Despite the abundancy of angiogenic factors present in different tissues, endothelial cell turnover in a healthy adult organism is remarkably low with a turnover in the order of thousands of days. The maintenance of endothelial quiescence is thought to be due to the presence of endogenous negative regulators. Moreover, positive and negative regulators often co-exist in tissues with extensive angiogenesis. These observations have led to the hypothesis that activation of the endothelium depends on a balance between these opposing regulators (115). If positive angiogenic factors dominate, the endothelium will be activated, whereas quiescence Thus, the angiogenic process can be divided in an activation phase (initiation and progression of the angiogenic process) and a phase of resolution (termination and stabilization of the vessels). It is not yet clear whether the resolution phase is due to upregulation of endogenous inhibitors or exhaustion of positive regulators.

A number of endogenous angiogenesis inhibitors have been described (115, 129) that act on endothelial cells to block their migration, proliferation and/or their ability to form functional capillaries (Table 3). These include proteins such as angiostatin or endostatin, platelet factor-4 (PF-4), thrombospondin-1 (TSP-1), IFN-a, TIMPs, PAIs and others. Some of these endogenous inhibitors with clinical potential will be described in page "Inhibiton of angiogenesis".

PHYSIOLOGICAL ANGIOGENESIS

Besides during embryogenesis, angiogenesis is also activated in the female reproductive system (75, 155) during the development of follicles, corpus luteum formation and embryo implantation. During these processes, angiogenesis is mediated mainly by VEGF (66, 69). Neovascularization also plays a critical role in successful wound healing that is probably regulated by IL-8 and the growth factors FGF-2 (177) and VEGF (170). Macrophages, known cellular components of the accompanying inflammatory response, may contribute to the healing process by releasing these angiogenic factors (228).

With respect to activated endothelium, an important distinction must be made between physiological and pathological settings (75). Although many positive and negative regulators operate in both, endothelial cell proliferation is tightly controlled in the former, whereas in the latter, the uncontrolled growth of microvessels may lead to several "angiogenic diseases" in different tissues (Table 4).

Hemangiomas are angiogenic diseases, characterized by the proliferation of capillary endothelium with accumulation of mast cells, fibroblasts and macrophages. They represent the most frequent tumors of infancy, occurring more frequently in females than males (3:1 ratio). Hemangiomas are characterized by rapid neonatal growth (proliferating phase). By the age of 6 to 10 months, the hemangioma’s growth rate becomes proportional to the growth rate of the child, followed by a very slow regression for the next 5 to 8 years (involuting phase) (55, 160, 187). Most hemangiomas occur as single tumors whereas about 20% of the affected infants have multiple tumors, which may appear at any body site. Approximately 5% produce life-, sight-, or limb-threatening complications, with high mortality rates (160). The pathogenesis of hemangiomas has not yet been elucidated. However, several immunohistochemical studies have provided insight into the histopathology of these lesions. In particular, proliferating hemangiomas express high levels of proliferating cell nuclear antigen (PCNA, a marker for cells in the S phase), type IV collagenase, VEGF and FGF-2 (230). During the involuting phase of hemangiomas, expression of these angiogenic factors decreases. Furthermore, urinary levels of FGF-2 are elevated during the proliferating phase of hemangioma, but become normal during involution or after therapy with IFN-a (37).

 

Other proliferative disorders of the skin include psoriasis and Kaposi’s sarcoma (75). Hypervascular psoriatic lesions express high levels of the angiogenic inducer IL-8, whereas the expression of the endogenous inhibitor TSP-1 is decreased (168). Kaposi’s sarcoma (KS) is the most common tumor associated with human immunodeficiency virus (HIV) infection and is in this setting almost always associated with human herpes virus 8 (HHV-8) infection (165). Typical features of KS are proliferating spindle-shaped cells, considered to be the tumor cells and endothelial cells forming blood vessels. KS is a cytokine-mediated disease, highly responsive to different inflammatory mediators like IL-1b, TNF-a and IFN-g and angiogenic factors (206). In particular, FGF-2 was found to synergize with HIV-tat to promote angiogenesis and KS development (14). Finally, growth of KS, both in vitro and in vivo, could be blocked by an antisense oligonucleotide targeting FGF-2 (60).

KS skin lesion

 

Diabetic retinopathy is the leading cause of blindness in the working population, but ocular neovascularization can also occur upon exposure of preterm babies to oxygen. It is assumed that both forms are induced by hypoxia in the retina. Elevated levels of the hypoxia-inducible angiogenic factor VEGF were detected in the aqueous and vitreous of eyes with proliferative retinopathy (1, 252). The photograph shows a tuft of neovascularization (arrowhead) extending from the optic nerve head into the vitreous cavity. These new vessels take on a frond-like configuration as they grow, similar in appearance to a sea fan. The photograph on the right is a fluorescein angiogram of a different patient showing a frond of vessels (arrowhead) extending from the disc. Neovascularization leaks fluorescein dye (white in this photograph), giving it a fluffy appearance.  Click here for more.

Excessive production of angiogenic factors from infiltrating macrophages, immune cells or inflammatory cells may also trigger the formation of pannus, an extensively vascularized tissue, that invades and destroys the cartilage, as seen in rheumatoid arthritis (133). Moreover, uncontrolled angiogenesis may underlie various female reproductive disorders (75), such as prolonged menstrual bleeding or infertility, and excessive endothelial cell proliferation has been observed in the endometrium of women with endometriosis (102).

Angiogenesis also contributes to atherosclerosis, a major cause of death of Western populations. Atherosclerosis is the main cause of heart attack. The walls of the coronary artery are normally free of microvessels except in the atherosclerotic plaques, where there are dense networks of capillaries, known as the vasa vasorum. These fragile microvessels can cause hemorrhages, leading to blood clotting, with a subsequent decreased blood flow to the heart muscle and heart attack (116). Finally, angiogenesis is thought to be indispensable for solid tumor growth and metastasis (see further).  All the above mentioned pathologies may benefit from anti-angiogenic therapy.

In contrast, the inability to build up a good angiogenic response may also lead to vascular disorders, including impaired wound healing or chronic ulcers. In gastric ulcers in humans, the level of FGF-2 was found to be 23 times lower than in normal mucosa (75). Oral administration of FGF-2 to rats bearing ulcers, induced angiogenesis and accelerated ulcer healing (229), an observation which has led to the development of clinical trials. Therapeutic angiogenesis may also be favorable in the treatment of myocardial and peripheral ischemia (250). The objective is to reduce local hypoxia in areas of hypovascularization by the administration of exogenous growth factors. In different animal models for myocardial ischemia, administration of VEGF, acidic fibroblast growth factor (FGF-1) or FGF-2 resulted in a significant beneficial effect, including reduction in infarct size, improved coronary blood flow and increased microvessel density in infarcted and non-infarcted regions (144, 179, 258).

THE PREVASCULAR PHASE

Tumor growth is often a multi-step process that starts with the loss of control of cell proliferation. The cancerous cell then begins to divide rapidly, resulting in a microscopically small, spheroid tumor: an in situ carcinoma (76). As the tumor mass grows, the cells will find themselves further and further away from the nearest capillary. Finally the tumor stops growing and reaches a steady state, in which the number of proliferating cells counterbalances the number of dying cells. The restriction in size is caused by the lack of nutrients and oxygen. In tissues, the oxygen diffusion limit corresponds to a distance of 100 µm between the capillary and the cells, which is in the range of 3-5 lines of cells around a single vessel (85). In situ carcinomas may remain dormant and undetected for many years and metastases are rarely associated with these small (2 to 3 sq.mm), avascular tumors (76).
 
 

THE ANGIOGENIC SWITCH Yet, several months or years later, an in situ tumor may switch to the angiogenic phenotype, induce the formation of new capillaries and start to invade the surrounding tissue. The "angiogenic switch" depends on a net balance of positive and negative angiogenic factors in the tumor. Thus, the angiogenic phenotype may result from the production of growth factors, such as FGF-2 and VEGF by tumor cells and/or the downregulation of negative modulators, like TSP-1, in tissues with a quiescent vasculature (180). Experiments with transgenic mice bearing spontaneous tumors have shown that only a subset (10%) of tumor cells acquire the angiogenic phenotype and that the non-angiogenic cells are sustained by capillaries recruited by neighboring cells (145). Similarly, human tumors consist of areas with intense neovascularization next to areas with significantly less capillary growth (251). Tumor growth and invasion of the tissue are mediated by proteinases, such as uPA and MMPs, produced by the tumor, which in turn induce the release of growth factors from the degraded ECM. Thus, positive angiogenic factors are produced by tumor cells and endothelial cells, released by attracted inflammatory cells such as mast cells (43) and macrophages (41) and/or mobilized from the ECM. So, different self-amplifying loops exist to maintain the angiogenic phenotype in the tumor.

TUMOR METASTASIS

The final step in the progression of a tumor is metastasis (74, 75, 85). Neovascularization of a primary tumor increases the possibility that cancer cells will enter the blood stream and spread to other organs and is also necessary for the growth of metastases in distant organs (130). Most of the micrometastases have a high death rate and are not vascularized until they switch to the angiogenic phenotype (75). Mice experiments have shown that for certain tumors, like Lewis lung carcinoma (LLC), this switch is dependent on the removal of the primary tumor, which releases an angiogenesis inhibitor: angiostatin (175). However, this is not a general rule, as metastases of B16 melanoma are not affected by removal of the primary tumor. In this case, the switch to the angiogenic phenotype does not depend on a decrease in endogenous circulating inhibitors, but on an intrinsic program in the metastatic cells (75).
 
 

CHARACTERISTICS OF THE TUMOR VESSELS Several data suggest that there are major differences between normal blood vessels and those associated with tumors (85). The final events of maturation of the newly formed capillaries with the recruitment of pericytes and smooth muscle cells are not taking place in tumors. Because of the lack of a complete and organized basement membrane, as well as accessory cells, tumor vessels maintain the aspect of developing vasculature. Tumor vessels often display an abnormal architecture, characterized by collapsing or poorly differentiated, fragile and leaky vessels, which are frequently unable to meet the rapid growth of tumor cells, resulting in local hypoxia and necrosis. Moreover, tumor vessels express specific markers, such as endoglin (CD105/EDG), a homodimeric cell surface component of the transforming growth factor-b (TGF-b) receptor complex that is expressed at high density on tumor endothelium, but not on normal blood vessels. An antibody directed against CD105 was able to quantitatively distinguish between tumor and preexisting vessels, which may be important in the assessment of tumor angiogenesis (137).

REGULATION OF TUMOR ANGIOGENESIS

In both normal and pathological angiogenesis, hypoxia is the main force initiating the angiogenic process. Hypoxia induces the expression of VEGF and its receptor via hypoxia-inducible factor-1a (HIF-1a) (35, 122) and is also an attractant for macrophages. In a tumor, the angiogenic phenotype can be triggered by hypoxia resulting from the increasing distance of the growing tumor cells to the capillaries or from the inefficiency of the newly formed vessels. Also, several oncogenes such as v-ras, K-ras, v-raf, src, fos and v-yes induce the upregulation of angiogenic factors like VEGF, insulin-like growth factor-1 (IGF-1) and TGF-a (122, 128, 162, 172). Moreover, oncogene products may act directly as angiogenic factors. This is the case for the protein product of FGF-4/hst-1 (233). In addition, oncogenes can indirectly promote angiogenesis by increasing the production of cytokines and proteolytic enzymes (5). In contrast, the tumor suppressor gene p53 was found to cause degradation of HIF-1a (192), inhibition of VEGF production (161), and stimulation of the inhibitor TSP-1 (46). Finally, the von Hippel-Lindau (VHL) gene product inhibits tumor growth and suppresses the expression of hypoxia-inducible genes VEGF, PDGF and the glucose transporter GLUT1 (110, 111). Consequently, inactivation of the VHL gene, as seen in von Hippel-Lindau (VHL) disease, an inherited cancer syndrome, characterized by extensively vascularized tumors, results in stabilization and activation of HIF-1a (150).