+ 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 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 dose-dependent manner (Fig. 5A). FACS analysis confirmed a 2-fold up-regulation in CXCR4 expression induced by 2 μg/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 Lin−c-kit+ stem cell migration in response to SDF-1 (Fig. 5C), as determined by counting migrated BMNCs by hemocytometry and Lin−ckit+ stem cells by FACS (Fig. 5C). Importantly, AMD3100 significantly inhibited Plg-stimulated BM cell migration and completed abolished Plg-mediated Lin−ckit+ 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). GFP+ 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 Plg+/+ 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 (GFP+ cells) in infarcted heart tissue of Plg−/− mice (Fig. 6D). Similarly, recruitment of BM-derived stem cells, detected as GFP+c-kit+ 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.
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 Plg+/+ 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 Lin−c-kit+ stem cells (Fig. 7D), indicating that MMP-9 is essential for Plg-mediated 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.
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 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 CXCR4+ 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 CXCR4+ 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 MMP-9 activity in BM cells, but no stem cell mobilization in CXCR4−/− mice, and by the fact that transplantation of CXCR4+VEGFR2+ 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.
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.
The authors thank Drs. Marc Penn and Edward Plow for their helpful discussions.
For a supplemental Methods section, please see the online version of this article.
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.
- Abbreviations and Acronyms
- bone marrow
- bone marrow mononuclear cell
- C-X-C chemokine receptor type 4
- endothelial cells
- fluorescence activated cell sorting
- granulocyte colony-stimulating factor
- green fluorescent protein
- left ventricle/ventricular
- matrix metalloproteinase
- stromal cell-derived factor
- vascular smooth muscle cells
- Received August 7, 2013.
- Revision received November 5, 2013.
- Accepted November 26, 2013.
- American College of Cardiology Foundation
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