Charles D. Little

The development of the coronary vessels and their differentiation into arteries and veins in the embryonic quail heart.

Research concerning the embryologic development of the coronary plexus has enriched our understanding of anomalous coronary vessel patterning. However, the differentiation of the coronary vessel plexus into arteries, veins, and a capillary network is still incomplete. Immunohistochemical techniques have been used for whole mounts and serial sections of quail embryo hearts to demonstrate endothelium, vascular smooth muscle cells, and fibroblasts. From HH35 onward, the lumen of the coronary plexus was visualized by injecting India ink into the aorta. In HH17, branches from the sinus venosus plexus expand into the proepicardial organ to reach the dorsal side of the atrioventricular sulcus. From HH25 onward, vessel formation proceeds toward the ventral side and the apex of the heart. After lumenized connections of the coronary vessels with the aorta and right atrium are established, a media composed of smooth muscle cells and an adventitia composed of procollagen‐producing fibroblasts are formed around the coronary arteries. In the early stage, bloodflow through the coronary plexus is possible, although connections with the aorta have yet to be established. After the coronary plexus and the aorta and right atrium are interconnected, coronary vessel differentiation proceeds by media and adventitia formation around the proximal coronary arteries. At the same time, the remodeling of the vascular plexus is manifested by disappearance of arteriovenous anastomoses, leaving only capillaries to connect the arterial and venous system. Dev. Dyn. 208:338–348, 1997.

Morphogenesis of the First Blood Vessels

ABSTRACT: The initial phase of vessel formation is the establishment of nascent endothelial tubes from mesodermal precursor cells. Development of the vascular epithelium is examined using the transcription factor TAL1 as a marker of endothelial precursor cells (angioblasts), and a functional assay based on intact, whole‐mounted quail embryos. Experimental studies examining the role(s) of integrins and vascular endothelial growth factor (VEGF) establish that integrin‐mediated cell adhesion is necessary for normal endothelial tube formation and that stimulation of embryonic endothelial cells with exogenous VEGF results in a massive “fusion” of vessels and the obliteration of normally avascular zones.

Organized type I collagen influences endothelial patterns during “spontaneous angiogenesis in vitro”: Planar cultures as models of vascular development

SummarySelected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries—a phenomenon referred to as “spontaneous angiogenesis in vitro”. We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to indentify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.

VEGF and Vascular Fusion: Implications for Normal and Pathological Vessels

The avian embryo is well suited for the study of blood vessel morphogenesis. This is especially true of investigations that focus on the de novo formation of blood vessels from mesoderm, a process referred to as vasculogenesis. To examine the cellular and molecular mechanisms regulating vasculogenesis, we developed a bioassay that employs intact avian embryos. Among the many bioactive molecules we have examined, vascular epithelial growth factor (VEGF) stands out for its ability to affect vasculogenesis. Using the whole-embryo assay, we discovered that VEGF induces a vascular malformation we refer to as hyperfusion. Our studies showed that microinjection of recombinant VEGF165 converted the normally discrete network of embryonic blood vessels into enlarged endothelial sinuses. Depending on the amount of VEGF injected and the time of postinjection incubation, the misbehavior of the primordial endothelial cells can become so exaggerated that for all practical purposes the embryo contains a single enormous vascular sinus; all normal vessels are subsumed into a composite vascular structure. This morphology is reminiscent of the abnormal vascular sinuses characteristic of certain neovascular pathologies.

Differences in development of coronary arteries and veins

OBJECTIVE The differentiation of the coronary vasculature was studied to establish in particular the formation of the coronary venous system. METHODS Antibody markers were used to demonstrate endothelial, smooth muscle, and fibroblastic cells in serial sections of embryonic quail hearts. The anti-beta myosin heavy chain and the neuronal marker HNK-1 were added to our incubation protocol. RESULTS In HH32, the coronary vascular network has developed into a circulatory system with connections to the sinus venosus, the aorta and the right atrium. The connections between the aorta and the right atrium allow for direct arteriovenous shunting. Subsequently, differentiation into coronary arteries and veins occurs with an interposed capillary network. The smooth muscle cells of the coronary arterial media derive from the subepicardial layer, whereas the subepicardially located cardiac veins recrute atrial myocardium, as these cells express the beta-myosin heavy chain antigen. Ganglia are located in the subepicardium close to the vessels, while nerve fibres tend to colocalize with the formed vessel channels. CONCLUSIONS A new finding is presented in which the subepicardial coronary veins have a media that consists of myocardial cells. The close positional relationship of neural tissue and coronary vessels that penetrate the heart wall is explained as inductive for vessel wall differentiation, but not for invasion into the heart.

Identification of the developmental marker, JB3-antigen, as fibrillin-2 and its de novo organization into embryonic microfibrous arrays

The monoclonal antibody JB3 was previously shown to react with a protein antigen present in the bilateral primitive heart‐forming regions and septation‐stage embryonic hearts; in addition, primary axial structures at primitive streak stages are JB3‐immunopositive (Wunsch et al. [1994] Dev. Biol. 165:585–601). The JB3 antigen has an overlapping distribution pattern with fibrillin‐1, and a similar molecular mass (Gallagher et al. [1993] Dev. Dyn. 196:70–78; Wunsch et al. [1994] Dev. Biol. 165:585–601). Here we present immunoblot and immunoprecipitation data showing that the JB3 antigen is secreted into tissue culture medium by day 10 chicken embryonic fibroblasts, from which it can be harvested using JB3‐immunoaffinity chromatography. A single polypeptide (Mr = 350,000), which was not immunoreactive with an antibody to fibrillin‐1, eluted from the affinity column. Mass spectroscopy peptide microsequencing determined the identity of the JB3 antigen to be an avian homologue of fibrillin‐2. Live, whole‐mounted, quail embryos were immunolabeled using a novel microinjection approach, and subsequently fixed. Laser scanning confocal microscopy indicated an elaborate scaffold of fibrillin‐2 filaments encasing formed somites. At more caudal axial positions, discrete, punctate foci of immunofluorescent fibrillin‐2 were observed; this pattern corresponded to the position of segmental plate mesoderm. Between segmental plate mesoderm and fully‐formed somites, progressively longer filamentous assemblies of fibrillin‐2 were observed, suggesting a developmental progression of fibrillin‐2 fibril assembly across the somite‐forming region of avian embryos. Extensive filaments of fibrillin‐2 connect somites to the notochord. Similarly, fibrillin‐2 connects the mesoderm associated with the anterior intestinal portal to the midline. Thus, fibrillin‐2 fibrils are organized by a diverse group of cells of mesodermal or mesodermally derived mesenchymal origin. Fibrillin‐2 microfilaments are assembled in a temporal and spatial pattern that is coincident with cranial‐to‐caudal segmentation, and regression of the anterior intestinal portal. Fibrillin‐2 may function to impart physical stability to embryonic tissues during morphogenesis of the basic vertebrate body plan. Dev. Dyn. 1998;212:461–471.

Fibulin‐1, vitronectin, and fibronectin expression during avian cardiac valve and septa development

Extracellular matrix (ECM) proteins have been implicated as mediators of events important to valvuloseptal development (reviewed by Little and Rongish, Experentia, 51:873–882, 1995). The aim of this study was to identify connective tissue ECM proteins present at sites of valvuloseptal morphogenesis, and to determine how their patterns of expression change during the developmental process.

Vascular Morphogenesis: In Vivo, In Vitro, In Mente

Part 1 Vascular morphogenesis in vivo. Part 2 Vascular morphogenesis in vitro. Part 3 Vascular morphogenesis in mente.

PROLIFERATION AND DIFFERENTIATION OF SMOOTH MUSCLE CELL PRECURSORS OCCURS SIMULTANEOUSLY DURING THE DEVELOPMENT OF THE VESSEL WALL

Formation of the blood vessel wall depends on the recruitment, proliferation, and differentiation of smooth muscle cell (SMC) precursors. The temporal events associated with the onset of expression of several SMC proteins have been well characterized in mouse and avian species. However, the timing of cell proliferation during this process has not been explored. More importantly, it has not been clear whether commitment to the smooth muscle pathway precludes proliferation during development. In the present study, we have determined the kinetics of replication in developing chick aortae between days 2.5 and 19 and have correlated these data with the expression of various SMC differentiation markers. We found that proliferation of aortic SMC precursors occurs in two waves; an early phase of rapid proliferation (15–17%; between days 4 and 12), and a second phase, when replication was reduced to less than 5% (days 16 to hatching). Proliferation of SMC during the first wave occurred concomitantly with the progressive accumulation of SMC contractile proteins, such as SM α‐actin, calponin, myosin heavy chain, and the 1E12 antigen. We also found that the relative proliferation capacity within each compartment of the vessel wall, ie., intima, media, and adventitia varies throughout development. Approximately, 55–63% of all replicating cells were found in the tunica adventitia from days 6 to 12, whereas 35% were found in the tunica media (tunica media:adventitia = 1:2). This ratio was inverted after day 12, when most of the replicating cells were located in the tunica media (tunica media:adventitia = 2:1). In addition, we observed a ventral‐to‐dorsal gradient in the proliferation of SMC precursors between days 2.5 and 5. The ventral‐to‐dorsal proliferation gradient was similar to the previously described differential expression of two early SMC markers: α‐actin and the 1E12 antigen. These data support the concept that a polarity exists either in the pool of SMC precursors or, in expression of factors that regulate recruitment of presumptive SMC. Dev. Dyn. 209:342–352, 1997.

Distribution of connective tissue proteins during development and neovascularization of the epicardium

OBJECTIVE The epicardium is the site of initial cardiac neovascularization and formation of the coronary circulatory system. Recent evidence indicates that vascular progenitor cells are influenced by the connective tissue proteins of their extracellular environment, yet little is known about the composition or function of the embryonic epicardial extracellular matrix (ECM). This study examines the distribution of ECM proteins during the migration, growth and maturation of epicardial cells and also during the development of the coronary vascular network. METHODS Immunofluorescence microscopy was used to determine the distributions of vitronectin, fibronectin and a newly described fibrillin-like protein, the JB3 antigen, in the embryonic chicken heart. Immunoblot analysis was performed to compare the relative electrophoretic mobilities of the JB3 antigen and fibrillin-1. RESULTS The data show that vitronectin and fibronectin are present at sites of initial migration of the epicardial cells. The expression of vitronectin (and also fibronectin) becomes more pronounced as the epicardium thickens, undergoes remodeling and differentiates. The JB3 antigen is prominently expressed in the coronary arteries, allowing visualization of their connection to the systemic circulation and to the heart muscle, as well as vessel wall formation and organization. Immunoblot analysis suggests that the JB3 antibody recognizes a fibrillin-like polypeptide that is distinct from fibrillin-1. CONCLUSIONS The observed distributions of vitronectin and fibronectin are consistent with roles in migration of epicardial cells, in remodeling of the epicardium and as substratum components during blood vessel formation. The observed distribution of the JB3 antigen indicates a structural/organizational role in coronary arterial wall assembly and suggests that the JB3 antibody be considered an early marker for maturing coronary arteries.