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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.

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