Rita Carmona

Hyperplastic Conotruncal Endocardial Cushions and Transposition of Great Arteries in Perlecan-Null Mice

Perlecan is a heparan-sulfate proteoglycan abundantly expressed in pericellular matrices and basement membranes during development. Inactivation of the perlecan gene in mice is lethal at two developmental stages: around E10 and around birth. We report a high incidence of malformations of the cardiac outflow tract in perlecan-deficient embryos. Complete transposition of great arteries was diagnosed in 11 out of 15 late embryos studied (73%). Three of these 11 embryos also showed malformations of semilunar valves. Mesenchymal cells in the outflow tract were abnormally abundant in mutant embryos by E9.5, when the endocardial-mesenchymal transformation starts in wild-type embryos. At E10.5, mutant embryos lacked well-defined spiral endocardial ridges, and the excess of mesenchymal cells obstructed sometimes the outflow tract lumen. Most of this anomalous mesenchyme expressed the smooth muscle cell-specific &agr;-actin isoform, a marker of the neural crest in the outflow tract of the mouse. In wild-type embryos, perlecan is present in the basal surface of myocardium and endocardium, as well as surrounding presumptive neural crest cells. We suggest that the excess of mesenchyme at the earlier stages of conotruncal development precludes the formation of the spiral ridges and the rotation of the septation complex in order to achieve a concordant ventriculoarterial connection. The observed mesenchymal overpopulation might be due to an uncontrolled migration of neural crest cells, which would arrive prematurely to the heart. Thus, perlecan is involved in the control of the outflow tract mesenchymal population size, underscoring the importance of the extracellular matrix in cardiac morphogenesis.

Antiangiogenic activity of aeroplysinin-1, a brominated compound isolated from a marine sponge

(+)‐Aeroplysinin‐1, an antibacterial brominated compound produced by certain sponges, was selected during a blind high‐throughput screening for new potential antiangiogenic compounds obtained from marine organisms. In a variety of experimental systems, representing the sequential events of the angiogenic process, aeroplysinin‐1 treatment of endothelial cells resulted in strong inhibitory effects. Aeroplysinin‐1 inhibited the growth of endothelial cells in culture and induced endothelial cell apoptosis. Capillary tube formation on Matrigel was completely abrogated by addition of aeroplysinin‐1 at the low micromolar range. Aeroplysinin‐1 also exhibited a clear inhibitory effect on the migration capabilities of endothelial cells. Zymographic assays showed that aeroplysinin‐1 treatment produced a decrease in the concentration of matrix metalloproteinase‐2 and urokinase in conditioned medium from endothelial cells. Finally, aeroplysinin‐1 exhibited a dose‐dependent inhibitory effect on the in vivo chorioallantoic membrane assay, showing potent apoptosis‐inducing activity in the developing endothelium. The in vivo inhibition of angiogenesis by aeroplysinin‐1 was confirmed by the Matrigel plug assay. Together, our data indicate that aeroplysinin‐1 is a compound that interferes with key events in angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis‐related pathologies.

Contribution of mesothelium‐derived cells to liver sinusoids in avian embryos

The developing liver is vascularized through a complex process of vasculogenesis that leads to the differentiation of the sinusoids. The main structural elements of the sinusoidal wall are endothelial and stellate (Ito) cells. We have studied the differentiation of the hepatic sinusoids in avian embryos through confocal colocalization of differentiation markers, in ovo direct labeling of the liver mesothelium, induced invasion of the developing chick liver by quail proepicardial cells, and in vitro culture of chimeric aggregates. Our results show that liver mesothelial cells give rise to mesenchymal cells which intermingle between the growing hepatoblast cords and become incorporated to the sinusoidal wall, contributing to both endothelial and stellate cell populations. We have also shown that the proepicardium, a mesothelial tissue anatomically continuous with liver mesothelium, is able to form sinusoid‐like vessels into the hepatic primordium as well as in cultured aggregates of hepatoblasts. Thus, both intrinsic or extrinsic mesothelium‐derived cells have the developmental potential to contribute to the establishment of liver sinusoids. Developmental Dynamics 229:465–474, 2004.

The origin of the endothelial cells: an evo-devo approach for the invertebrate/vertebrate transition of the circulatory system

Summary Circulatory systems of vertebrate and invertebrate metazoans are very different. Large vessels of invertebrates are constituted of spaces and lacunae located between the basement membranes of endodermal and mesodermal epithelia, and they lack an endothelial lining. Myoepithelial differentation of the coelomic cells covering hemal spaces is a frequent event, and myoepithelial cells often form microvessels in some large invertebrates. There is no phylogenetic theory about the origin of the endothelial cells in vertebrates. We herein propose that endothelial cells originated from a type of specialized blood cells, called amoebocytes, that adhere to the vascular basement membrane. The transition between amoebocytes and endothelium involved the acquisition of an epithelial phenotype. We suggest that immunological cooperation was the earliest function of these protoendothelial cells. Furthermore, their ability to transiently recover the migratory, invasive phenotype of amoebocytes (i.e., the angiogenic phenotype) allowed for vascular growth from the original visceral areas to the well‐developed somatic areas of vertebrates (especially the tail, head, and neural tube). We also hypothesize that pericytes and smooth muscle cells derived from myoepithelial cells detached from the coelomic lining. As the origin of blood cells in invertebrates is probably coelomic, our hypothesis relates the origin of all the elements of the circulatory system with the coelomic wall. We have collected from the literature a number of comparative and developmental data supporting our hypothesis, for example the localization of the vascular endothelial growth factor receptor‐2 ortholog in hemocytes of Drosophila or the fact that circulating progenitors can differentiate into endothelial cells even in adult vertebrates.

Immunolocalization of the transcription factor Slug in the developing avian heart.

Slug is a transcription factor involved in processes such as the formation of mesoderm and neural crest, two developmental events that imply a transition from an epithelial to a mesenchymal phenotype. During late cardiac morphogenesis, mesenchymal cells originate from two epithelia – epicardial mesothelium and cushion endocardium. We aimed to check if Slug is expressed in these systems of epithelial-mesenchymal transition. We have immunolocated the Slug protein in the heart of quail embryos between Hamburger and Hamilton stages HH16 and HH30. In the proepicardium (the epicardial primordium), Slug was detected in most cells, mesothelial as well as mesenchymal. Slug immunoreactivity was strong in the mesenchyme of the endocardial cushions and subepicardium from its inception until HH24, but the immunoreactivity disappeared in later embryos. Only a small portion of the endocardial cells located in the areas of epithelial-mesenchymal transition (atrioventricular groove and outflow tract) were immunolabelled, mainly between HH16 and HH20. Endocardial cells from other cardiac segments were always negative, except for a transient, weak immunoreactivity that coincided with the development of the intertrabecular sinusoids of the ventricle. In contrast, virtually all cells of the epicardial mesothelium were immunoreactive until stage HH24. The mesenchymal cells that migrate to the heart through the spina vestibuli were also conspicuously immunoreactive. The myocardium was not labelled in the stages studied. Our results stress the involvement of Slug in the epithelial to mesenchymal transition. We suggest that Slug can constitute a reliable marker of the cardiac epithelial cells that are competent to transform into mesenchyme as well as a transient marker of the epithelial-derived mesenchymal cells in the developing heart.

El epicardio y las células derivadas del epicardio: múltiples funciones en el desarrollo cardíaco

Durante el desarrollo cardiaco, el epicardio deriva de un primordio externo al corazon, denominado proepicardio, que esta formado por un acumulo de celulas mesoteliales situado en la superficie ventral y cefalica del limite higado-seno venoso (aves) o en la cara pericardica del septo transverso (mamiferos). El proepicardio entra en contacto con la superficie miocardica y da lugar a un mesotelio que crece y recubre progresivamente al miocardio. El epicardio genera, por un proceso localizado de transicion epitelio-mesenquima, una poblacion de celulas mesenquimaticas, las celulas derivadas de epicardio (CDEP). Las CDEP contribuyen al desarrollo del tejido conectivo del corazon y tambien dan lugar a los fibroblastos y las celulas musculares lisas de los vasos coronarios. Existen evidencias que sugieren la diferenciacion de las CDEP en celulas endoteliales del plexo subepicardico primitivo. De confirmarse esto, las CDEP mostrarian propiedades similares a los precursores vasculares bipotenciales derivados de celulas madre recientemente descritos, cuya diferenciacion en endotelio y musculo liso se regula por exposicion a VEGF y PDGF-BB, respectivamente. Ademas de las funciones senaladas en la formacion de los tejidos vascular y conectivo del corazon, las CDEP podrian desempenar un papel modulador esencial para la formacion de la capa compacta ventricular del miocardio, un papel que podria estar regulado por el factor de transcripcion WT1 y la produccion de acido retinoico.

Development of the coronary arteries in a murine model of transposition of great arteries

Transposition of great arteries in humans is associated with a wide spectrum of coronary artery patterns. However, no information is available about how this pattern diversity develops. We have studied the development of the coronary arteries in mouse embryos with a targeted mutation of perlecan, a mutation that leads to ventriculo-arterial discordance and complete transposition in about 70% of the embryos. The perlecan-deficient embryos bearing complete transposition showed a coronary artery pattern consisting of right and left coronary arteries arising from the morphologically dorsal and ventral sinuses of Valsalva, respectively. The left coronary artery gives rise to a large septal artery and runs along the ventral margin of the pulmonary root. In the earliest embryos where transposition could be confirmed (12.5 d post coitum), a dense subepicardial vascular plexus is located in this ventral margin. In wild-type mice, however, capillaries are very scarce on the ventral surface of the pulmonary root and the left coronary artery runs dorsally to this root. We suggest that the establishment of the diverse coronary artery patterns is determined by the anatomical arrangement and the capillary density of the peritruncal vascular plexus, a plexus that spreads from the atrio-ventricular groove and grows around the aortic or pulmonary roots depending on the degree of the short-axis aortopulmonary rotation. This simple model, based on very few assumptions, might explain all the observed variation of the coronary artery patterns in humans with transposition, as well as our observations on the perlecan-deficient and the normal mice.

Epicardial development in lamprey supports an evolutionary origin of the vertebrate epicardium from an ancestral pronephric external glomerulus

SUMMARY The epicardium is the outer layer of the vertebrate heart. Both the embryonic epicardium and its derived mesenchyme are critical to heart development, contributing to the coronary vasculature and modulating the proliferation of the ventricular myocardium. The embryonic epicardium arises from an extracardiac, originally paired progenitor tissue called the proepicardium, a proliferation of coelomic cells found at the limit between the liver and the sinus venosus. Proepicardial cells attach to and spread over the cardiac surface giving rise to the epicardium. Invertebrate hearts always lack of epicardium, and no hypothesis has been proposed about the origin of this tissue and its proepicardial progenitor in vertebrates. We herein describe the epicardial development in a representative of the most basal living lineage of vertebrates, the agnathan Petromyzon marinus (lamprey). The epicardium in lampreys develops by migration of coelomic cells clustered in a paired structure at the roof of the coelomic cavity, between the pronephros and the gut. Later on, these outgrowths differentiate into the pronephric external glomerulus (PEG), a structure composed of capillary networks, mesangial cells, and podocytes. This observation is consistent with the conclusion that the primordia of the most anterior pair of PEG in agnathans have been retained and transformed into the proepicardium in gnathostomes. Glomerular progenitor cells are highly vasculogenic and probably allowed for the vascularization of a cardiac tube primarily devoid of coronary vessels. This new hypothesis accounts for the striking epicardial expression of Wt1 and Pod1, two transcription factors essential for development of the excretory system.

Immunoreactivity of the ets-1 transcription factor correlates with areas of epithelial-mesenchymal transition in the developing avian heart

Abstract Cardiac morphogenesis involves substantial remodeling processes that include cell transdifferentiation and migration. The c-ets-1 protooncogene codes for a transcription factor that can transactivate a number of genes involved in developmental processes such as degradation of extracellular matrices and cell migration. We have immunolocated the ets-1 protein in the heart of quail and chick embryos between the Hamburger and Hamilton stages HH16 and HH37. In HH16–17 embryos, the ets-1 transcription factor was only detected in some endocardial cells and in most mesothelial and mesenchymal cells of the proepicardium. Ets-1 immunoreactivity increased markedly in the developing endocardial cushions, myocardium, epicardium and early subepicardial mesenchyme of HH18–19 embryos. By HH20–24 the immunoreactivity was found throughout the heart, with a stronger intensity in the areas of epithelial-mesenchymal transition of the endocardium and epicardium. In embryos between HH26 and HH33, ets-1 immunoreactivity increased in the cushion mesenchyme, atrioventricular endocardium, ventricular epicardium and subepicardial mesenchyme cells, but not in other areas of the heart. The immunoreactivity declined in the innermost part of the endocardial cushions. The subepicardial mesenchyme was particularly immunoreactive in these stages, coinciding with the development of the subepicardial vascular network. In fact, ets-1 colocalized with the quail vascular marker QH1 in the subepicardial mesenchymal cells. Ets-1-negative cells were abundant in the subepicardium and valvuloseptal tissue of the HH37 embryos. The results suggest that ets-1, probably through transactivation of genes such as urokinase-type plasminogen activator and matrix metalloproteinases, might play a crucial role in the differentiation of the cushion and subepicardial mesenchyme, the formation of the intratrabecular sinusoids and the early development of the cardiac vessels.

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

Significance Here we show, for the first time to our knowledge, that septum transversum/proepicardium (ST/PE)-derived endothelial cells are required for proper coronary blood vessel morphogenesis. We used different mouse transgenic lines to show that the ST/PE contributes to coronary endothelium and that the endocardium is not the only developmental origin of this tissue. Our results indicate that ST/PE-derived endothelial cells preferentially incorporate into prospective coronary arteries and capillaries but not veins. Deletion of the epicardial and coronary developmental regulator Wilms’ tumor suppressor gene from both the ST/PE and embryonic endothelial cells reveals that ST/PE endothelial cells are required for the establishment of coronary arterio–venous connections through the ventricular wall and thus are necessary for the completion of coronary vascularization. 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 (Wt1Cre) and previously undescribed (G2-Gata4Cre) 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 ofG2-Gata4Cre mice and in the endothelium of Tie2Cre 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.