Heart-Infiltrating Prominin-1+/CD133+ Progenitor Cells Represent the Cellular Source of Transforming Growth Factor β–Mediated Cardiac Fibrosis in Experimental Autoimmune Myocarditis
Rationale: Myocardial fibrosis is a hallmark of inflammation-triggered end-stage heart disease, a common cause of heart failure in young patients.
Objective: We used CD4+ T-cell–mediated experimental autoimmune myocarditis model to determine the parameters regulating cardiac fibrosis in inflammatory heart disease.
Methods and Results: α-Myosin heavy chain peptide/complete Freund’s adjuvant immunization was used to induce experimental autoimmune myocarditis in BALB/c mice. Chimeric mice, reconstituted with enhanced green fluorescence protein (EGFP)+ bone marrow, were used to track the fate of inflammatory cells. Prominin-1+ cells were isolated from the inflamed hearts, cultured in vitro and injected intracardially at different stages of experimental autoimmune myocarditis. Transforming growth factor (TGF)-β–mediated fibrosis was addressed using anti–TGF-β antibody treatment. Myocarditis peaked 21 days after immunization and numbers of cardiac fibroblasts progressively increased on follow-up. In chimeric mice, >60% of cardiac fibroblasts were EGFP+ 46 days after immunization. At day 21, cardiac infiltrates contained ≈30% of prominin-1+ progenitors. In vitro and in vivo experiments confirmed that prominin-1+ but not prominin-1− cells isolated from acutely inflamed hearts represented the cellular source of cardiac fibroblasts at late stages of disease, characterized by increased TGF-β levels within the myocardium. Mechanistically, the in vitro differentiation of heart-infiltrating prominin-1+ cells into fibroblasts depended on TGF-β–mediated phosphorylation of Smad proteins. Accordingly, anti–TGF-β antibody treatment prevented myocardial fibrosis in immunized mice.
Conclusions: Taken together, heart-infiltrating prominin-1+ progenitors are the major source of subsequent TGF-β–triggered cardiac fibrosis in experimental autoimmune myocarditis. Recognizing the critical, cytokine-dependent role of bone marrow–derived progenitors in cardiac remodeling might result in novel treatment concepts against inflammatory heart failure.
Cardiac inflammation is most commonly triggered by viral infections. Virus-triggered myocarditis leading to inflammatory cardiomyopathy represents the most common cause of chronic heart failure in young patients. Heart-infiltrating inflammatory cells include granulocytes, monocytes, T cells, B cells, and mast cells, releasing various cytokines such as interleukin (IL)-17, -6, -1, -10, -12, interferon-γ, transforming growth factor (TGF)-β, and tumor necrosis factor-α; chemokines; and matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases.1–6 Inflamed hearts are at increased risk to undergo pathological remodeling, which may finally result in heart failure and the phenotype of inflammatory dilated cardiomyopathy. This process is associated with disruption of normal myocardial structures, and extracellular matrix deposition promoting tissue fibrosis.7,8
Experimental autoimmune myocarditis (EAM) is a CD4+ T-cell–mediated mouse model of inflammatory cardiomyopathy. EAM can be induced in susceptible mouse strains by immunization with cardiac specific peptides derived from α myosin heavy chain (αMyHC),9 or cardiac troponin I10, together with a strong adjuvant or by injection of activated bone marrow (BM)–derived dendritic cells loaded with heart-specific self peptide.9,11 In general, grading of EAM severity scores bases on the extent of inflammatory infiltrates at the peak of inflammation. In the ensuing late phase of disease, cardiac inflammation slowly resolves. However, many of the affected hearts show progressive dilation and increasing tissue fibrosis on follow-up.2
Insight from ischemic heart disease models suggests that the transition from acute inflammation to fibrosis is mediated by TGF-β.8 Along this line, resolution of inflammation and progressive remodeling are associated with high levels of TGF-β in the myocardium.12,13 TGF-β promotes the synthesis of various cytokines and growth factors, that are involved in formation/progression of cardiac fibrosis.8 In contrast, the cause of cardiac fibrosis in the context of inflammatory heart disease remains unclear. BM-derived progenitor cells recruited to the inflamed heart represent a potential cellular source for fibrosis,14,15 but this idea is largely speculative. In particular, the role of BM-derived progenitor cells in the process of regeneration/pathological remodeling in EAM is unknown.
Here, we show that prominin-1+ progenitor cells represent a subpopulation of heart-infiltrating BM-derived CD45+ cells at the peak of inflammation in EAM. These cells represent a common progenitor source for both fibroblasts and monocytes/macrophages, and play a critical role in the pathogenesis of cardiac fibrosis, a hallmark of end-stage heart failure. Fibroblast differentiation of prominin-1+ cells was promoted during the late stage of EAM because of a progressive shift of the intracardiac cytokine balance, reflected by increasing TGF-β levels.
BALB/c and C57BL/6-EGFP mice (enhanced green fluorescent protein [EGFP] under the control of β-actin promoter) were purchased from The Jackson Laboratory. C57BL/6-EGFP mice were backcrossed onto the BALB/c background for >10 generations (hereafter referred to as BALB/c-EGFP). The local authorities approved the animal protocol and all experiments were performed in accordance with Swiss Federal Law.
In Vitro Expansion/Differentiation of Heart-Infiltrating Prominin-1+ Cells
Mouse hearts were processed as described previously.16 Prominin-1+ cells were isolated by magnetic cell sorting and further expanded in vitro in culture expansion medium (Online Data Supplement, available at http://circres.ahajournals.org). To generate single-cell–derived clones, a single prominin-1+/EGFP+ cell was coplated with prominin-1+/EGFP− feeder cells derived from the healthy heart and cultured for 2 to 3 weeks. Cardiac differentiation was induced with 100 μmol/L oxytocin (Sigma, Basel, Switzerland); macrophage differentiation was induced with 10 ng/mL macrophage colony-stimulating factor (M-CSF) (PeproTech, London, UK); fibroblast differentiation was induced with 10 ng/mL TGF-β (PeproTech).
Immunization and Treatment Protocols
Six- to 8-week-old mice were injected subcutaneously with 150 μg/mouse of αMyHC (Ac-HN-SLKLMATLFSTYASAD-OH, Caslo) emulsified 1:1 with complete Freund’s adjuvant (CFA)/PBS on days 0 and 7, as described.6 Control mice were immunized with CFA/PBS only. Prominin-1+/EGFP+ cells were injected either directly into the left ventricle of anesthetized animals (5×104 cells per mouse) 0, 14, or 32 days after immunization using a Hamilton microsyringe; or intravenously (1x106 cells per mouse) at days 7 and 14 after immunization.
A neutralizing monoclonal antibody α-TGF-β (clone1D11; R&D Systems) was used at 15 μg/mL for in vitro experiments. For in vivo neutralization, mice received intraperitoneal injections of α-TGF-β (20 μg in 100 μL of PBS) or mouse IgG1 isotype control antibody every third day between days 17 to 46 after αMyHC/CFA immunization.
Generation of Chimera
Six- to 8-week-old BALB/c mice were lethally irradiated with 2×6.5 Gy using a Gammatron (Co-60) system and reconstituted with 2×107 donor BM cells from BALB/c-EGFP mice (hereafter referred to as EGFP->BALB/c) (Online Data Supplement).
Depending on the experiment, animals were euthanized at day 21, 32, and 46 after the first immunization. Hearts were removed and stained with hematoxylin/eosin to assess myocarditis severity and with Masson’s trichrome staining to detect fibrosis. Myocarditis and fibrosis severity scores were assessed using semiquantitative scale (Online Data Supplement).
RT-PCR and Real-Time RT-PCR
RT-PCR was performed as described (Online Data Supplement).16 For real-time RT-PCR analysis, cDNA was amplified using the Power SYBR Green PCR Master Mix (Applied Biosystems) and oligonucleotides complementary to transcripts of the analyzed genes (Online Table I).
Cells were cultured on gelatin-coated cover slips. Hearts were harvested and cell engraftment was analyzed on cryo- and paraffin-fixed sections. Fixation and immunostaining procedures were performed as described previously (Online Data Supplement).16
Prominin-1+ cells were cultivated in vitro with TGF-β (PeproTech) for 1 hour and 3 and 7 days. Control cells were cultivated in the absence of TGF-β. Cell lysates were blotted and incubated with appropriate antibodies (Online Data Supplement).
Cell suspensions were stained using fluorochrome-conjugated mouse-specific antibodies (Online Data Supplement) and analyzed with a CyAn-ADP analyzer (Beckman Coulter) using FlowJo software (Tree Star). Heart infiltrates were identified as CD45+ cells from heart tissue suspensions gated on CD45/side scatter plots as described.3
Heart-infiltrating mononuclear cells were isolated as described3 with slight modifications (Online Data Supplement). Cytokine levels of interferon-γ, tumor necrosis factor-α, IL-17 and TGF-β were measured by ELISA in supernatants of heart-infiltrating mononuclear cells (Online Data Supplement).
The Mann–Whitney U test was used for the evaluation of nonparametric data. Normally distributed data were compared using Student t test. Differences were considered as statistically significant for P<0.05.
BM Cells Represent the Cellular Source of Cardiac Fibrosis in Late EAM
Accumulation of collagen-producing fibroblasts is a hallmark of pathological remodeling in end-stage heart failure. For addressing the contribution of the BM compartment to tissue fibrosis during the late stage of EAM, we created chimeric mice using lethally irradiated BALB/c mice reconstituted with EGFP+ syngeneic BM. Six weeks after BM reconstitution, only a few EGFP+ cells were found in healthy hearts (data not shown). Immunization of chimeric mice with αMyHC/CFA, however, resulted in severe myocarditis at day 21 (Figure 1A). Flow cytometry revealed that nearly all EGFP+ cells isolated from the heart of chimeric mice at this time point coexpressed CD45, suggesting an inflammatory phenotype (Figure 1B). We also identified a significant fraction of prominin-1+ cells among the EGFP+/CD45+ heart-infiltrating cells (Figure 1B). Nonetheless, in CFA-only immunized control chimeric mice, just few EGFP+ cells were observed (Figure 1B).
After day 21, inflammatory infiltrates largely resolved and progressive fibrosis developed by day 46 (Figure 1C through 1F). Analysis of these fibrotic hearts demonstrated that >60% of collagen-producing, α smooth muscle actin (αSMA)-producing, and fibronectin-producing fibroblasts expressed EGFP (Figure 1C through 1E). Importantly, we observed no EGFP signal in αMyHC+ cardiomyocytes (Figure 1F). CFA-only immunized control chimeric mice did not develop any fibrosis as expected (data not shown).
These findings indicate that the BM compartment represents the cellular source for both heart-infiltrating progenitor cells at the peak, as well as fibroblasts during the late stage of disease.
Inflammatory Prominin-1+ Progenitor Cells Showed Bilineage Differentiation Potential
Next, we aimed to identify the potential fibroblast progenitors within the inflammatory infiltrates at the peak of disease. Analysis of inflamed hearts of immunized chimeric mice revealed a fraction of ≈30% of EGFP+ cells coexpressing the progenitor cell marker prominin-1 (Figure 1B). Nearly all prominin-1+ cells coexpressed CD45 and stem/progenitor cell markers c-kit, Sca-1, and Cxcr4 (Figure 2A and 2B). Instead, after transition from the acute to the chronic phase of EAM, the number of inflammatory infiltrates dramatically decreased, and we found only few prominin-1+ cells in the fibrotic myocardium (data not shown).
Given the progenitor phenotype of prominin-1+ cells, we addressed their specific differentiation capacity. To this aim, we isolated inflammatory prominin-1+ cells from acutely inflamed hearts at day 21 and expanded them in vitro for 2 to 3 weeks. The cultured cells expressed prominin-1, CD45, c-kit, Sca-1, and Cxcr4 antigens and nanog, c-kit, sca-1, islet-1, and nestin mRNA transcripts (Figure 3A, 3B, and 3G). In the presence of TGF-β, prominin-1+ cells acquired a fibroblast-like phenotype, expressed αSMA and fibronectin (Figure 3C) and downregulated the expression of genes and proteins characteristic for stem/progenitor cells (Figure 3F and 3H). These findings demonstrate the capacity of heart-infiltrating prominin-1+ cells to differentiate into fibroblasts in the presence of TGF-β.
Alternatively, prominin-1+ cells gained a large, flat morphology with granular cytoplasm and showed high phagocytic activity in the presence of M-CSF (Figure 3D), suggesting a monocyte/macrophage phenotype. However, the prominin-1+ cells failed to upregulate cardiomyocyte-specific genes and proteins (Figure 3E and 3H). Unlike prominin-1+/CD45+ cells, the prominin-1−/CD45+ cell fraction isolated from inflamed hearts showed only macrophage phenotype in the presence of M-CSF, but failed to differentiate into fibroblasts and cardiomyocytes (Online Figure I).
Notably, prominin-1+ cells isolated from the inflamed hearts prevented EAM development when intravenously injected before the onset of acute inflammation, at days 7 and 14, into αMyHC/CFA immunized mice (Online Figure II).
Prominin-1 Identifies a Common Fibroblast/Macrophage Progenitor Cell in EAM
To address whether heart-infiltrating prominin-1+ cell represents a common progenitor for fibroblasts and monocytes/macrophages, we isolated prominin-1+ cells from inflamed heart of immunized BALB/c-EGFP mouse. Coculture of 1 to 5 of prominin-1+/EGFP+ cells on a nontransgenic heart-derived feeder layer gave rise to single-cell–derived EGFP+ cell-colony (Figure 4A), positive for prominin-1 (Figure 4B). Next, cocultures containing single-cell–derived EGFP+ clones were divided into 3 dishes containing either TGF-β, M-CSF or oxytocin. In the presence of TGF-β, EGFP+ cells expressed fibronectin (Figure 4C), whereas exposure to M-CSF gave rise to a macrophage phenotype with phagocytic activity (Figure 4D). In the presence of oxytocin, however, cells failed to assume cardiomyocyte-like phenotype, ie, stained negative for αMyHC (Figure 4E). Thus, the prominin-1+ cells represent a common progenitor for fibroblasts and monocytes/macrophages but not for cardiomyocytes in the acutely inflamed heart.
To test whether heart-infiltrating prominin-1+ cells undoubtedly originate from BM, we isolated and expanded prominin-1+/EGFP+ cells from the inflamed hearts of chimeric mice reconstituted with EGFP+ BM. EGFP+ cells retained an undifferentiated phenotype expressing prominin-1 and CD45 (Online Figure III, A and B), and on induction of differentiation, differentiated into fibroblasts and monocytes/macrophages but not into cardiomyocyte-like cells (Online Figure III, C through F).
Taken together, prominin-1 expression characterizes a common fibroblast/macrophage progenitor among heart-infiltrating BM-derived cells at the peak of inflammation in the EAM model. Consequently these prominin-1+ cells represent the potential cellular source of fibroblasts mediating cardiac fibrosis during the late stage of disease.
TGF-β Signaling Mediates Fibroblast Activation via Smad Proteins
So far, we showed that TGF-β mediates fibroblast differentiation of heart-infiltrating prominin-1+ cells. In fact, TGF-β–treated prominin-1+ cells significantly upregulated the expression of genes characteristic for fibroblasts such as collagen I, αsma, and fibronectin (Figure 5A). Activation of the TGF-β receptor complex involves phosphorylation of Smad proteins, which subsequently regulate transcription.17 TGF-β–induced in vitro fibroblast differentiation of heart-infiltrating prominin-1+ cells was associated with phosphorylation of Smad proteins (Figure 5B and 5C). Of note, phosphorylation of Smad2 (P-Smad2) occurred already 1 hour after TGF-β exposure. P-Smad2 expression was reduced in cells cultivated with TGF-β for 7 days (Figure 5B) and maintained at low levels following differentiation (data not shown).
Microenvironment Defines the Fate of Heart-Infiltrating Prominin-1+ Progenitors in EAM
So far, we demonstrated that in vitro differentiation of heart-infiltrating prominin-1+ cells into fibroblast-like cells critically depended on TGF-β. To characterize the stage-specific microenvironment of the inflamed heart that dictates the fate of prominin-1+ cells in vivo, we measured cytokine production by heart-infiltrating cells at various time points after immunization. Release of the proinflammatory cytokines: IL-17, interferon-γ, and tumor necrosis factor-α by heart-infiltrating mononuclear infiltrates was significantly reduced at day 32 (beginning of fibrotic phase) compared with day 21 (inflammatory phase; Figure 6A). In contrast, levels of TGF-β were markedly increased in heart tissue during the late stage of disease (Figure 6A). Subsequently, we isolated the inflammatory heart-infiltrating prominin-1+ cells from EGFP+ mice at day 21, expanded in vitro and injected intracardially into healthy (d0), inflamed (d14), and fibrotic (d32) hearts. Seven to 14 days after intracardiac injection the mice were euthanized and the hearts analyzed.
Prominin-1+/EGFP+ cells maintained an undifferentiated phenotype after injection into the healthy hearts (Figure 6B). Injection of prominin-1+/EGFP+ cells at the onset of inflammation (day 14) resulted in EGFP+ monocyte/macrophage differentiation at day 21 (Figure 6C). Of note, some EGFP+ cells already expressed also fibronectin at this time point (Figure 6C). Injection of prominin-1+/EGFP+ cells at day 32, characterized by enhanced tissue fibrosis, resulted in the differentiation of EGFP+ cells into collagen I-, fibronectin-, and αSMA-expressing fibroblasts, analyzed at day 46 (Figure 6D).
Taken together, these findings suggest that the stage-specific cytokine milieu dictates the fate of BM-derived progenitor cells in the diseased heart during EAM.
TGF-β Is a Critical Mediator for Cardiac Fibrosis in EAM
So far, our data suggest that TGF-β promotes the fibroblast differentiation of heart-infiltrating prominin-1+ progenitor cells in the inflammatory microenvironment in the EAM model. Given the myriad of mediators/cytokines released during cardiac inflammation, the in vivo experiments described above cannot exclude that other mediator than TGF-β promotes tissue fibrosis independent from the prominin-1+ cells. To address the overall role of TGF-β in the development of cardiac fibrosis in EAM, we therefore repetitively injected α-TGF-β blocking antibody in mice between 17 to 46 days after αMyHC/CFA immunization. Control mice were treated with a nonspecific isotype antibody. As illustrated in Figure 7, we observed markedly reduced fibrosis measured as collagen I-positive area in heart tissues isolated from mice treated with α-TGF-β antibody (Figure 7A and 7C) compared to sham-treated control animals (Figure 7B and 7C). In addition, heart weight/body weight ratios were lower in α-TGF-β compared to sham-treated mice (Figure 7D). Furthermore, fibroblast-specific differentiation was abrogated if heart-infiltrating prominin-1+/EGFP+ cells were injected into fibrotic myocardium of αMyHC/CFA-immunized mice treated with serially administrated α-TGF-β blocking antibody (Online Figure IV). These findings complete our observation that α-TGF-β antibody blocked fibroblast differentiation of inflammatory heart-infiltrating prominin-1+ cells, cultivated in the presence of TGF-β (Figure 7E and 7F).
Taken together, TGF-β critically modulates fibroblast differentiation of heart-infiltrating prominin-1+ progenitor cells and tissue fibrosis in the EAM model.
Myocarditis predisposes to dilated cardiomyopathy, the most common cause of heart failure in young patients. Cardiac fibrosis is a hallmark of the failing heart, but little is known about the cellular origins and the underlying mechanisms of accumulating fibroblasts. Using a mouse model of EAM, we demonstrate that prominin-1 expression defines a specific population of heart-infiltrating BM-derived progenitor cells. Regulated by TGF-β, they differentiate into fibroblasts, thereby promoting tissue fibrosis during the late stages of disease. Importantly, however, these cells showed no ability to differentiate into cardiomyocytes.
Fibrogenic cells can expand from at least 3 sources: (1) from local activation and proliferation of heart-resident mesenchymal cells, (2) by epithelial-to-mesenchymal transition, and (3) from BM-derived cells.18 Using immunized chimera reconstituted with EGFP+ donor BM, we illustrated that the majority (>60%) of cardiac fibroblasts promoting fibrosis in the EAM model originate from BM. Given the facts that EGFP expression was detected in 80% to 90% of EGFP transgenic cells (K Gabriela, E Urs, unpublished observations, 2009) and that BM-derived fibroblasts were not observed before the onset of cardiac inflammation, we conclude that inflammatory infiltrates represent the major source of cardiac fibroblasts in EAM. Nevertheless, we cannot exclude that endogenous cardiac fibroblasts also participate in the fibrotic process at late stages of EAM. Some authors have postulated heart resident fibroblasts as the major cellular source of tissue fibrosis associated with hypertrophy and ischemic heart failure.19,20 Moreover, El-Helou et al21 identified heart-resident nestin-expressing fibroblast progenitors, migrating to the infarcted region in the response to ischemia, suggesting involvement of these cells in the scarring processes of the injured myocardium.
Injured hearts are believed to dispose of a pool of endogenous/recruited progenitors playing a relevant role in myocardial regeneration and pathological remodeling,22 but nothing is known about a potential role of any progenitors in EAM. We demonstrated here that at the peak of EAM prominin-1+ progenitors denote a significant fraction among CD45+ heart-infiltrating inflammatory cells. Prominin-1 is a well-established marker of hematopoietic, embryonic and adult progenitors.16,23,24 We illustrated that heart-infiltrating prominin-1+ cells represent a common progenitor for fibroblasts and macrophages in EAM. However, heart-infiltrating prominin-1+ cells showed impaired regenerative capacity (new cardiomyocytes formation). This is in strong contrast to prominin-1+ cells derived from healthy heart,16 despite the high similarity regarding the expression of stem/progenitor cell markers and immunomodulatory properties. Thus, our findings point to careful evaluation of stem/progenitor cells isolated from injured organs for use in regenerative medicine. In the EAM model, inflammation in the myocardium disables the expansion of progenitors with regenerative capacity from cardiac tissue. It remains still to be elucidated whether genetic or epigenetic manipulations can revert/induce regenerative potential in prominin-1+ cells derived from inflamed heart or even BM.
Heart injury results in leukocyte recruitment and a specific inflammatory environment, promoting healing processes and scar formation. Accordingly, proinflammatory cytokines/chemokines become rapidly induced in the injured myocardium. In parallel, counter regulatory mechanisms result in release and activation of inhibitory mediators, such as profibrotic growth factor TGF-β regulating the transition from inflammation to fibrosis.25 Our experiments demonstrate that the stage-specific microenvironment dictates the fate of transplanted progenitor cells at various time points during the EAM course. Accordingly, prominin-1+ cells derived from inflamed and healthy (Online Figure V) heart gained a fibroblast phenotype after intracardiac injection during late stages of EAM when profibrotic cytokine/chemokine production predominates. In contrast, prominin-1+ cells, injected into healthy heart, did not acquire fibroblast phenotype.
Our observations are in line with studies on infarcted26 or hypertrophic14 hearts, which suggest a decisive role of the myocardial microenvironment in promoting fibroblast-specific differentiation and scar formation of BM-derived cells. Similarly, it has been shown in the myocardial infarction model that the microenvironment promoted the differentiation of BM-derived, recruited, or administered progenitor cells into hematopoietic cell phenotypes, but not cardiomyocytes.27,28 Together with our findings in an inflammatory heart disease model, the present evidence supports the view that the specific microenvironment of an injured heart does not allow cardiac regeneration but promotes either the accumulation of monocytes/macrophages or tissue fibrosis/scarring. Thus, it is not surprising that most studies evaluating BM-derived stem cell therapies in mouse models of myocardial infarction showed minimal in vivo cardiomyocyte differentiation only14,29 or questioned the regenerative potential of stem cell–based therapies at all.27,28,30,31 Accordingly, BM-derived CD133+ progenitors transplanted after myocardial infarction did not form new cardiomyocytes but nevertheless improved cardiac function.32 Similarly, we showed that prominin-1+ progenitor cells expanded from both, healthy16 and inflamed hearts efficiently prevent EAM development but do not contribute to the regeneration of damaged cardiac tissue. Therefore, the development of clinically relevant cell-based therapies requires either ex vivo manipulation of progenitor cells, rendering them resistant to the inflammatory environment, or the combined in vivo targeting of specific cytokines/chemokines, impairing the regenerative capacity of progenitor cells. In fact, ex vivo pretreatment of autologous BM-derived progenitor cells with cardiomyogenic growth factors potentates their cardiac differentiation and their functional regenerative capacity in vivo.33
To date, several studies on models of noninflammatory heart disease suggested a critical role of TGF-β in cardiac fibrosis.8 A broad range of different cells recruited because of cardiac issue injury, ie, macrophages, T cells, and mast cells produce TGF-β.2,5 In EAM, intracardiac TGF-β levels progressively increase in parallel with steadily resolving cardiac infiltrates. TGF-β modulates the behavior of fibroblast progenitors/mature fibroblasts by stimulating the synthesis of various extracellular matrix proteins including collagens, fibronectin, tenascin, and proteoglycans,25 factors that affect the fate of progenitor cells. In our model, the fibroblast-specific differentiation of prominin-1+ cells depended on TGF-β, suggesting that this cytokine is the key mediator of cardiac fibrosis late during the EAM course. In fact, TGF-β signaling mediated the phosphorylation of Smad2 proteins in prominin-1+ progenitors, pointing to the involvement of canonical Smad-dependent signaling pathways19,34 in the transition of prominin-1+ progenitors to fibroblasts. Given the fact that TGF-β strongly promotes fibroblast differentiation of heart-infiltrating prominin-1+ cells in vitro, it was not surprising that prominin-1+ cells differentiated into fibroblasts after intracardiac injection at late stage of EAM characterized by high levels of TGF-β release within the heart. Accordingly, TGF-β depletion efficiently blocked cardiac fibrosis in our model. Similarly, anti–TGF-β treatment markedly decreased collagen deposition in infarcted hearts.35 Given the high frequency of heart-infiltrating progenitors in EAM, further studies are needed to develop strategies that specifically block the fibroblast-specific differentiation of progenitor cells. Based on our data, we would expect that TGF-β–dependent signaling pathways might become a potential target against progressive cardiac fibrosis and end-stage heart failure in the future.
In conclusion, we identified for the first time prominin-1+ bilineage fibroblast and monocyte/macrophage progenitors among heart-infiltrating CD45+ cells as the major cellular source of cardiac fibrosis in postinflammatory heart disease. Furthermore, we proved that the developmental fate of heart-infiltrating prominin-1+ progenitors was dictated by a specific, disease stage–dependent myocardial microenvironment. Finally, we identified TGF-β as the key cytokine promoting fibroblast differentiation of prominin-1+ progenitors during the transition from acute myocarditis to inflammatory cardiomyopathy in the EAM model. The critical role of prominin-1+ progenitors in the progression of cardiac inflammation to myocardial fibrosis identifies these cells as a promising target for novel treatment approaches for postinflammatory cardiac fibrosis during nonischemic heart failure in the future.
We thank Heidi Bodmer and Marta Bachman for technical assistance and Ulrich Schneider and Monika Hermle for animal care.
Sources of Funding
U.E. acknowledges support from the Gebert-Rüf Foundation and the Swiss Life Foundation. G.K. acknowledges support from the Swiss Heart Foundation. U.E. holds a Swiss National Foundation professorship.
Original received February 25, 2009; revision received June 23, 2009; accepted July 14, 2009.
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