Robert E. Poelmann

Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis

Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.

Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras.

The endothelium of the coronary vascular system has been described in the literature as originating from different sources, varying from aortic endothelium for the main coronary stems, endocardium for the intramyocardial network, and sinus venosus lining for the venous part of the coronary system. Using an antibody against quail endothelial cells (alpha-MB1), we investigated the development of the coronary vascular system in the quail (Hamburger and Hamilton stages 15 to 35) and in a series of 36 quail-chicken chimeras. In the chimeras, pieces of quail epicardial primordium and/or liver tissue were transplanted into the pericardial cavity of a chicken host. The results showed that the coronary vascular endothelial distribution closely followed the formation of the epicardial covering of the heart. However, pure epicardial primordium transplants did not lead to endothelial cell formation, whereas a liver graft with or without an epicardial contribution did have this capacity. The first endothelial cells were seen to reach the heart at the sinus venosus region, subsequently spreading through the inner curvature to the atrioventricular sulcus and the outflow tract and, last of all, over the ventricular surfaces. At these sites, the precursor cells and small vessels were seen to invade the sinus venosus wall, the ventricular and atrial myocardium, and the mesenchymal border of the aortic orifice. Connections with the endocardium of the heart tube were only observed in the right ventricular outflow region. Initially, the connections with the aortic endothelium were multiple, but later in development only two of these connections persisted to form the proximal part of the two main coronary arteries. Connections to the pulmonary orifice were never observed. Our transplantation data showed that the entire coronary endothelial vasculature originated from an extracardiac source. Moreover, using the developing subepicardial layer as a matrix, we showed that the endothelial cells reached the heart from the liver region. Ingrowth into the various cardiac segments was also observed. Implications for the relation to specific congenital cardiac malformations are discussed.

Double-Outlet Right Ventricle and Overriding Tricuspid Valve Reflect Disturbances of Looping, Myocardialization, Endocardial Cushion Differentiation, and Apoptosis in TGF-β2–Knockout Mice

Background—Transforming growth factor-β2 (TGF-β2) is a member of a family of growth factors with the potential to modify multiple processes. Mice deficient in the TGF-β2 gene die around birth and show a variety of defects of different organs, including the heart. Methods and Results—We studied the hearts of TGF-β2–null mouse embryos from 11.5 to 18.5 days of gestation to analyze the types of defects and determine which processes of cardiac morphogenesis are affected by the absence of TGF-β2. Analysis of serial sections revealed malformations of the outflow tract (typically a double-outlet right ventricle) in 87.5%. There was 1 case of common arterial trunk. Abnormal thickening of the semilunar valves was seen in 4.2%. Associated malformations of the atrioventricular (AV) canal were found in 62.5% and were composed of perimembranous inlet ventricular septal defects (37.5%), AV valve thickening (33.3%), overriding tricuspid valve (25.0%), and complete AV septal defects (4.2%). Anomalies of the aorta and its...

Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium.

 Previous research has revealed that cells contributing to coronary vascular formation are derived from the dorsal mesocardium, however, the fate of these cells during consecutive stages of heart development is still unclear. We have conducted a study regarding the recruitment of vascular components and the subsequent differentiation into mature vessel wall structures with the aid of immunohistochemical markers directed against endothelium, smooth muscle cells, and fibroblasts. The proepicardial organ including an adhering piece of primordial liver of quail embryos (ranging from HH15 to HH18) was transplanted into the pericardial cavity of chicken embryos (ranging from HH15 to HH18). The chicken-quail chimeras (n=16) were harvested from the early stage of endothelial tube formation (HH25) to the late stage of mature vessel wall composition (HH43). Before HH32 endothelial cells have invaded the myocardium to give rise to yet undifferentiated coronary vessels. These endothelial cells are not accompanied by other non-endothelial cells. The superficial epicardial layer changes from a squamous mesothelium into a cuboid epithelium preceding media and adventitia formation. Subsequently, a condensed area of mesenchymal cells delaminates from the cuboidal lining extending toward the vessel plexus. Around the coronary arteries, these mesenchymal cells differentiate into smooth muscle cells or fibroblasts as shown by immunohistochemical markers. We conclude that epithelial-mesenchymal transformation of the epicardial lining delivers the smooth muscle cells and fibroblasts of the coronary arterial vessel wall. Molecules involved in epithelial transformation processes elsewhere in the embryo are also expressed within the subepicardial layer, and are considered to participate in inducing this process.

Neural Crest Cell Contribution to the Developing Circulatory System Implications for Vascular Morphology

In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular morphogenesis. For this purpose, we performed long-term lineage tracing using quail-chick chimera techniques and premigratory NCC infection with a replication-incompetent retrovirus containing the LacZ reporter gene in combination with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the anterior cardinal veins was also determined to be NC-derived. NC-derived elastic arteries differed from non-NC elastic vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating a muscular or elastic artery. Boundaries between NC-infested areas and mesodermal vessel structures were mostly very sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in the aortic and pulmonary trunks.

Development of the origin of the coronary arteries, a matter of ingrowth or outgrowth?

SummaryInconsistencies still exist with regard to the exact mode of development of proximal coronary arteries and coronary orifices. In this regard 15 quail embryos were investigated using a monoclonal anti-endothelium antibody, enabling a detailed study of the development of endothelium-lined vasculature. Coronary orifices emerged at 7–9 days of incubation (Zacchei stages 24–26) and were invariably present at 10 days of incubation (Zacchei stage 27).We never observed more than 2 coronary orifices; these were always single in either of the facing sinuses of the aorta. A coronary orifice was always observed being connected to an already developed proximal coronary artery, which belonged to a peritruncal ring of coronary arterial vasculature. We did not find any coronary orifice without a connection to a proximal coronary artery. Moreover, at 7–9 days of incubation (Zacchei stages 24–26) we observed coronary arteries from the peritruncal ring penetrating the aortic media. In 2 specimen this coronary artery, with a lumen, was in contact with the still intact endothelial lining of the aorta.We conclude that coronary arteries do not grow out of the aorta, but grow into the aorta from the peritruncal ring of coronary arterial vasculature. This throws new light on normal and abnormal development of proximal coronary arteries and coronary orifices.

Evolution of an Arsenal Structural and Functional Diversification of the Venom System in the Advanced Snakes (Caenophidia)

Venom is a key innovation underlying the evolution of advanced snakes (Caenophidia). Despite this, very little is known about venom system structural diversification, toxin recruitment event timings, or toxin molecular evolution. A multidisciplinary approach was used to examine the diversification of the venom system and associated toxins across the full range of the ∼100 million-year-old advanced snake clade with a particular emphasis upon families that have not secondarily evolved a front-fanged venom system (∼80% of the 2500 species). Analysis of cDNA libraries revealed complex venom transcriptomes containing multiple toxin types including three finger toxins, cobra venom factor, cysteine-rich secretory protein, hyaluronidase, kallikrein, kunitz, lectin, matrix metalloprotease, phospholipase A2, snake venom metalloprotease/a disintegrin and metalloprotease, and waprin. High levels of sequence diversity were observed, including mutations in structural and functional residues, changes in cysteine spacing, and major deletions/truncations. Morphological analysis comprising gross dissection, histology, and magnetic resonance imaging also demonstrated extensive modification of the venom system architecture in non-front-fanged snakes in contrast to the conserved structure of the venom system within the independently evolved front-fanged elapid or viperid snakes. Further, a reduction in the size and complexity of the venom system was observed in species in which constriction has been secondarily evolved as the preferred method of prey capture or dietary preference has switched from live prey to eggs or to slugs/snails. Investigation of the timing of toxin recruitment events across the entire advanced snake radiation indicates that the evolution of advanced venom systems in three front-fanged lineages is associated with recruitment of new toxin types or explosive diversification of existing toxin types. These results support the role of venom as a key evolutionary innovation in the diversification of advanced snakes and identify a potential role for non-front-fanged venom toxins as a rich source for lead compounds for drug design and development.

Smooth Muscle Cell Origin and Its Relation to Heterogeneity in Development and Disease

Smooth muscle cells (SMC) of the vascular system form an intriguing population of cells that are relevant for maintaining vascular tone and function. They also play a key role in pathological processes in the vessel wall. If we focus on the development of intimal thickening in the latter function, it is clear that even this pathological subset presents itself in various forms. That is, arteriosclerosis after hypertension,1 atherosclerosis,2 and restenosis after percutaneous transluminal coronary angioplasty or coronary artery bypass grafting surgery3 have features in common as well as characteristics selective for each disease. Relevant to an understanding of the above processes is the basic question of whether we are dealing either with a SMC heterogeneity in origin or with a spatiotemporal heterogeneity in expression of differentiation markers. To add to this complexity there is an increasing evidence that already committed and differentiated cells can transdifferentiate into another cell type. In studying SMC heterogeneity, a combination of these factors is likely. It has been shown by several research groups that SMC heterogeneity exists within the vessel wall, varying from the adult rat4 5 to the human fetal population.6 These data are mainly based on in vitro cell culture studies. A different approach is to study the intact vascular wall and expression of differentiation markers.7 8 9 10 11 This approach shows a change in gene expression patterns with normal maturation and with development of intimal thickening of the vessel wall. During development of intimal thickening in various settings, including physiological circumstances,11 thickening experimentally induced by a perivascular cuff,12 and atherosclerosis in humans,13 reexpression of fetal genes11 has been observed as well as altered migration and proliferation patterns as compared with normal. The most recent addition to characteristics in development of intimal …

Unilateral Vitelline Vein Ligation Alters Intracardiac Blood Flow Patterns and Morphogenesis in the Chick Embryo

To study the role of blood flow in normal and abnormal heart development, an embryonic chicken model was developed. The effect of altered venous inflow on normal intracardiac blood flow patterns was studied by visualization of blood flow with India ink. At stage 17, India ink was injected into a capillary or small venule within a specific yolk sac region. After determination of the normal intracardiac flow pattern, the right lateral vitelline vein was ligated, and the new intracardiac flow pattern was studied. Ligation resulted in disturbance of normal intracardiac flow patterns, which was most obvious in the conotruncus. The long-term effect of these abnormal intracardiac flow patterns on the development of the heart and pharyngeal arch arteries was investigated by permanent ligation in ovo with a microclip at stage 17 and subsequent evaluation at stages 34, 37, and 45. These experiments revealed anomalies of the vascular system in 58 of the 91 ligated embryos studied. We observed intracardiac malformations consisting of subaortic ventricular septal defects (n = 52), semilunar valve anomalies (n = 19), atrioventricular anomalies (n = 7), and pharyngeal arch artery malformations (n = 32). It is concluded that abnormal intracardiac blood flow, resulting from hampered venous inflow, may result in serious intracardiac and pharyngeal arch artery malformations comparable to defects observed in embryonic chicken models subjected to neural crest ablation, cervical flexure experiments, and excessive retinoic acid treatment.

Early development of quail heart epicardium and associated vascular and glandular structures

As in the other vertebrates the epicardium of the quail embryo develops from proepicardial tissue located between the sinus horns and the liver primordium. The cuboidal cells of the coelomic lining above the proepicardium are transformed into mesothelial cells which in cooperation with the underlying mesenchymal cells elaborate a large quantity of extracellular matrix, so producing the villous outgrowths of the proepicardium. The mesenchymal cells of this area are attached to each other with typical desmosomes and have anti-α cytokeratin-stained tonofilament bundles. These cells resemble keratinocytes and are designated as proepicardial matrix keratinocytes. The proepicardium proliferates first in the sulci of the U-shaped tubular heart, and within 2 days (between stages 15–25) establishes the visceral layer of the epicardium. The proliferating proepicardium consists of gland-like tubular strands, formed by the invaginations of the surface mesothelial cells, mesenchymal cells, fibroblasts, angioblasts, blood cells and capillaries. Because of its heterogeneous structure and multiple functions, the proepicardium is considered a transitory organ of the developing heart. In the quail embryo the forerunners of the coronary vessels grow from the perihepatic area into the proepicardial organ, and when the epicardial covering is completed, but before the coronary artery orifices open, these primordial vessels form a subepicardial and intramural vascular network in the ventricular myocardium. After the completion of the epicardial covering the proepicardium involutes and is not seem from stage 26 onward.