Raymond B. Runyan

Cell Biology of Cardiac Cushion Development

The valves of the heart develop in the embryo from precursor structures called endocardial cushions. After cardiac looping, endocardial cushion swellings form and become populated by valve precursor cells formed by an epithelial-mesenchymal transition (EMT). Endocardial cushions subsequently undergo directed growth and remodeling to form the valvular structures and the membranous septa of the mature heart. The developmental processes that mediate cushion formation include many prototypic cellular actions including adhesion, signaling, migration, secretion, replication, differentiation, and apoptosis. Cushion morphogenesis is unique in that these cellular possesses occur in a functioning organ where the cushions act as valves even while developing into definitive valvular structures. Cardiovascular defects are the most common congenital defects, and one of the most common causes of death during infancy. Thus, there is significant interest in understanding the mechanisms that underlie this complex developmental process. In this regard, substantial progress has been made by incorporating an understanding of cardiac morphology and cell biology with the rapidly expanding repertoire of molecular mechanisms gained through human genetics and research using animal models. This article reviews cardiac morphogenesis as it relates to heart valve formation and highlights selected growth factors, intracellular signaling mediators, and extracellular matrix components involved in the creation and remodeling of endocardial cushions into mature cardiac structures.

Epithelial-mesenchymal cell transformation in the embryonic heart can be mediated, in part, by transforming growth factor β

Progenitor cells of the valves and membranous septa of the vertebrate heart are formed by transformation of a specific population of endothelial cells into mesenchyme. Previous studies have shown that this epithelial-mesenchymal cell transformation is mediated by a signal produced by the myocardium of the atrioventricular (AV) canal and transferred across the extracellular matrix. Data are presented here that transforming growth factor beta (TGF beta 1 or TGF beta 2), in combination with an explant of ventricular myocardium, will produce an epithelial-mesenchymal transformation by cultured AV canal endothelial cells in vitro. Alone, neither component is capable of producing this effect. The factor provided by the ventricular explant cannot be substituted by either epidermal growth factor or basic fibroblast growth factor. Further experiments show that an antibody that blocks TGF beta activity is effective in preventing the epithelial-mesenchymal cell transformation normally produced by AV canal myocardium. Control antibodies are without effect. By immunological criteria, a member of the TGF beta family of molecules can be demonstrated in the chicken embryo and heart at the time overt valvular formation begins. Together, these data show that TGF beta 1 can produce mesenchymal cell formation in vitro and provide evidence that a member of the TGF beta family is present and plays a role in the process of epithelial-mesenchymal cell transformation in the embryonic heart.

Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue☆

In normal heart development the endothelium of the atrioventricular canal, but not the ventricle, produces mesenchymal cells which seed (invade) into the intervening extracellular matrix toward the myocardium at around 64-69 hr of development. We have utilized three-dimensional collagen substrates to examine the initiation of seeding by atrioventricular canal endothelia in vitro and to compare and contrast the responses of the ventricular endothelia. Explants of atrioventricular canals and ventricles from staged embryos were placed on the surfaces of collagen gels prior to the onset of seeding in situ. At varied intervals of incubation, the explant was removed, leaving behind a monolayer on the surface of the gel which consisted of endothelial cells. Subsequently, the endothelial outgrowths were examined for seeded cells. The results confirm the regional endothelial differences seen in vivo. They also show that invasion of the collagen gels is due to an alteration in phenotype mediated by interaction with other components of embryonic heart explant. Lastly, the time course of this tissue interaction in vitro mimics the onset of seeding in vivo.

Protein extracts from early embryonic hearts initiate cardiac endothelial cytodifferentiation

Prior to the formation of multiple chambers, the embryonic heart consists of two epithelial tubes, one within the other. As development proceeds, portions of the inner epithelium, i.e., the endothelium, undergo a morphological transformation into a migrating mesenchymal cell population. Our results show that this transformation is affected by proteins secreted by the outer epithelium, i.e., the myocardium, into the extracellular matrix between these two tissues. This conclusion is based on tissue autoradiographic studies of whole embryo cultures with 3H-amino acids. Continuous labeling conditions generated an apparent gradient of proteins extending away from the myocardium and contacting the endothelium just prior to the formation of mesenchyme, i.e., activation of the transformation sequence. Pulse/chase studies confirmed this directional movement of matrix protein. By performing sequential extractions of preactivation staged embryonic hearts with EDTA and testicular hyaluronidase followed by ammonium sulfate precipitation we obtained an enriched preparation of cardiac extracellular matrix. This fraction was capable of eliciting several of the events characteristic of endothelial activation in vitro. These events included: (i) cell-cell separation, (ii) lateral cell mobility, and (iii) hypertrophy and polarization of intracellular PAS staining (Golgi apparati). The biological activity of the extract was sensitive to heat denaturation: a homogenate of the remaining extracted tissue would not substitute for the matrix extract. Morphologically the extracted hearts appeared intact, however, the extracellular matrix space was significantly diminished. No more than 6% of the total lactic dehydrogenase activity, a cytosolic enzyme, was found in the extract. Preliminary electrophoretic characterization of the extract (metabolically labeled with 14C-amino acids) indicated that it may contain as many as 35 proteins or subunits. The relationship of ECM to endothelial differentiation in cardiac morphogenesis is discussed as a model for other developmental systems.

Multiple Transforming Growth Factor-β Isoforms and Receptors Function during Epithelial-Mesenchymal Cell Transformation in the Embryonic Heart

Epithelial-mesenchymal cell transformation (EMT) is a critical process during development of the heart valves. Transition of endothelial cells into mesenchymal cells in the atrioventricular (AV) canal and the outflow tract regions of the heart form the cardiac cushions that eventually form the heart valves. Collagen gel invasion assay has aided in the identification of molecules that regulate EMT. Among those, transforming growth factor-β (TGF-β) ligands and receptors demonstrate a critical role during EMT. In the chick, TGF-β ligands and some receptors have specific functions during EMT. TGF-β2 mediates endothelial cell-cell activation and separation, and TGF-β3 mediates cell invasion into the extracellular matrix. Receptors involved in the EMT process include TGF-β receptor type II (TBRII), TBRIII, endoglin and the TBRI receptors, ALK2 and ALK5. In contrast, in the mouse model, TGF-β2 is the only ligand involved in EMT. The TGF-β2 null mouse has either increased EMT or a mesenchymal cell proliferation after EMT. However, functional studies of TGF-β1 in vivo and in vitro showed that TGF-β1 functions in the EMT of the mouse AV canal. Latent TGF-β-binding protein (LTBP-1) and endoglin have a role in the EMT process. Therefore, TGF-βs mediate cardiac EMT in both embryonic species. Further studies will reveal the identification of ligand and receptor-specific activities.

Ligand-specific function of transforming growth factor beta in epithelial-mesenchymal transition in heart development.

The ligand specificity of transforming growth factor beta (TGFβ) in vivo in mouse cardiac cushion epithelial‐to‐mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGFβ in cushion EMT, we analyzed Tgfb1−/−, Tgfb2−/−, and Tgfb3−/− mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1−/− and Tgfb3−/− mice. However, analysis of Tgfb2−/− AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2−/− embryos, and not Tgfb1−/− or Tgfb3−/− embryos, develop enlarged cushions at E14.5 with elevated levels of well‐validated indicators of EMT. Collectively, these data indicate that TGFβ2, and not TGFβ1 or TGFβ3, mediates cardiac cushion EMT by promoting both the initiation and cessation of EMT. Developmental Dynamics 238:431–442, 2009.

Epithelial‐mesenchymal transformation in the embryonic heart is mediated through distinct pertussis toxin‐sensitive and TGFβ signal transduction mechanisms

During early development, progenitors of the heart valves and septa are formed by epithelial‐mesenchymal transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Previously, we showed that pertussis toxin, a specific inhibitor of a subset of G proteins, inhibited EMT in chick AV canal cultures. This study examines in detail the effects of pertussis toxin on the process of EMT. One of the major mediators of EMT is Transforming Growth Factor beta 3 (TGFβ3) which acts through the TGFβ Type II receptor. To determine whether pertussis toxin affects EMT via the TGFβ Type II receptor pathway, we compared AV cultures treated with pertussis toxin and TGFβ Type II receptor blocking antibody. Pertussis toxin inhibited several elements of EMT. At all stages tested, pertussis toxin blocked endothelial cell‐cell separation, cell hypertrophy, and the cellular polarization associated with endothelial activation. These activities were unaffected by TGFβ Type II receptor antibodies. Pertussis toxin also reduced transformed mesenchymal cell migration by 61%. The expression patterns of several proteins (as markers of EMT) were analyzed in untreated, pertussis toxin‐treated, and TGFβ Type II receptor blocking antibody‐treated cultures. These markers were α‐smooth muscle actin, Mox‐1, fibrillin 2, tenascin, cell surface β 1,4 galactosyltransferase (GalTase), and integrin α6. Clear differences in marker expression were found between the two inhibitors. For example, in all cells, pertussis toxin inhibited expression of α‐smooth muscle actin and GalTase while TGFβ Type II receptor antibody treatment increased expression of these two proteins. These data suggest that G protein‐mediated signaling is required for several elements of EMT. Furthermore, distinct G protein and TGFβ signal transduction pathways mediate discrete components of EMT. Dev Dyn 1999;214: 81–91.

A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300-1000 micrograms/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300-1000 micrograms/ml), which mimics a binding domain of LM, and alpha-lactalbumin (1-10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and alpha-lactalbumin inhibited cell migration 50%. Antibodies against integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.

TGFβ‐mediated RhoA expression is necessary for epithelial‐mesenchymal transition in the embryonic chick heart

Endothelia in the atrioventricular canal (AVC) of the embryonic heart undergo an epithelial‐mesenchymal transition (EMT) and migrate into the underlying extracellular matrix. We explore here whether RhoA mediates this EMT. RhoA was detected in all cells of the chick heart during the stages studied. Expression was elevated when EMT was actively occurring. Explants treated with C3 exoenzyme in collagen gel cultures showed a significant decrease in mesenchymal cell numbers. siRNA was used to inhibit RhoA mRNA, and both activated endothelial and mesenchymal cells decreased significantly with treatment. Loss of RhoA produced a reduction of RhoB, cyclin‐b2, and β‐catenin messages showing that these genes are regulated downstream of RhoA. In contrast, runx‐2 was not reduced. Inhibition of TGFβ3 or TGFβ2 activity caused a large reduction of RhoA message. These data place RhoA in TGFβ regulated pathways for both endothelial activation and mesenchymal invasion and demonstrate a functional requirement during EMT. Developmental Dynamics 235:1589–1598, 2006.

Transforming growth factor beta signaling in adult cardiovascular diseases and repair

The majority of children with congenital heart disease now live into adulthood due to the remarkable surgical and medical advances that have taken place over the past half century. Because of this, adults now represent the largest age group with adult cardiovascular diseases. It includes patients with heart diseases that were not detected or not treated during childhood, those whose defects were surgically corrected but now need revision due to maladaptive responses to the procedure, those with exercise problems and those with age-related degenerative diseases. Because adult cardiovascular diseases in this population are relatively new, they are not well understood. It is therefore necessary to understand the molecular and physiological pathways involved if we are to improve treatments. Since there is a developmental basis to adult cardiovascular disease, transforming growth factor beta (TGFβ) signaling pathways that are essential for proper cardiovascular development may also play critical roles in the homeostatic, repair and stress response processes involved in adult cardiovascular diseases. Consequently, we have chosen to summarize the current information on a subset of TGFβ ligand and receptor genes and related effector genes that, when dysregulated, are known to lead to cardiovascular diseases and adult cardiovascular deficiencies and/or pathologies. A better understanding of the TGFβ signaling network in cardiovascular disease and repair will impact genetic and physiologic investigations of cardiovascular diseases in elderly patients and lead to an improvement in clinical interventions.