PART I Biology of Blood Vessels 1 Vascular Embryology and Angiogenesis Aglaia Ntokou, Inamul Kabir, Fatima Zahra Saddouk, and Daniel M. Greif In simple terms, the cardiovascular system consists of a sophisticated Finally, the outermost layer of the vessel wall is the adventitia, a collec- pump (i.e., the heart) and a remarkable array of tubes (i.e., the blood tion of loose connective tissue, fibroblasts, macrophages, cells express- and lymphatic vessels). Arteries and arterioles (the efferent blood ing stem-cell markers, and small vessels, known as the vasa vasorum, vessels in relation to the heart) deliver oxygen, nutrients, paracrine that perfuse the cells of larger arteries. hormones, blood and immune cells, and many other products to the This chapter summarizes many of the key molecular and cellular capillaries, small-caliber, thin-walled vascular tubes. These substances processes and underlying signals in the morphogenesis of the different are then transported through the capillary wall into the extravascu- layers of the blood vessel wall and of the circulatory system in general. lar tissues, where they participate in critical physiological processes. Specifically, for intimal development, it concentrates on early EC pat- In turn, waste products are transported from the extravascular space terning, specification and differentiation, lumen formation, branching, back into the blood capillaries and returned by the venules and veins metabolism, and lymphatic vessel morphogenesis. In the second sec- (the afferent vessels) to the heart. Alternatively, approximately 10% of tion, the development of the tunica media is divided into subsections the fluid returned to the heart courses via the lymphatic system to the examining the components of the media, VSMC origins, smooth mus- large veins. To develop normally, the embryo requires the delivery of cle cell (SMC) differentiation, and patterning of the developing VSMC nutrients and removal of waste products beginning early in develop- layers and the ECM. Finally, the chapter concludes with a succinct ment, and, indeed, the cardiovascular system functions early during summary of morphogenesis of the adventitia, adventitial stem cells, morphogenesis. and macrophages. Understanding these fundamental vascular devel- The fields of vascular embryology and angiogenesis have been opmental processes is important from a pathophysiological and ther- revolutionized through experimentation with model organisms. In apeutic standpoint because many diseases almost certainly involve the particular, this chapter focuses on key studies using common vas- recapitulation of developmental programs. For instance, in many vas- cular developmental models, including the mouse, zebrafish, chick, cular disorders, mature VSMCs dedifferentiate and exhibit increased and chick-quail transplants, each of which has its advantages. Among rates of proliferation, migration, and ECM synthesis through a process mammals, the most powerful genetic-engineering tools and the great- termed phenotypic switching.1 est breadth of mutants are readily available in the mouse. Furthermore, the mouse is a good model of many aspects of human vascular de- TUNICA INTIMA: ENDOTHELIUM velopment, and, in particular, the vasculature of the mouse retina is a powerful model as it develops postnatally and is visible externally. The Early Development zebrafish is a transparent organism that develops rapidly with a well- Development begins with the fertilization of the ovum by the sperm. described pattern of cardiovascular morphogenesis and sophisticated Chromosomes of the ovum and sperm fuse, and then a mitotic pe- genetic manipulations that are readily available. The chick egg is large riod ensues. The early 16- to 32-cell embryo, or morula, consists of with a yolk sac vasculature that is easily visualized and develops rapidly. a sphere of cells with an inner core termed the inner cell mass. The And finally, the coupling of chick-quail transplants with species-specific first segregation of the inner cell mass generates the hypoblast and antibodies allows for cell tracing experiments. The combination of stud- epiblast. The hypoblast gives rise to the extraembryonic yolk sac and ies with these powerful model systems, as well as others, has yielded key the epiblast to the amnion and the three germ layers of the embryo, insights into human vascular embryology and angiogenesis. known as the endoderm, mesoderm, and ectoderm. The epiblast is Although blood vessels are composed of three tissue layers, the vast divided into these layers in the process of gastrulation when many majority of the vascular-development literature has focused on the of the embryonic epiblast cells invaginate through the cranial-ca udal morphogenesis of the intima, or inner, layer. This intima consists of primitive streak and become the mesoderm and endoderm, while the a single layer of flat endothelial cells (ECs) that line the vessel lumen cells that remain in the embryonic epiblast become the ectoderm. and are elongated in the direction of flow. Moving radially outward, the Most of the cardiovascular system derives from the mesoderm, in- next layer is the media consisting of layers of circumferentially oriented cluding the initial ECs, which are first observed during gastrulation. vascular smooth muscle cells (VSMCs) and extracellular matrix (ECM) A notable exception to the mesodermal origin is the SMCs of the components, including elastin and collagen. In smaller vessels, such aortic arch and cranial vessels, which instead derive from the neural as capillaries, the mural cells consist of pericytes instead of VSMCs. crest cells of the ectoderm.2 1 2 PART I Biology of Blood Vessels Although ECs are thought to derive exclusively from mesodermal angiogenesis is often initiated by EC proliferation, which results in lu- origins, the other germ layers may play an important role in regulating men widening.14 The lumen then splits through transcapillary ECM the differentiation of the mesodermal cells to an EC fate. In a classic pillars or fusion and splitting of capillaries to generate more vessels.14 study of quail-chick intracelomic grafts, host ECs invaded limb bud In addition, the developing vascular tree is fine-tuned by the pruning grafts, whereas in internal organ grafts, EC precursors derived from of small vessels. Although not involved in the construction of the initial the graft itself.3 Thus the authors hypothesized that the endoderm (i.e., vascular plan, flow is an important factor in shaping the maturation from internal organ grafts) stimulates the emergence of ECs from asso- of the vascular system, determining which vessels mature and which ciated mesoderm, whereas the ectoderm (i.e., from the limb bud grafts) regress. For instance, unperfused vessels will regress. may have an inhibitory influence.3 Yet, the endoderm does not appear to be absolutely required for the initial formation of EC precursors.4,5 Arterial and Venous Endothelial Cell Differentiation The initial primitive vascular system is formed prior to the first car- Classically, it was thought that arterial and venous blood vessel iden- diac contraction, and the early development of ECs involves the inter- tity was established as a result of oxygenation and hemodynamic fac- play of multiple signaling pathways.6,7 This early vasculature develops tors, such as blood pressure, shear stress, and the direction of flow. through vasculogenesis, a two-step process in which mesodermal cells However, over the past two decades, it has become increasingly evi- differentiate into angioblasts in situ, which, in turn, subsequently co- dent that arterial-specific and venous-specific markers are segregated alesce into blood vessels.8 Early in this process, many EC progenitors to the proper vessels quite early in the program of vascular morpho- apparently pass through a bipotential hemangioblast stage in which genesis. For instance, ephrinB2, a transmembrane ligand, and one of they can give rise to endothelial or hematopoietic cells. Fibroblast its receptors, the EphB4 tyrosine kinase, are expressed in the mouse growth factor (FGF) 2 and bone morphogenetic protein (BMP) 4 sig- embryo in an arterial-specific and relatively venous-specific man- naling are required for mesoderm specification and its differentiation ner, respectively, prior to the onset of angiogenesis (Fig. 1.1).15–18 toward endothelial and hematopoietic cell fates.7 In addition, Indian EphrinB2 and EphB4 are each required for normal angiogenesis of hedgehog is secreted by the yolk sac visceral endoderm during vascu- both arteries and veins.16,17 However, in mice homozygous for a tau- logenesis and promotes the differentiation of posterior epiblast cells lacZ knock-in into the ephrinB2 or EphB4 locus (which renders the into both endothelial and hematopoietic cells.9,10 The visceral endo- mouse null for the gene of interest), lacZ staining is restricted to ar- derm also secretes vascular endothelial growth factor (VEGF), which is teries or veins, respectively.16,17 This result indicates that neither of widely implicated in EC biology. The ligand VEGF-A signals predomi- these signaling partners is required for the arterial and venous spec- nantly through receptor VEGFR2, and Veg fr2-null embryos lack blood ification of ECs. vessel islands and vasculogenesis and die in utero.11 Importantly, most Furthermore, even before initial ephrinB2 and EphB4 expression genes that specify an EC fate contain binding sites for the E-twenty-six and prior to the first heartbeat,