Although the vasculature or blood supply of neoplasms has been investigated since the nine- teenth century, it is only in the past few decades that the morphology and mechanisms of blood vessel formation in neoplasms have been studied extensively. Blood vessels in neoplas- tic tissue appear to differ from blood vessels in normal tissues with respect to their growth and neogenesis, especially at the areas of greatest growth of the neoplasm along its borders with normal tissue (cf. Beckner, 1999). In 1971 Folkman proposed a theory that neoplasms lie dormant yet viable and unable to grow beyond 2 to 3 mm3 in size in the absence of vascular-
ization from the host. He proposed that neoplasms produced a diffusible product that stimu- lated angiogenesis by host vessels to supply the vasculature for the neoplasm. Another term, vasculogenesis, refers to the formation of a primary vascular network during embryonic development of the vascular system (cf. Malonne et al., 1999). Angiogenesis, on the other hand, develops from preexisting vessels from which capillary sprouts migrate to produce new vessels.
Angiogenesis may occur in the organism as a result of a number of different stimuli. A simplified diagram of the “angiogenic cascade” is seen in Figure 18.12. As noted, the phase of initiation of angiogenesis may be stimulated by vascular injury, wounds, neoplastic growth, and/or local inflammation. Proliferation of the new vascular sprout with “invasion” into the ex- tracellular matrix (ECM, Chapter 10) occurs with many of the characteristics seen with neoplas- tic invasion. Finally, maturation with lumen formation and differentiation of the new vessel occurs. As noted in the figure, the initiation phase of angiogenesis as well as to some extent the proliferation phase depend on the presence of growth factors and other hormones, while the mat- uration phase in normal tissues depends to a great extent on various inhibitors of angiogenesis, allowing for differentiation and normal vascular formation in the healing sequence. A list of var- ious angiogenic stimulators and inhibitors is seen in Table 18.7.
Figure 18.12 The angiogenic cascade. Angiogenesis is depicted as a continuum of three phases or stages as shown. Initiation of angiogenesis may occur from vascular injury, neoplastic growth, or activation by a variety of cytokines. The proliferative/invasive stage is characterized by an increased release of pro- teolytic enzymes, differential regulation of cell adhesion molecules, endothelial cell migration, prolifera- tion, and invasion of adjacent tissues. The maturation/differentiation phase occurs on the basis of cell-cell and cell-ECM interactions, as well as the release of various angiogenesis inhibitors and maturation compo- nents resulting in differentiation into mature blood vessels. (Adapted from Brooks, 1996, with permission of the author and publisher.)
Growth Factors and Angiogenesis
Of all of the growth factors listed in Table 18.7 as angiogenesis stimulators, only two, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), influence the behavior and replication of endothelial cells directly (cf. Norrby, 1997). Many of the other stimulators appear to act indirectly, possibly by stimulating other cells such as macrophages and mast cells as well as endothelial cells themselves to produce VEGF (Norrby, 1997). VEGF itself is a highly specific mitogen for vascular endothelial cells, and at least five isoforms have been described, generated as a result of alternative splicing from a single VEGF gene (cf. Neufeld et al., 1999). At least three VEGF receptors have been identified, each showing some specificity for VEGF and PlGF. A diagram of the receptors and their ligand specificities in seen in Figure 18.13. Note that the VEGFR-3 is also involved in lymphangiogenesis as well as angiogenesis. The lymphat- ics are extremely important in the overall circulation of any tissue and in neoplasms as well, offering another pathway for the release of neoplastic cells into the lymphatic circulation. VEGF-mediated angiogenesis is very important in the developing embryo but is relatively rare in the normal adult, the exceptions being the female reproductive system (in the ovary) as well as wound healing and tissue repair (cf. Klagsbrun and Moses, 1999). Some of the other angiogene- sis stimulators, such as the angiopoietins, appear to play a major role in angiogenesis in the de- veloping embryo (cf. Klagsbrun and Moses, 1999).
Just as in neoplastic invasion, VEGF induces significant increases in specific integrins (Senger et al., 1997), and others have identified integrin αvβ3 as a marker of angiogenic and proliferating vessels (Brooks et al., 1994a). Antagonists to this integrin actually induce apoptosis in angiogenic blood vessels (Brooks et al., 1994b). Another very important factor regulating the expression of VEGF is hypoxia (Shweiki et al., 1992). This phenomenon may actually mediate
hypoxia-initiated angiogenesis through the induction of hypoxia-inducible factor (HIF)-1α, a transcription factor regulating specific genes involved in cell cycle control and other important cell functions (Carmeliet et al., 1998).
Angiogenesis in Neoplastic Growth
In accord with Folkman’s (1971) prediction that neoplasms produce factors stimulating angio- genesis by the host, Denekamp and Hobson (1982) demonstrated that endothelium in neoplasms had an extremely high proliferative index, in marked contrast to the reportedly low DNA label- ing of normal tissue endothelium. On the basis of the large number of angiogenesis stimulators listed in Table 18.7, as well as the fact that all neoplasms produce at least one or more such factors ectopically or as a result of the differentiation program of their tissue of origin, the result of Denekamp and Hobson can be readily explained. While a number of normal tissues— especially heart, skeletal muscle, ovary, and intestine—produce significant amounts of one or more of the isoforms of VEGF, one or more isoforms of this growth factor are likely produced by virtually every malignant neoplasm (Nicosia, 1998). Furthermore, there is now ample evi- dence that several cellular oncogenes may modulate angiogenesis via indirect effects on VEGF production by neoplastic cells (Bouck, 1993; Rak et al., 1995; Schlessinger, 2000).
The Angiogenic Switch During Carcinogenesis
Folkman and his colleagues (Folkman et al., 1989) were among the first to report that angio- genic activity as assayed in an in vitro system first appeared in “hyperplastic” islet cells of the pancreas in transgenic mice expressing the SV40 large T antigen in these cells prior to the onset of formation of neoplasms. They extended this observation to other transgenic models and have applied it to the development of human neoplasms, as noted in Figure 18.14. In this figure, the
Figure 18.13 The three known VEGF receptors, VEGFR-1, VEGFR-2, and VEGFR-3, and their inter- action with specific VEGF isoforms. Ligand binding induces receptor signal transduction leading to vari- ous responses, some of which are listed below the figure. While all ligands are capable of mediating signals for angiogenesis, at least VEGF-C induces lymphangiogenesis. sVEGFR-1, soluble VEGFR-1, encoded by alternatively spliced mRNA; HSPG, heparan sulfate proteoglycan; NP-1, neuropilin-1, a cell surface glyco- protein involved in the central nervous system. (Adapted from Veikkola and Alitalo, 1999, with permission of the authors and publisher.)
early expression during carcinogenesis of genes whose products are involved in angiogenesis, such as VEGF, occurs at the morphological stage of dysplasia or carcinoma in situ (CIS). Fol- lowing that, angiogenesis itself occurs with ultimate invasive malignancy. This model is com- pletely in accord with the stages of initiation, promotion, and progression, as indicated in the figure. The preneoplastic lesion is the hyperplastic ductular proliferation, while progression be- gins with carcinoma in situ, extending to invasive carcinoma. Thus, in this model, one of the critical genetic alterations occurring in the transition from the reversible stage of promotion to the irreversible stage of progression is activation of angiogenesis. As expected, overproduction of VEGF by neoplastic cells contributes to malignant progression in model systems (Aonuma et al., 1999). Recently, another accompanying genetic change in the angiogenic switch is the acti- vation of a metalloproteinase also involved early in the stage of progression (Fang et al., 2000).
Thus, it becomes apparent that angiogenesis is not only critical for the growth of malig- nant neoplasms but also may well be an important marker in the critical transition from the stage of promotion to that of progression in epithelial and probably other neoplasms. Mechanisms in- volved in the exact activation of these genes—such as altered methylation, gene amplification, translocation, or direct enhanced transcription—are not apparent as yet.
As discussed in Chapter 20, antiangiogenesis has now developed into a field with potential chemotherapeutic effectiveness in the treatment of solid neoplasms. Although, as with any such therapeutic modality, there are difficulties (Westphal et al., 2000), there is also considerable promise, since the therapy is not related to the neoplasm directly, with its unstable karyotype, allowing for a variety of mechanisms of drug resistance (Chapter 20), but rather the therapy is directed at a normal tissue with a stable karyotype, namely the endothelium. While studies have
Figure 18.14 The angiogenic switch in neoplasia: reference to the stages of initiation, promotion, and progression. The diagram is of the carcinogenesis of ductal epithelium of the mammary gland going through the various morphological stages noted. The appearance of darkened cells at the dysplasia/carci- noma in situ (CIS) phase is the transition from the stage of promotion to that of progression. (Adapted from Hanahan and Folkman, 1996, with permission of the authors and publisher.)
demonstrated the effectiveness of antiangiogenesis factors (Kim et al., 1993; Kerbel, 2000), it will be only after extensive studies in the human that the effectiveness of this form of therapy for the treatment of neoplasia will be realized.