The epicardium plays an important role in cardiomyogenesis during development, while it becomes quiescent in adult heart during homeostasis. This study investigates the efficiency of thymosin β4 (Tβ4) release with RPRHQGVM conjugated to the C-terminus of RADA16-I (RADA-RPR), the functionalized self-assembling peptide (SAP), to activate the epicardium and repairing the infarcted myocardium. Methods: The functionalized SAP was constituted with self-assembling motif, Tβ4-binding site, and cell adhesive ligand. Myocardial infarction (MI) models of the transgenic mice were established by ligation of the left anterior descending coronary artery. At one week after intramyocardial injection of Tβ4-conjugated SAP, the activation of the epicardium was assessed. At four weeks after implantation, the migration and differentiation of epicardium-derived cells (EPDCs) as well as angiogenesis, lymphangiogenesis and myocardial regeneration were examined. Results: We found that the designer RADA-RPR bound Tβ4 and adhered to EPDCs and that Tβ4 released from the functionalized SAP could effectively activate the epicardium and induce EPDCs to differentiate towards cardiovascular cells as well as lymphatic endothelial cells. Moreover, SAP-released Tβ4 (SAP-Tβ4) promoted proliferation of cardiomyocytes. Furthermore, angiogenesis, lymphangiogenesis and myocardial regeneration were enhanced in the MI models at 4 weeks after delivery of SAP-Tβ4 along with attenuation of adverse myocardial remodeling and significantly improved cardiac function. Conclusions: These results demonstrate that sustained release of Tβ4 from the functionalized SAP can activate the epicardium and effectively enhance the repair of infarcted myocardium. We believe the delivery of SAP-Tβ4 may be a promising strategy for MI therapy.

Myocardial infarction (MI) leads to activation of cardiac fibroblasts (aCFs) and at the same time induces the formation of epicardium-derived cells at the heart surface. To discriminate between the two cell populations, we elaborated a fast and efficient protocol for the simultaneous isolation and characterization of aCFs and epicardial stromal cells (EpiSCs) from the infarcted mouse heart.
For the isolation of aCFs and EpiSCs, infarcted hearts (50 min ischaemia/reperfusion) were digested by perfusion with a collagenase-containing medium for only 8 min, while EpiSCs were enzymatically removed from the outside by applying mild shear forces via a motor driven device. Cardiac fibroblasts (CFs) isolated from unstressed hearts served as control. Viability of isolated cells was >90%. Purity of EpiSCs was confirmed by immunofluorescence staining and qPCR of various mesenchymal markers including Wilms-tumor-protein-1. Microarray analysis of CFs, aCFs, and EpiSCs on day 5 post-MI revealed a unique gene expression pattern in the EpiSC fraction, which was enriched for epithelial markers and epithelial to mesenchymal transition-related genes. Compared to aCFs, 336 significantly altered gene entities were identified in the EpiSC fraction. qPCR analysis showed high expression of Serpinb2, Cxcl13, Adora2b, and Il10 in EpiSCs relative to CFs and aCFs. Furthermore, microarray data identified Ddah1 and Cemip to be highly up-regulated in aCFs compared to CFs. Immunostaining of the infarcted heart revealed a unique distribution of Dermokine, Aquaporin-1, Cytokeratin, Lipocalin2, and Periostin within the epicardial cell layer.
We describe the simultaneous isolation of viable, purified fractions of aCFs and EpiSCs from the infarcted mouse heart. In this study, several differentially expressed markers for aCFs and EpiSCs were identified, underlining the importance of cell separation to study heterogeneity of stromal cells in the healing process after MI.

Human epicardium-derived cells (hEPDCs) transplanted in the NOD-SCID mouse heart after myocardial infarction (MI) are known to improve cardiac function, most likely orchestrated by paracrine mechanisms that limit adverse remodeling. It is not yet known, however, if hEPDCs contribute to preservation of cardiac function via the secretion of matrix proteins and/or matrix proteases to reduce scar formation. This study describes the ability of hEPDCs to produce human collagen type I after transplantation into the infarct border zone, thereby creating their own extracellular environment. As the in vivo environment is too complex to investigate the mechanisms involved, we use an in vitro set-up, mimicking biophysical and biochemical cues from the myocardial tissue to unravel hEPDC-induced matrix remodeling. The in vivo contribution of hEPDCs to the cardiac extracellular matrix (ECM) was assessed in a historical dataset of the NOD-SCID murine model of experimentally induced MI and cell transplantation. Analysis showed that within 48 h after transplantation, hEPDCs produce human collagen type I. The build-up of the human collagen microenvironment was reversed within 6 weeks. To understand the hEPDCs response to the pathologic cardiac microenvironment, we studied the influence of cyclic straining and/or transforming growth beta (TGFβ) signaling in vitro. We revealed that 48 h of cyclic straining induced collagen type I production via the TGFβ/ALK5 signaling pathway. The in vitro approach enables further unraveling of the hEPDCs ability to secrete matrix proteins and matrix proteases and the potential to create and remodel the cardiac matrix in response to injury.

The proepicardium (PE) is a transitory extracardiac embryonic structure which plays a crucial role in cardiac morphogenesis and delivers various cell lineages to the developing heart. The PE arises from the lateral plate mesoderm (LPM) and is present in all vertebrate species. During development, mesothelial cells of the PE reach the naked myocardium either as free-floating aggregates in the form of vesicles or via a tissue bridge; subsequently, they attach to the myocardium and, finally, form the third layer of a mature heart-the epicardium. After undergoing epithelial-to-mesenchymal transition (EMT) some of the epicardial cells migrate into the myocardial wall and differentiate into fibroblasts, smooth muscle cells, and possibly other cell types. Despite many recent findings, the molecular pathways that control not only proepicardial induction and differentiation but also epicardial formation and epicardial cell fate are poorly understood. Knowledge about these events is essential because molecular mechanisms that occur during embryonic development have been shown to be reactivated in pathological conditions, for example, after myocardial infarction, during hypertensive heart disease or other cardiovascular diseases. Therefore, in this review we intended to summarize the current knowledge about PE formation and structure, as well as proepicardial cell fate in animals commonly used as models for studies on heart development. Anat Rec, 302:893-903, 2019. © 2018 Wiley Periodicals, Inc.

The epicardium plays a key role during cardiac development, homeostasis and repair, and has thus emerged as a potential target in the treatment of cardiovascular disease. However, therapeutically manipulating the epicardium and epicardium-derived cells (EPDCs) requires insights into their developmental origin and the mechanisms driving their activation, recruitment and contribution to both the embryonic and adult injured heart. In recent years, studies of various model systems have provided us with a deeper understanding of the microenvironment in which EPDCs reside and emerge into, of the crosstalk between the multitude of cardiovascular cell types that influence the epicardium, and of the genetic programmes that orchestrate epicardial cell behaviour. Here, we review these discoveries and discuss how technological advances could further enhance our knowledge of epicardium-based repair mechanisms and ultimately influence potential therapeutic outcomes in cardiovascular regenerative medicine.

During embryonic heart development, the transcription factors Tcf21, Wt1, and Tbx18 regulate activation and differentiation of epicardium-derived cells, including fibroblast lineages. Expression of these epicardial progenitor factors and localization of cardiac fibrosis were examined in mouse models of cardiovascular disease and in human diseased hearts. Following ischemic injury in mice, epicardial fibrosis is apparent in the thickened layer of subepicardial cells that express Wt1, Tbx18, and Tcf21. Perivascular fibrosis with predominant expression of Tcf21, but not Wt1 or Tbx18, occurs in mouse models of pressure overload or hypertensive heart disease, but not following ischemic injury. Areas of interstitial fibrosis in ischemic and hypertensive hearts actively express Tcf21, Wt1, and Tbx18. In all areas of fibrosis, cells that express epicardial progenitor factors are distinct from CD45-positive immune cells. In human diseased hearts, differential expression of Tcf21, Wt1, and Tbx18 also is detected with epicardial, perivascular, and interstitial fibrosis, indicating conservation of reactivated developmental mechanisms in cardiac fibrosis in mice and humans. Together, these data provide evidence for distinct fibrogenic mechanisms that include Tcf21, separate from Wt1 and Tbx18, in different fibroblast populations in response to specific types of cardiac injury.