1.3.1. Types of Angiogenesis

Sprouting angiogenesis and intussusceptive angiogenesis both occur in utero and in adults. Sprouting angiogenesis is better understood having been discovered nearly 200 years ago: intussusceptive angiogenesis was discovered by Burri [19,20] about two decades ago. Figure 1.3 shows the basic morphological events for both types of angiogenesis. As implied by its name, sprouting angiogenesis is characterized by sprouts composed of endothelial cells, which usually grow toward an angiogenic stimulus such as VEGF-A. Sprouting angiogenesis can therefore add blood vessels to portions of tissues previously devoid of blood vessels. On the other hand, intussusceptive angiogenesis involves formation of blood vessels by a splitting process in which elements of interstitial tissues invade existing vessels, forming transvascular tissue pillars that expand. Both types of angiogenesis are thought to occur in virtually all tissues and organs.

FIGURE 1.3

Basic types of primary vascular growth. Redrawn after Carmeliet and Collen (2000) [21].

1.3.2. Sprouting Angiogenesis

The basic steps of sprouting angiogenesis include enzymatic degradation of capillary basement membrane, endothelial cell (EC) proliferation, directed migration of ECs, tubulogenesis (EC tube formation), vessel fusion, vessel pruning, and pericyte stabilization. Sprouting angiogenesis is initiated in poorly perfused tissues when oxygen sensing mechanisms detect a level of hypoxia that demands the formation of new blood vessels to satisfy the metabolic requirements of parenchymal cells (Figure 1.4). Most types of parenchymal cells (myocytes, hepatocytes, neurons, astrocytes, etc.) respond to a hypoxic environment by secreting a key proangiogenic growth factor called vascular endothelial growth factor (VEGF-A). There does not appear to be redundant growth factor mechanisms that can replace the role of VEGF-A in hypoxia-induced angiogenesis.

FIGURE 1.4

VEGF-A directed capillary growth to poorly perfused tissues. (A) Endothelial cells exposed to the highest VEGF-A concentration become tip cells (green). Hypoxic tissue is indicated by the circular blue fade. (B) The tip cells lead the developing sprout (more...)

An endothelial tip cell guides the developing capillary sprout through the ECM toward an angiogenic stimulus such as VEGF-A [22–25]. Long, thin cellular processes on tip cells called filopodia secrete large amounts of proteolytic enzymes, which digest a pathway through the ECM for the developing sprout [26,27]. The filopodia of tip cells are heavily endowed with VEGF-A receptors (VEGFR2), allowing them to “sense” differences in VEGF-A concentrations and causing them to align with the VEGF-A gradient (Figure 1.5). When a sufficient number of filopodia on a given tip cell have anchored to the substratum, contraction of actin filaments within the filopodia literally pull the tip cell along toward the VEGF-A stimulus. Meanwhile, endothelial stalk cells proliferate as they follow behind a tip cell causing the capillary sprout to elongate. Vacuoles develop and coalesce, forming a lumen within a series of stalk cells. These stalk cells become the trunk of the newly formed capillary. When the tip cells of two or more capillary sprouts converge at the source of VEGF-A secretion, the tip cells fuse together creating a continuous lumen through which oxygenated blood can flow. When the local tissues receive adequate amounts of oxygen, VEGF-A levels return to near normal. Maturation and stabilization of the capillary requires recruitment of pericytes and deposition of ECM along with shear stress and other mechanical signals [28].

FIGURE 1.5

Microanatomy of a capillary sprout and tip cell selection. (A) An interstitial gradient for VEGF-A and an endothelial cell gradient for VEGFR2 are shown. Tip cell migration is thought to depend upon the VEGF-A gradient and stalk cell proliferation is (more...)

Delta-Notch signaling is a key component of sprout formation (Figure 1.5). It is a cell–cell signaling system in which the ligand, Delta-like-4 (Dll4) mates with its notch receptor on neighboring cells. Both the receptor and ligand is cell bound and thus act only through cell–cell contact. VEGF-A induces Dll4 production by tip cells, which leads to activation of notch receptors in stalk cells. Notch receptor activation suppresses VEGFR2 production in stalk cells, which dampens migratory behavior compared with that of tip cells. Hence, endothelial cells exposed to the highest VEGF-A concentration are most likely to become tip cells [24,25,30]. Although tip cells are exposed to the highest VEGF-A concentration, their rate of proliferation is far less compared with that of stalk cells.

Not all aspects of the Delta-Notch signaling pathway are fully understood, but it is clear that production of a normal vasculature is heavily dependent upon the concentration of VEGF-A in the tissues. A 50% reduction of VEGF-A expression is lethal embryonically because of vascular defects [31,32], and excess VEGF-A in tumors induces overproduction of tip cells leading to a disorganized vasculature [33]. This critical dependence on physiological concentrations of VEGF-A for construction of viable blood vessels might help explain why attempts to induce angiogenesis in poorly perfused tissues with VEGF-A administration and gene therapy have not been highly successful.

1.3.3. Intussusceptive Angiogenesis

Intussusceptive angiogenesis is also called splitting angiogenesis because the vessel wall extends into the lumen causing a single vessel to split in two. This type of angiogenesis is thought to be fast and efficient compared with sprouting angiogenesis because, initially, it only requires reorganization of existing endothelial cells and does not rely on immediate endothelial proliferation or migration. Intussusceptive angiogenesis occurs throughout life but plays a prominent role in vascular development in embryos where growth is fast and resources are limited [34–36]. However, intussusception mainly causes new capillaries to develop where capillaries already exist.

Evidence for the occurrence of intussusceptive angiogenesis is based upon the presence of transcapillary tissue pillars (Figure 1.6). Identification of tissue pillars requires scanning electron micrographs of vascular casts or three-dimensional reconstruction of serial micrographs. This type of angiogenesis was discovered in postnatal lungs of rats and humans [19,20], but it also occurs in many other tissues and organs, especially in capillary networks that abut an epithelial surface, e.g., choroid of the eye, vascular baskets around glands, intestinal mucosa, kidney, ovary, and uterus [37,38]. It also occurs in skeletal muscle, heart, and brain. In addition to forming new capillary structures, intussusceptive growth plays a major role in the formation of artery and vein bifurcations as well as pruning of larger microvessels.

FIGURE 1.6

Scanning electron micrographs of Mercox casts. (a) Fetal chicken lung microvasculature. (b) Rat lung microvasculature at postnatal day 44. The small holes indicated by arrows have diameters of about 2 µM. The holes correspond to tissue pillars (more...)

The control of intussusceptive angiogenesis is poorly understood compared with sprouting angiogenesis. This difference is only partly due to its recent discovery in 1986 [20]. A rate-limiting step in intussusceptive growth research can be pinned to the laborious methods required to prove its presence, which, again, involve determining the frequency of tissue pillars from scanning electron micrographs of vascular casts. However, it is known that intussusceptive angiogenesis can be stimulated in the chick chorioallantoic membrane (CAM) with application of VEGF-A (Figure 1.7), and there is little doubt that many growth factors and signaling systems are involved [34,37]. Mechanical stresses related to increases in blood flow can initiate intussusceptive growth in some high flow regions of the circulation, as discussed in Chapter 4 [34,35].

FIGURE 1.7

Intussusceptive angiogenesis in three dimensions (a–d) and two dimensions (a'–d'). (a,b,a',b') The process begins with protrusion of opposing endothelial cells into the capillary lumen. (c,c') An interendothelial contact is established (more...)