A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction1. A therapeutic ideal—relative to cell transplantation—would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells2 with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards residentcell-based therapy in human ischaemic heart disease.

The lymphatic vasculature is a blind-ended network crucial for tissue fluid homeostasis, immune surveillance and lipid absorption from the gut. Recent evidence has proposed an entirely venous-derived mammalian lymphatic system. In contrast, we reveal here that cardiac lymphatic vessels have a heterogeneous cellular origin, whereby formation of at least part of the cardiac lymphatic network is independent of sprouting from veins. Multiple cre-lox based lineage tracing revealed a potential contribution from the hemogenic endothelium during development and discrete lymphatic endothelial progenitor populations were confirmed by conditional knockout of Prox1 in Tie2+ and Vav1+ compartments. In the adult heart, myocardial infarction (MI) promoted a significant lymphangiogenic response, which was augmented by treatment with VEGF-C resulting in improved cardiac function. These data prompt the re-evaluation of a century-long debate on the origin of lymphatic vessels and suggest that lymphangiogenesis may represent a therapeutic target to promote cardiac repair following injury.

Cardiovascular disease remains the major cause of mortality, and cardiac cell therapy has recently emerged as a paradigm for heart repair. The epicardium is a layer of mesothelial cells covering the heart that during development contributes to different cardiovascular lineages, including cardiomyocytes, but which becomes quiescent after birth. We previously revealed that the peptide thymosin beta 4 (Tβ4) can reactivate adult epicardium-derived cells (EPDCs) after myocardial infarction (MI), to proliferate, and differentiate into cardiovascular derivatives. The aim of this study was to provide a lineage characterization of the adult EPDCs relative to the embryonic epicardial lineage and to determine prospective cell fate biases within the activated adult population during cardiovascular repair. Wt1(GFPCre/+) mice were primed with Tβ4 and MI induced by ligation of the left anterior descending coronary artery. Adult WT1(+) GFP(+) EPDCs were fluorescence-activated cell sorted (FACS) at 2, 4, and 7 days after MI. Embryonic WT1(+) GFP(+) EPDCs were isolated from embryonic hearts (E12.5) by FACS, and sorted cells were characterized by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunostaining. Adult WT1(+) GFP(+) EPDCs were highly heterogeneous, expressing cardiac progenitor and mesenchymal stem markers. Based on the expression of stem cell antigen-1 (Sca-1), CD44, and CD90, we identified different subpopulations of EPDCs of varying cardiovascular potential, according to marker gene profiles, with a molecular phenotype distinct from the source embryonic epicardial cells at E12.5. Thus, adult WT1(+) GFP(+) cells are a heterogeneous population that when activated can restore an embryonic gene programme, but do not revert entirely to adopt an embryonic phenotype. Potential biases in cardiovascular cell fate suggest that discrete subpopulations of EPDCs might be clinically relevant for regenerative therapy.

Epicardium-derived cells (EPDCs) contribute cardiovascular cell types during development and in adulthood respond to Thymosin β4 (Tβ4) and myocardial infarction (MI) by reactivating a fetal gene programme to promote neovascularization and cardiomyogenesis. The mechanism for epicardial gene (re-)activation remains elusive. Here we reveal that BRG1, the essential ATPase subunit of the SWI/SNF chromatin–remodelling complex, is required for expression of Wilms’ tumour 1 (Wt1), fetal EPDC activation and subsequent differentiation into coronary smooth muscle, and restores Wt1 activity upon MI. BRG1 physically interacts with Tβ4 and is recruited by CCAAT/enhancer-binding protein β (C/EBPβ) to discrete regulatory elements in the Wt1 locus. BRG1-Tβ4 co-operative binding promotes optimal transcription of Wt1 as the master regulator of embryonic EPDCs. Moreover, chromatin immunoprecipitation-sequencing reveals BRG1 binding at further key loci suggesting SWI/SNF activity across the fetal epicardial gene programme. These findings reveal essential functions for chromatin–remodelling in the activation of EPDCs during cardiovascular development and repair.

Rationale: Compromised development of blood vessel walls leads to vascular instability that may predispose to aneurysm with risk of rupture and lethal hemorrhage. There is currently a lack of insight into developmental insults that may define the molecular and cellular characteristics of initiating and perpetrating factors in adult aneurismal disease.Objective: To investigate a role for the actin-binding protein thymosin ␤4 (T␤4), previously shown to be proangiogenic, in mural cell development and vascular wall stability. Methods and Results:Phenotypic analyses of both global and endothelial-specific loss-of-function T␤4 mouse models revealed a proportion of T␤4-null embryos with vascular hemorrhage coincident with a reduction in smooth muscle cell coverage of their developing vessels. Mechanistic studies revealed that extracellular T␤4 can stimulate differentiation of mesodermal progenitor cells to a mature mural cell phenotype through activation of the transforming growth factor-beta (TGF␤) pathway and that reduced TGF␤ signaling correlates with the severity of hemorrhagic phenotype in T␤4-null vasculature.Conclusions: T␤4 is a novel endothelial secreted trophic factor that functions synergistically with TGF␤ to regulate mural cell development and vascular wall stability. These findings have important implications for understanding congenital anomalies that may be causative for adult-onset vascular instability. (Circ Res. 2012; 111:e89-e102.) Key Words: thymosin Ⅲ vasculature Ⅲ mural cell Ⅲ aorta Ⅲ mouse Ⅲ mouse mutants Ⅲ smooth muscle differentiation Ⅲ vascular biology Ⅲ vascular smooth muscle T he development of a functional vasculature is an essential process during embryogenesis, perturbations in which result in fetal lethality or vascular disease after birth. The formation of systemic blood vessels occurs in a stereotypical fashion -endothelial tubes form through a number of mechanisms (angiogenesis, vasculogenesis, or intussusception). 1 Endothelial cells then recruit mural cells comprising the subsets of vascular smooth muscle cells (VSMCs) and pericytes to the external wall of the vessel. [2][3][4] These mural cells are required to provide structural support for the blood vessel and probably play a role in maintaining endothelial health and integrity. The establishment of a vessel wall is accomplished either through the differentiation of de novo mural cells from precursor populations or recruitment from a proliferating pool of mature cells. The former is thought to occur chiefly through the actions of endothelial secreted transforming growth factor-beta (TGF␤) and the latter through paracrine platelet-derived growth factor-B (PDGF-B). 4 Typically, in the embryo, mural cells originate from the in situ differentiation of mesodermal tissues, which surround endothelial tubes. 3,5 The exception to this is in the central nervous system, where blood vessels recruit to their outer layer via the migration of neurectodermal-derived mature mural cells, as typified by the development of the postnatal retinal vasc...