Abstract
The cardiac microenvironment includes cardiomyocytes, fibroblasts and macrophages, which regulate remodeling after myocardial infarction (MI). Targeting this microenvironment is a novel therapeutic approach for MI. We found that the natural compound derivative, BIO ((2′Z,3′E)-6-Bromoindirubin-3′-oxime) modulated the cardiac microenvironment to exert a therapeutic effect on MI. Using a series of co-culture studies, BIO induced proliferation in cardiomyocytes and inhibited proliferation in cardiac fibroblasts. BIO produced multiple anti-fibrotic effects in cardiac fibroblasts. In macrophages, BIO inhibited the expression of pro-inflammatory factors. Significantly, BIO modulated the molecular crosstalk between cardiac fibroblasts and differentiating macrophages to induce polarization to the anti-inflammatory M2 phenotype. In the optically transparent zebrafish-based heart failure model, BIO induced cardiomyocyte proliferation and completely recovered survival rate. BIO is a known glycogen synthase kinase-3β inhibitor, but these effects could not be recapitulated using the classical inhibitor, lithium chloride; indicating novel therapeutic effects of BIO. We identified the mechanism of BIO as differential modulation of p27 protein expression and potent induction of anti-inflammatory interleukin-10. In a rat MI model, BIO reduced fibrosis and improved cardiac performance. Histological analysis revealed modulation of the cardiac microenvironment by BIO, with increased presence of anti-inflammatory M2 macrophages. Our results demonstrate that BIO produces unique effects in the cardiac microenvironment to promote recovery post-MI.
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Introduction
Myocardial infarction (MI) is a leading cause of death, irrespective of socioeconomic status and ethnicity1. Medical interventions have been developed to stabilize patients with MI, which has improved survival rates, but there is currently no clinically approved method to reverse the loss of cardiac muscle (cardiomyocytes). Patients that survive acute MI can develop heart failure because of remodeling, due to the poor capacity of heart regeneration2.
The small molecule compound, BIO ((2′Z,3′E)-6-Bromoindirubin-3′-oxime) is a cell permeable derivative of the natural product, 6-bromoindirubin3. This compound is produced by predatory rock snails, such as Hexaplex trunculus, and has been used since ancient times to produce the famous ‘Tyrian purple’ dye3. BIO was shown to inhibit the multifunctional enzyme, glycogen synthase kinase-3β (GSK-3β). This inhibition reduced β-catenin phosphorylation and activated the Wnt signaling pathway, which maintains the undifferentiated state of stem cells4. Interestingly, it was shown that BIO treatment could induce proliferation in post-mitotic adult rat cardiomyocytes5. However, to our knowledge, BIO has not been tested as a drug that can modulate cardiac remodeling to improve heart function after MI.
The cardiac cellular composition of the microenvironment post-MI comprises: cardiomyocytes, cardiac fibroblasts, macrophages, endothelial cells and smooth muscle cells6. Cardiomyocytes account for only 30–40% of the total cell population in the heart7. Non-cardiomyocyte cell types carry out important roles to maintain cardiac homeostasis. For example, cardiac fibroblasts are the predominant cell type in terms of cell number and are located alongside cardiomyocytes throughout the heart8. Cardiac fibroblasts maintain myocardial architecture, contribute to cardiac electrophysiology, and communicate with cardiomyocytes via gap junctions. In pathological states, fibroblasts form a coupled network to regulate the inflammatory response, nutrient/metabolite homeostasis and spread of the infarcted region9. Moreover, fibroblast-cardiomyocyte networks communicate the signals from infarcted cardiomyocytes to produce a wave of cell death that spreads beyond the initial site of cardiac damage10.
Cardiac macrophages are another significant cell type in the cardiac microenvironment and carry out pivotal regulatory functions during remodeling after MI11. Cardiac macrophages can be broadly classified into two phenotypes: 1) M1, which are pro-inflammatory and remove debris from dead cells, and 2) M2, which are anti-inflammatory and promote wound resolution. M1 and M2 macrophages can be distinguished using specific markers, such as aginase-1 (Arg), inducible nitric oxide synthase (iNOS) and interleukin-10 (IL-10)12. Generally, M1 macrophages are iNOShigh Arglow and M2 macrophages are iNOSlow Arghigh. After MI, circulating monocytes are recruited to the infarction and converted to macrophages by signaling through cytokine receptors, such as chemokine receptor 213. Studies have shown that increasing the numbers of M2 macrophages improved cardiac recovery14.
In this study, we evaluated the effect of the BIO on the cardiac microenvironment. Interestingly, we observed that BIO induces opposing effects in cardiomyocytes and cardiac fibroblasts, which could promote cardiac functional recovery during remodeling. Significantly, BIO can shift macrophages from an M1 to M2 phenotype. These positive effects on cardiac recovery were validated in a zebrafish cardiomyocyte depletion system and confirmed in a rat MI model. The effects of BIO were characterized as regulation of IL-10 expression and modulation of p27. Our results indicate a significant reappraisal of the biological activity of BIO in the context of cardiac remodeling after MI and establish this compound as an attractive candidate for drug development.
Results
BIO Selectively Induces Cardiomyocyte Proliferation and Blocks Cardiac Fibroblast Proliferation
The chemical structure of BIO and the snail, Muricidae, is shown in Fig. 1A. Primary neonatal rat cardiomyocytes were treated with BIO for 5 days and showed a significant increase in proliferation (Fig. 1B). In contrast, BIO treatment reduced neonatal rat cardiac fibroblast proliferation (Fig. 1C). The increased proliferation of cardiomyocytes treated with BIO for 4 days was also confirmed by MTT assay (Fig. 1D). To model the cardiac microenvironment, co-cultures of cardiomyocytes and cardiac fibroblasts were treated with BIO, which increased cardiomyocyte proliferation and inhibited cardiac fibroblast proliferation (Fig. 1E,F).
BIO Inhibits Pro-fibrotic Mechanisms
The effect of BIO on cardiac fibroblast motility was assessed using the scratch assay. BIO treatment inhibited fibroblast motility (Fig. 2Ai). BIO is used as a GSK-3β inhibitor and LiCl is one of the most widely used GSK-3β inhibitors15. However, LiCl did not reduce fibroblast motility (Fig. 2Aii).
In consequence of the scratch test results, the effect of BIO on fibrotic factors was assessed. BIO treatment blocked pro-fibrotic CCL11 upregulation in AngII-stimulated cardiac fibroblasts (Fig. 2Bi,Bii). BIO also increased anti-fibrotic IL-10 expression in AngII-stimulated cardiac fibroblasts (Fig. 2Bi,Bii). In addition, the expression of pro-fibrotic factors CTGF and TGF-β was reduced by BIO treatment (Fig. 2Ci,Cii).
BIO Increases p27 Expression and Reduces Akt Signaling
To investigate the effect of BIO on proliferation in cardiac fibroblasts, immunoblotting was carried out for known regulators. BIO increased expression of the cell cycle inhibitor, p27 (Fig. 2Di,Dii). BIO treatment also increased p27 mRNA levels, but did not affect p21 mRNA (Fig. 2Di,Dii). In contrast, treatment with BIO decreased the phosphorylation of Akt, which regulates PI3K/Akt/mTOR signaling, and reduced expression of the cell cycle inhibitor, p21 (Fig. 2E). Immunofluorescence staining also confirmed that phosphorylated Akt and p21 were significantly reduced, while p27 was increased in BIO-treated cardiac fibroblasts (Fig. 2F).
BIO Modulates Macrophage Polarization
Murine macrophage cell line RAW264.7 were stimulated with lipopolysaccharide (LPS) to induce pro-inflammatory M1 polarization, which is measured by increased expression of iNOS. Treatment with BIO blocked iNOS expression, and induced expression of the M2 anti-inflammatory macrophage marker, arginase-1 (Arg1) (Fig. 3A). In contrast, LiCl (20 mM) was less effective than BIO (5 μM) at reducing iNOS induction. Arg1 expression was also increased by BIO treatment, but not by LiCl treatment (Fig. 3B). Immunofluorescence staining demonstrated upregulation of Arg1 and recovered expression of the M2 marker, CD206. LPS-stimulated iNOS induction was also blocked by BIO treatment (Fig. 3C).
Resident fibroblasts are known regulators of monocyte differentiation into macrophages. In co-cultures of THP-1 human monocytes and rat cardiac fibroblasts, increased monocyte adherence was observed, indicating macrophage differentiation (Fig. 4A,B). IL-10 is a M2 macrophages marker16, and co-cultured monocytes treated with BIO increased IL-10 expression (Fig. 4C). The increase in IL-10 expression was similar to co-cultured monocytes treated with IL-4, which is known to induce differentiation into M2 macrophages (Fig. 4C). In contrast, LiCl did not increase IL-10 expression. Treatment with BIO in the presence of LPS still produced an increase in IL-10 expression (Fig. 4D). Treatment of co-cultured monocytes with BIO or IL-4 was shown to increase Arg activity, which is a marker of M2 polarization (Fig. 4E). Treatment with LiCl did not increase Arg activity. In addition, treatment with BIO and LPS still produced an increase in Arg activity.
To verify that the effects of BIO are relevant in vivo, an ex vivo co-culture system was established using mouse bone marrow-derived monocytes (BMDMs) (Fig. 4F). BMDMs were cultured with conditioned media from rat cardiac fibroblasts, which induced macrophage differentiation (Fig. 4G). Treatment with BIO or IL-4 increased the expression of IL-10 (Fig. 4H). LPS produced an increase in IL-10 expression compared to BMDMs, but this increase was significantly less than observed with BIO treatment. LiCl did not increase IL-10 expression. BIO also increased IL-10 expression in the presence of LPS, unlike treatment with LPS and LiCl (Fig. 4I). As a positive control for M2 polarization, BMDMs were treated with a combination of TGF-β and IL-4. Treatment of differentiating BMDMs with BIO or IL-4, but not LiCl, increased Arg activity (Fig. 4J). Moreover, BIO increased Arg expression in differentiating BMDMs, even in the presence of LPS (Fig. 4J).
BIO Rescues Cardiomyocyte Loss in Zebrafish Heart Failure
To investigate whether BIO produces therapeutic effects after cardiac injury in vivo, an optically transparent zebrafish model of heart failure was employed17. Zebrafish provide an ideal vertebrate system for initial testing of candidate drugs18,19. Tg(cmlc2:GFP) transgenic zebrafish showed reduced ventricular size after treatment with the cardiac toxin, aristolochic acid (AA) (Fig. 5A). Treatment with BIO, but not LiCl, recovered ventricular size and completely recovered survival rate (Fig. 5B). To test if BIO can induce cardiomyocyte proliferation, Tg(cmlc2:GFP) transgenic zebrafish were stained for proliferating cells using BrdU. A higher number of BrdU positive cells were present in the cardiac region of BIO-treated fish (Fig. 5C).
BIO Promotes Favorable Cardiac Remodeling in Rat MI and Increases M2 Macrophages in the Infarction Zone
To test the effect of BIO on cardiac injury in mammals, a rat MI model was used. Male rats were treated with 0.2 mg/kg BIO or vehicle every day for 2 weeks after MI. BIO treated rats showed reduced ventricular fibrosis (Fig. 6A). Moreover, echocardiography analysis showed that BIO treatment significantly improved cardiac recovery and the indices of cardiac function at 2 weeks post-MI (Fig. 6B,C, Online Table 2). Left ventricular ejection fraction (EF %) was considerably improved (30.67% ± 1.659 in the Veh group vs. 41.31 ± 6.823 in the BIO group, p < 0.05), and fractional shortening (FS %) was also notably increased (12.62% ± 0.755 in the Veh group vs. 18.25 ± 3.525 in the BIO group, p < 0.05) by BIO treatment. Measurement of organ and body weight indicated that BIO produced no toxic effects in the rats (Online Table 3).
Our results indicated that BIO increased the expression of M2 macrophage markers (Fig. 4). Higher numbers of M2 macrophages were observed in the infarction zone of the BIO-treated group, as detected using the M2 marker, Arg (Fig. 6D,E). IL-6 is linked to the development of cardiac fibrosis and serum levels negatively correlate with prognosis in heart failure20. Using ELISA, it was observed that BIO treatment reduced serum IL-6 after MI (Fig. 6F).
Discussion
Drug candidates that improve cardiac remodeling would possess significant therapeutic potential. The natural compound derivative, BIO, has been used as a GSK-3β inhibitor/Wnt pathway activator for numerous cardiac research applications, such as inducing cardiomyocyte differentiation in cardiac stem/precursor cells21. In this study, we establish BIO as a new drug that modulates the cardiac microenvironment to reduce scarring and improve cardiac performance after MI. Moreover, novel mechanisms of action for BIO were identified that are not observed for other GSK-3β inhibitors, such as LiCl. These novel mechanisms of BIO are directly related to the therapeutic effects observed in animal models.
Our results show that BIO induces proliferation in cardiomyocytes and inhibits proliferation in cardiac fibroblasts (Fig. 1). This important effect of BIO is emphasized by a previous study, which showed that adult cardiac fibroblasts induce only hypertrophy, not proliferation, in co-cultured cardiomyocytes22. The previous report by Tseng et al., showed that BIO increased cardiomyocyte proliferation by downregulating p275. Our data showed that BIO increased p27 protein expression in fibroblasts, which explains the different effects of BIO in cardiomyocytes and fibroblasts. Immunoblotting showed that BIO decreased p21 protein expression in cardiac fibroblasts (Fig. 2E). These results indicate that p27 up-regulation after BIO treatment in cardiac fibroblasts overrides p21 to block proliferation. BIO treatment also reduced Akt activation (Fig. 2E). Akt activation induces cell proliferation and it has been shown that BIO treatment activates Akt in mesangial cells23. Thus, BIO differentially affects proliferation in various cell types.
In addition to inhibiting proliferation in cardiac fibroblasts, BIO also reduces motility (Fig. 2Ai). Interestingly, the known GSK-3β inhibitor, LiCl, did not reduce motility (Fig. 2Aii), indicating that BIO has significant additional effects in cardiac fibroblasts that are not related to GSK-3β inhibition. The role of GSK-3β inhibition in fibroblasts is controversial; increased fibrosis has been reported in numerous disease contexts, such as dermal and venous wall fibrosis24,25 and deletion of GSK-3β in cardiac fibroblasts increases scarring in the ischemic heart26. However, GSK-3β inhibition by over-expression of a dominant-negative isoform was protective in a murine model of heart failure27. Our data shows that BIO induces anti-fibrotic effects in cardiac fibroblasts, via reduced proliferation, inhibition of motility, decreased expression of the pro-fibrotic factors, CCL11 and CTFG, and increased expression of the anti-fibrotic factor, IL-10. Additionally, BIO also produced a small but significant reduction in the expression of TGF-β, which is considered a ‘master regulator’ of the fibrosis program (Fig. 2)28. Although our results show positive effects of BIO on modulating profibrotic factors, the signaling pathways that regulate fibrosis and myofibroblast differentiation are highly complex. Therefore, to further clarify the potential anti-fibrotic effects of BIO, additional analysis of key regulators is required, such as endothelin-1 and scleraxix29,30,31.
Resident macrophages are a component of the cardiac microenvironment and regulators of the remodeling process after MI11. M1 polarized macrophages clear wound debris and regulate the inflammatory response, whereas M2 polarized macrophages promote wound resolution and benefit cardiac remodeling when present in greater numbers14. Our results show that BIO reduced expression of the M1 marker, iNOS, in macrophages (Fig. 3). Moreover, the known GSK-3β inhibitor, LiCl, could not reduce iNOS expression to the same degree as BIO (Fig. 3B). Of note, it has previously been shown that increased iNOS expression after MI is linked to cardiac dysfunction and increased mortality32. In addition, treatment with iNOS inhibitors after experimental MI can reduce infarct size33.
During wound healing and remodeling post-MI, circulating monocytes ‘home’ to the infarction site and differentiate into macrophages. In other disease states, such as cancer, the role of microenvironment fibroblasts in modulating macrophage phenotype has been widely studied34. However, these cellular interactions are less studied in the cardiac microenvironment. Using a co-culture system, our results show that cardiac fibroblasts can induce monocyte differentiation into macrophages (Fig. 4B). To our knowledge, this is the first demonstration that cardiac fibroblasts secrete factors that induce monocyte differentiation. This ‘molecular crosstalk’ between different cell types is a major regulator of disease progression in cancer35 and our results show that crosstalk between cardiac fibroblasts and monocytes regulates macrophage differentiation. BIO treatment in our co-culture system increased expression of the M2 macrophage markers, IL-10 and Arg1 (Fig. 4C–E). IL-10 is a key anti-inflammatory cytokine and positively regulates cell-based repair after MI36. Moreover, the ratio of IL-10 expression compared to pro-inflammatory IL-6 is linked to the development of MI in patients37. To our knowledge, there is only one previous report investigating the interactions between cardiac fibroblasts and macrophages in co-culture, in which it was shown that murine macrophages stimulate pro-inflammatory IL-6 production and pro-fibrotic signaling in cardiac fibroblasts38. Our results show that BIO modulates the cellular ‘crosstalk’ between cardiac fibroblasts and differentiating macrophages to dramatically increase expression of IL-10 at a similar degree to the major M2 macrophage inducer, IL-4 (Fig. 4C,D). IL-10 is known to suppress the expression of iNOS in macrophages and induce expression of the M2 macrophage marker, Arg1. Our results show that BIO treatment also induced Arg1 expression in differentiating bone-marrow derived and human macrophages (Fig. 4E,J). Arg1-expressing macrophages are known to inhibit liver fibrosis in mice.
The zebrafish model has proved an invaluable resource for the primary validation of new bioactive compounds in vivo. The optically transparent zebrafish heart failure model used in our study demonstrated that BIO induces cardiomyocyte proliferation to effectively compensate for toxicity induced by AA. In contrast, the GSK-3β inhibitor LiCl was not as effective as BIO at rescuing the zebrafish. The zebrafish data represents the first demonstration that BIO induces cardiomyocyte proliferation in vivo (Fig. 5C). Moreover, the lack of toxicity in BIO-treated zebrafish supports the further drug development of BIO, because mammals and zebrafish show strong similarities in their response to toxic agents39.
Our zebrafish-based data validated the testing of BIO in a mammalian model of MI. BIO treatment improved cardiac remodeling and reduced fibrotic scar formation (Fig. 6). The improvement in cardiac fibrosis produced by BIO compared favorably with other experimental interventions that enhance remodeling. For example, mesenchymal-derived cell (MSC) therapy for MI produced an average reduction of 6.67% in fibrosis level40, compared to 11.72% for BIO treated rats. Increasing the numbers of M2 macrophages is known to improve cardiac remodeling and recovery after MI14. In the context of cardiac fibrosis, IL-6 acts as a pro-inflammatory cytokine that is both produced by M1 macrophages and cardiac fibroblasts to induce M1 polarization41. BIO treatment reduced serum levels of pro-inflammatory IL-6 (Fig. 6F), which provides a potential mechanism to explain the increased numbers of M2 macrophages in the infarction zone.
BIO was effective in the rat and zebrafish models without producing noticeable toxicity. However, to translate the BIO compound into clinical applications, its potential toxicity in different organs should be investigated more rigorously in animal models. It should be noted that many patients have received long-term treatment with the GSK-3β inhibitor, lithium, without producing significant effects on the cardiovascular system. However, recent studies have indicated that GSK-3 isoform selective (α- or β-) small molecule inhibitors should be developed to prevent myocardial fibrotic remodeling42.
Our study indicates that BIO has potential to be developed as a therapy for improving remodeling post-MI, although the effects are only partial. There are currently few drug options available for patients, despite numerous lead compounds showing promise in animal models. Angiotensin-converting enzyme (ACE) inhibitors, aldosterone inhibitors and the beta blocker, carvedilol, have shown effectiveness in MI patients, but may not actually reverse the remodeling process43,44. Therefore, although BIO has shown promising results in this study, it is still at an early stage of development as a therapeutic for attenuating remodeling post-MI.
Our study shows that BIO can increase proliferation in zebrafish and rat cardiomyocytes. The ability for cardiomyocytes to re-enter the cell cycle varies amongst species and is very limited in humans45. For example, zebrafish cardiomyocytes analyzed in our study readily re-enter the cell cycle after heart trauma. Moreover, rat neonatal cardiomyocytes used for our mammalian assays of BIO treatment show greater proliferative potential than adult cardiomyocytes5. Therefore, further study of the effects of BIO on cardiomyocyte proliferation in higher mammalian species is warranted to assess whether this compound has the potential to be effective in human cardiomyocytes.
A recent study demonstrated that activation of Notch 1 by its ligand, Jagged1, produced distinct responses that are dependent on cell types in the stressed adult mouse heart46. Activated Notch1 in Jagged1 transgenic mice reduced both hypertrophy and fibrosis in response to pressure overload. Interestingly, upregulated Notch1 increased the number of stem cell antigen-1-posivite cardiac precursor cells, and decreased the number of myofibroblasts in the stressed heart. In the Notch1 activated heart, pressure overload-induced Akt phosphorylation was reduced, which could explain the decrease in cardiac hypertrophy. Further identification of the relationship between BIO and the Notch1 signaling pathway in cardiomyocytes and cardiac fibroblasts may further clarify mechanism of action of BIO in the damaged cardiac microenvironment.
Adult cardiac precursor cells (CPCs) have been identified as an important cell population that contributes to repair after damage to the myocardium47. The Wnt signaling pathway has been shown to be regulate the contribution of CPCs to cardiac repair48. Wnt pathway inhibition after chronic left anterior descending coronary artery ligation reduced mortality and improved remodeling. However, BIO activates the Wnt signaling pathway via the inhibition of GSK-3β. To our knowledge, there is no published data concerning the effects of BIO on adult CPC renewal or differentiation into cardiomyocytes. In light of our results showing the beneficial effect of BIO on remodeling post-MI, an investigation of how BIO modulates CPC phenotype would be an interesting area for further research.
In summary, this study has shown that BIO induces multiple, beneficial effects on the major cell types comprising the cardiac microenvironment to reduce fibrosis and improve remodeling after MI. Many of these effects were not observed after treatment with the commonly used GSK-3β inhibitor, LiCl, confirming that BIO produces pleiotropic effects in these cells types that are additional to its reported activity against GSK-3β. We show that BIO treatment enhances the proliferation of cardiomyocytes and reduces the proliferation of cardiac fibroblasts via differential regulation of p27 expression. BIO treatment produces anti-fibrotic effects in cardiac fibroblasts, such as reduced motility, down-regulation of CCL11 and up-regulation of IL-10. In monocytes, BIO treatment promotes monocyte differentiation into anti-inflammatory M2 macrophages by strongly inducing IL-10 expression in cardiac fibroblast co-cultures. These beneficial effects of BIO can be observed in a zebrafish heart failure model and a rat MI model. The pleiotropic effects of BIO in the cardiac microenvironment are summarized in Fig. 7. To our knowledge, no previously reported compound has been shown to produce this combination of differential, positive effects on the cardiac microenvironment. Thus, our study represents a significant reappraisal of the biological activity and therapeutic potential of BIO. With ischemic heart disease remaining a leading case of mortality/morbidity and health care expense, this study supports the further development of BIO as a drug to target the cardiac microenvironment and improve remodeling after MI.
Materials and Methods
All experiments conformed the NIH Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication, 8th edition, 2011) and were approved by the Chonnam National University Institutional Animal Care and Use Committee (study approval code: CNU IACUC-H-2014-23). A description of the following methods are available in the Online Supplement: Antibodies and reagents, cell lines, isolation and culture of neonatal left ventricular cardiomyocytes and cardiac fibroblasts, measurement of cell proliferation, cardiomyocyte: cardiac fibroblast co-culture system, immunocytochemistry, monolayer scratch assay, RT-PCR, quantitative real-time PCR, isolation and culture of primary bone marrow-derived monocytes, cardiac fibroblast and monocyte co-culture, arginase assay, zebrafish model of heart failure, rat model of acute MI, histological staining of fibrosis, echocardiography of ventricular function, immunohistochemistry of cardiac tissue, interleukin-6 enzyme-linked immunosorbent assay, statistical analysis.
Additional Information
How to cite this article: Kim, Y. S. et al. Natural product derivative BIO promotes recovery after myocardial infarction via unique modulation of the cardiac microenvironment. Sci. Rep. 6, 30726; doi: 10.1038/srep30726 (2016).
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Acknowledgements
This research was supported by the Bio & Medical Technology Development Program of the NRF funded by the Korean government (2015M3A9C6031684, 2015M3A9C6030838, and 2015M3A9B4051063). This research was also supported by a fund from Ministry of Health and Welfare, Republic of Korea (HI15C0498) and by the GIST Research Institute (GRI) in 2016. Figure 1A and the heart in Fig. 7 were obtained from Wikipedia and used under the Creative Commons Attribution-Share Alike 4.0 International license.
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Y.S.K., D.-W.J., D.R.W. and Y.A. conceived the project, designed the experiments and analyses; H.-y.J., A.R.K., W.-H.K., H.C., J.U., Y.S., W.S.K., S.-W.J., M.C.K. and Y.-C.K. carried out the experiments and analyzed the data; Y.S.K., D.-W.J., D.R.W. and Y.A. wrote the main manuscript. All authors reviewed the manuscript.
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Kim, Y., Jeong, Hy., Kim, A. et al. Natural product derivative BIO promotes recovery after myocardial infarction via unique modulation of the cardiac microenvironment. Sci Rep 6, 30726 (2016). https://doi.org/10.1038/srep30726
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DOI: https://doi.org/10.1038/srep30726
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