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Epithelial-Mesenchymal Transformation (EMT) and the subsequent invasion of epicardial and endocardial cells during cardiac development is critical to the development of the coronary vessels and heart valves. The transformed cells give rise to cardiac fibroblasts and vascular smooth muscle cells or valvular interstitial cells, respectively. The Type III Transforming Growth Factor β (TGFβR3) receptor regulates EMT and cell invasion in both cell types, but the signaling mechanisms downstream of TGFβR3 are not well understood. Here we use epicardial and endocardial cells in in vitro cell invasion assays to identify common mechanisms downstream of TGFβR3 that regulate cell invasion. Inhibition of NF-κB activity blocked cell invasion in epicardial and endocardial cells. NF-κB signaling was found to be dysregulated in Tgfbr3(-/-) epicardial cells which also show impaired cell invasion in response to ligand. TGFβR3-dependent cell invasion is also dependent upon Activin Receptor-Like Kinase (ALK) 2, ALK3, and ALK5 activity. A TGFβR3 mutant that contains a threonine to alanine substitution at residue 841 (TGFβR3-T841A) induces ligand-independent cell invasion in both epicardial and endocardial cells in vitro. These findings reveal a role for NF-κB signaling in the regulation of epicardial and endocardial cell invasion and identify a mutation in TGFβR3 which stimulates ligand-independent signaling.
Copyright © 2016 Elsevier Inc. All rights reserved.
Epithelial-to-mesenchymal transition (EMT) of endocardial cells is a critical initial step in the formation of heart valves. The collagen gel in vitro model has provided significant information on the role of growth factors regulating EMT but has not permitted investigation of mechanical factors. Therefore we sought to develop a system to probe the effects of mechanical inputs on endocardial EMT by incorporating hyaluronic acid (HA), the primary component of endocardial cushions in developing heart valves, into the gel assay. This was achieved using a combination collagen and crosslinkable methacrylated HA hydrogel (Coll-MeHA). Avian atrioventricular canal explants on Coll-MeHA gels showed increased numbers of transformed cells. Analysis of the mechanical properties of Coll-MeHA gels shows that stiffness does not directly affect EMT. Hydrogel deformation from the beating myocardium of explants directly led to higher levels of regional gel deformation and larger average strain magnitudes associated with invaded cells on Coll-MeHA gels. Inhibition of this contraction reduced EMT on all gel types, although to a lesser extent on Coll-MeHA gels. Using the system we have developed, which permits the manipulation of mechanical factors, we have demonstrated that active mechanical forces play a role in the regulation of endocardial EMT.
Copyright © 2013 Elsevier Ltd. All rights reserved.
RATIONALE - The spatial distribution of blood flow in the hearts of genetically modified mice is a phenotype of interest because derangements in blood flow may precede detectable changes in organ function. However, quantifying the regional distribution of blood flow within organs of mice is challenging because of the small organ volume and the high resolution required to observe spatial differences in flow. Traditional microsphere methods in which the numbers of microspheres per region are indirectly estimated from radioactive counts or extracted fluorescence have been limited to larger organs for 2 reasons; to ensure statistical confidence in the measured flow per region and to be able to physically dissect the organ to acquire spatial information.
OBJECTIVE - To develop methods to quantify and statistically compare the spatial distribution of blood flow within organs of mice.
METHODS AND RESULTS - We developed and validated statistical methods to compare blood flow between regions and with the same regions over time using 15-µm fluorescent microspheres. We then tested this approach by injecting fluorescent microspheres into isolated perfused mice hearts, determining the spatial location of every microsphere in the hearts, and then visualizing regional flow patterns. We demonstrated application of these statistical and visualizing methods in a coronary artery ligation model in mice.
CONCLUSIONS - These new methods provide tools to investigate the spatial and temporal changes in blood flow within organs of mice at a much higher spatial resolution than currently available by other methods.
RATIONALE - Formation of heart valves requires early endocardial to mesenchymal transformation (EMT) to generate valve mesenchyme and subsequent endocardial cell proliferation to elongate valve leaflets. Nfatc1 (nuclear factor of activated T cells, cytoplasmic 1) is highly expressed in valve endocardial cells and is required for normal valve formation, but its role in the fate of valve endocardial cells during valve development is unknown.
OBJECTIVE - Our aim was to investigate the function of Nfatc1 in cell-fate decision making by valve endocardial cells during EMT and early valve elongation.
METHODS AND RESULTS - Nfatc1 transcription enhancer was used to generate a novel valve endocardial cell-specific Cre mouse line for fate-mapping analyses of valve endocardial cells. The results demonstrate that a subpopulation of valve endocardial cells marked by the Nfatc1 enhancer do not undergo EMT. Instead, these cells remain within the endocardium as a proliferative population to support valve leaflet extension. In contrast, loss of Nfatc1 function leads to enhanced EMT and decreased proliferation of valve endocardium and mesenchyme. The results of blastocyst complementation assays show that Nfatc1 inhibits EMT in a cell-autonomous manner. We further reveal by gene expression studies that Nfatc1 suppresses transcription of Snail1 and Snail2, the key transcriptional factors for initiation of EMT.
CONCLUSIONS - These results show that Nfatc1 regulates the cell-fate decision making of valve endocardial cells during valve development and coordinates EMT and valve elongation by allocating endocardial cells to the 2 morphological events essential for valve development.
Specific cell and tissue interactions drive the formation and function of the vertebrate cardiovascular system. Although much attention has been focused on the muscular components of the developing heart, the endocardium plays a key role in the formation of a functioning heart. Endocardial cells exhibit heterogeneity that allows them to participate in events such as the formation of the valves, septation of the outflow tract, and trabeculation. Here we review, the contributions of the endocardium to cardiovascular development and outline useful approaches developed in the chick and mouse that have revealed endocardial cell heterogeneity, the signaling molecules that direct endocardial cell behavior, and how these insights have contributed to our understanding of cardiovascular development and disease.
Copyright © 2011 Wiley-Liss, Inc.
Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-β receptor (TGFβR3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGFβR3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGFβ signaling pathways in TGFβR3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGFβR3-dependent EMT stimulated by TGFβ2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGFβR3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGFβ2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGFβR3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGFβR3-dependent endocardial cell EMT stimulated by either TGFβ2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.
Copyright © 2011 S. Karger AG, Basel.
Utilization of MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) for tissue imaging is a relatively new proteomic technique that simultaneously maps the spatial distribution of multiple proteins directly within a single frozen tissue section. Here, we report the development of a methodology to apply MALDI tissue imaging to chick heart tissue sections acquired from fixed and paraffin-embedded samples. This protocol produces molecular images that can be related to the high-quality histological tissue sections. Perfused term chick hearts were fixed in acidic ethanol and embedded in paraffin wax. Tissue sections (15 microm) were collected onto conductive slides, deparaffinized with xylene, and transitioned into water with graded ethanol washes and allowed to air dry. In separate experiments, three different MALDI matrices were applied to chick heart tissue sections through repeated cycles from a glass nebulizer. Tissue sections were then analyzed by MALDI mass spectrometry using a raster step-size of 75-100 microm, and molecular images for specific m/z ratios reconstituted. MALDI tissue imaging revealed spatially resolved protein signals within single heart sections that are specific to structures or regions of the heart, for example, vessels, valves, endocardium, myocardium, or septa. Moreover, no prior knowledge of protein expression is required as is the case for immunohistochemistry and in situ hybridization methodologies. The ability to simultaneously localize a large number of unique protein signals within a single tissue section, with good preservation of histological features, provides cardiovascular researchers a new tool to give insight into the molecular mechanisms underlying normal and pathological conditions.
Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal-spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.
Valvular heart disease due to congenital abnormalities or pathology is a major cause of mortality and morbidity. Understanding the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. In the developing heart, epithelial-mesenchymal transformation (EMT) in a subpopulation of endocardial cells in the atrioventricular cushion (AVC) is an important step in valve formation. Transforming growth factor-beta (TGFbeta) has been shown to be an important regulator of AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Recently Par6c (Par6) has been shown to function downstream of TGFbeta to recruit Smurf1, an E3 ubiquitin ligase, which targets RhoA for degradation to control apical-basal polarity and tight junction dissolution. We tested the hypothesis that Par6 functions in a pathway that regulates endocardial cell EMT. Here we show that the Type I TGFbeta receptor ALK5 is required for endocardial cell EMT. Overexpression of dominant negative Par6 inhibits EMT in AVC endocardial cells, whereas overexpression of wild-type Par6 in normally non-transforming ventricular endocardial cells results in EMT. Overexpression of Smurf1 in ventricular endocardial cells induces EMT. Decreasing RhoA activity using dominant negative RhoA or small interfering RNA in ventricular endocardial cells also increases EMT, whereas overexpression of constitutively active RhoA in AVC endothelial cells blocks EMT. Manipulation of Rac1 or Cdc42 activity is without effect. These data demonstrate a functional role for Par6/Smurf1/RhoA in regulating EMT in endocardial cells.
It is readily apparent that the process of heart development is an intricate one, in which cells derived from many embryonic sources coalesce and coordinate their behaviors and development, resulting in the mature heart. The behaviors and mechanisms of this process are complex, and still incompletely understood. However, it is readily apparent that communication between diverse cell types must be involved in this process. The signaling that emanates from epicardial and endocardial sources is the focus of this review.