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Home , Angiogenesis, Blood islands, Dorsal aorta, Posterior cardinal vein, Vasculogenesis

PART I Biology of Blood Vessels 1

Vascular and

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 ) and a remarkable array of tubes (i.e., the blood tion of loose connective tissue, fibroblasts, , 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 , 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 , small-caliber, thin-walled vascular tubes. These substances processes and underlying signals in the morphogenesis of the different are then transported through the wall into the extravascu- layers of the wall and of the 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 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, , 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: velopment, and, in particular, the vasculature of the mouse 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 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, , 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 cells (VSMCs) and (ECM) A notable exception to the mesodermal origin is the SMCs of the components, including elastin and . In smaller vessels, such aortic arch and cranial vessels, which instead derive from the neural as capillaries, the mural cells consist of 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 , 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 , and one of they can give rise to endothelial or hematopoietic cells. Fibroblast its receptors, the EphB4 , are expressed in the mouse (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 VEGFR2, and Veg fr2-null 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, Notch pathway members, delta C and (ETS) family of transcription factors, and ETS variant 2 regulates the gridlock, mark presumptive ECs in the zebrafish.19–21 The zebrafish differentiation of mesodermal progenitors toward an EC fate.12,13 gene deltaC is a homologue of the Notch ligand gene Delta, and gridlock Following the formation of the initial vascular plexus, more capil- (grl) encodes a basic helix-loop-helix protein that is a member of the laries are generated through sprouting and nonsprouting angiogenesis, Hairy-related transcription factor family and is downstream of Notch. and the vascular system is refined through pruning and regression.14 In The lateral plate mesoderm (LPM) contains artery and vein precur- the most well studied form of angiogenesis, existing blood vessels sprout sors,22 and prior to vessel formation, the grl gene is expressed as two new vessels, usually into areas of low , through a process in- bilateral stripes in the LPM.21 Subsequently, gridlock expression is lim- volving proteolytic degradation of surrounding ECM, EC proliferation ited to the trunk artery (dorsal ) and excluded from the trunk vein and migration, lumen formation, and EC maturation. Nonsprouting (cardinal vein).21 Lineage-tracking experiments of the zebrafish LPM

Fig. 1.1 Endothelial Cell Arterial-venous Specification. (A) Notch promotes arterial fate of Flk-1+ (i.e., VEGFR2+) angioblasts. (B) In the embryo, angioblasts aggregate directly into the or cardinal vein, a process mediated by vascular endothelial growth factor (VEGF), Sonic hedgehog (SHH), and Notch signaling via the delta-like ligand (DLL). In the yolk sac, angioblasts fuse to form the vascular plexus with expression of arterial (-B2) and venous (EphB4) markers. (Redrawn with permission from Chung AS, Ferrara N. Developmental and pathological angiogenesis. Annu Rev Cell Dev Biol. 2011;27:563–584.) CHAPTER 1 Vascular Embryology and Angiogenesis 3 suggest that by the 7- to 12-somite stage, an individual angioblast is ­invariably uses sensors that detect external stimuli.29 This information destined to contribute in a mutually exclusive fashion to the arterial or is then integrated and translated into a biological response. Important venous system.20 examples of such biological sensors include the growth cones of neu- In addition to being an early marker of arterial ECs, the Notch rons and the terminal cells of the Drosophila tracheal system. Both of pathway is a key component of a signaling cascade that regulates ar- these sensors have long dynamic filopodia that sense and respond to terial EC fate (see Fig. 1.1).18 In zebrafish, downregulating the Notch external guidance cues and are critical in determining the ultimate pat- pathway through genetic means or injection of mRNA encoding a tern of their respective tubular structures. dominant-negative Suppressor of Hairless, a known intermediary in Similarly, endothelial tip cells are located at the end of angiogenic the Notch pathway, results in reduced ephrinB2 expression with loss of sprouts and are polarized with long filopodia that play both a sensory regions of the dorsal aorta.20,23 Reciprocally, contiguous regions of the and motor role (Fig. 1.2).29,30 In a classic study published approximately cardinal vein expand and EphB4 expression increases.20 By contrast, 40 years ago, Ausprunk and Folkman reported that on the day after V2 activation of the Notch pathway results in reduced expression of flt4, carcinoma implantation into the rabbit cornea, ECs of the host limbal a marker of venous cell identity, without an effect on arterial marker vessels display surface projections that resemble “regenerating ECs,”31 expression or dorsal aorta size, suggesting that Notch is not sufficient consistent with what is now classified as tip cell filopodia. Tip cells are to induce arterial EC fate.20,23 Further studies demonstrated that Sonic highly migratory leading cells, are rarely proliferative, and are enriched hedgehog (SHH) is upstream of VEGF, and VEGF-A binds to VEGFR2 in platelet-derived growth factor (PDGF)-B, VEGFR-2, the Notch li- and its coreceptor 1 (NRP1), leading to activation of Notch gand delta-like ligand 4 (DLL4), and apelin.29,32 Proximal to tip cells are signaling.24,25 The specific expression of NRP1 and NRP2 in arteries stalk cells which also express VEGFR-2 but, unlike tip cells, are highly and veins, respectively, is established prior to blood flow.26 Importantly, proliferative (see Fig. 1.2).29,32 During the initiation of sprouting angio- the fate of venous ECs is mediated primarily by the chicken ovalbumin genesis, endothelial tip cells develop initial projections prior to stalk upstream promoter-transcription factor II (COUP-TFII).7 Deletion of cell proliferation.31 the gene encoding COUP-TFII in ECs leads to veins expressing NRP1 The mouse retina model has been widely used in studies of angio- and Notch signaling molecules.27 Taken together, these results suggest genesis and is an excellent model for studying different aspects of blood that the SHH-VEGF-Notch axis is necessary for arterial EC differen- vessel development: the retinal vasculature is visible externally and de- tiation and that venous identity is not simply a default pathway in the velops postnatally through a stereotyped sequence of well-described absence of Notch signaling but is actively maintained by COUP-TFII. steps. In addition, at most time points, the retina simultaneously in- A study of the origins of the coronary vascular endothelium high- cludes sprouting at the vascular front and remodeling at the core. The lights the plasticity of ECs during early mouse development.28 This VEGF pathway is critical for guiding angiogenic sprouts, and in the study suggests that ECs sprout from the , the structure retina, the expression of the ligand VEGF-A is limited to astrocytes which returns blood to the embryonic heart, and dedifferentiate as with the highest levels at the leading edge of the front of the extending they migrate over and through the myocardium.28 ECs that invade the EC plexus,29 suggesting that the astrocytes lay down a road map for the myocardium differentiate into the coronary arterial and capillary ECs, ECs to follow.33 VEGF-A signals through VEGFR-2 on tip and stalk whereas those that remain on surface of the heart will redifferentiate cells. Interestingly, the proper distribution of VEGF-A is required for into the coronary veins.28 tip cell filopodia extension and tip cell migration, whereas the absolute concentration, but not the gradient, of VEGF-A appears to be critical Endothelial Tip and Stalk Cell Specification in Sprouting for stalk cell proliferation.29 Angiogenesis VEGF induces DLL4 expression, and Notch pathway-mediated lat- Tubular structures are essential for diverse physiological processes, and eral inhibition is critical for assigning ECs in sprouting angiogenesis the proper construction of these tubes is critical. Tube morphogenesis to tip and stalk positions (Fig. 1.3).30 DLL4 is specifically expressed in requires the coordinated migration and growth of cells that comprise arterial and capillary ECs, and in development, DLL4 is enriched in tip the tubes, and the intricate modulation of the biology of these cells cells, whereas Notch activity is greatest in stalk cells.30,34,35 Attenuation

isolectin B4 (EC) PECAM-1 phosphohistone

* Tip cell

Stalk cells

Lumen A B C Fig. 1.2 Endothelial Tip and Stalk Cells. (A) Graphic illustration of tip and stalk cells of an endothelial sprout. (B) Endothelial tip cell with filopodia from a mouse retina stained to mark endothelial cells (EC) (isolectin B4, green) and nuclei (blue). (C) Vascular sprout labeled with markers for ECs (PECAM-1, red), mitosis (phosphohistone, green) and nuclei (blue). Arrow indicates a mitotic stalk cell nucleus; asterisk indicates tip cell nucleus. (Redrawn with permission from Gerhardt H, Golding M, Fruttiger M, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 2003;161:1163–1177.) 4 PART I Biology of Blood Vessels

Fig. 1.3 Notch-mediated Lateral Inhibition in Tip/Stalk Cell Specification. VEGF - A binds VEGFR2, expressed at the endothelial cell surface, and neuropilin (NRP) modulates the VEGF signaling output. VEGF stimulation upregulates DLL4 in tip cells, which in turn, activates Notch signaling in the stalk, suppressing the tip cell phenotype. Notch signaling activation reduces VEGFR2 expression and increases levels of VEGFR1 and soluble VEGFR1 (sVEGFR1), as well as that of Notch target genes (e.g., Notch-regulated ankyrin repeat protein [Nrarp]). In contrast, the tip cell has low Notch signaling, facilitating elevated expression of VEGFR2 and NRP but low VEGFR1. In contrast to DLL4, the Jagged1 ligand is expressed by stalk cells. Jagged1 antagonizes DLL4-Notch signaling in the sprouting front when the Notch receptor is modified by the glycosyltransferase Fringe, thereby enhancing differential Notch activity between tip and stalk cells. The duration and amplitude of the Notch signal are modulated by the histone deacetylase SIRT1. (Redrawn with permission from Blanco R, Gerhardt H. VEGF and Notch in tip and stalk cell selection. Cold Spring Harb Perspect Med. 2013;3:a006569.) of Notch activity through genetic (i.e., Dll4(+/−)) or pharmacological ­analysis usually is complementary to experiments with total knockouts (i.e., γ-secretase inhibitors) approaches results in increased capillary and often can, in fact, be more informative as complete removal of a sprouting and branching, filopodia formation, and tip cell marker gene may impair interpretation by grossly distorting the tissue archi- expression.30,36 tecture or eliminating competition between cells that harbor differing In addition to DLL4, Jagged1 is another Notch ligand that is in- levels of a gene product. volved in regulating tip and stalk cell fate in a related but contrast- Experiments using mosaic analysis of Notch pathway mutants in ing manner. Stalk cells express higher levels of Jagged1, and Jagged1 a wild-type background indicate that the Notch pathway acts in a cell levels are inversely proportional to the amount of EC sprouting.30 autonomous fashion to limit the number of tip cells. In comparison to Interestingly, Jagged1 antagonizes DLL4-Notch signaling, reducing wild-type ECs in the mouse retina, ECs that are genetically engineered overall EC Notch activity.30 Thus high stalk expression of Jagged1 en- to have reduced or no Notch1 receptor expression are enriched in the hances the differential Notch activity in stalk (high activity) versus tip tip cell population.34 In addition, mosaic studies of Notch signaling (low activity) cells by competitively inhibiting against DLL4-mediated components in the developing zebrafish intersegmental vessels (ISVs) Notch activity in adjacent tip cells.30 are informative. The ISVs traverse between the somites from the dor- Mosaic analyses indicate that competition between cells (in this sal aorta to the dorsal longitudinal anastomotic vessel (DLAV) and are case for Notch activity) is critical in determining the division of labor widely used in investigation of blood vessel development. The ISV has in sprouting angiogenesis. Genetic mosaic analysis involves the mix- been classified as consisting of three (or four) cells in distinct positions: ing of at least two populations of genetically distinct cells frequently a base cell that contributes to the dorsal aortic cell, a connector cell that in the early embryo and, subsequently, comparing the contribution of courses through the somites, and the most dorsal cell that contributes each cell population to a specific structure or process. Notably, mosaic to the DLAV.37,38 LPM angioblasts contribute to the ECs of all the trunk CHAPTER 1 Vascular Embryology and Angiogenesis 5 vasculature, including the dorsal aorta, , ISVs, The most well-studied molecular determinants of vascular branch- DLAV, and the subintestinal venous vessels. Precursors destined for the ing are the VEGF family of ligands (VEGF-A, -B, -C, and -D) and ISVs and DLAV initially migrate to the midline dorsal aorta and then endothelial receptor tyrosine kinases (VEGFR1, 2, and 3).48 VEGF between somites to their ultimate positions.37,38 Siekmann and Lawson is a potent EC mitogen, motogen, and vascular permeability factor, generated mosaic zebrafish by transplanting into early wild-type em- and the level of VEGF is strictly regulated in development, because bryos marked cells from embryos either lacking the key Notch signal- VEGF heterozygous mice die at approximately E11.5 with impaired ing component recombining protein suppressor of hairless (Rbpsuh) or angiogenesis and blood island formation.49,50 During embryogene- expressing an activated form of Notch.38 Interestingly, rbpsuh-­deficient sis, VEGFRs are expressed in proliferating ECs and the ligands in cells were excluded from the dorsal aorta and enriched in the DLAV adjacent tissues. For instance, the secretion of VEGF by the ventric- position.38 In turn, transplanted cells harboring activated Notch muta- ular neuroectoderm is thought to induce capillary ingrowth from tions were excluded from the DLAV in mosaics and instead preferen- the perineural vascular plexus.51 Mice null for Veg fr2 or Veg fr1 die tially localized to the base cell and dorsal aorta positions.38 at approximately E9.0 with Veg fr2 (−/−) mice lacking yolk-sac blood Taken together, the findings indicate that in sprouting angiogene- islands and vasculogenesis,11 and Veg fr1 (−/−) mice displaying disor- sis, ECs compete for the tip position through Notch-mediated lateral ganized vascular channels and .52 Although VEGFR3 inhibition of neighboring cells (see Fig. 1.3).30 High levels of DLL4 and expression eventually restricts to lymphatic ECs, its broad vascular Jagged1 are expressed on tip and stalk cells, respectively, resulting in endothelial expression early in development is critical for embryonic high relative Notch activity in stalk cells and thereby promoting dis- morphogenesis. Indeed, Veg fr3-null mice undergo vasculogenesis tinct tip and stalk cell identity. Furthermore, in the developing retina, and angiogenesis; however, the lumens of large vessels are defec- the expression of DLL4 is regulated by VEGF-A, which is secreted by tive, resulting in pericardial effusion and cardiovascular failure by astrocytes in response to hypoxia. E9.5.53 Low oxygen levels induce vascular EC branching through hy- poxia inducible factor-1 alpha–mediated expression of VEGFR2.54 Molecular Determinants of Branching VEGFR1 largely functions as a negative regulator of VEGF signaling The pattern of many branched structures such as the vasculature is crit- by sequestering VEGF-A. The affinity of VEGFR1 for VEGF-A is ical for function, and diverse branched structures use similar signaling higher than that of VEGFR2, and VEGFR1 kinase domain mutants pathways to generate their specific patterns. A number of well-studied are viable.55 systems, such as the Drosophila trachea, mammalian lung, ureteric bud Although not as well studied as the role of the VEGF pathway in (UB), and the vasculature, consist of hierarchical tubes, progressing from vessel branching, other signaling pathways, such as those mediated larger to smaller diameter, that transport important gas and/or fluid by FGF, Notch, and other guidance factors, are also likely to play im- constituents. The molecular strategies underlying the morphogenesis of portant roles. For instance, EC-specific deletion of Fgfr1 on a global these patterns often includes receptor tyrosine kinase–mediated signal- Fgfr3-null background attenuates branching of the skin vasculature.56 ing, as well as fine-tuning with inhibitors of these signaling pathways.39,40 In addition, transgenic FGF expression in myocardium augmented Similar to its key role in Drosophila tracheogenesis, the FGF path- coronary artery branching and blood flow, whereas expression of a way is essential for determining branch patterning in the mammalian dominant-negative FGFR1 in retinal-pigmented epithelium reduced lung. In the mouse, the trachea and lung bronchi bud from the epithe- the density and branching of retinal vessels.39 The role of the Notch lium of the gut wall at approximately E9.5,41 and, subsequently, three pathway is discussed earlier in the section of endothelial tip and distinct branching subroutines are repeated in various combinations to stalk cells. Finally, the maturation of branches to a more stable state generate a highly stereotyped, complex treelike structure42 that facili- that is resistant to pruning is thought to largely be regulated by sig- tates gas exchange. In early embryogenesis, the visceral mesenchyme naling pathways that modulate EC branch coverage by mural cells. adjacent to the heart expresses FGF10, and FGF10 binds endodermal Interestingly, two of the most important such pathways involve re- FGFR2b.39 Fgf10 null mice lack lungs and have a blind trachea,43 and ceptor tyrosine kinases such as the -Tie and the PDGF similarly, Fgfr2b(−/−) mice form underdeveloped lungs that undergo ligand-receptor pathways. apoptosis.44 As an additional level of regulation, the inhibitor of FGFR signaling, sprouty, is a key component of an FGF-induced negative Vascular Lumenization feedback loop in the lung. In response to FGF10, FGFR2b induces ECs at the tip of newly formed branches do not create lumens, but sprouty2 tyrosine phosphorylation and activation, and active sprouty2 as the vasculature matures, formation of a lumen is an essential step inhibits signaling downstream of FGFR2b.39 In addition, carefully reg- in generating tubes that can transport products. Angioblasts initially ulated levels of the morphogens SHH and BMP4 modulate the branch- migrate and coalesce to form a solid cord that is subsequently hol- ing of lung airways.39 lowed out to generate a lumen through a mechanism that is contro- As with the lung, generation of the metanephric kidney requires versial. Approximately 100 years ago, researchers first suggested that signals conveyed through epithelial receptor tyrosine kinase. The vascular lumenization in the embryo occurs through an intracellular metanephric mesenchyme secretes glial-derived neurotrophic fac- process involving vacuole formation.57 Seventy years later, Folkman tor (GDNF), which activates the receptor tyrosine kinase RET and its and Haudenschild developed the first method for long-term cul- ­membrane-anchored coreceptor Gdnf family receptor α 1 (GFRα1), ture of ECs, and bovine or human ECs cultured in the presence of thereby inducing the UB to evaginate from the nephric duct.45,46 These ­tumor-conditioned medium were shown to form lumenized tubes.58 components are required for UB branching, because UB outgrowth fails In this and similar in vitro approaches, an individual cell forms in mice null for Gdnf, Gfrα1, or Ret.45 Furthermore, Ret is frequently CDC42+ pinocytic vacuoles that coalesce, extend longitudinally, and mutated in humans with renal agenesis.47 In addition, FGFR2b is also then join the vacuole of neighboring ECs to progressively generate highly expressed on UB epithelium and FGFR2b-mediated signaling an extended lumen.58–60 Subsequently, a study using two-photon regulates UB branching.39 FGF7 and FGF10 are expressed in mesenchy- high-resolution time-lapse microscopy suggested that the lumens mal tissue surrounding the UB, and FGFR2b binds with comparable af- of zebrafish ISVs are generated through a similar mechanism of finity to these ligands.39 Similar to lung development, BMP4-mediated ­endothelial intracellular vacuole coalescence followed by intercellu- signaling modulates the branching of the renal system.39 lar vacuole fusion.61 6 PART I Biology of Blood Vessels

More recently, however, a number of studies have called this intra- Metabolism cellular vacuole coalescence model into question and, instead, support Although ECs line the vessel lumen and are thereby in contact with an alternate model in which the lumen is generated extracellularly.62 sufficient oxygen for oxidative respiration, surprisingly, up to 85% One such investigation63 suggests that in contrast to what has been of their adenosine triphosphate (ATP) is generated from glycoly- thought previously,37,38 ECs are not arranged serially along the lon- sis.70 Of note, ECs have a low mitochondrial content, and when glu- gitudinal axis of the zebrafish ISV but, instead, overlap one another cose is unlimited, high glycolytic flux can produce more ATP than substantially; the circumference of an ISV at a given longitudinal po- oxidative metabolism in a shorter time, which is critical for rapid sition usually traverses multiple cells. If the lumen of a vessel were EC sprouting.71 Furthermore, VEGF enhances the kinetics of gly- derived intracellularly in a unicellular tube, then the tube would be colysis by increasing levels of phosphofructokinase-2/fructose-2,6-­ “seamless” (as in the terminal cells of the Drosophila airways)64 and bisphosphatase3 (PFKFB3).70 PFKFB3 catalyzes the synthesis of have intercellular junctions only at the proximal and distal ends of fructose-2-6-­bisphosphate, which, in turn, allosterically activates the cells. However, in the 30-hour postfertilization zebrafish, the junc- 6-phosphofructo-1-kinase, a rate-limiting glycolytic . EC ex- tional proteins zona occludens 1 (ZO-1) and VE-cadherin are coex- pression of another key glycolytic enzyme, hexokinase, is regulated pressed, often in two medial “stripes” along the longitudinal axis of by FGF-mediated signaling, and EC deletion of this enzyme impairs the ISV, suggesting that ECs align and overlap along extended regions lymphatic EC branching and migration.56 In addition to the rapid ki- of the ISV.63 Thus the lumen is exclusively an extracellular structure netics, anaerobic glycolysis has other potential advantages for ECs, in- developmentally (i.e., between adjacent cells, and not within the cyto- cluding preserving oxygen levels for transfer to perivascular cells and plasm of a single cell). priming ECs for growth into hypoxic regions.71 EC polarization is a prerequisite for lumen formation, and the Par3 The enzyme PFKFB3 is essential for tip EC phenotype. Lamellipodia complex, VE-cadherin, and microtubule dynamics play a critical role are thin structures which lack mitochondria, whereas PFKFB3 is pres- in establishing polarity.65 Endothelial-specific deletion of the gene ent in membrane ruffles of lamellipodia in migrating ECs.70 Silencing encoding β1 reduces levels of Par3 and leads to a multilay- of Pfkfb3 in ECs diminishes proliferation, lamellipodia formation, ered endothelium with cuboidal-shaped ECs and frequent occlusion sprouting, and directional migration.70 Moreover, -induced of mid-sized vascular lumens.65 VE-cadherin is a transmembrane neonatal VE-cadherin-CreERT2, Pfkfb3(flox/flox) mice have impaired reti- EC-specific cell adhesion molecule that fosters homotypic interac- nal vascularization.70 The importance of this pathway is emphasized by tions between neighboring ECs, and in vascular cords, VE-cadherin studies with both EC spheroids and zebrafish, indicating that PFKB3 is distributed broadly in the apical membrane.62 VE-cadherin dele- trumps Notch activity in regard to specifying tip cell fate.70 tion is lethal in the embryonic mouse, because the development of In addition to glycolysis, fatty acid oxidation (FAO) is another VE-cadherin(−/−) embryonic vessels arrests at the cord stage and does metabolic process that is critical for angiogenesis. A key enzyme con- not proceed to lumenization.62,66,67 Under normal conditions, during trolling FAO rates is carnitine palmitoyltransferase 1 (CPT1), which polarization, junctions form at the lateral regions of the apical mem- imports fatty acids (FAs) into the mitochondria, where they are oxi- brane as VE-cadherin translocates to these regions, which also har- dized to acetyl coenzyme A for the Krebs cycle.72 CPT1a is essential bor ZO-1.62 VE-cadherin is required for the apical accumulation of for EC proliferation and thus sprouting, but not for EC migration.72 de-adhesive­ molecules, such as the highly glycosylated podocalyxin/ FA-derived carbons are incorporated into aspartate (a nucleotide pre- gp135, which likely contributes to lumen formation through cell-cell cursor), uridine monophosphate (a precursor of pyrimidine nucleoside repulsion. In addition to anchoring neighboring ECs, VE-cadherin triphosphate), and DNA, and EC silencing of CPT1a depletes stores of 72 also is linked through β-catenin, plakogobin, and α-catenin to the aspartate and deoxyribonucleoside trisphosphates. This DNA synthe- F-actin cytoskeleton.62 In reparative angiogenesis of ischemic hind- sis through FAO is a specialized characteristic of ECs and fibroblasts.72 limbs, the RAS homolog R-RAS activates AKT and stabilizes mi- crotubules, augmenting lumen formation.68 VEGF-A treatment also Lymphatic Vessel Development activates AKT but, in contrast, does not induce microtubule stabiliza- Complementing the veins, the lymphatic system plays a critical role tion or lumenogenesis.­ 68 Mechanistically, R-RAS distributes activated in transporting lymph (i.e., fluid, macromolecules, and cells) from the AKT along microtubule fibers, all the way to the (+) end, whereas interstitial space to the subclavian veins and thereby back to heart. VEGF-A induces perinuclear localization of activated AKT.68 Lymphatic capillaries are highly permeable by virtue of their structure: Although establishing polarity of the ECs is a critical step, it is not a single layer of discontinuous lymphatic endothelial cells (LECs) with- sufficient to induce lumen formation. Indeed, in Veg fa (+/−) mice, ECs out mural cells or . Lymph drains from lymphatic of the dorsal aorta polarize but this vessel does not develop a lumen.67 capillaries into precollector vessels and then into collecting lymphatic VEGF-A activates Rho-associated protein kinases (ROCKs), which vessels, which have valves, continuous inter-EC junctions, basement induce nonmuscle myosin II light chain phosphorylation, thereby membrane, and an SMC layer. These collecting vessels drain into the enhancing the recruitment of nonmuscle myosin to the apical mem- right lymphatic trunk or thoracic duct and then into the right or left brane.67 Actomyosin complexes at the apical surface are thought to play subclavian vein, respectively. an important role in pulling the apical membranes of neighboring cells Based on her experiments more than 100 years ago, Florence Sabin apart, thus generating an extracellular lumen.65 proposed the “centrifugal model” in which lymphatic sacs derive from Another important component of the process of EC cord lumeni- veins, and vessels sprouting from these sacs give rise to the lymphatic zation is the dynamic dissolution and formation of inter-EC junctions. vasculature.73,74 Subsequently, histological, marker, and lineage studies EGFL7 is an EC-derived secreted protein that promotes EC motility yielded findings supportive of Sabin’s model.75 The tran- and is required for tube formation.69 The knockdown of Egfl7 in ze- scription factor SOX18 (sex determining region Y box 18) is a molecu- brafish impairs angioblasts from dissolving their junctions, thus pre- lar switch that turns on the differentiation of venous ECs to a lymphatic venting them from separating, which is required for tube formation.69 EC fate,76 and in SOX18 underlie lymphatic abnormalities Interestingly, the excessive cell-cell junctions in migratory angioblasts in the human disorder hypotrichosis lymphedema telangectasia.77 may explain the delayed migration of these cells in endodermless SOX18 induces expression of a number of lymphatic markers, includ- zebrafish.4 ing the homeobox gene Prox1,76 which is absolutely required to initiate CHAPTER 1 Vascular Embryology and Angiogenesis 7

­lymphatic vessel morphogenesis.75 Lymphatic development begins in (PDGFR)-β, neuron glial 2 (NG2), and not αSMA. The controversy the lateral parts of the cardinal veins with EC expression of Sox18, fol- largely stems from a simple question: what is a ?90 However, lowed by Prox1 expression, and subsequently these SOX18+PROX1+ the field continues to struggle to define this cell type, and molecu- ECs sprout laterally and form .75 The peripheral lymphatic lar markers of pericytes and SMCs are overlapping. The commonly vasculature then results from centrifugal sprouting from the lymph used markers of pericytes include PDGFR-β, NG2, CD13, desmin, sacs and remodeling of the LEC capillary plexus. Interestingly, the ve- and vimentin.84 A study by Betsholtz’s group using RNA sequenc- nous identity of lymphatic precursors is critical, because deletion of ing identified a new set of genes to study in pericytes and validated the gene encoding COUP-TFII in ECs results in arterialization of veins pericyte-enriched expression of two genes of this set, vitronectin and and inhibition of LEC specification of cardinal vein ECs.27,78 -induced transmembrane protein1.91 VSMC markers include Although PROX1 is critical for formation of the lymphatic vascu- αSMA, transgelin (SM22α), calponin, and the more specific markers lature, the regulation of PROX1 expression in LECs remains elusive. smooth muscle myosin heavy chain (SMMHC) and smoothelin.1 The zinc finger transcription factor GATA2 binds a Prox1 enhancer element, but it is not required for the onset of PROX1 expression or Vascular Smooth Muscle Cell Origins specification and early migration of LEC progenitors.79 Yet, GATA2 The origins of VSMCs are diverse and differ among blood vessels and does play an essential role in lymphovenous valve development and even within specific regions of individual blood vessels. Neural crest separation of the blood and lymphatic vasculatures.79 Ablation of cells of the ectoderm give rise to SMCs of the cranial vessels, aorti- Gata2 with Prox1-CreERT2 confirms that GATA2 is pivotal for the copulmonary septum, and the proximal aorta from the root to the sub- initiation of lymphatic valve development and in adults for lymphatic clavian artery (Fig. 1.4).2,92,93 The inner layers (on the luminal side) of vessel structure and transport.79 Histone acetyltransferase 3 (HDAC3) the ascending aortic media derive from neural crest cells, and on the regulates GATA2 expression epigenetically, and EC-specific Hdac3 de- dorsal aspect of the ascending aorta, neural crest cells also contribute letion mimics the phenotype of mice lacking Gata2 in ECs.79,80 to SMCs in the outer layers of the media (on the adventitial side).92 Second heart field cells give rise to aortic SMCs from the root to the TUNICA MEDIA: SMOOTH MUSCLE AND innominate artery and in the ascending aorta are limited to the outer EXTRACELLULAR MATRIX layers.92,94 SMCs of the originate from the mesoderm.2 Cellular and Extracellular Matrix Components Using HoxB6-Cre to mark cells derived from the LPM, Wasteson and In large- and medium-sized vessels, radially outward from the EC colleagues suggest that these cells are the source of descending aortic layer is the tunica media consisting of VSMCs and ECM components ECs and that the ventral wall of the descending aorta is temporarily including elastin and collagen. The dynamic contraction and relax- inhabited at E9.5 for approximately 1 day with early SMCs that derive ation of VSMCs allows for the tone of the blood vessel to be adjusted from the LPM.95 Subsequently, Meox1-Cre, which labels cells derived to the physiological demands of the relevant tissue and to maintain from both the presomitic paraxial mesoderm and the somites, marks blood pressure and perfusion. Collagen provides strength to the vessel SMCs that replace the LPM-derived aortic wall cells.95 Thus, in the wall, and elastin is largely responsible for its elasticity such that upon adult descending aorta, ECs and SMCs derive from distinct mesoder- receiving the cardiac output in systole, the arterial wall stretches to mal populations, the LPM and the presomitic/somitic mesoderm, re- increase the lumen volume, and subsequently, in diastole, it recoils spectively. Importantly, a distinct study previously showed that aortic to help maintain blood pressure. The capillary wall is substantially SMCs share a lineage with paraxial mesoderm-derived skeletal mus- thinner than that of larger vessels, thus facilitating the transfer of cle cells.96 Finally, Topouzis and Majesky suggest that the lineage of substances to and from the vascular compartment. Capillary mural SMC populations has important functional implications.97 In response cells consist of pericytes instead of VSMCs. Pericytes, VSMCs, and to transforming growth factor (TGF)-β stimulation, ectodermally de- the ECM play critical roles in many vascular diseases; however, there rived E14 chick-embryo aortic-arch SMCs increase DNA synthesis, are strikingly few studies of the development of these components in whereas the growth of mesodermally derived abdominal aortic SMCs comparison with the vast number of investigations of the morphogen- was inhibited.97 esis of EC networks and tubes. Coronary artery SMCs are critical players in atherosclerotic heart Although differences exist between pericytes and VSMCs, these disease, and there have been significant investigations into their or- mural cell types are generally considered to exist along a continuum igin from the proepicardium/epicardium. The proepicardium is a and lack firm distinctions.81 Pericytes are imbedded in the basement transient tissue that forms on the pericardial surface of the septum membrane of capillary ECs and thus may be characterized as having transversum in the E9.5 mouse and, through a fascinating process, an intimal location, whereas VSMCs are separated from the basement gives rise to ­epicardial cells that migrate as a mesothelial sheet over membrane in the media. VSMCs are oriented circumferentially around the myocardium. Signals emanating from the myocardial cells induce the vessel, whereas pericytes have an irregular orientation with elon- epithelial-to-­mesenchymal transition in which some epicardial cells gated cytoplasmic processes contacting multiple ECs.82,83 lose their cell-cell adhesion and invade the myocardium. Furthermore, VSMCs regulate vascular tone and blood flow distribution, whereas lineage labeling has illustrated that the proepicardium and epicardium pericytes have diverse functions including intercellular communica- contribute to the coronary artery SMC lineage.98,99 Recently, based tion, microvessel structure, phagocytosis, and perhaps vasoconstric- on the results of lineage tracing and clonal analyses, it was suggested tion.84 In the brain, pericytes play important roles in the formation and that developing coronary artery SMCs, at least partly, come from maintenance of the blood-brain barrier,85–87 and their involvement in ­epicardial-derived NG2+ pericytes via a Notch3-dependent process.100 the regulation of blood flow has recently become controversial. Hall Related to these studies of the coronary artery, investigations of et al.88 suggest that in response to neuronal activity, brain pericytes in- other organs suggest that the mesothelium could more generally be duce capillary dilation and increase blood flow, whereas a study by Hill an important source of VSMCs. For instance, MSLN is a membrane et al.89 indicates that cerebral blood flow is regulated predominantly glycoprotein expressed on the mesothelium, and a recent study found by arteriole SMCs expressing α-smooth muscle actin (αSMA) but through lineage tracing with a Msln-Cre that the mesothelium contrib- not by pericytes that express platelet-derived growth factor receptor utes to SMCs of the trunk vasculature.101 Previously, Wilm et al. showed 8 PART I Biology of Blood Vessels

Fig. 1.4 Distribution of Neural Crest– and Second Heart Field–derived Cells in the Proximal Thoracic Aorta. Representative ventral views of β-galactosidase (β-gal) activity in proximal thoracic from Wnt1-Cre (A) and Mef2c-Cre (B) mice in tissues acquired at 12 weeks of age, n = 3 to 4 for each group. Representative images of β-gal activity and eosin staining from sagittal sections of the aortic root and arch in Wnt1- (C) and Mef2c-Cre (D) male mice, n = 3 for each group. Magnified images were taken from the anterior (blue box) and posterior region (green box). Cross-sections of mid-ascending aortas from Wnt1-Cre (E) and Mef2c-Cre (F) mice were stained with X-gal and eosin B, n = 3 for each group. Magnified images were taken from the anterior region (blue box). Representative histograms measured β-gal activity from internal to external elastic lamina in the anterior region of ascending aortas from Wnt1-Cre (G) and Mef2c-Cre (H) mice, n = 3 for each group. Blue color is positive staining for distribution of Cre excision. Yellow dotted lines depict location of IEL and EEL. A, Adventitia; Ao, aorta; AR, anterior region; CA, common carotid artery; DA, ; EEL, external elastin lamina; IA, innominate artery; IEL, internal elastin lamina; L, lumen; LV, left ventricle; M, media; PA, ; PR, posterior region; SA, ; S T- J , sinotubular junction. (From Sawada H, Rateri DL, Moorleghen JJ, et al. Smooth muscle cells derived from second heart field and cardiac neural crest reside in spatially distinct domains in the media of the ascending aorta-brief report. Arterioscler Thromb Vasc Biol 2017;37:1722–1726.) CHAPTER 1 Vascular Embryology and Angiogenesis 9 that expression of the Wt1 protein in the developing gut is limited to the many vascular diseases, extracellular cues are implicated in inducing serosal mesothelium, and a Wt1-Cre yeast artificial chromosome (YAC) VSMCs to assume a dedifferentiated state through a process termed transgene marked a lineage of cells that includes the SMCs of the major phenotypic switching.117 mesenteric blood vessels.102 Tremendous efforts and advances have been made by numerous The etiology of pulmonary artery SMCs is controversial. Using the laboratories to elucidate the molecular mechanisms regulating phe- Wt1-Cre YAC transgene and a panel of Cre reporters, the mesothelium notypic modulation, and the model that has emerged depends on of the lung was implicated as the source of approximately one-third of combinatorial interactions of multiple factors that are either ubiqui- all pulmonary vascular cells expressing αSMA103; however, Morimoto tously expressed or selective for smooth muscle (Fig. 1.5).1 The most et al. subsequently reported that embryos carrying the same Wt1-Cre well-characterized underlying regulatory paradigm is the CArG-serum YAC transgene and a ROSA26R-YFP Cre reporter have only rare YFP+ response factor (SRF)-dependent system. Expression of almost all lung VSMCs.104 Furthermore, using the Tie1-Cre, these authors sug- smooth muscle contractile and cytoskeletal genes is modulated by the gested that most SMCs of the proximal pulmonary arteries arise from ubiquitous transcription factor SRF. SRF binds the 10-base-pair DNA 104 ECs. Transdifferentiation of ECs into VSMCs has been raised in de- consensus sequence CC(A/T)6GG known as the CArG box (i.e., C, AT velopmental and disease contexts.105−108 For instance, embryonic stem rich, G box), which is found in the regulatory regions of virtually all cell–derived Flk1+ cells have the potential to differentiate into ECs or smooth muscle genes. In fact, for most SMC genes, there are at least two mural cells.107 However, our results with the VE-cadherin-Cre109 and CArG boxes. However, the CArG box sequence is also found within mTomato/mGFP Cre reporter110 indicated that ECs are not a significant the 23-base-pair serum response enhancer element of early growth re- source of the E18.5 pulmonary arterial SMCs, and additional experi- sponse genes. Because SRF is ubiquitous and the cis-regulatory CArG ments demonstrated that, instead, these cells largely derive from the element is present in both growth and differentiation genes, a higher local mesenchyme.111 Studies by another group of ROSA26R-tdTomato order of control is required to determine which of these disparate gene mice also carrying Gli-CreERT2, Axin2-CreERT2, or Acta2-CreERT2, in- sets are expressed in a specific cell at a given time point. duced with tamoxifen at E11.5 and analyzed 7 days later, suggest that Control of expression of contractile and cytoskeletal SMC genes is GLI+ and AXIN2+ mesenchymal cells are a major, but not specific, pool regulated through a competition for SRF between the transcriptional of progenitors for lung vascular SMCs; however, SMA+ cells do not give coactivator myocardin and ternary complex factors.118 Myocardin is rise to many distal vascular SMCs. Thus VSMCs in the lung predomi- a master regulator of SMC differentiation, because ectopic expression nantly derive from αSMA- lung mesenchyme that differentiate locally. of this factor in nonmuscle cells is sufficient to induce activation of the SMC differentiation gene program.119 In addition, murine embryos Smooth Muscle Cell Differentiation null for myocardin lack VSMC differentiation and die at midgesta- A critical component of characterizing the morphogenesis of any tissue tion.120 Many studies have focused on factors that counterbalance the (e.g., vascular smooth muscle) is defining morphologic and molecular effect of myocardin to promote dedifferentiation, including Kruppel- criteria which constitute the differentiated phenotype of specific cell like factor 4 (),121 E26 ETS-like transcription factor 1 (ELK-1),118 types (e.g., VSMCs) that comprise the tissue. Early undifferentiated and FOXO4.122 cells that are presumed to be destined to the VSMC fate have promi- In addition, short and long noncoding RNA (lncRNA) genes mod- nent endoplasmic reticulum and Golgi, a euchromatic nucleus and lack ulate SMC phenotype.123 Cordes et al. showed that expression of miR- a distinctly filamentous cytoplasm.112 In contrast, mature VSMCs have 143 and miR-145 is restricted to cardiac and SMCs and that these a heterochromatic nucleus, myofilaments, and decreased synthetic or- miRs target Klf4, Elk-1, and myocardin transcripts, inhibiting their ganelles.112 In addition to these morphological changes, the differenti- expression.124 The miR-143/145 gene cluster is a direct transcriptional ation of SMCs is marked by the expression of a number of contractile target of SRF, myocardin, and NKX2-5, suggesting a positive feedback and cytoskeletal proteins. αSMA is the most abundant protein of SMCs, loop mechanism to maintain the differentiated SMC phenotype.124,125 comprising 40% of the total protein in a differentiated SMC.113 αSMA Beyond miR-143/145, additional miRNAs are implicated in the con- is an early marker of SMCs but is not specific, because it is expressed trol of SMC differentiation state including miR-21,126 miR-24,127 miR- in skeletal muscle and a variety of other cell types, and is temporarily 26a,128 and miR-133a.129 Emerging evidence points to the importance expressed in cardiac muscle during development.113,114 The actin- and of lncRNAs, such as the SMC-specific MYOSLID,130 as novel SMC reg- tropomyosin-binding protein SM22α is another early marker of SMCs ulators.123 The expression of MYOSLID is transcriptionally regulated and a more specific marker of adult SMCs; however, it also is expressed by SRF-myocardin and promotes SMC differentiation through a mech- 114 in the other muscle types during development. The two isoforms of anism that may involve modulating the TGF-β pathway.130 SMMHC are expressed slightly later during development than αSMA Studies have emerged investigating the role of epigenetics in and SM22α, and in contrast to these other markers, SMMHC expres- transcriptional regulation of SMC plasticity.131,132 Epigenetics refers sion is ostensibly limited to the SMC lineage.115 Smoothelin is another to mechanisms that regulate heritable changes in gene expression cytoskeletal protein that is also specific for SMCs but is not expressed without altering DNA sequence but rather modifying DNA bases by until very late in the differentiation process when the cells are part of a methylation and hydroxymethylation, as well as posttranslational mod- contractile tissue.116 ifications of histones. Epigenetic regulation of chromatin structure of Studies of VSMCs in development or in mature blood vessels are CArG-containing regions in SMC genes is critical for modulating SMC challenging because these cells can assume a variety of phenotypes differentiation state. For instance, the histone modification H3K4me2 depending on their environment.113 During the early stages of blood is enriched on SMC marker genes Myh11, Acta2, and Tagln in mature vessel development, many VSMCs rapidly proliferate, migrate substan- SMCs and progenitor cells committed to differentiate into SMCs.1 tial distances, and synthesize large amounts of ECM components. In Furthermore, the H3K4me2 mark persists through SMC phenotype contrast, more mature VSMCs are predominantly sedentary and non- modulation, and H3K4me2 at the Myh11 locus is restricted to the SMC proliferative and express contractile proteins but do not generate sig- lineage.131 This result is of major significance, because it has been used nificant ECM. However, the distinctions between these synthetic and as a method for identifying whether SMC marker-cells in human tis- contractile states are not always firm. In contrast to cardiac and skeletal sues derive from cells that previously expressed SMC markers.131,133 muscle, VSMCs in adults are not terminally differentiated, and thus, in In addition to histone methylation, studies by Kathleen Martin and 10 PART I Biology of Blood Vessels

Fig. 1.5 Complex Interactions of Multiple Conserved cis-Regulatory Elements and Transcription Factors Govern Expression of Smooth Muscle–Enriched Genes. Myocardin promotes interactions of serum response factor (SRF) with CArG boxes within the promoters of SMC contractile and cytoskeletal genes to induce the recruitment of RNA polymerase II (Pol II) and drive expression of these genes. Additional factors support myocardin-SRF-CArG interactions and drive SMC gene expression, including paired-related homeobox gene 1 (Prx1) and complexes of the protein inhibitor of activated Stat1 (PIAS1) with basic helix-loop-helix (bHLH) factors at E-box cis-regulatory elements. In contrast, transcription factors such as Kruppel-like factors (KLFs), E26 ETS-like transcription factor 1 (Elk-1), and HES-related repressor protein 1 (Herp1) repress SMC gene expression, at least in part by disrupting myocardin-SRF-CArG interactions. Elk-1 binds to ternary complex factor (TCF) sites, whereas KLFs bind to TGFβ control elements (TCEs). TBP, TATA binding protein. (Redrawn with permission from Alexander MR, Owens GK. Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease. Annu Rev Physiol. 2012;74:13–40.) colleagues134 provide evidence that mature SMCs are enriched in ten- ­recruitment of SMCs and/or their precursors to the vascular wall, the eleven translocation-2 (TET2), a member of a family of DNA demeth- investment of these cells around the nascent EC tube, and the pattern ylases that converts 5-methylcytosine to 5-­hydroxymethylcytosine of differentiation of VSMC precursors within or in proximity to the (5-hmc), resulting in DNA demethylation and gene activation. TET2 vascular wall. Limited relevant studies have mostly focused on the his- induces SMC differentiation and binds to CArG-rich regions of Myocd tology and αSMA expression in the developing aortic wall. Early in (encoding myocardin), Srf, and Myh11, and its product 5-hmC is en- development, the dorsal aortae exist as parallel tubes that subsequently riched in these regions.134 fuse to generate the single descending aorta. The early EC tube is sur- Beyond dedifferentiating, VSMCs undergoing phenotypic switching rounded by loose undifferentiated mesenchymal cells, and as the aorta can also acquire characteristics of other distinct cell types. In cultured matures, the expression of αSMA proceeds in a cranial to caudal di- SMCs, cholesterol loading induces downregulation of SMC markers rection.138,139 Within a cross-section of the descending aorta, the loca- and upregulation of markers.135,136 Seminal in vivo fate tion of initial mesenchymal cell consolidation and αSMA expression mapping studies in the Apoe(−/−) atherosclerosis mouse model indicate depends on the cranial-caudal position: proximally these processes that most smooth muscle–derived cells lose their classical SMC mark- initially occur on the dorsal aspect of the aorta, whereas more distally ers in plaques and many adopt phenotypic characteristics of other cell they are first noted on the ventral side.138,139 Studies published 45 years types, including macrophages and mesenchymal stem cells.133,137 ago indicate that within the chick aortic media, the outer layers mature initially with condensation and elongation of early presumptive SMCs Patterning of the Developing Vascular Smooth Muscle and accumulation of elastic tissue.140,141 In contrast, in rodent or quail Cells Layers aortas, cells immediately adjacent to the EC layer are the first to con- Although a number of recent investigations describe the molecu- solidate and express SMC markers; subsequently, additional layers of lar mechanisms regulating SMC differentiation, there are relatively SMCs are added.138,139,142,143 few studies of the patterning of the morphogenesis of SMC layers of We conducted a meticulous investigation of murine pulmonary ar- a developing blood vessel.112 Consequently, little is known about the tery morphogenesis and found that the medial and adventitial wall of CHAPTER 1 Vascular Embryology and Angiogenesis 11 this vessel is constructed radially from inside out, by sequential induc- for expression of structural matrix components has been documented tion and recruitment of successive layers.111 The inner layer undergoes in other animals and in humans.159 SMCs are thought to secrete most a series of morphological and molecular transitions that lasts approxi- of the elastin and in the tunica media.160 mately 3 days to build a relatively mature SMC layer. After this process The mechanical strength and viscoelastic properties of the aortic commences in the first layer, the next layer initiates and completes a wall result from fibrillar collagens, elastic fibers, and associated pro- similar process. Finally, this developmental program arrests midway teins. In a healthy aorta, these molecules form a scaffolding allowing through the construction of the outer layer to generate a relatively “un- the aorta to withstand the pulsatile flow and high pressure of blood differentiated” adventitial cell layer.111 delivered by the heart. Collagen fibers have high tensile strength and This inside-outside radial patterning is likely to involve an EC- bear most of the stressing forces at or above physiological blood pres- derived signal and result from one or more potential mechanisms. For sures, shielding SMCs from excessive stress.158,161 Seventeen different instance, in the morphogen gradient model,144 an EC-derived signal collagen types have been identified in the developing murine aortic diffuses through the media and adventitia and, depending on discrete wall with collagens I, III, IV, V, and VI having the highest expression.158 concentration thresholds, induces responses in the cells of these com- Deletions and/or mutations in a number of collagens results in vascu- partments, such as changes in morphology, gene expression, and/or lar phenotypes; for instance, COLLAGEN3A1 mutations in humans are proliferation. Alternatively, in the relay mechanism,145 a short-range responsible for Ehlers-Danlos syndrome type IV, with vascular man- or plasma membrane–bound EC signal induces adjacent cells, which, ifestations that include vessel fragility and large-vessel aneurysm and in turn, propagate the signal through either secreting a morphogen or rupture.158 inducing their neighbors and so on (i.e., “the bucket brigade model”). In contrast to collagen, elastin has low tensile strength, is disten- Such a bucket brigade mediated by Jagged1 on SMCs is implicated in sible, and distributes stress throughout the wall, including onto the regulating ductus arteriosus closure.146 Finally, our results suggest a collagen fibers.158 Elastin is the major protein of the arterial wall, third mechanism, which we have termed “the conveyer belt model,” composing up to 50% of the dry weight of the aorta.162 VSMCs secrete in which some of the progeny of inner layer SMCs migrate radially tropoelastin monomers that undergo posttranslational modifications outward to contribute to the next layer(s) of SMCs.111 and cross-linking, and are organized into circumferential elastic lamel- A number of signaling pathways involving an EC-derived signal and lae in the tunica media.160 These elastic lamellae alternate with rings mesenchymal receptors have been implicated in vascular wall morpho- of VSMCs to form lamellar units. Eln(+/−) mice have a normal life genesis.147 The PDGF pathway is perhaps the most well-studied path- span despite being hypertensive and having a 50% reduction in elas- way in vascular mural cell development with a ligand that is expressed tin mRNA.163,164 In comparison with wild type, the Eln(+/−) aorta has in ECs (PDGF-B) and receptors that are expressed in undifferentiated thinner elastic lamellae but a 35% increase in the number of lamellar mesenchyme (PDGFR-α and -β) and pericytes (PDGFR-β). Mice null units, which results in a similar tension per lamellar unit.164,165 More for Pdgfb or Pdgfrb have reduced SMC coverage of medium-sized dramatically, humans hemizygous for the ELN null mutant have a 2.5- arteries and reduced pericytes which results in microvascular hem- fold increase in lamellar units and suffer an obstructive arterial dis- orrhages and perinatal lethality.148–151 In addition, when cocultured ease, supravalvular aortic stenosis.164 Similarly, at the end of gestation with ECs, undifferentiated embryonic mesenchymal 10T1/2 cells are in the mouse, cells that are located subendothelium in Eln(−/−) arteries induced to express SMC markers and elongate in a TGF-β–dependent are hyperproliferative, resulting in increased number of αSMA+ cells manner.152 Similar changes are also induced by directly treating 10T1/2 and reduced luminal diameter, with lethality by approximately P4.5.166 cells with TGF-β1.152 Furthermore, the Notch pathway plays important We recently reported that integrin β3 expression and activation are roles in arterial SMC differentiation in vivo, and EC-derived Jagged1 is increased in the aortic media of Eln(−/−) mice.167 Moreover, genetic required for normal aortic and yolk sac vessel SMC differentiation.153 or pharmacological inhibition of integrin β3 in elastin-null mice at- In human adults, the receptor NOTCH3 is specifically expressed in tenuates aortic SMC misalignment, hyperproliferation, and stenosis, arterial SMCs, and, at birth, blood vessels of Notch3-null mice and suggesting inhibition of integrin β3–mediated signaling is a poten- of wild-type mice are indistinguishable.154,155 However, Notch3 is re- tial therapeutic strategy for supraventricular aortic stenosis patients quired for the postnatal maturation of the tunica media of small ves- (Fig. 1.6).167 sels in mice.154 Furthermore, NOTCH3 mutations in humans cause Finally, microfibrils are fibrous structures that are intimately asso- CADASIL (cerebral autosomal dominant arteriopathy with stroke and ciated with elastic fibers surrounding the elastin core. Fibrillin1 is the dementia) syndrome, characterized clinically by adult-onset recurrent major structural component of microfibrils, and its temporal pattern subcortical ischemic strokes and vascular dementia, and pathologically of expression during aortic development is similar to that of most by degeneration and eventual loss of VSMCs.155,156 Finally, it is import- structural proteins, such as elastin, except the peak expression of fibril- ant to note that other signaling pathways, such as those mediated by lin1 occurs at P0.157 In contrast, fibrillin2 expression is highest in the ­angiopoietin-Tie and sphingosine-1-phosphate ligand-receptor pairs, early embryonic period and then decreases linearly throughout mat- do not involve an EC-derived ligand and/or mesenchymal receptors uration.157 Mutations in the human FBN1 gene result in Marfan syn- but play important roles in SMC development.147 drome, with vascular manifestations that include aortic root aneurysm and dissection.168 Extracellular Matrix: Collagen and Elastic Fibers In addition to maturation of the cellular constituents of the blood vessel TUNICA ADVENTITIA wall, proper formation of the ECM is also critical for vascular function. Gene expression profiling of the developing mouse aorta demonstrates Components of the Adventitia dynamic expression of most structural matrix proteins: an initial major Owing to a paucity of studies, little is known about the develop- increase of expression at E14 is often followed by a brief decrease at ment of the outer layer of blood vessels, which is referred to as the postnatal day 0 (P0), then a steady rise for approximately 2 weeks, and tunica adventitia or tunica externa. The tunica externa is composed finally a decline to low levels at 2 to 3 months that persist into adult- of loose connective tissue (mostly collagen), and the cellular constit- hood.157,158 Fibrillar collagens, elastin, and most of the structural ma- uents include the fibroblast, which is the predominant cell type, as trix proteins of the vascular wall follow this pattern. A similar pattern well as marker–positive cells and macrophages. Diffusion 12 PART I Biology of Blood Vessels

Cells expressing SCA1 are located in the adventitia of the mouse be- tween the aortic and pulmonary trunks initially in the late embryonic stages and persisting into adulthood, and SHH signaling appears to be critical for this population of cells, because the number of adventitial SCA1+ cells is greatly diminished in Shh null mice.174 In addition, in SCA1+ cells isolated from murine arteries, knockdown of the pluripo- tency factor KLF4 reduces SCA1 levels and conversely overexpression of KLF4 induces SCA1 expression.175 Interestingly, mature SMCs are apparently an important source of these adventitial stem cells. Fate mapping of adult SMCs in Myh11- CreERT2 mice also carrying the ROSA26R-YFP Cre reporter demon- strates that 8 weeks after tamoxifen induction, a subset of SMA- cells expressing SCA1 or CD34 in the adventitia are YFP+.175 This result suggests that some SMMHC+ cells dedifferentiate and migrate radially outward from the media to the adventitia.175 In sum, the adventitia is Fig. 1.6 The Effects of Integrin β3 Inhibition in Elastin-Null Mice. After apparently an important tissue in vascular development and disease; E15.5, the pathology of the elastin-null aorta develops as characterized however, its role in these processes is critically understudied. by subendothelial smooth muscle cells (SMC) that have increased integrin β3 levels and are misaligned (radially elongated). In addition, Macrophages smooth muscle myosin heavy chain (SMMHC) expression is reduced, It is well accepted that macrophages reside in the adventitia; how- whereas SMC proliferation and radial migration are increased, resulting ever, using fate mapping, the ontogeny of vascular wall macrophages in hypermuscularization. Genetic or pharmacological inhibition of integrin was only recently revealed. Ensan et al. showed that arterial macro- 3 attenuates most of this pathobiology. (Redrawn with permission from β phages develop embryonically from early and late erythro-myeloid Mazurek R, Dave JM, Chandran RR, et al. Vascular cells in blood vessel 176 wall development and disease. Adv Pharmacol. 2017;78:323–350.) progenitors generated in the yolk sac. In the early postnatal period, macrophage colonization of arteries occurs during a brief period of cir- culating monocyte recruitment, whereas in adulthood, arterial macro- of ­nutrients from the lumen to the adventitia and outer media is phages are replenished by local proliferation and not from circulating inadequate in larger vessels, and hence the adventitia of these ves- monocytes.176 Similar to macrophages residing in the brain or kidney sels also includes small arteries, known as the vasa vasorum, which and to Ly-6Clow monocytes, arterial macrophage survival is promoted supply a capillary network extending through the adventitia and by interactions between the receptor CX3CR1 and its li- into the media. The adventitia of coronary vessels is thought to arise gand CX3CL1.176 from the epicardium based on experiments with quail-chick trans- Given that macrophages are present in developing vessels, the plants.169 Quail epicardial cells grafted into the pericardial space of question arises: what, if any, are the roles of macrophages in vascular the E2 chick ­undergo epithelial-to-­mesenchymal transition and con- morphogenesis? During development of the vasculature, macrophages tribute to both coronary vascular SMCs (consistent with the findings engage in a number of heterotypic cell-cell interactions with other vas- discussed previously in the section above on tunica media, VSMC cular cell types, including ECs, pericytes, and VSMCs. Macrophages origin) and coronary perivascular fibroblasts.169 Our results suggest direct neovessel pruning via phagocytosis during the maturation of that adventitial cells of the pulmonary artery derive from PDGFR-β+ microvessel networks. For example, in organogenesis of the testes, undifferentiated mesenchymal cells.111 macrophages arise from primitive yolk sac–derived hematopoietic progenitors, and nearly all of them express CD206, a marker of the Adventitial Cells Expressing Stem Cell Markers M2 macrophage state which is characteristic of angiogenic and tissue More recently, a number of studies have investigated a population remodeling.177 Macrophage depletion in this tissue results in disrupted of adventitial cells expressing stem cell markers (e.g., SCA1, CD34). vascular patterning due to inadequate remodeling.177 Similar results These investigations are largely a result of a paradigm shift: classically, were found in terms of the role of macrophages in pruning of the hy- the adventitia was considered a passive supportive tissue; however, ad- aloid vasculature of the developing eye.178 Finally, macrophages have ventitial fibroblast and progenitor cells are now implicated in playing been shown to induce fusion of EC tip cells, linking distinct angiogenic important roles in neointimal formation during vascular disease.170,171 sprouts.179 A population of cells expressing CD34 but not expressing markers of other cell types including ECs (CD31) and leukocytes (CD45) reside SUMMARY in the adventitia of stromal vessels of human white adipose tissue and express mesenchymal stem cell markers.172 When isolated, these cells Morphogenesis of the vascular system initiates shortly after gastru- give rise to clonogenic multipotent progenitor cells in culture, as do lation. The mesoderm gives rise to most vascular cells; however, the standard –derived mesenchymal stem cells.172 In human ectoderm contributes to SMCs of the aortic root, ascending aorta, and internal thoracic arteries, a niche for CD34+CD31− cells has been iden- cranial vessels. The early vasculature develops through vasculogene- tified at the interface between the media and adventitia of human inter- sis in which mesodermal cells differentiate into angioblasts and then nal thoracic arteries.173 coalesce into blood vessels, and, in general, capillaries are generated SHH is expressed in this vascular “stem cell” niche of medium- thereafter predominantly through sprouting angiogenesis. Migratory and large-sized arteries of the perinatal mouse.174 Patched-1 (Ptc1) tip and proliferative stalk ECs are crucial for sprouting angiogenesis. and Patched-2 (Ptc2) are SHH target genes, and their gene products This early development of the tunica intima involves many molecular are SHH receptors. β-Galactosidase staining in SHH reporter mice, signaling pathways, including those mediated by VEGF and Notch, as Ptc1lacZ or Ptc2lacZ, suggests that SHH signaling is active in the adven- well as metabolic processes. In addition to the blood vasculature, the titia during the late embryonic period and early postnatal period.174 lymphatic system is composed of lymphatic ECs, which derive from CHAPTER 1 Vascular Embryology and Angiogenesis 13 venous ECs. In large-caliber blood vessels, radially outward from ECs 22. Noden DM. Embryonic origins and assembly of blood vessels. Am Rev is the tunica media consisting of SMCs, elastin, and collagen. 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