A novel angiogenesis model for screening anti-angiogenic compounds: the chorioallantoic membrane/feather bud assay.

Enhanced angiogenesis is a hallmark of solid tumors and hematological malignancies. Anti-angiogenic therapeutic approaches have recently been shown to be effective for the treatment of certain cancers. Endothelial cells migrating to tumors provide them with new blood vessels that are critical for their growth and survival. We have developed a novel and rapid method to evaluate the anti-angiogenic activity of new agents consisting of a combined chorioallantoic membrane (CAM) and feather bud (FB) assay. Unlike previous assays, this new assay assesses the effects of drugs on the ability of tissues to attract and develop their own blood supply. The CAM already has a well-developed vascular network that is capable of providing blood vessels to the non-vascularized FB, allowing for this tissue to develop feathers. As a result, the exposure of the FB to drugs for 2 days followed by attachment to the CAM for 4 days allows evaluation of the compound's ability to impact blood vessel and feather formation within the CAM-attached FB tissue. Feather formation is determined as well as expression of endothelial cell genes and proteins analyzed. Using agents with known anti-angiogenic activity including fumagillin, minocycline, zoledronic acid, doxorubicin and agents lacking anti-angiogenic activity such as melphalan, we have shown that the CAM/FB assay can accurately and rapidly assess the ability of agents to prevent blood vessel and feather development within non-vascularized tissues.

The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf.

Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, we provide evidence for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.

Analysis of talpid3 and wild-type chicken embryos reveals roles for Hedgehog signalling in development of the limb bud vasculature.

Chicken talpid(3) mutant embryos have a wide range of Hedgehog-signalling related defects and it is now known that the talpid(3) gene product encodes a novel protein essential for Hedgehog signalling which is required for both activator and repressor functions of Gli transcription factors (Davey, M.G., Paton, I.R., Yin, Y., Schmidt, M., Bangs, F.K., Morrice, D.R., Gordon-Smith, T., Buxton, P., Stamataki, D., Tanaka, M., Münsterberg, A.E., Briscoe, J., Tickle, C., Burt, D.W. (2006). The chicken talpid(3) gene encodes a novel protein essential for Hedgehog signalling. Genes Dev 20 1365-77). Haemorrhaging, oedema and other severe vascular defects are a central aspect of the talpid(3) phenotype (Ede, D.A. and Kelly, W.A (1964a). Developmental abnormalities in the head region of the talpid(3) mutant fowl. J. Embryol. exp. Morp. 12:161-182) and, as Hedgehog (Hh) signalling has been implicated in every stage of development of the vascular system, the vascular defects seen in talpid(3) are also likely to be attributable to abnormal Hedgehog signalling. Gene expression of members of the VEGF and Angiopoietin families of angiogenic growth factors has been linked to haemorrhaging and oedema and we find widespread expression of VEGF-D, rigf and Ang2a in the talpid(3) limb. Furthermore, ectopic expression of these genes in talpid(3) limbs points to regulation via Gli repression rather than activation. We monitored specification of vessel identity in talpid(3) limb vasculature by examining expression of artery-specific genes, Np1 and EphrinB2, and the vein-specific genes, Np2a and Tie2. We show that there are supernumerary subclavian arteries in talpid(3) limb buds and abnormal expression of an artery-specific gene in the venous submarginal sinus, despite the direction of blood flow being normal. Furthermore, we show that Shh can induce Np1 expression but has no effect on Np2a. Finally, we demonstrate that induction of VEGF and Ang2a expression by Shh in normal limb buds is accompanied by vascular remodelling. Thus Hedgehog signalling has a pivotal role in the cascade of angiogenic events in a growing embryonic organ which is similar to that proposed in tumours.

Vasculogenesis and angiogenesis in the mouse embryo studied using quail/mouse chimeras.

Using quail/chick chimeras, we have previously shown that different embryonic territories are vascularized through two distinct mecanisms, angiogenesis and vasculogenesis. Angiogenesis occurs in tissues of somatopleural origin, vasculogenesis occurs in territories of splanchnopleural origin. The aim of this work was to establish if these modes of vascularization were conserved in the mammalian embryo. Since in vivo manipulations with mammalian embryos are difficult to perform, we used a quail/mouse chimera approach. Mouse limb buds of somatopleural origin, and visceral organ rudiments of splanchnopleural origin, were grafted into the coelomic cavity of 2.5 day-old quail embryos. After four to seven days, the hosts were killed and the origin of the endothelial cells in the mouse tissues was determined by double staining with the quail endothelial and hematopoietic cell-specific marker, QH1 and mouse-specific VEGFR2 and VEGFR3 probes. Our findings show that the great majority of vessels which developed in the mouse limbs was QH1+, indicating that these tissues were vascularized by angiogenesis. Conversely, visceral organs were vascularized through the vasculogenesis process by mouse endothelial cells which differentiated in situ. These results demonstrate for the first time that in the mouse embryo, as previously shown in avian species, the tissues from somatopleural origin are vascularized by angiogenesis, while rudiments of a splanchnopleural origin are vascularized by vasculogenesis, both at vascular and lymphatic levels.

Expression of rigf, a member of avian VEGF family, correlates with vascular patterning in the developing chick limb bud.

In a differential display screening for genes regulated by retinoic acid in the developing chick limb bud, we have isolated a novel gene, termed rigf, retinoic-acid induced growth factor, that encodes a protein belonging to the vascular endothelial growth factor (VEGF) family. Rigf transcripts were found in the posterior region of the limb bud in a region-specific manner as well as in other embryonic tissues and regions, including the notochord, head and trunk mesenchyme, retinal pigment epithelium, and branchial arches. Several manipulations revealed that retinoic acid and sonic hedgehog signaling pathways regulate rigf expression in the limb bud. VEGF family members, which promote the migration, differentiation and proliferation of endothelial cells in both blood and lymphatic vessels, are important factors for the formation of blood and lymphatic vasculatures during development. We demonstrated that the anterior border of the rigf expression domain in the limb bud corresponds with the position of the primary central artery (the subclavian artery in the forelimb), which is a main artery for supplying blood to the limb. These observations taken together with results from some experimental manipulations suggest that the limb tissue attracts blood vessels into the limb bud and that rigf is involved in the pattern formation of blood vessels in the limb.

Neuroectodermal origin of brain pericytes and vascular smooth muscle cells.

The origin of vascular pericytes (PCs) and smooth muscle cells (vSMCs) in the brain has hitherto remained an open question. In the present study, we used the quail-chick chimerization technique to elucidate the lineage of cranial PCs/vSMCs. We transplanted complete halves of brain anlagen, or dorsal (presumptive neural crest [NC]) or ventral cranial neural tube. Additional experiments included transplantations of neuroectoderm into limb mesenchyme, and of head mesoderm or limb mesenchyme into paraxial head mesoderm. After interspecific transplantation of quail brain rudiment, graft-derived vSMCs were found in the vessel walls of the grafted brain. Notably, transplanted ventral neural tube also gave rise to vSMCs. After grafting of quail head mesoderm, quail endothelial cells were found in the host brain, but no vSMCs of donor origin. Grafting of quail whole or ventral neural tube into the limb bud led to endowment of graft and host vessels with graft-derived vSMCs. Quail limb bud mesenchyme contributed to vSMCs in the ectopic neural graft, but, when transplanted into paraxial head mesenchyme, it did not form intraneural vSMCs. After orthotopic transplantation of cranial NC, graft-derived vSMCs were not only found in meninges and brain of the operated side, but also on the contralateral side. Our results show that 1) avian cranial neuroectoderm is able to differentiate into vSMCs of the brain; 2) this potential is not restricted to the prospective NC; and 3) neither cranial mesoderm nor cranially transplanted limb bud mesoderm can give rise to brain vSMC.

The roles of the marginal veins in the differentiation of the rat hind limb bud: an immunocytochemical study.

Rat hind limb buds, aged between prenatal days 14 and 18, were used for electron microscopy and immunocytochemistry of fibronectin, laminin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining indicative of DNA fragmentation. Fibronectin and laminin were actively synthesized in the rough endoplasmic reticulum of epidermal cells in the apical ectodermal ridge between prenatal days 14 and 15, but most cells underwent apoptosis after prenatal day 15. As the regression of the apical ectodermal ridge progressed, mesenchymal cells associated with the marginal veins were successively incorporated into the endothelium devoid of the basal lamina. No mitotic figures of endothelial cells were recognized either in the marginal vein or in the surrounding growing capillaries. Extracellular matrix components connected the adjacent mesenchymal cells, with the endothelium of such vessels immunoreacting to fibronectin and laminin. In addition, fibronectin-immunoreactive networks among the interstices of the mesenchymal cell cords developed in the avascular zone between the epidermis and the growing capillaries at prenatal day 15, but became inconspicuous at prenatal day 16. These results indicate that the apoptosis of the epidermal cells is the major reason for the regression of the apical ectodermal ridge, and that the capillary ingrowth from the marginal veins to the avascular zone is accelerated by transformation of mesenchymal cells to endothelial ones. Fibronectin and laminin seem to play crucial roles in capillary growth, especially in the adhesion between endothelial cells of the pre-existing vessels and mesenchymal cells.

Development of pericyte-like cells during angiogenesis in quail chick chimeras as detected by combined Feulgen reaction and immunohistochemistry.

Perivascular fibroblasts have been proposed as possible precursor cells for microvascular pericytes. To investigate the development of pericytes during angiogenesis we examined interspecific grafts between chick and quail embryos. Limb buds of three-day old quail embryos were transferred to the chorioallantoic membrane (CAM) of ten to fourteen day-old chick embryos. Six days after grafting, the limb buds were explanted and histologically examined by combined Feulgen reaction and immunohistochemistry using an antibody to quail endothelial and hemopoietic cells (QH-1). Limb buds were found to be vascularized by a network of capillaries which were partially derived from sprouts of the chick CAM microvasculature. Numerous hybrid capillaries were detected, consisting of host endothelial cells (chick) and graft pericytes (quail). These results provide further support for the idea that microvascular pericytes can evolve from perivascular fibroblasts.