|Home | About | Journals | Submit | Contact Us | Français|
The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.
Myocardial infarction (MI) causes cardiomyocyte loss that far exceeds the limited regenerative capacity of mammalian myocardium (1), resulting in substantial morbidity and mortality. Progress in developing new therapies hinges on understanding the myocardial injury response elicited by MI. Recent studies in zebrafish, a vertebrate model capable of heart regeneration, suggested that the epithelial cell sheet covering the heart, the epicardium, plays a pivotal role in its regenerative response (2). In mammals, the fetal epicardium secretes factors that promote growth of myocardium. Epicardium also makes essential cellular contributions to the fetal myocardium, undergoing epithelial-to-mesenchymal transition (EMT) to form epicardium-derived cells (EPDCs) that differentiate into cardiomyocyte, coronary EC, smooth muscle cell, and interstitial fibroblast lineages (3–7). These data raise the tantalizing possibility that adult mammalian epicardium might be recruited for use in therapeutic myocardial regeneration. However, little is known about the roles of epicardium in the adult mammalian heart, either in organ homeostasis or in response to myocardial injury. Improved understanding of the pathophysiology of the myocardial injury response is fundamental for development of novel regenerative approaches for heart disease.
A major block to gaining greater insight into the function of adult epicardium has been an inability to specifically trace these cells in vivo and to isolate a pure population of EPDCs for further characterization and analysis in vitro. Here, we overcame this hurdle using 2 independent Cre-loxP–based approaches to specifically label epicardium and its derivatives. We tested the hypothesis that adult epicardium differentiates into other myocardial lineages in the adult heart. Furthermore, we isolated and characterized genetically marked EPDCs, permitting further analysis of their function using in vitro and in vivo models. Our results indicated that epicardial cells were activated by myocardial injury and formed an expanded layer of EPDCs. These EPDCs remained mesenchymal and did not adopt cardiomyocyte or coronary EC fates. However, they engaged in the myocardial injury response, promoting coronary vessel growth by conditioning the subepicardial region through paracrine mechanisms. Remarkably, injection of EPDC-conditioned media (EPDC-CM) in a MI model reduced infarct size and improved cardiac function. These results suggest that augmentation of properties of native epicardium may be an attractive therapeutic strategy in cardiac repair and regeneration.
We used inducible Cre-loxP technology to selectively and irreversibly label adult epicardial cells and their derivatives. CreERT2, a tamoxifen-activated (tam-activated) fusion of Cre recombinase to an engineered hormone binding domain of the estrogen receptor (ESR1), was selectively expressed in epicardium by knocking it into the Wt1 locus (Wt1CreERT2) (4). In fetal heart, cardiac WT1 expression was largely restricted to the epicardium (refs. 4, 5, Figure Figure1A,1A, and Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI45529DS1). In postnatal mouse and human heart, WT1 was also detected in epicardial cells (Figure (Figure1,1, B and C), although at a reduced frequency compared with fetal epicardium (Figure (Figure1D).1D). Activation of Cre by tam treatment of uninjured adult Wt1CreERT2/+ mice allowed us to track the fate of adult epicardial cells during heart homeostasis. Epicardial cells with a history of Cre activity were identified using the reporter Rosa26mTmG/+, which heritably switched from RFP to GFP expression upon Cre recombination (Figure (Figure1E).1E). We did not detect GFP in the absence of tam (Figure (Figure1E1E and Supplemental Figure 4A), which is indicative of tight control of Cre activity in this system. From 1 to 8 weeks after tam treatment, a subset of epicardial cells expressed GFP (Figure (Figure1E).1E). These cells resided on the surface of the heart, did not migrate into myocardium, and did not express cardiomyocyte, endothelial, or smooth muscle cell markers (data not shown). We did detect rare GFP+ cells within the myocardium that expressed EC markers (Supplemental Figure 2, A–E), but these were likely caused by endogenous expression of Wt1 in rare ECs (Supplemental Figure 2, F–H) rather than by differentiation of epicardial cells into ECs.
The lack of cells within uninjured myocardium lineage-marked by Wt1CreERT2 might reflect technical limitations of this approach. Therefore, we used an independent lineage-tracing method based on injection of Ad:Msln-Cre, an adenovirus in which the epicardially restricted mesothelin (Msln) promoter (8, 9) drives Cre expression. MSLN was expressed in adult epicardium and cultured epicardial cells, and cardiac expression remained confined to epicardium after MI (Supplemental Figure 3A and data not shown). Ultrasound-guided delivery of Ad:Msln-Cre to Rosa26mTmG/+ heart specifically labeled epicardium (Supplemental Figure 3, B and C). At 4 weeks after virus injection, nearly all Cre-dependent GFP lineage tracer was on the surface of the heart (Supplemental Figure 3, D and E), in WT1+ and RALDH2+ epicardial cells. We did not detect any GFP+ cells that expressed EC markers in this model (Supplemental Figure 3F).
We investigated the participation of epicardium in the cardiac injury response. Fetal epicardial genes Wt1, Tbx18, and Raldh2 were dynamically upregulated after MI, peaking between 1 and 5 days and declining to near-baseline levels by 4 weeks (Figure (Figure2A).2A). Organ-wide, the epicardial region increased to several cells thick. The thickening was most pronounced in the vicinity of the infarct (Figure (Figure2B).2B). We investigated in detail the expression map of WT1 in infarcted heart to facilitate interpretation of epicardial lineage tracing using the Wt1CreERT2/+ allele. WT1 was upregulated in a region overlying the myocardium containing epicardium and its derivatives (Figure (Figure2B),2B), so that the fraction of WT1+ cells increased from approximately 25% in normal heart to approximately 75% after MI (Figure (Figure2D).2D). We independently confirmed expansion of this population by staining for the ESR1 epitope of the CreERT2 fusion protein in Wt1CreERT2/+ hearts (Figure (Figure2C)2C) and by fluorescence-activated cell sorting (FACS) analysis, which showed a 12-fold increase of Wt1CreERT2-labeled cells after MI (normal, 6.3 × 103 cells/heart; after MI, 78.1 × 103 cells/heart; Figure Figure2E).2E). Pericardial opening was insufficient to stimulate Wt1 upregulation and epicardial expansion, as neither was observed with sham operation (Figure (Figure2B).2B). Consistent with expansion of Wt1-derived epicardial cells after MI, phospho-histone H3 (pH3) and BrdU staining showed that Wt1CreERT2-marked cells in the epicardial region were highly proliferative 2 days after MI (Figure (Figure2,2, F–H, and data not shown). To determine whether proliferation and expansion of the epicardial region occurs in larger mammals, we also examined canine myocardium obtained at baseline and between 5 days and 2 weeks after MI. As in the mice, MI induced cells in the canine epicardial region to become highly proliferative, and the epicardial region substantially increased in thickness in canine-infarcted heart (Figure (Figure2,2, I and J). These data indicate that MI reactivates a fetal program, including increased proliferation and upregulation of fetal genes.
A hallmark of fetal epicardium is the formation of mesenchymal cells by EMT. Due to the lack of lineage-tracing tools, there has been a paucity of direct in vivo evidence to evaluate whether this process occurs in adult heart injury. Here we used our genetic mouse models to directly track the fate of epicardial cells in infarcted heart and to isolate pure populations of post-MI EPDCs for further analysis. In the normal adult heart, epicardial EMT appeared quiescent (Figure (Figure1).1). We asked whether it was reactivated by myocardial injury. To selectively label epicardial cells and their derivatives with GFP and track their fate after MI, we treated adult Wt1CreERT2/+;Rosa26mTmG/+ mice with tam, then induced MI by coronary artery ligation (Figure (Figure3A).3A). In tam-treated MI hearts, 24.6% ± 2.5% of epicardial cells were GFP+, which indicated labeling of a substantial fraction of cells. Control experiments established the specificity of this labeling protocol (Supplemental Figure 4, A–H). In mice not treated with tam and subjected to MI, we detected very rare GFP+ cells (0.6% ± 0.5% of epicardial cells) that were confined to the epicardial layer. Because these cells were rare and limited to the epicardial layer, this “leaky” Cre activity induced by MI stress did not impair interpretation of subsequent experiments.
The GFP+ cells, composed of epicardium and its derivatives, are hereafter collectively referred to as EPDCs. To evaluate whether EPDCs arise from adult Wt1+ epicardial cells through EMT, we used FACS to isolate cells highly enriched for EPDCs. Validation studies showed that the FACS-purified EPDCs were highly enriched for epicardial markers and depleted for myocardial markers (Supplemental Figure 5A). We next measured expression of EMT-related genes in FACS-purified EPDCs. Compared with non-EPDCs, EPDCs were enriched for Snail, Slug, Twist, and Smad1 (Supplemental Figure 5B). Cardiac expression of these transcripts changed dynamically following MI, with maximal expression observed 5 days after MI (Supplemental Figure 5C). Immunostaining of infarcted mouse heart for EMT-regulatory proteins further confirmed this result, demonstrating upregulation of pSMAD1/5/8, pSMAD2, SNAIL, and SLUG in the epicardial region (Supplemental Figure 5, D–G). Consistent with their origin via EMT, FACS-sorted EPDCs had a mesenchymal rather than epithelial morphology (Supplemental Figure 5H). Collectively, these data support reactivation of epicardial EMT after MI.
We used genetic lineage tracing to examine the fate of EPDCs after MI (Figure (Figure3A).3A). GFP-marked EPDCs remained in the epicardial region overlying the heart and did not migrate into myocardium (Figure (Figure3,3, B–F). In 24 MI hearts, after excluding autofluorescence signals by spectral analysis (Supplemental Figure 6, A–G), we did not detect EPDCs that coexpressed cardiomyocyte markers TNNT2 or ACTN2 (Figure (Figure3,3, C and D), which indicates that EPDCs do not give rise to cardiomyocytes in the adult mouse heart after MI. Similarly, within the thickened epicardial region, we did not detect EPDCs that coexpressed endothelial markers (Figure (Figure3E).3E). We did find rare GFP+ cells within the myocardium, and all of these rare cells expressed endothelial markers (Figure (Figure3F).3F). GFP labeling of these cells was likely due to Wt1-driven CreERT2 expression in some coronary ECs (10), rather than EPDCs that had migrated into the myocardium (Supplemental Figure 6, H–K), as there was no spatial relationship of these cells to the epicardium even shortly after MI. We conclude that EPDCs did not adopt cardiomyocyte or EC fates after MI.
In fetal heart, EPDCs largely differentiate into smooth muscle and fibroblast lineages (5–7). In adult heart after MI, EPDCs also expressed fibroblast-specific protein 1 (FSP1), procollagen I (ProCol), collagen III (ColIII), fibronectin (FN1), α-SMA, SM22α, and smooth muscle myosin heavy chain (SM-MHC), markers of mesenchymal lineages including fibroblasts, myofibroblasts, and smooth muscle cells (Figure (Figure3,3, G–M). Differentiation of EPDCs into these mesenchymal lineages was independently confirmed by analysis of FACS-purified EPDCs, which were highly enriched for markers of these lineages by immunostaining (Supplemental Figure 7, A–L) and by quantitative RT-PCR; Supplemental Figure 7M). FACS-purified EPDCs also lacked expression of cardiomyocyte markers ACTN2 and TNNT2 and of endothelial marker PECAM (Supplemental Figure 7, F–H). These data indicate that adult EPDCs differentiate into smooth muscle, myofibroblast, and fibroblast lineages after MI, but do not adopt a cardiomyocyte fate as previously described (11).
To confirm the above result, we used ultrasound-guided delivery of Ad:Msln-Cre to selectively label epicardium by an independent method. We injected Ad:Msln-Cre into adult Rosa26mTmG/+ heart to label epicardial cells, induced experimental MI, and then analyzed hearts 3 days to 4 weeks after MI (Supplemental Figure 8A). Consistent with Wt1CreERT2 labeling results, Ad:Msln-Cre–labeled epicardial cells were observed in the epicardial region covering the heart (Supplemental Figure 8, B–E). GFP+ cells within the epicardial region expressed the mesenchymal cell marker FSP1 and did not express endothelial or cardiomyocyte markers (Supplemental Figure 8, F and G). We also did not observe any GFP+ ECs within the myocardium, which again indicated that Wt1CreERT2-labeled GFP+ ECs observed in the post-MI heart are likely to arise from Wt1CreERT2 expression in these cells. We concluded that after MI, epicardial cells undergo EMT and adopt mesenchymal cell fates. These mesenchymal cells remain in the epicardial region and differentiate primarily into fibroblast and smooth muscle cells, but do not reconstitute cardiomyocyte or coronary ECs.
Whereas EPDCs within the thickened epicardial region did not adopt EC fates, ECs were common in this area and were frequently located adjacent to EPDCs (Figure (Figure3E).3E). Similarly, ECs were frequently observed in the epicardial region in canine heart after MI (data not shown). These observations are reminiscent of the close relationship between epicardium and coronary vasculature during heart development (12, 13), where coronary vasculature first develops immediately below the epicardium. To directly test the proangiogenic role of the expanded epicardial region in adult post-MI heart, we FACS-purified EPDCs from infarcted hearts and expanded them in culture (Supplemental Figure 9). After amplification for 5 passages, EPDCs continued to exhibit mesenchymal morphology (Figure (Figure4A)4A) and retained their high purity (>99.5% GFP+, Supplemental Figure 9, C and D). Expression of selected surface markers were stable between initial culture (i.e., passage 0 [P0]) and P5 (Supplemental Figure 9E). Epicardial markers Wt1 and Tbx18 continued to be expressed at P5, although their levels decreased approximately 50% (Supplemental Figure 9F).
EPDC-CM from P5 EPDCs stimulated growth of multiple types of ECs — human cord blood ECs (cbECs), HUVECs, cardiac microvasculature ECs, immortalized lung ECs, and bovine capillary ECs — to a degree comparable to that stimulated by 5% FBS or media conditioned by mouse bone marrow mesenchymal stem cells (MSCs; Figure Figure4,4, B–D, Supplemental Figure 10, A–F, and data not shown), which have established angiogenic activity (14). EPDC-CM from 8 independent EPDC cultures significantly increased EC number, measured by both direct cell counting and MTT assays, and the growth-promoting effect was observed at up to 72 hours of incubation and 1:80 dilution (Figure (Figure4,4, B–D, and Supplemental Figure 10, A–F). Boiling abolished the growth-promoting effect (Figure (Figure4D),4D), consistent with essential heat-labile factors such as proteins. EPDC-CM promoted growth by both reducing apoptosis (Figure (Figure4E)4E) and stimulating proliferation (EPDC-CM, 5.8% BrdU+ cells; control media, 1.4% BrdU+ cells, P < 0.05; Figure Figure4,4, E and F). Together, these data indicate that EPDC-secreted factors promote EC growth.
In addition to stimulating EC growth, EPDC secreted factors stimulated vessel assembly. When cocultured with ECs, EPDCs incorporated into the EC vessel network (Figure (Figure4G4G and Supplemental Figure 11, A and B), suggestive of potential cellular contribution as pericytes or smooth muscle cells to promote vessel formation and maturation. This is consistent with the frequent position of EPDCs next to ECs in both mouse and dog heart after MI (Figure (Figure3E3E and data not shown). To further study the effect of EPDCs on vessel assembly in an in vivo model, we used the Matrigel plug assay (Figure (Figure4H).4H). Whereas cbECs formed scant vessels in Matrigel plugs lacking EPDCs, they formed robust vessels in the presence of EPDCs, comparable to what was observed by the combination of cbECs with MSCs (Figure (Figure4,4, I and J, and Supplemental Figure 11, C and D). EPDCs, marked by GFP, were located adjacent to human ECs, labeled with UEA, a lectin that specifically labels human ECs (Figure (Figure4K).4K). EPDCs expressed NG2, CNN, SM22, and SMA, markers of pericytes and smooth muscle cells (Figure (Figure4,4, L and M, and Supplemental Figure 11, E and F). As we observed in heart tissue (Figure (Figure3),3), EPDCs did not adopt an EC fate in this assay (Figure (Figure4K4K and Supplemental Figure 11G). Blood vessels assembled in the Matrigel plugs formed functional connections to the host circulatory system, as tail vein injection of UEA labeled vessels within the plugs (Figure (Figure4N).4N). Moreover, Matrigel plugs containing only EPDCs without implanted ECs developed blood vessels (Supplemental Figure 11, H and I), which indicates that EPDCs alone stimulate host angiogenesis. Additionally, the cornea micropocket assay provided further in vivo evidence of angiogenic activity of EPDC-secreted factors. Corneal implantation of EPDC-CM–loaded pellets promoted vessel growth on the normally avascular cornea, whereas pellets loaded with unconditioned media did not (Supplemental Figure 11J). Together, these data show the proangiogenic activity of EPDC-secreted factors.
To identify molecules responsible for CM proangiogenic activity, we screened EPDCs for enriched expression of angiogenic factors by qRT-PCR. A large number of proangiogenic transcripts were highly enriched in EPDCs compared with non-EPDCs, including Vegfa, Angpt1, Ang, Fgf1, Fgf2, Fgf9, Pdgfa, Pdgfc, Pdgfd, Adamts1, Sdf1, Mcp1, and Il6 (Figure (Figure5A).5A). These genes were also expressed in cultured EPDCs, and their expression was maintained between P0 and P5 (Supplemental Figure 10G). To investigate whether MI alters expression of these genes in Wt1CreERT2 lineage cells, we compared the angiogenic gene expression profile of EPDCs after MI with the small subset of epicardial cells labeled by Wt1CreERT2 in non-MI heart. Several angiogenic genes were either up- or downregulated, although there was no systematic change in the angiogenic profile (Supplemental Figure 12). These data suggest that amplification of EPDC number after MI is an important contributor to the epicardium’s effects on angiogenesis. However, Sdf1 and Mcp1, chemotactic factors for circulating cells active in angiogenesis, were highly upregulated by MI, which indicates that MI significantly alters expression of additional epicardial genes relevant to the myocardial injury response.
An angiogenesis antibody array confirmed post-MI EPDC secretion of VEGFA, SDF1, MCP1, IGFPBP2, IGFPBP3, IGFPBP9, and IGFPBP10 at the protein level (Figure (Figure5B).5B). Blocking antibodies to these factors indicated that FGF2 and VEGFA accounted for approximately half the growth-promoting activity of CM on ECs (Figure (Figure5C).5C). However, EPDC-CM treated with both FGF2 and VEGFA blocking antibodies retained some growth-stimulating activity (Figure (Figure5C),5C), suggestive of additional factors or combined effects of multiple factors. Consistent with these cell culture data, we observed strong expression of VEGFA and FGF2 in the epicardial region of the post-MI heart (data not shown).
MI creates a hypoxic environment, which can regulate angiogenic gene transcription and translation (15). To determine whether hypoxia influences EPDC production of angiogenic factors, we obtained CM from post-MI EPDCs cultured in normoxia or hypoxia. Hypoxia upregulated VEGFA levels, as determined by quantitative ELISA (Figure (Figure5D).5D). We also detected FGF2 upregulation under hypoxic conditions, and similar observations were made using an epicardial cell line (ref. 16 and data not shown). These data suggest that hypoxia may dynamically upregulate secretion of proangiogenic factors from the epicardial layer after MI.
Collectively, our data support a role for EPDCs as a source of angiogenic factors in the infarcted heart. This effect is partially mediated by VEGFA and FGF2, secretion of which increases under hypoxic conditions.
To test the hypothesis that EPDC-secreted factors have a beneficial effect on the myocardial response to MI, we injected EPDC-CM or unconditioned media into the ischemic border zone immediately after inducing experimental MI. At 5–6 days after MI, triphenyltetrazolium chloride (TTC) staining showed that CM significantly reduced infarct size compared with control (P < 0.05; Figure Figure6A).6A). Correspondingly, we observed increased vessel density in EPDC-CM–treated myocardium (P < 0.05; Figure Figure6B).6B). To assess the effect of CM on heart function, we used both MRI and hemodynamic measurements 5–6 days after MI. MRI showed that LV ejection fraction, a measure of systolic heart function, was significantly increased in CM compared with control (Figure (Figure6C).6C). In Langendorff-perfused, isovolumetric heart preparations, systolic function improved with CM treatment, as measured by significant increases in peak and developed systolic pressure under baseline conditions and with dobutamine stress (P < 0.05; Figure Figure6,6, D–F). Emax, a load-independent measure of contractility, was also significantly improved by CM (Figure (Figure6F).6F). LV stiffness, which is inversely related to diastolic function, was reduced with CM injection (P = 0.015; Figure Figure6G),6G), indicating that CM also beneficially affects diastolic function. Taken together, our data demonstrate that at an early time point, CM improves cardiac function of infarcted hearts both at baseline and upon increased workload.
Finally, we tested the effects of CM on cardiac function at a more distant time point. At 9 weeks after MI, EPDC-CM treatment at the time of left anterior descending coronary artery (LAD) ligation significantly reduced adverse cardiac remodeling, as shown by decreased LV systolic and diastolic volume in the CM-treated group (Supplemental Figure 14, A and B). There was a trend toward higher ejection fraction with CM treatment (P = 0.076; Supplemental Figure 14C). However, hemodynamic measurements in Langendorff-perfused hearts were not significantly different between groups (Supplemental Figure 14, D–G). Thus, CM treatment at the time of MI reduced cardiac remodeling, but did not significantly improve measures of cardiac function.
Better understanding of the endogenous myocardial injury response will illuminate mechanisms that can be therapeutically modulated to minimize myocardial loss after MI and to amplify the limited regenerative capacity of mammalian myocardium. Using 2 independent genetic lineage-tracing methods, we investigated the cellular and paracrine contributions of epicardium to the MI injury response (Supplemental Figure 13). Whereas epicardium was quiescent in the normal heart, MI reactivated a fetal-like state, including proliferation, embryonic gene expression, and EMT. After MI, the epicardial layer expanded substantially, and a large fraction of cells expressed the Wt1 epicardial marker. The increased number of WT1+ cells was likely due to both proliferation of these cells and reactivation of WT1 expression in previously nonexpressing cells. However, the injury-activated adult EPDCs appeared to incompletely recapitulate the properties of fetal EPDCs. Unlike fetal EPDCs (which migrate into myocardium and differentiate into cardiomyocytes, smooth muscle cells, interstitial cells, and ECs), post-MI adult EPDCs largely remain on the surface of the heart. Furthermore, as determined by both Wt1CreERT2 and Ad:Msln-Cre labeling approaches, adult EPDCs no longer differentiate into cardiomyocytes or ECs. A prior lineage-tracing study based on lentiviral labeling of epicardial cells using CMV-GFP suggested that epicardial cells differentiate into both ECs and cardiomyocytes after MI (11). The discrepancy with our results indicates that either the lentiviral labeling approach marked an epicardial population distinct from that labeled by Wt1CreERT2, or the lentiviral label was not precisely confined to the epicardium. The molecular mechanisms underlying diminished mobilization and plasticity of adult epicardium are unknown, but may partially contribute to the reduced regenerative ability of adult heart. Enabling adult epicardium to more fully recapitulate its fetal properties may enhance the adult heart’s regenerative capacity.
Another important function of fetal epicardium is to regulate myocardial growth and coronary development through secretion of paracrine factors. Therefore, we investigated whether EPDCs affect the adult myocardial injury response through paracrine mechanisms. Our data indicated that EPDC paracrine factors promote enhanced survival and growth of coronary vessels after MI. During development, coronary vessels form immediately under epicardium, stimulated by epicardial secretion of proangiogenic factors including VEGFA, FGF9/16/20, Sonic hedgehog, and WNT9b (17–21). MI reactivates fetal epicardial properties, and post-MI EPDCs secreted factors with potent proangiogenic activity in vitro and in vivo. We identified a number of factors with substantial enrichment in epicardium, including VEGFA and FGF2, and showed that these 2 factors contributed to angiogenic activity of EPDC-CM. However, other factors and their synergy are also important, as blocking both VEGFA and FGF2 only partially inhibited CM activity. Candidates include PDGF-CC (22), placental growth factor (PLGF; ref. 23), and PDGF-BB (24).
Our present work showed that EPDCs secrete paracrine factors that participate in the myocardial injury response. Remarkably, injection of EPDC-CM reduced infarct size and improved heart function at 1 week after MI. At 9 weeks after MI, the single CM treatment reduced adverse cardiac remodeling, but the improvement in cardiac contractile performance was not sustained. MI reactivates fetal-like epicardial properties and stimulates dramatic expansion of EPDCs that amplifies epicardial paracrine signaling. The beneficial effect of EPDC-CM injected at the time of MI may result in part from immediate availability of protective EPDC factors that normally would not approach maximal availability for several days. Consistent with our findings of beneficial EPDC-CM activity, myocardial injection of human EPDCs in a murine MI model improved ventricular function, vascularization, wall thickness, and survival to 6 weeks, although there was no significant EPDC engraftment at this time point (25). More prolonged availability of EPDC-secreted factors with EPDC injection may account for the sustained functional benefit seen in this model. Thus, effective translation of the beneficial activity of EPDC-CM will require defining its active components and developing improved methods for sustained delivery.
We showed that one beneficial effect of EPDC-CM is to protect and augment coronary vasculature, which may promote survival of tenuous myocardium in the peri-infarct zone. Additional mechanisms, such as protection of cardiomyocytes from apoptosis or stimulation of cardiomyocyte progenitor expansion and differentiation, may also contribute to the protective effects of EPDC-CM. Further beneficial effects of EPDC-CM may arise from recruitment of other cell types. Consistent with marked upregulation of Mcp1 and Sdf1 in infracted EPDCs, we observed substantial infiltration of monocytes into the thickened post-MI epicardium (B. Zhou and W.T. Pu, unpublished observations), where they may contribute to the angiogenic response after MI (26). Further studies will be required to dissect the likely pleiotropic effects of EPDC-CM.
In this study, we described and validated approaches to genetically label and track EPDCs in the adult heart. This allowed us to assess, for the first time to our knowledge, the fate of adult epicardial cells after myocardial injury. We also developed methods to isolate adult post-MI EPDCs and to amplify them in culture. Amplification in culture was essential to make investigation of EPDC functional properties experimentally feasible. Expression of most EPDC gene and surface markers that we measured did not change substantially through 5 passages in culture, but we cannot exclude the possibility that culture-induced changes in EPDC-secreted factors contribute to the observed activity of EPDC-CM. Identification of the factors responsible for EPDC-CM activity will be needed to fully address this point and to facilitate studies of the long-term benefits of EPDC paracrine factors after MI.
In summary, our data suggest that post-MI EPDCs condition a subepicardial niche and that one action of EPDC-secreted factors is to promote survival and growth of blood vessels. Studies in zebrafish heart regeneration support the concept of a subepicardial niche. As we saw in mice, zebrafish heart apex amputation stimulated organ-wide fetal reactivation of epicardium that contributed to vascular development of the regenerate (2). Moreover, apex amputation stimulated proliferation of subepicardial cardiomyocytes, which subsequently replaced the amputated cardiomyocytes (27, 28). Our data suggest that a mammalian subepicardial niche, conditioned by factors secreted from the expanded EPDC layer, supports blood vessel survival and growth. We hypothesize this niche also favors survival, growth, and differentiation of cardiomyocytes and their progenitors; future experiments will test this hypothesis. The EPDC-conditioned subepicardial niche provides what we believe to be a new paradigm to study the myocardial injury response in mammals, and augmentation of the subepicardial niche may represent a novel therapeutic strategy to improve outcome from MI.
Further information can be found in Supplemental Methods.
All experiments with mice were performed according to protocols approved by the Institutional Animal Care and Use Committees of Children’s Hospital Boston or Beth Israel Deaconess Medical Center. Wt1GFPCre/+, Wt1CreERT2/+, Rosa26fsLz/+, iTNT-Cre, and Rosa26mTmG/+ mice were described previously (4, 29–32). We administered tam (1 mg/10 g body weight) by gavage to pregnant mice at E10.5 to induce Cre. For adult mice, 4 mg tam (Sigma-Aldrich) was administered by gavage to 6- to 9-week-old mice twice weekly for 2–3 weeks.
MI was induced by ligation of LAD through a thoracotomy, as described previously (33). Sham operation included opening of the pericardium, but not ligation of the coronary artery. Where indicated, concentrated CM or unconditioned media were injected into the infarct border zone at the time of LAD ligation. Measurement of heart function by MRI was performed on anesthetized mice using gated Flash-CINE sequences. Langendorff perfusion was performed as described previously (34).
EPDCs were isolated from apical regions of hearts by collagenase digestion to obtain single-cell suspensions, followed by FACS sorting as described previously (35). After recovery, cells were amplified in MSC growth media (Lonza) with 10% FBS. For preparation of CM, cells were switched to EBM2 (Lonza) without serum for 24 hours. Media was then collected and concentrated 30-fold.
EC growth was measured by MTT assay (Promega) and direct counting. EC apoptosis was measured by TUNEL and cleaved caspase-3 assays (Roche; Cell Signaling Technology). Tubule formation, Matrigel plug, and corneal angiogenesis assays were performed as described previously (36–38).
Immunohistochemistry and spectral emission analyses were performed as described previously (39, 40), using antibodies listed in Supplemental Table 1. Gene expression was analyzed by qRT-PCR using the ΔΔCt method and primers listed in Supplemental Table 2.
Statistical analysis was performed using 2-tailed Student’s t test. A P value less than 0.05 was considered significant. Values are reported as mean ± SEM.
The authors thank Elizabeth Boush for aiding FACS procedures and Reza Akhavan for mouse cardiac MRI. This work was supported by funding from NIH (RO1 HL094683 and U01 HL100401 to W.T. Pu and P01 CA045548 to M.A. Moses), an American Heart Association Postdoctoral Fellowship (to B. Zhou), a Career Development Award from the Translational Research Program at Children’s Hospital Boston (to W.T. Pu), and charitable support from James Smith and Gail Federici-Smith (to W.T. Pu) and the Simeon Burt Wolbach Research Fund (to B. Zhou). H. He was supported in part by the Children’s Hospital Boston Anesthesia Foundation.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2011;121(5):1894–1904. doi:10.1172/JCI45529.