Epicardial Progenitor Cells Research Articles

Harnessing the regenerative potential of interleukin11 to enhance heart repair

Balancing between regenerative processes and fibrosis is crucial for heart repair, yet strategies regulating this balance remain a barrier to developing therapies. The role of Interleukin 11 (IL11) in heart regeneration remains controversial, as both regenerative and fibrotic functions have been reported. We uncovered that il11a, an Il11 homolog in zebrafish, can trigger robust regenerative programs in zebrafish hearts, including cardiomyocytes proliferation and coronary expansion, even in the absence of injury. Notably, il11a induction in uninjured hearts also activates the quiescent epicardium to produce epicardial progenitor cells, which later differentiate into cardiac fibroblasts. Consequently, prolonged il11a induction indirectly leads to persistent fibroblast emergence, resulting in cardiac fibrosis. While deciphering the regenerative and fibrotic effects of il11a, we found that il11-dependent fibrosis, but not regeneration, is mediated through ERK activity, suggesting to potentially uncouple il11a dual effects on regeneration and fibrosis. To harness the il11a’s regenerative ability, we devised a combinatorial treatment through il11a induction with ERK inhibition. This approach enhances cardiomyocyte proliferation with mitigated fibrosis, achieving a balance between regenerative processes and fibrosis. Thus, we unveil the mechanistic insights into regenerative il11 roles, offering therapeutic avenues to foster cardiac repair without exacerbating fibrosis.

Role of inflammation and macrophages in the formation of the atrial cardiomyopathy associated with obesity

Abstract Introduction The atrial fibrillation (AF), the most frequent cardiac arrhythmia in clinical practice is often associated with an atrial cardiomyopathy (ACM) which is favoured by several clinical conditions including metabolic diseases such as obesity. Recently, the epicardial adipose tissue (EAT) has emerged as an important component of ACM notably in the context of metabolic diseases. Epicardial progenitor cells (EPC) are the main source of EAT. The replacement of EAT by fibrosis is arrhythmogenic and involved inflammation but not yet characterized. The hypothesis tested here is that macrophages are central players for both the recruitment of EPC and replacement of EAT by fibrosis. Purpose-Aims First to characterize subpopulation of macrophages involves in the progression of ACM. Second, to study how the inflammatory environment regulates EPC differentiation. Method A spatial gene expression was used to analyze human samples (n=4) (Agreement, CODECOH, Declaration or authorization of activities involving the preservation and preparation of human body parts for scientific purposes DC-2020-4183). A well-characterized mouse model of atrial cardiomyopathy associated with obesity induced by high fat diet (HFD) was used to characterize macrophages associated with progression of ACM by using immunolabeling together with single nuclei RNA sequencing approaches. ACM was characterized by using echocardiography and atrial burst pacing to evaluate determine AF vulnerability. Transgenic RGB-MAC mice with macrophages tagged with McApple-Csf1R and ECGFP-CX3CR1 or with a knock-out for CCR2 were used to follow infiltration of myeloid cells in the atrial at the different stages of the ACM. Bi-photon microscopy was used to visualize cell subpopulations in the distinct area of the atrial wall. In some experiments EPC and macrophages were cocultured with transwell. Results Macrophages were detected in subepicardium of human atrial samples in area of fibro-fatty infiltrates. After 2 months of HFD, monocytes were infiltrated atria and macrophages were overexpressed in atria from 1 to 4 months of HFD. Using Bi-photon microscopy it was possible to visualise resident ECGFP-CX3CR1+ macrophages at the contact of EAT whereas McApple-Csfr1r+ macrophages were at the junction with fibrosis (figure 1). Preliminary data generated by single nuclei RNA sequencing showed clusters of different subpopulations of macrophages at different time point of the ACM (figure 2). We will present results on the effects of suppression of the CCR2 subpopulation of macrophages on characteristics of the ACM. Conclusion Distinct populations of macrophages i.e resident and monocyte-derived macrophages, participate to the distinct stages of the ACM caused by obesity in mice.Myeloid cells infiltration in epicardiumCCR2+cells infiltration in EAT-fibrosis

Cardiac Development and Factors Influencing the Development of Congenital Heart Defects (CHDs): Part I.

The traditional description of cardiac development involves progression from a cardiac crescent to a linear heart tube, which in the phase of transformation into a mature heart forms a cardiac loop and is divided with the septa into individual cavities. Cardiac morphogenesis involves numerous types of cells originating outside the initial cardiac crescent, including neural crest cells, cells of the second heart field origin, and epicardial progenitor cells. The development of the fetal heart and circulatory system is subject to regulatation by both genetic and environmental processes. The etiology for cases with congenital heart defects (CHDs) is largely unknown, but several genetic anomalies, some maternal illnesses, and prenatal exposures to specific therapeutic and non-therapeutic drugs are generally accepted as risk factors. New techniques for studying heart development have revealed many aspects of cardiac morphogenesis that are important in the development of CHDs, in particular transposition of the great arteries.

Abstract P2102: The Basic Helix-loop-helix Transcription Factor Tcf21 Regulates Fibroblast Cell States In The Adult Murine Heart

Objective: Transcription factor 21 (Tcf21) is a basic helix-loop-helix protein that is essential for normal mesodermal organogenesis and its loss is incompatible with life. In the developing heart, epicardial progenitor cells differentiate to either vascular smooth muscle cells or fibroblasts, the latter of which is dependent on Tcf21 expression. In the adult heart, Tcf21 is expressed by the majority of fibroblasts but is downregulated in response to fibrotic stimuli as the fibroblast differentiates to a myofibroblast. In atherosclerosis, smooth muscle cells that undergo phenotypic modulation towards a fibroblast-like cell acquire expression of Tcf21. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Methods and Results: To test this hypothesis, we generated tamoxifen-inducible genetic mouse models to study cardiac fibroblast Tcf21 deletion and overexpression in vivo . Acute and long-term Tcf21 ablation in the adult heart did not promote differentiation to myofibroblasts. After long-term ablation, no differences were detected on cardiac interstitial cell populations, cardiac structure or function and extracellular matrix deposition. In contrast, in response to fibrotic stimuli, overexpression of Tcf21 suppressed differentiation to myofibroblasts with reduced expression of contractile proteins. This was accompanied by reduced cardiac fibrosis. Conclusions: The expression of Tcf21 in the healthy fibroblast is not required to maintain a ‘resting’ fibroblast cell-state. In disease, Tcf21 functions as a suppressor of fibroblast differentiation to the contractile myofibroblast and its overexpression reduces the fibrotic burden on the heart.

Long chains fatty acids induce differentiation of epicardial progenitor cells into adipocytes through preadipocyte marker DLK1

The atrial fibrillation (AF), the most frequent cardiac arrhythmia is most often the clinical expression of the atrial cardiomyopathy. The epicardial adipose tissue (EAT) is now considered as an important component of the atrial cardiomyopathy notably when AF is associated metabolic disorders such as obesity. We previously showed that the epicardial progenitor cells (EPC) are the main source of EAT. Here we tested the hypothesis that long chain free fatty acids (LCFFA) present in the serum of patients with metabolic disorders can induce the differentiation of EPC into adipocytes. First, characterization of the effects LCFFA oleate (OLE) and palmitate (PAL) on differentiation of EPC; second, to examine the involvement of DLK1/Pref-1, a key actor of adipogenesis in the differentiation of EPC into adipocytes. In a rat model of atrial cardiomyopathy secondary to ischemic heart failure, EPC were harvested and cultured in presence of OLE (500 μM) and PAL (250 μM) for 7 days. EPC-derived adipocytes were characterized with oil red-O staining and Bodipy labelling. The transcriptomic of EPC was performed with single cell RNA sequencing together with a gene expression of EPC-derived adipocytes performed with qPCR at 3 and 7days after treatment. Soluble form of Pref-1 was measured in supernatant of EPC treated by using ELISA assay. The Notch/Pref-1 signaling pathway was investigated with biochemistry approach. After 3 days, the subpopulation of EPC expressing pref1 accumulated lipid droplets evidenced by red oil. At day 7, DLK1 expression was decreased whereas that of the adipogenic marker PPARγ was increased at the transcriptomic level (n = 5; P < 0.05). Furthermore, the expression of short soluble form of Pref-1 accumulated in supernatant of EPC indicated the shedding of the protein. Preliminary data indicated an effect of the short soluble form of Pref-1 on EPC proliferation. Single cell RNA sequencing revealed Notch expression in cluster of EPC engaged in the adipocyte differentiation pathway. Serum long chain fatty acid can trigger the adipogenic differentiation of EPC and DLK1/Notch signaling pathway appears to be an early step during this process. It remains to determine mechanisms of lipidic receptor GPR40 in EPC differentiation and the contribution to the expansion of atrial EAT during metabolic diseases.

Clinical Signs of Kawasaki Disease from the Perspective of Epithelial-to-Mesenchymal Transition Recruiting Erythrocytes: A Literature Review.

Kawasaki disease (KD) is a systemic vasculitis affecting children younger than 5 years of age. Early period in life is marked by rapid somatic growth with cell proliferation and immaturity of the immunity with dominant innate immune system. Coronary complications in KD are the most common acquired heart disease in children, yet the diagnosis of KD still depends on the clinical diagnostic criteria. Glossy red lips and conjunctival injection are characteristic signs enabling pediatricians to make the initial diagnosis of KD; however, little is known why these are so characteristic. The diagnostic criteria of KD seem to be scattered in seemingly irrelevant body systems such as the eyes, lips, skin, and heart. KD is classified as a connective tissue disease. Recently, red blood cells (RBCs) have emerged as important modulators in innate immune response. RBCs are reported to participate in extracellular matrix remodeling and upregulating matrix metalloproteinase (MMP) expression in dermal fibroblasts. Also, fibroblast growth factors and microRNAs associated with fibrosis are drawing attention in KD. The cardinal signs of KD appear at the border of muco-cutaneous junction. Head and neck regions are abundant in tissues undergoing epithelial-to-mesenchymal transition (EMT). Interstitial carditis and valve insufficiency as well as coronary arterial lesions may complicate KD, and these lesions present in tissues that originated from epicardial progenitor cells by EMT. Having reviewed the recent research on KD, we presume that the signs of KD present at borders between keratinized and non-keratinized stratified squamous epithelium where the EMT is still ongoing for the rapid somatic growth where RBCs are recruited as an innate immune response and to prevent excessive fibrosis in mucosa. KD presents scarcely in adults with somatic growth and immune maturation completed. In this review, we attempted to explain the reasons for the clinical manifestations of KD and to search for a link among the diagnostic clues in the perspective of EMT during the somatic growth and immune system maturation in children with KD.

Activation of a transient progenitor state in the epicardium is required for zebrafish heart regeneration

The epicardium, a mesothelial cell tissue that encompasses vertebrate hearts, supports heart regeneration after injury through paracrine effects and as a source of multipotent progenitors. However, the progenitor state in the adult epicardium has yet to be defined. Through single-cell RNA-sequencing of isolated epicardial cells from uninjured and regenerating adult zebrafish hearts, we define the epithelial and mesenchymal subsets of the epicardium. We further identify a transiently activated epicardial progenitor cell (aEPC) subpopulation marked by ptx3a and col12a1b expression. Upon cardiac injury, aEPCs emerge from the epithelial epicardium, migrate to enclose the wound, undergo epithelial-mesenchymal transition (EMT), and differentiate into mural cells and pdgfra+hapln1a+ mesenchymal epicardial cells. These EMT and differentiation processes are regulated by the Tgfβ pathway. Conditional ablation of aEPCs blocks heart regeneration through reduced nrg1 expression and mesenchymal cell number. Our findings identify a transient progenitor population of the adult epicardium that is indispensable for heart regeneration and highlight it as a potential target for enhancing cardiac repair.

In vivo and in vitro cartilage differentiation from embryonic epicardial progenitor cells

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Universities and Consejería de Salud y Familias, Junta de Andalucía. The presence of cartilage tissue in embryonic and adult hearts of various vertebrate species is a well-recorded fact. However, while the embryonic neural crest has been historically considered as the main source of cardiac cartilage, recently reported results on the wide connective potential of epicardial lineage cells suggest they could also differentiate into chondrocytes. During heart embryogenesis, the epicardial epithelium forms over the originally bare myocardial surface from epicardial progenitor (proepicardial) cells to then give rise to a large population of mesenchymal Epicardial-Derived Cells (EPDCs) that will crucially contribute to the building, growth and maturation of the ventricle and atrioventricular cardiac structures. In this work, we describe the formation of cardiac cartilage clusters from proepicardial cells, both in vivo and in vitro. Our findings report, for the first time, cartilage formation from epicardial progenitor cells in the embryonic heart, and strongly support the concept of proepicardial cells as multipotent connective progenitors. These results are relevant to our understanding of cardiac cell complexity and responses to pathologic stimuli.

In Vivo and In Vitro Cartilage Differentiation from Embryonic Epicardial Progenitor Cells.

The presence of cartilage tissue in the embryonic and adult hearts of different vertebrate species is a well-recorded fact. However, while the embryonic neural crest has been historically considered as the main source of cardiac cartilage, recently reported results on the wide connective potential of epicardial lineage cells suggest they could also differentiate into chondrocytes. In this work, we describe the formation of cardiac cartilage clusters from proepicardial cells, both in vivo and in vitro. Our findings report, for the first time, cartilage formation from epicardial progenitor cells, and strongly support the concept of proepicardial cells as multipotent connective progenitors. These results are relevant to our understanding of cardiac cell complexity and the responses of cardiac connective tissues to pathologic stimuli.

Abstract 11266: Murine Adult Epicardium Requires Hypoxia for Progenitor and Multipotency Ability

Introduction: The epicardium is a source of the coronary vasculature and stroma during heart development. Although quiescent in the physiological adult heart, increasing evidences indicate the potential epicardium activation and differentiation into its progeny after cardiac injury; however, this remains debated. Hypothesis: We hypothesize that in vivo hypoxia exposure can mimic the embryonic cardiac profile and prime the adult epicardial progenitor cell to a more immature profile. Methods: Perinatal and adult BL6 and Pw1 nLacz hearts were used to track the expression of PW1 - progenitor cell marker - together with cell surface proteins. In vivo hypoxia (10% O 2 ) exposure was used to stimulate the adult heart. Histology, protein expression profiles and single cell analysis were assessed. Freshly FACS isolated Gp38 + PW1 + epicardial cells were cultured to determine their progenitor cell potential (clonogenicity, self-renewal and cell fate potential). Results: Here, we report that PW1 expression decreases during postnatal life, but is maintained in the cardiac hypoxic niche epicardium and subepicardium. Adult mice exhibit increased PW1 expression in epicardial/subepicardial cells and endothelial cells in response to hypoxia. The epicardium and subepicardium are activated to undergo cell proliferation and re-express a subset of embryonic genes in response to hypoxia. We established a new in vitro model to study purified PW1 + /Gp38 + epicardial cells, and we found properties of clonogenicity, self-renewal and multipotency (fibroblast, smooth muscle and endothelial cells) at single cell level in the developing epicardial cells. Cell fate potential towards an endothelial profile is dependent upon low levels of oxygen, and the in vitro ability to grow in culture is restore after in vivo priming of the adult heart. Conclusions: In conclusion our data support that the resident PW1 + /Gp38 + epicardial cells are a reminiscent progenitor population in adult mammalian heart that can be reactivated by exposure to hypoxia, highlighting the potential therapeutic role of hypoxia in regenerative medicine.

Epicardial origin of cardiac arrhythmias: clinical evidences and pathophysiology.

Recent developments in imaging, mapping, and ablation techniques have shown that the epicardial region of the heart is a key player in the occurrence of ventricular arrhythmic events in several cardiac diseases, such as Brugada syndrome, arrhythmogenic cardiomyopathy, or dilated cardiomyopathy. At the atrial level as well, the epicardial region has emerged as an important determinant of the substrate of atrial fibrillation, pointing to common underlying pathophysiological mechanisms. Alteration in the gradient of repolarization between myocardial layers favouring the occurrence of re-entry circuits has largely been described. The fibro-fatty infiltration of the subepicardium is another shared substrate between ventricular and atrial arrhythmias. Recent data have emphasized the role of the epicardial reactivation in the formation of this arrhythmogenic substrate. There are new evidences supporting this structural remodelling process to be regulated by the recruitment of epicardial progenitor cells that can differentiate into adipocytes or fibroblasts under various stimuli. In addition, immune-inflammatory processes can also contribute to fibrosis of the subepicardial layer. A better understanding of such ‘electrical fragility’ of the epicardial area will open perspectives for novel biomarkers and therapeutic strategies. In this review article, a pathophysiological scheme of epicardial-driven arrhythmias will be proposed.

Inhibition of Notch Signaling Promotes the Differentiation of Epicardial Progenitor Cells into Adipocytes.

Background The role of Notch signaling pathway in the differentiation of epicardial progenitor cells (EPCs) into adipocytes is unclear. The objective is to investigate the effects of Notch signaling on the differentiation of EPCs into adipocytes. Methods Frozen sections of C57BL/6J mouse hearts were used to observe epicardial adipose tissue (EAT), and genetic lineage methods were used to trace EPCs. EPCs were cultured in adipogenic induction medium with Notch ligand jagged-1 or γ-secretase inhibitor DAPT. The adipocyte markers, Notch signaling, and adipogenesis transcription factors were determined. Results There was EAT located at the atrial–ventricular groove in mouse. By using genetic lineage tracing methods, we found that EPCs were a source of epicardial adipocytes. EPCs had lipid droplet accumulation, and the expression of adipocyte markers FABP-4 and perilipin-1 was upregulated under adipogenic induction. Activating the Notch signaling with jagged-1 attenuated the adipogenic differentiation of EPCs and downregulated the key adipogenesis transcription factor peroxisome proliferator activated receptor-γ (PPAR-γ), while inhibiting the signaling promoted adipogenic differentiation and upregulated PPAR-γ. When blocking PPAR-γ, the role of Notch signaling in promoting adipogenic differentiation was inhibited. Conclusions EPCs are a source of epicardial adipocytes. Downregulation of the Notch signaling pathway promotes the differentiation of EPCs into adipocytes via PPAR-γ.

TCF21 variant is a risk factor for coronary artery disease and will it be a prognostic marker?

Abstract Background TCF21 gene, encodes a basic-helix- loop- helix transcription factor, playing a critical action in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Recent data suggest that TCF21 may play a role in the state of differentiation of SMC precursor cells that migrate to vascular lesions and contribute to fibrous cap. Purpose Investigate the association of TCF21 rs12190287G&amp;gt;C variant with coronary artery disease (CAD) in a Portuguese population and its role on the prognosis. Methods Case-control study with 3120 participants, 1687 coronary patients with at least 75% obstruction of a major coronary artery and 1433 controls. Genotyping used the TaqMan technique (Applied Biosystems) and then a univariate and multivariate logistic regression analysis were performed. After a mean follow-up of 5.01±4.2 years (interquartile range 1.96–7.57), the occurrence of the combined Major Adverse Cardiovascular Events (MACE) (Cardiovascular Mortality, non-fatal Myocardial Infarction, new Revascularization, Cerebrovascular Disease and Peripheric Vascular Disease) were registered and analysed by Cox regression. Finally, Kaplan-Meier survival estimate was performed. Results In the total population, GC+CC genotype was found to be associated with CAD with an OR of 1.285; CI: 1.022–1.614; p=0.031. After multivariate logistic regression, adjusted to traditional risk factors, the association with CAD remained significant for this genotype (OR=1.340; CI: 1.042–1.723; p=0.022).After Cox regression adjusted for confounding variables (age and sex, hypertension, diabetes, smoking, dyslipidemia, eGFR, Ejection fraction &amp;lt;55) the mutated genotype remained a significant predictor of MACE (HR=1.420; CI: 1.032–1.953; p=0.031). The individuals carrying the mutated allele (GC+CC) at the mean follow-up showed an event probability of 36.1%, whereas the wild population (GG) presented only 23.4%. The Log-Rank test showed significant differences between the two curves (p=0.019). Conclusion The mutated TCF21 variant can provide a new marker to identify patients at high cardiovascular risk and may representa potential target for gene therapy in future. Figure 1 Funding Acknowledgement Type of funding source: None

Epicardium as a new target for regenerative technologies in cardiology

Epicardium is actively involved in the embryonic heart development and its repair after injury, which allows it to be considered as a potential target for the treatment of heart diseases. In this regard, the study of the mechanisms of its development, the components of the microenvironment, as well as the signals regulating the behavior of epicardial progenitor cells, is the most important area of modern cardiology. This review considers the results of recent studies of homeostasis of epicardial cells and technological advances to modulate their activity, which is essential for the development of new therapeutic agents.

NFATc1+CD31+CD45− circulating multipotent stem cells derived from human endocardium and their therapeutic potential

Many studies have shown the existence of cardiac stem cells in the myocardium and epicardial progenitor cells in the epicardium. However, the characteristics of stem cells in the endocardium has not been fully elucidated. In this study, we investigated the origin of newly identified cells in the blood and their therapeutic potential. The new population of cells, identified from human peripheral blood, was quite different from previously reported stem cells. These newly identified cells, which we named Circulating Multipotent Stem (CiMS) cells, were multipotent, and therefore differentiated into multiple lineages in vitro and in vivo. In order to determine the origin of these cells, we collected peripheral blood from a group of patients who underwent bone marrow, liver, heart, or kidney transplantation. We identified the endocardium as the origin of these cells because the Short Tandem Repeat profile of CiMS cells from the recipient had changed from the recipient's profile to the donor's profile after heart transplantation. CiMS cells significantly increased after stimuli to the endocardium, such as catheter ablation for arrhythmia or acute myocardial infarction. CiMS cells circulate in human peripheral blood and are easily obtainable, suggesting that these cells could be a promising tool for cell therapy.

Sphingosine 1-phosphate induces epicardial progenitor cell differentiation into smooth muscle-like cells.

Epicardial progenitor cells (EpiCs) which are derived from the proepicardium have the potential to differentiate into coronary vascular smooth muscle cells during development. Whether sphingosine 1-phosphate (S1P), a highly hydrophobic zwitterionic lysophospholipid in signal transduction, induces the differentiation of EpiCs is unknown. In the present study, we demonstrated that S1P significantly induced the expression of smooth muscle cell specific markers α-smooth muscle actin and myosin heavy chain 11 in the EpiCs. And the smooth muscle cells differentiated from the EpiCs stimulated by S1P were further evaluated by gel contraction assay. To further confirm the major subtype of sphingosine 1-phosphate receptors (S1PRs) involved in the differentiation of EpiCs, we used the agonists and antagonists of different S1PRs. The results showed that the S1P1/S1P3 antagonist VPC23019 and the S1P2 antagonist JTE013 significantly attenuated EpiCs differentiation, while the S1P1 agonist SEW2871 and antagonist W146 did not affect EpiCs differentiation. These results collectively suggested that S1P, principally through its receptor S1P3, increases EpiCs differentiation into VSMCs and thus indicated the importance of S1P signaling in the embryonic coronary vasculature, while S1P2 plays a secondary role.

Bone morphogenetic protein 4 promotes the differentiation of Tbx18-positive epicardial progenitor cells to pacemaker-like cells.

Clarifying the mechanisms via which pacemaker- like cells are generated is critical for identifying novel targets for arrhythmia-associated disorders and constructing pacemakers with the ability to adapt to physiological requirements. T-box 18 (Tbx18)+ epicardial progenitor cells (EPCs) have the potential to differentiate into pacemaker cells. Although bone morphogenetic protein 4 (Bmp4) is likely to contribute, its role and regulatory mechanisms in the differentiation of Tbx18+ EPCs into pacemaker-like cells have remained to be fully elucidated. In the present study, the association between Bmp4, GATA binding protein 4 (Gata4) and hyperpolarization- activated cyclic nucleotide gated potassium channel 4 (Hcn4) to regulate NK2 homeobox 5 (Nkx2.5), which is known to be required for the differentiation of Tbx18+ EPCs into pacemaker-like cells, was assessed. Tbx18+ EPCs were isolated from Tbx18:Cre/Rosa26Renhanced yellow fluorescence protein (EYFP) murine embryos at embryonic day 11.5 and divided into the following four treatment groups: Control, Bmp4, Bmp4+LDN193189 (a Bmp inhibitor) and LDN193189. In vitro Bmp4 promoted the expression of Hcn4 in Tbx18+ EPCs via lineage tracing of Tbx18:Cre/Rosa26REYFP mice, which was likely due to upregulation of Gata4 expression. Gata4 knockdown experiments were then performed using the following five treatment groups: Control, control small interfering RNA (siRNA), Bmp4, Bmp4+siRNA targeting Gata4 (siGata4) and siGata4 group. Knockdown of Gata4 caused a downregulation of Hcn4 and an upregulation of Nkx2.5, but had no effect on Bmp4 expression. In conclusion, it was indicated that in Tbx18+ EPCs, the expression of Nkx2.5 was regulated by Bmp4 via Gata4. Taken together, these results provide important information on regulatory networks of pacemaker cell differentiation and may serve as a basis for further studies.

Autophagy is involved in the differentiation of epicardial progenitor cells into vascular smooth muscle cells in mice

Most coronary smooth muscle cells(CoSMCs) are differentiated from epicardial progenitor cells(EPCs), but the specific mechanism is not fully investigated. Previous studies have shown that autophagy plays an important role for smooth muscle cells(SMCs) differentiation, yet whether autophagy is involved in the differentiation of EPCs into CoSMCs remains unclear. In the present study, We first isolated and cultured EPCs and continuously cultured them for 72 h. Then the autophagy induction and inhibition experiment was established by using the autophagy inducer Rapamycin(RAPA) and the inhibitor 3-Methyladenine(3-MA). And further animal experiments were conducted to observe the effects of autophagy on the development of coronary arteries. Our data showed that autophagy occurred in the differentiation of EPCs into SMCs. Over activation of autophagy may lead to early transient differentiation of EPCs, enhanced migration ability and weakened systolic function, but overall, CoSMC development is still inhibited. However, inhibition of autophagy may delay the differentiation of EPCs, thus reducing the number of coronary arteries. Together, all these processes indicate that autophagy may regulate the differentiation of EPCs into CoSMCs by affecting the time point of differentiation, and appropriate autophagy intensity is required during the development of CoSMCs.

Towards a Novel Patch Material for Cardiac Applications: Tissue-Specific Extracellular Matrix Introduces Essential Key Features to Decellularized Amniotic Membrane.

There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining it with cell-free amniotic membrane via a dry-coating procedure. Cardiac biocompatibility and immunogenicity were tested in vitro using human cardiac fibroblasts, epicardial progenitor cells, murine HL-1 cells, and human immune cells derived from buffy coat. Processing of the ECM preserved important matrix proteins as demonstrated by mass spectrometry. ECM coating did not alter the mechanical characteristics of decellularized amniotic membrane but did cause a clear increase in adhesion capacity, cell proliferation and viability. Activated monocytes secreted less pro-inflammatory cytokines, and both macrophage polarization towards the pro-inflammatory M1 type and T cell proliferation were prevented. We conclude that the incorporation of human cardiac ECM hydrogel shifts and enhances the bioactivity of decellularized amniotic membrane, facilitating its use in future cardiac applications.

Intramiocardial administration of resident c-kit+ cardiac progenital cells activates epicardial progenitor cells and promotes myocardial vascularation after the infarction

Resident cardiac progenitor cells reside in the adult heart and govern myocardial homeostasis and repair after injury. Many experimental and clinical studies are being completed with encouraging results. However, the mechanisms of the therapeutic action of CPC remain poorly understood. Initially they were explained by the ability of CPC to differentiate into cardiomyocytes and vascular cells, recently their regenerative effects are mainly explained by secretion biologically active molecules and the release of exosomes, which promote activation of the regenerative program in the heart cells. The aim of the present study is to assess the effect of intramyocardial CPC transplantation on the activation of the vasculogenic pool of epicardial cells. In our study we ligated the anterior descending coronary artery in the hearts of male Wistar rats and intramyocardial injections of a fluorescently labeled (CM-DIL+) CPC or control medium were performed. Fourteen days after transplantation, CPC retained viability, proliferation properties and some cells showed signs of vasculogenic differentiation. We did not find significant differences in the infarct size between two groups assessed by morphometric studies. However, CPC transplantation attenuated adverse remodeling: we found reduction in left ventricular dilatation, severity of transmural injury and activation of arteriogenesis in the border zone. By immunofluorescence staining of myocardial sections, obtained after CPC transplantation, we found a significant increase the number of Wt1+ cells in the epicardium, indicating activation of the epithelial-mesenchymal transition and the formation of epicardial progenitor cells (EPC). EPC migrated to the myocardium, some of them coexpressed markers CD31 (Pecam), alpha-smooth muscle actin (a-SMA), and participated in the new vessels formation. Thus, intramyocardial CPC transplantation increased the vascularization of the myocardium by differentiation of the transplanted cells, as well as the activation of vasculogenic epicardial cells, which can contribute the reduction of negative cardiac remodeling.