Currently, the field of tumor angiogenesis is undergoing more explosive growth than any other field in cancer research. More than 860 papers were published on various aspects of tumor angiogenesis in 1999, and scientists, clinicians, patients, and the media have closely scrutinized this research. Regrettably, trials of anti-angiogenic agents thus far have produced mostly toxicity data. Given the complexity of this process, the basic biology of angiogenesis also must be better understood before more effective anti-angiogenic therapy can be developed.
By definition, "angiogenesis" is the establishment of a neovascular blood supply derived from preexisting blood vessels, whereas "vasculogenesis" is the embryonic establishment of a blood supply from mesodermal precursors such as angioblasts or hemangioblasts. "Tumor angiogenesis" more accurately refers to a combination of angiogenesis and vasculogenesis, in which the main blood supply to a tumor is derived from preexisting blood vessels, but circulating endothelial cell precursors may contribute to the growing endothelial cell mass.
Angiogenesis is an essential step in both the growth of primary tumors and metastasis.[1,2] A neovascular blood supply is also essential for increasing the chance that tumor cells will gain access to the circulation and subsequently begin the process of growth at a different site. Once a tumor establishes an invasive phenotype in the organ of metastasis, it must then establish its own neovascular blood supply for growth. Numerous investigators have established the association between tumor angiogenesis and tumor metastasis.
Pathologic angiogenesis occurs when the effect of stimulatory molecules outweighs the effect of inhibitory molecules (Table 1). Intensive study led to the realization that the angiogenic process involves more than simple proliferation of endothelial cells; rather, it requires endothelial cells to divide, invade the basement membrane, migrate, and undergo differentiation and capillary-tube formation (Figure 1). This process is driven by angiogenic molecules and also by other factors, such as degradative enzymes, which mediate the above processes. Interestingly, tumor angiogenesis and tumor-cell invasion are very similar processes.
The best characterized of the stimulatory angiogenic factors is the vascular endothelial growth factor (VEGF). VEGF has also been associated with the aggressive phenotype in numerous solid malignancies.[4-9] VEGF is a 32- to 44-kDa protein secreted by nearly all cells. VEGF is expressed as four isoforms derived from alternate splicing of the mRNA. The smaller isoforms, VEGF-121 and VEGF-165 (the numbers denote the number of amino acids), are secreted from cells. The larger isoforms, VEGF-189 and VEGF-205, are cell-associated, and their functions are not well known at this time.
One distinguishing factor of VEGF is its ability to induce vascular permeability. In fact, this factor was originally named the vascular permeability factor (VPF) and was subsequently found to be homologous to VEGF. [11-13] The extent of vascular permeability induced by VEGF is 50,000 times that of histamine, the gold standard for induction of permeability. This action by VEGF allows proteins to diffuse into the interstitium and to form the lattice network onto which endothelial cells migrate.
Endothelial Growth Factor-Receptor Family
Receptors for VEGF are expressed almost exclusively on endothelial cells. VEGF receptors have been found on cells of neural origin, Kaposi’s sarcoma cells, hematopoietic precursor cells, and other rare tumor cell types.[14,15] The current nomenclature for the VEGF receptors lists three receptors: VEGFR-1/Flt-1, VEGFR-2 KDR/Flk-1, and VEGFR-3/Flt-4. These tyrosine-kinase receptors require dimerization to induce intracellular signaling upon binding to specific ligands. The receptors for VEGF may mediate distinct functions within the endothelial cell; for example, VEGFR-1 may be important in migration, whereas VEGFR-2 may be important in the induction of permeability and cell proliferation.
Recently, the angiopoietin family of ligands has been found to play an important role in homeostasis of tumor vasculature. The angiopoietins are proteins involved in angiogenesis that bind to the endothelial-cell-specific tyrosine kinase receptor Tie-2. Angiopoietin-1 (Ang-1) acts as an agonist and is involved in endothelial-cell differentiation and stabilization. In contrast, Ang-2 binds to Tie-2 and blocks the binding of Ang-1 to this receptor.[17,18] This blockade leads to endothelial cell destabilization and vascular regression.
A recent theory of tumor angiogenesis suggests that this process involves the co-option of preexisting blood vessels in addition to vascular regression and subsequent neovascularization. Initially, tumors co-opt existing blood vessels within an organ for their nutrient blood supply. Shortly thereafter, the existing vasculature becomes destabilized, most likely through the release of Ang-2 by endothelial cells. This loss of vascular integrity leads to relative hypoxia within the tumor, which, in turn, leads to upregulation of VEGF in the tumor cells. These events then lead to a robust angiogenic response. At that stage, the newly developed endothelial cells require stabilization, achieved through release of Ang-1 by endothelial cells and possibly through continued response to VEGF. Thus, the process of angiogenesis depends on the temporal coordination of factors that regulate pathways for the establishment of stable conduits that provide a nutrient blood supply to the tumor.
Numerous nonspecific angiogenic molecules and factors affect the growth of cell types other than endothelial cells. These include fibroblast growth factors (acidic and basic); transforming growth factor-alpha and epidermal growth factor (EGF), both of which bind to the EGF receptor; platelet-derived growth factor (PDGF); platelet-derived endothelial cell growth factor (PD-ECGF); angiogenin; and the CXC chemokine IL-8, macrophage inflammatory protein (MIP), PF-4, and growth-regulated oncogene (GRO) (Table 1).
These factors are known to be angiogenic in in vivo models but are not specific for endothelial cells. However, as noted earlier, angiogenesis is not driven by a single molecule or family of molecules, but rather depends on the cooperation and integration of various factors that lead to endothelial cell proliferation, migration, invasion, differentiation, and capillary-tube formation. It has yet to be determined whether inhibiting the activity of a single angiogenic factor will lead to vascular compromise of significant duration. More likely, the redundancy in the angiogenic process will select for other angiogenic factors when a specific angiogenic factor is targeted.
In formulating antiangiogenic regimens, it is essential to understand the cascade of events that leads to upregulation of angiogenic factor expression and secretion. Signals that upregulate angiogenic factors include extracellular signals, intrinsic upregulation of signal transduction activity, and loss of tumor suppressor genes. Examples of these signals are discussed below.
External signals that lead to the induction of angiogenic factor expression include environmental stimuli such as hypoxia or a decrease in pH.[21-23] Hypoxia is the most potent stimulus for inducing angiogenic factors, especially VEGF. Hypoxic induction of VEGF is probably mediated through Src kinase activity, which then leads to downstream induction of signaling cascades and eventually to an increase in the activity of hypoxia-inducible factor-1 (HIF-1) alpha .[24,25] This factor then increases the transcription of the VEGF gene, which in turn leads to the induction of angiogenesis. Other external factors that increase the angiogenic response include various cytokines and growth factors. Insulin growth factors -I and -II, epidermal growth factor, hepatocyte growth factor, interleukin-1, and platelet-derived growth factor have all been shown to upregulate VEGF.[26-28] Thus, anti-angiogenic therapy could involve downregulation of upstream targets of the angiogenic factors rather than targeting the angiogenic factors themselves.[24,29]
Once a growth factor or a cytokine binds to its receptor, a cascade of intracellular signaling events is initiated. Two specific signal transduction pathways are well known to mediate the upregulation of angiogenic factors: the PI-3 kinase/Akt signal transduction pathway, which eventually leads to stabilization of HIF-1 alpha,[30,31] and the mitogen-activated protein (MAP) kinase pathway, in which activation of extracellular signal-regulated kinases-1/2 (Erk-1/2) activates factors that increase transcription of the VEGF gene. Activated Ras and Src also have been demonstrated in in vivo models to be associated with increased VEGF production and angiogenesis. Again, therapeutic strategies that target the upstream effector molecules in angiogenesis may be a rational means of preventing angiogenesis. Inhibitors of signal transduction molecules have been demonstrated to inhibit angiogenesis in in vivo tumor models.
Protein products of tumor suppressor genes such as the von Hippel-Lindau (VHL) or p53 genes also regulate angiogenesis. The wild-type VHL protein represses transcriptional regulation of the VEGF gene.[34-36] A loss of heterozygosity with a mutation in the remaining VHL allele leads to loss of transcriptional control of the VEGF gene and overexpression of VEGF. Mutant p53 has also been associated with an increase in angiogenesis. Reinsertion of the wild-type p53 gene into cells with mutant p53 can downregulate VEGF expression and angiogenesis. Thus, the process of angiogenesis is driven by external forces (including environmental stimuli), aberrations in internal signaling, and alterations in tumor suppressor gene function.