Epicardium and Coronary Vessels.

Congenital anomalies and acquired diseases of the coronary blood vessels are of great clinical relevance. The early diagnosis of these conditions remains, however, challenging. In order to improve our knowledge of these ailments, progress has to be achieved in the research of the molecular and cellular mechanisms that control development of the coronary vascular bed. The aim of this chapter is to provide a succint account of the key elements of coronary blood vessel development, especially in the context of the role played by the epicardium and epicardial cellular derivatives. We will discuss the importance of the epicardium in coronary blood vessel morphogenesis, from the contribution of the epicardially derived mesenchyme to these blood vessels to its role as an instructive signaling center, attempting to relate these concepts to the origin of coronary disease.

Congenital Coronary Blood Vessel Anomalies: Animal Models and the Integration of Developmental Mechanisms.

Coronary blood vessels are in charge of sustaining cardiac homeostasis. It is thus logical that coronary congenital anomalies (CCA) directly or indirectly associate with multiple cardiac conditions, including sudden death. The coronary vascular system is a sophisticated, highly patterned anatomical entity, and therefore a wide range of congenital malformations of the coronary vasculature have been described. Despite the clinical interest of CCA, very few attempts have been made to relate specific embryonic developmental mechanisms to the congenital anomalies of these blood vessels. This is so because developmental data on the morphogenesis of the coronary vascular system derive from complex studies carried out in animals (mostly transgenic mice), and are not often accessible to the clinician, who, in turn, possesses essential information on the significance of CCA. During the last decade, advances in our understanding of normal embryonic development of coronary blood vessels have provided insight into the cellular and molecular mechanisms underlying coronary arteries anomalies. These findings are the base for our attempt to offer plausible embryological explanations to a variety of CCA as based on the analysis of multiple animal models for the study of cardiac embryogenesis, and present them in an organized manner, offering to the reader developmental mechanistic explanations for the pathogenesis of these anomalies.

The Genetics of Human Congenital Coronary Vascular Anomalies.

The genetics of human congenital coronary vascular anomalies (hCCVA) remains largely underresearched. This is surprising, because although coronary vascular defects represent a relatively small proportion of human congenital heart disease (CHD), hCCVAs are clinically significant conditions. Indeed, hCCVA frequently associate to other congenital cardiac structural defects and may even result in sudden cardiac death in the adult. In this brief chapter, we will attempt to summarize our current knowledge on the topic, also proposing a rationale for the development of novel approaches to the genetics of hCCVA.

Bone marrow contribution to the heart from development to adulthood.

Cardiac chamber walls contain large numbers of non-contractile interstitial cells, including fibroblasts, endothelial cells, pericytes and significant populations of blood lineage-derived cells. Blood cells first colonize heart tissues a few days before birth, although their recruitment from the bloodstream to the cardiac interstitium is continuous and extends throughout adult life. The bone marrow, as the major hematopoietic site of adult individuals, is in charge of renewing all circulating cell types, and it therefore plays a pivotal role in the incorporation of blood cells to the heart. Bone marrow-derived cells are instrumental to tissue homeostasis in the steady-state heart, and are major effectors in cardiac disease progression. This review will provide a comprehensive approach to bone marrow-derived blood cell functions in the heart, and discuss aspects related to hot topics in the cardiovascular field like cell-based heart regeneration strategies.

Synthesis of Amino Terminal Clicked Dendrimers. Approaches to the Application as a Biomarker.

Herein, we present an easy and efficient synthesis of amino terminal dendrons, combining protection/deprotection reactions with copper-catalyzed azide alkyne cycloaddition in a convergent way. This new approach affords dendrons in gram scale with excellent yields and easy purification. By choosing the appropriate azido-functionalized core, these dendrons lead to a more efficient and controlled convergent synthesis of dendrimers with different sizes and shapes and multivalence. The amino terminal dendrimers were analyzed by diffusion-ordered spectroscopy experiments. The observed dendrimer size is in excellent correlation with the expected size and shape by molecular dynamic simulations. The construction of these kinds of nanostructures, in a simple and efficient way, opens new opportunities for biomedical applications. Moreover, by choosing the appropriate core, these versatile macromolecules become an excellent fluorescent biomarker.

Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio-venous connections.

Recent reports suggest that mammalian embryonic coronary endothelium (CoE) originates from the sinus venosus and ventricular endocardium. However, the contribution of extracardiac cells to CoE is thought to be minor and nonsignificant for coronary formation. Using classic (Wt1(Cre)) and previously undescribed (G2-Gata4(Cre)) transgenic mouse models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardium (ST/PE)-derived endothelial cells are required for the formation of ventricular coronary arterio-venous vascular connections. Our results indicate that at least 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE compartment. Moreover, we show that conditional deletion of the ST/PE lineage-specific Wilms' tumor suppressor gene (Wt1) in the ST/PE of G2-Gata4(Cre) mice and in the endothelium of Tie2(Cre) mice disrupts embryonic coronary transmural patterning, leading to embryonic death. Taken together, our results demonstrate that ST/PE-derived endothelial cells contribute significantly to and are required for proper coronary vascular morphogenesis.

Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the ß-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.

Avian embryonic coronary arterio-venous patterning involves the contribution of different endothelial and endocardial cell populations.

BACKGROUND: Coronary vasculature irrigates the myocardium and is crucial to late embryonic and adult heart function. Despite the developmental significance and clinical relevance of these blood vessels, the embryonic origin and the cellular and molecular mechanisms that regulate coronary arterio-venous patterning are not known in detail. In this study, we have used the avian embryo to dissect the ontogenetic origin and morphogenesis of coronary vasculature. RESULTS: We show that sinus venosus endocardial sprouts and proepicardial angioblasts pioneer coronary vascular formation, invading the developing heart simultaneously. We also report that avian ventricular endocardium has the potential to contribute to coronary vessels, and describe the incorporation of cardiac distal outflow tract endothelial cells to the peritruncal endothelial plexus to participate in coronary vascular formation. Finally, our findings indicate that large sinus venosus-independent sections of the forming coronary vasculature develop without connection to the systemic circulation and that coronary arterio-venous shunts form a few hours before peritruncal arterial endothelium connects to the aortic root. CONCLUSIONS: Embryonic coronary vasculature is a developmental mosaic, formed by the integration of vascular cells from, at least, four different embryological origins, which assemble in a coordinated manner to complete coronary vascular development. Developmental Dynamics 247:686-698, 2018. © 2017 Wiley Periodicals, Inc.

A chick embryo cryoinjury model for the study of embryonic organ development and repair.

Tissue ablation is a classic experimental approach to study early embryo patterning. However, ablation methods are less frequently used to assess the reparative or regenerative properties of embryonic tissues during organogenesis. Surgical procedures based on the removal of a significant amount of tissue during organ formation very much depend on the skills of the researcher, are difficult to reproduce, and often result in extensive tissue disruption leading to embryonic death. In this paper, we present a new protocol to generate discrete, locally-restricted and highly reproducible wounds in the developing chick embryo using a liquid N2-cooled metallic probe. This in ovo procedure allows for the study of organ-specific tissue responses to damage, such as compensatory cell growth, cell differentiation, and reparative/regenerative mechanisms throughout the embryonic lifespan.

Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart.

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.

Retinoic acid and VEGF delay smooth muscle relative to endothelial differentiation to coordinate inner and outer coronary vessel wall morphogenesis.

RATIONALE: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. OBJECTIVE: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. METHODS AND RESULTS: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. CONCLUSION: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels.

The expanding role of the epicardium and epicardial-derived cells in cardiac development and disease.

PURPOSE OF REVIEW: In this review, we aim at presenting and discussing the cellular and molecular mechanisms of embryonic epicardial development that may underlie the origin of congenital heart disease (CHD). RECENT FINDINGS: New discoveries on the multiple cell lineages that form part of the original pool of epicardial progenitors and the roles played by epicardial transcription factors and morphogens in the regulation of epicardial epithelial-to-mesenchymal transition, epicardial-derived cell (EPDCs) differentiation, coronary blood vessel morphogenesis and cardiac interstitium formation are presented in a comprehensive manner. SUMMARY: We have provided evidence on the critical participation of epicardial cells and EPDCs in normal and abnormal cardiac development, suggesting the implication of defective epicardial development in various forms of CHD.

A New Versatile Platform for Assessment of Improved Cardiac Performance in Human-Engineered Heart Tissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testing.

Understanding the Adult Mammalian Heart at Single-Cell RNA-Seq Resolution.

During the last decade, extensive efforts have been made to comprehend cardiac cell genetic and functional diversity. Such knowledge allows for the definition of the cardiac cellular interactome as a reasonable strategy to increase our understanding of the normal and pathologic heart. Previous experimental approaches including cell lineage tracing, flow cytometry, and bulk RNA-Seq have often tackled the analysis of cardiac cell diversity as based on the assumption that cell types can be identified by the expression of a single gene. More recently, however, the emergence of single-cell RNA-Seq technology has led us to explore the diversity of individual cells, enabling the cardiovascular research community to redefine cardiac cell subpopulations and identify relevant ones, and even novel cell types, through their cell-specific transcriptomic signatures in an unbiased manner. These findings are changing our understanding of cell composition and in consequence the identification of potential therapeutic targets for different cardiac diseases. In this review, we provide an overview of the continuously changing cardiac cellular landscape, traveling from the pre-single-cell RNA-Seq times to the single cell-RNA-Seq revolution, and discuss the utilities and limitations of this technology.

Indolenine-Based Derivatives as Customizable Two-Photon Fluorescent Probes for pH Bioimaging in Living Cells.

Novel pH probes based on 2-(6-methoxynaphthalen-2-yl)-3,3-dimethyl-3H-indole have been synthesized and characterized. These compounds display excellent "off-on" fluorescence responses to acidic pH especially under two-photon (TP) excitation conditions as well as strong selectivity and sensitivity toward H+. These features are supported by fluorescence quantum yields over 35%, TP cross sections ∼60 GM, and good resistance to photodegradation under acidic conditions. The synthetic versatility of this model allows subcellular targets to be tuned through minor scaffold modifications without affecting its optical characteristics. The effectiveness of the probes' innate photophysical properties and the structural modifications for different pH-related applications are demonstrated in mouse embryonic fibroblast cells.

Development of the Myocardial Interstitium.

The space between cardiac myocytes is commonly referred-to as the cardiac interstitium (CI). The CI is a unique, complex and dynamic microenvironment in which multiple cell types, extracellular matrix molecules, and instructive signals interact to crucially support heart homeostasis and promote cardiac responses to normal and pathologic stimuli. Despite the biomedical and clinical relevance of the CI, its detailed cellular structure remains to be elucidated. In this review, we will dissect the organization of the cardiac interstitium by following its changing cellular and molecular composition from embryonic developmental stages to adulthood, providing a systematic analysis of the biological components of the CI. The main goal of this review is to contribute to our understanding of the CI roles in health and disease. Anat Rec, 302:58-68, 2019. © 2018 Wiley Periodicals, Inc.

Platinum-Doped Dendritic Structure as a Phosphorescent Label for Bacteria in Two-Photon Excitation Microscopy.

Herein, we present a water-soluble dendritric Pt(II) complex as a phosphorescent label for bacterial cells. The dendritic moiety endows the Pt(II) complex with unique properties such as water solubility, shielding from quenching by dioxygen, and binding to bacterial surfaces. The new biosensor was employed for two-photon excitation microscopy, and the binding was confirmed by electron microscopy, which demonstrates that such hybrid arrays can provide orthogonal yet complementary readouts.

Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells.

Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (pro)epicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (pro)epicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (pro)epicardial-like cells displaying functional properties (adhesion and spreading over the myocardium) of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (pro)epicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration.

In vitro self-assembly of proepicardial cell aggregates: an embryonic vasculogenic model for vascular tissue engineering.

Proepicardial/epicardial-derived cells are the main origin of the early embryonic coronary vascular bed. In vivo coronary vasculogenesis, which is a fast-occurring event, can be mimicked in vitro by culturing proepicardial tissue in different ways. The in vitro vasculogenic model presented in this study (a proepicardial suspension culture assay) partially reproduces coronary vascular development from its cellular precursors, a process known to be highly dependent on cell migration, cell differentiation, cell adhesion/sorting, and tissue fusion phenomena. The main aim of this study is to study the triggering signals and the cellular dynamics that regulate the differentiation of proepicardial cells into the angioblastic/endothelial lineage and their in vitro vasculogenic potential. Our results indicate that hanging drop-cultured proepicardia, which have an intrinsic vascular potential, behave like self-assembling cell aggregates or spheroids that can fuse to give rise to complex vascularized 3D structures. We believe that these self-assembling cell aggregates are an optimal choice to study the differentiation of coronary angioblasts, as well as a good method to reproduce vascular development in vitro. Finally, we propose the proepicardium as a suitable cellular source for vascular tissue engineering.