Mauricio González-Iriarte

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.

Localization of the Wilms' tumour protein WT1 in avian embryos

Abstract. The Wilms tumour suppressor gene WT1 encodes a zinc-finger transcription factor which is essential for the development of kidney, gonads, spleen and adrenals. WT1-null embryos lack all of these viscerae and they also show a thin ventricular myocardium and unexpectedly die from cardiac failure between 13 and 15xa0days post coitum. We studied the localization of the WT1 protein in chick and quail embryos between stages HH18 and HH35. In early embryos, WT1 protein was located in specific areas of the coelomic mesothelium adjacent to the nephric ducts, the myocardium or the primordia of the endodermal organs (gut, liver and lungs). These mesothelial areas also showed localized expression of Slug, a zinc-finger transcription factor involved in epithelial-mesenchymal transitions. WT1+ mesenchymal cells were always found below the immunoreactive mesothelial areas, either forming a narrow band on the surface of the endodermal organs (gut, liver and lungs) or migrating throughout the mesodermal organs (mesonephros, metanephros, gonads, spleen and heart). In the developing heart, the invasion of WT1+ cells started at stage HH26, and all the ventricular myocardium was pervaded by these cells, presumably derived from the epicardium, at HH30. We suggest that WT1 is not required for the epithelial-mesenchymal transition of the coelomic mesothelium, but it might be a marker of the mesothelial-derived cells, where this protein would be acting as a repressor of the differentiation.

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.

STUDY OF PUUPEHENONE AND RELATED COMPOUNDS AS INHIBITORS OF ANGIOGENESIS

Puupehenone, a sesquiterpene produced by certain sponges, was selected in the course of a blind screening for new potential inhibitors of angiogenesis. In our study, we compare the potential anti‐angiogenic activities of puupehenone and another 11 related compounds that were either natural products from marine origin or their synthetic derivatives. The effects of these compounds were determined with cell growth and differentiation assays on bovine aorta endothelial cells. Our results show that these compounds are weak inhibitors to cell growth and are not selective for endothelial cells. However, contrary to cell growth, the differentiation of endothelial cells into tubular structures was completely inhibited by 7 of these compounds at concentrations equal or lower than 3 μM. Three of these compounds, isozonarol, 8‐epipuupehedione and 8 epi‐9,11‐dihydropuupehedione, completely inhibited the in vivo angiogenesis in the CAM assay at doses equal or lower than 30 nmol/egg. Further characterisation showed that these 3 terpenes also inhibited endothelial cell production of urokinase and invasion. One compound (8‐epipuupehedione) inhibited endothelial cell migration in a dose‐dependent manner. The anti‐angiogenic properties of the selected compounds, the simplicity of their structures and the feasibility of their synthesis make them attractive drugs for further evaluation in the treatment of angiogenesis‐related pathologies.

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.

A modified chorioallantoic membrane assay allows for specific detection of endothelial apoptosis induced by antiangiogenic substances

Current in vivo angiogenesis assays allow for the assessment of vascular growth inhibition induced by a test substance, but they usually do not provide information about the mechanisms underlying such an inhibition. A potential antiangiogenic mechanism is the triggering of endothelial apoptosis in the growing vessels. Apoptogenic substances can be of interest for antiangiogenic therapy specially if they specifically perform their action on the angiogenic endothelium. We have developed a modification of the chorioallantoic membrane (CAM) assay using embryos of quail (Coturnix coturnix japonica). This novel assay allows to elucidate whether an antiangiogenic substance is specifically triggering an apoptotic response in endothelial cells. We have used a quail-specific monoclonal endothelial marker (QH1), a standard TUNEL technique of apoptotic cell labelling together with a general nuclear counterstaining with propidium iodide. Through laser confocal microscopy, paraffin sections of chorioallantoic membranes treated with test substances are stained in three colours: red for normal cell nuclei, yellow—green for apoptotic nuclei and blue for endothelial cells and endothelial progenitors. In a test experience, our assay showed significant differences in the apoptogenic properties of two antiangiogenic substances, camptothecin and aeroplysinin-1.