I. General Aspects of Angiogenesis

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The formation of new blood vessels out of pre-existing capillaries, or angiogenesis, is a sequence of events that is of key importance in a broad array of physiologic and pathologic processes. Normal tissue growth, such as in embryonic development, wound healing, and the menstrual cycle, is characterized by dependence on new vessel formation for the supply of oxygen and nutrients as well as removal of waste products. Also, a large number of different and nonrelated diseases is associated with formation of new vasculature. Among these pathologies are diseases, such as tissue damage after reperfusion of ischemic tissue or cardiac failure, where angiogenesis is low and should be enhanced to improve disease conditions (Carmeliet et al., 1999; Ferrara and Alitalo, 1999). In several diseases, excessive angiogenesis is part of the pathology. These diseases include cancer (both solid and hematologic tumors), cardiovascular diseases (atherosclerosis), chronic inflammation (rheumatoid arthritis, Crohn's disease), diabetes (diabetic retinopathy), psoriasis, endometriosis, and adiposity. These diseases may benefit from therapeutic inhibition of angiogenesis (Folkman, 1995; Hanahan and Folkman, 1996).

The initial recognition of angiogenesis being a therapeutically interesting process began in the area of oncology in the early 1970s, when Drs. Folkman and Denekamp put forward the idea that tumors are highly vascularized and thereby vulnerable at the level of their blood supply. In those early years, it was already hypothesized that the process of angiogenesis might be a target for therapy. It was only after the discovery of the first compounds with specific angiostatic effects in the early 1990s (Ingber et al., 1990; O'Reilly et al., 1994) that the research field of angiogenesis rapidly expanded and provided an increasing body of evidence that inhibition of angiogenesis could attenuate tumor growth. More recently, novel angiogenesis inhibitors have shown great potential in the treatment of cancer in preclinical studies. Several of those compounds are currently being tested in clinical trials (Molema and Griffioen, 1998). With increasing insight into the role of angiogenesis in other diseases as well, modulation of vascular outgrowth is now also regarded as a therapeutic target in these diseases.

To date, antiangiogenesis therapy is considered, worldwide, a promising approach, supposedly leading to the desperately needed breakthrough in cancer therapy and other proangiogenic diseases. Nevertheless, many questions remain unanswered and many concepts unverified at present. For example, it has to be established whether the exciting effects seen in preclinical investigations using xenogeneic and syngeneic tumor transplant models and transgenic systems also prevail in the human situation. Furthermore, it has been shown that the angiogenesis inhibitors angiostatin and endostatin (O'Reilly et al., 1994, 1997) do not elicit drug-induced resistance on prolonged treatment in tumor-bearing animals, although being highly effective in tumor growth reduction. This observation is of extreme importance, because it opens possibilities for long-term treatment or the development of treatment modalities for the prevention of disease in high-risk populations prone to develop tumors. It remains to be seen whether this scenario can be extended to other angiogenesis inhibitors as well as to other proangiogenic diseases of interest. Another important issue is the concept of cancer treatment with angiogenesis inhibitors as a single-compound strategy. Is this a feasible treatment strategy or should antiangiogenic therapy be used in combination with other treatment modalities such as immuno- or chemotherapy? Also, although antiangiogenesis therapy is considered to have low toxicity, there is as yet little information on the safety of therapeutic angiostatic strategies; there is little or no information to what extent inhibition of angiogenesis as tumor treatment will affect normal physiological processes in embryonic development or in wound healing and what the long-term side effects will be.

Although current interest in angiogenesis comes mainly from oncology researchers, also nononcological research fields have now recognized that modulation of angiogenesis may provide a tool for clinical interventions. This demonstrates that angiogenesis is a multidisciplinary theme from a pharmacologic target point of view. In addition, many disciplines of biomedical origin are contributing to basic angiogenesis research, because the processes involved are so complex. In this review, the molecular players of vessel growth, methodology of angiogenesis research, and preclinical and clinical use of angiogenesis as a target for therapy will be discussed.

Blood vessels consist of endothelial cells that are in direct contact with the blood, and subendothelially located pericytes, smooth muscle cells, fibroblasts, basement membrane (BM),¹ and extracellular matrix (ECM). Depending on the location in the body, the organ microenvironment, the cellular constituents, BM, and ECM of the vasculature differ in phenotype, composition, and function (Rajotte et al., 1998).

The endothelial cells form a monolayer in every single blood vessel in the circulation and are actively involved in several regulatory processes in the body (Fig. 1). Besides being metabolically active and selectively permeable for small solutes and peptides/proteins, they regulate blood coagulation. When their integrity is maintained, endothelial cells exert anticoagulative properties via the synthesis of thrombomodulin, tissue factor (TF) pathway inhibitor and tissue-type plasminogen activator (t-PA). On activation or damage, endothelial cells quickly release proteins like multimeric von Willebrand factor (vWF), which promotes platelet adhesion and aggregation, and plasminogen activator inhibitor-1, a member of the serpin family. In addition, TF expression by endothelium leads to initiation of the extrinsic blood coagulation pathway (Verstraete, 1995). Another important feature of endothelial cells is their ability to direct cells of the immune system to specific sites in the body. Constitutively expressed or cytokine-inducible cellular adhesion molecules [e.g., E-selectin and intercellular adhesion molecule-1 (ICAM-1)] and soluble factors such as chemoattractants, cytokines, and chemokines act in concert to recruit the immune cells to lymphoid organs or inflammatory sites (Carlos and Harlan, 1994). Last, endothelial cells are actively involved in vascular remodeling during, for example, ovulation, wound healing, tumor growth, and diabetic retinopathy. Although complex in regulation and sometimes difficult to functionally analyze in vitro, as well as during disease progression, data have become available that link (parts of) these endothelial cell functions to various steps in the angiogenic cascade.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Endothelial cells exert several important functions in the body. A, the endothelium forms a semipermeable barrier for the transport of blood-borne peptides, proteins, and other soluble molecules to underlying tissue; B, via the regulated expression of pro- and anticoagulative activities, endothelium actively participates in the hemostatic balance in the body; C, under the influence of proinflammatory cytokines, endothelial cells up-regulate a variety of cellular adhesion molecules to tether and activate leukocytes and facilitate leukocyte adhesion and transmigration from the blood into the tissue; D, during wound healing and tumor growth, among others, angiogenesis takes place. In this process, an active role exists for endothelial cells. EC, endothelial cells; IgSF, Ig superfamily; PAI-1, plasminogen activator inhibitor; PBMC, peripheral blood mononuclear cells; sLex, sialyl Lewis X; TFPI, Tissue Factor pathway inhibitor; TM, thrombomodulin.

In vasculogenesis during embryonic development, new endothelial cells differentiate from stem cells. In contrast, in angiogenesis new blood vessels mainly emerge from pre-existing ones (Risau, 1997). In adult life, physiologic stimuli during wound healing and the reproductive cycle in women lead to angiogenesis, whereas vasculogenesis is absent. Pathologic conditions such as tumor growth, rheumatoid arthritis, and diabetic retinopathy are characterized by abundant angiogenesis. The active vascular remodeling phase in tumors, e.g., is reflected by the fact that tumor endothelial cells proliferate 20 to 2000 times faster than normal tissue endothelium in the adult (Denekamp, 1984). In the last decade, several molecular players have been identified that significantly contribute to the molecular processes leading to new blood vessel formation. In the following sections, recent advances in this area of research are discussed.

1. Initiation of the Angiogenic Response. Angiogenesis is rapidly initiated in response to hypoxic or ischemic conditions. Vascular relaxation, for example, mediated by nitric oxide (NO) is a prerequisite for endothelial cells to enter the angiogenic cascade. Likely, morphologic changes of the endothelial cells lead to a decrease in confluency status to make them susceptible to mitogens (Folkman, 1997). In all types of angiogenesis, either under physiologic or pathologic conditions, endothelial cell activation is the first process to take place. Cytokines from various sources are released in response to hypoxia or ischemia. It is suggested that vascular endothelial growth factor (VEGF) is a major player in angiogenesis initiation based on its ability to induce vasodilation via endothelial NO production and its endothelial cell permeability increasing effect (Ziche et al., 1997). This allows plasma proteins to enter the tissue to form a fibrin-rich provisional network (Dvorak, 1986). The observation that VEGF production is under control of hypoxia inducible factor (HIF) strengthens the suggestion of an early involvement of VEGF in the angiogenic response. Moreover, VEGF receptor (VEGFR) expression is up-regulated under hypoxic or ischemic conditions as well (Forsythe et al., 1996).

3. Maturation of the Neovasculature. Endothelial cell interaction with ECM and mesenchymal cells is a prerequisite to form a stable vasculature. Therefore, after endothelial cell proliferation and maturation, and the formation of endothelial tube structures, surrounding vessel layers composed of mural cells (pericytes in small vessels and smooth muscle cells in large vessels), need to be recruited. Endothelial cells may accomplish this via the synthesis and secretion of platelet-derived growth factor (PDGF), a mitogen and chemoattractant for a variety of mesenchymal cells. Subsequent differentiation of the mural precursor cells into pericytes and smooth muscle cells is believed to be a cell-cell contact-dependent process. On endothelial cell-mural cell contact, a latent form of TGF-, produced by both endothelium and mural cells, is activated in a plasmin-mediated process. Activated TGF- can induce changes in myofibroblasts and pericytes, which may contribute to the formation of a quiescent vessel, ECM production, and maintenance of growth control. The coinciding investment of growing capillaries by pericytes with the deposition of BM and cessation of vessel growth during wound healing also indicates vessel growth regulation by pericytes (Hirschi and D'Amore, 1997). FGF-1 is also implicated in endothelial cell differentiation leading to vascular tube formation. Besides inducing plasminogen activator and endothelial cell proliferation and migration, FGF-1 receptor signaling resulted in endothelial tube formation in collagen (Kanda et al., 1996).

4. Other Mechanisms Implicated in Angiogenesis Control. Although the roles of several factors during angiogenesis have been discussed here separately, it is important to note that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other factors or cytokines in the environment of the responding endothelium (Pepper et al., 1998). For example, exogenous factors such as hormones can affect conditions leading to angiogenesis (Schiffenbauer et al., 1997). Isoforms of VEGF that bind to ECM-associated heparan sulfate proteoglycans can release ECM-stored FGF-2 in a bioactive form (Jonca et al., 1997), and angiopoietins potentiate the effects of VEGF (Asahara et al., 1998).

	II. Angiogenesis Stimulation

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Much attention has been payed to therapeutic strategies that are able to stop the angiogenic cascade in tumor growth (see Section III) and more recently, in chronic inflammatory situations such as rheumatoid arthritis (see Section IV). There are, however, various diseases affecting millions of people every year that would benefit from the induction of angiogenesis, so-called therapeutic angiogenesis (Takeshita et al., 1994). Although the number of studies reported in this area of research are not nearly as high as the number of studies on antiangiogenic therapies, the approach appeared to be quite successful in a preclinical setting as well as in the recently performed first clinical trials.

The disease conditions that may benefit from therapeutic angiogenesis encompass ischemic diseases such as ischemic coronary artery disease, critical limb ischemia with various etiologies, and decubitus. In these diseases, functional blood flow is partially lost in an organ or limb. For coronary artery disease, the leading cause of morbidity and mortality in Western countries, the therapeutic options (reducing the risk factors, restoration of the blood flow by angioplasty, or coronary bypass grafting), are insufficient. In critical limb ischemia, estimated to develop in 500 to 1000 individuals per million per year, the anatomic extent and the distribution of the arterial occlusions render the patients unsuitable for operative or percutaneous revascularization. At present, no pharmacologic treatment could favorably affect the ischemia. Often, loss of the limb by amputation is the recommended treatment for these patients (Baumgartner et al., 1998). A specific form of vascular occlusive disease that leads to critical limb ischemia, is thromboangiitis obliterans, or Buerger's disease. The disease afflicts arteries of young smokers and is characterized by the onset of distal extremity ischemic symptoms, leading to ulceration and gangrene (Isner et al., 1998). Gastroduodenal ulcers, also caused by local insufficient perfusion, have been subject of angiogenesis stimulation therapies (Wolfe et al., 1995). It has recently been suggested that for congestive heart failure, possibly a result of myocardial ischemia, stimulation of angiogenesis may also become a therapeutic option (Carmeliet et al., 1999; Isner and Losordo, 1999).

The treatment of arterial occlusions by balloon angioplasty is frequently associated with delinquent re-endothelialization and smooth muscle cell proliferation. One therapeutic option to reduce subsequent intimal thickening is the induction of apoptosis in infiltrating immune cells (Sata et al., 1998). Therapeutic angiogenesis to facilitate endothelial cell regeneration in this specific pathology has been proposed as well (Callow et al., 1994; Asahara et al., 1996).

In the case of organ transplantation, surgical procedures dictate loss of vessel integrity and function of the transplanted organ (Taub et al., 1998). Transplantation of encapsulated pancreatic islets as a treatment modality for type I diabetes, for example, may be more successful when prevascularized solid supports are used or solid supports are pretreated with proangiogenic factors (de Vos et al., 1997).

1. Vascular Endothelial Growth Factor. Angiogenesis is driven by numerous mediators produced by numerous cells under a variety of conditions. These mediators are either soluble, ECM or membrane bound growth factors, or components of the ECM itself. Of the soluble factors, one of the best studied and the most potent proangiogenic factor is VEGF, discovered in the early eighties by Dvorak and colleagues (Senger et al., 1983). VEGF (also known as VEGF-A) isoforms VEGF-121, -145, -165, -183, -189, and -205 are a result of alternative splicing from a single VEGF gene located on chromosome 6 (Mattei et al., 1996). Together with VEGF-B, -C, and -D, they belong to the VEGF/PDGF super family. Recently, a viral VEGF family member, designated VEGF-E, was described (Meyer et al., 1999). The two VEGF-specific tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), are expressed on vascular endothelium, and to a lesser extent on monocytes/macrophages and certain tumor cell types. VEGFR-3 (Flt-4), which binds VEGF-C and VEGF-D, is mainly expressed on lymphatic endothelium (Kaipainen et al., 1995). Interaction of VEGF with VEGFR-2 is a critical requirement to induce the full spectrum of VEGF biologic responses. Intracellular signal transduction pathways in endothelial cells through VEGFR-2 dimerization lead to permeability enhancement, cellular proliferation, and migration, as schematically shown in Fig. 2 (Abedi and Zachary, 1997; Kroll and Waltenberger, 1997; Wheeler Jones et al., 1997; Ziche et al., 1997; Gerber et al., 1998; Hood and Granger, 1998; Wellner et al., 1999; Doanes et al., 1999; Shen et al., 1999; Yu and Sato, 1999). All these studies were performed in vitro, exploiting either HUVEC or vascular endothelium from bovine or porcine origin. Evidence for at least partly similar VEGFR-mediated signal transduction pathways in vivo was recently provided using intact microvessels of mouse mesentery (Mukhopadhyay et al., 1998).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Dimerization of VEGFR-2 (KDR/flt-1) or VEGFR-1 (Flt-1) leads to a series of events in endothelial cells in vitro. Via a sequence of intracellular signal transduction steps, VEGFR signaling induces permeability increases, endothelial cell proliferation and migration, and cell survival. AA, arachidonic acid; AP-1, activator protein-1; cPLA2, cytoplasmic phospholipase A2; eNOS, endothelial NO synthase; FAK, focal adhesion kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PKG, protein kinase G; PLC, phospholipase C-; SAPK/JNK, stress-activated protein kinase/c-Jun NH2 terminal kinase.

2. Fibroblast Growth Factors. Members of the FGF family are also potent inducers of angiogenesis. Cellular responses mediated by FGFs include cell migration, proliferation, and differentiation (Kanda et al., 1997). The FGF family consists of nine structurally related polypeptides, of which FGF-1 (acidic FGF) and FGF-2 (basic FGF) are the most extensively studied. Both FGF-1 and FGF-2 are devoid of a signal sequence for secretion. Export from cells without compromising cell integrity or requiring cell death possibly follows a nonclassical, synaptotagmin-1-dependent exocytotic pathway (LaVallee et al., 1998).

3. Angiopoietin-1. Ang-1 is an endogenously secreted glycoprotein of approximately 75 kDa. Its receptor, Tie2, is generally restricted to the endothelium and of importance in angiogenesis during development, tumor growth, and wound healing (Sato et al., 1995; Lin et al., 1997; Wong et al., 1997; Stratmann et al., 1998). In vitro, Ang-1 stimulated tyrosine phosphorylation of Tie2 in endothelial cells, inhibited serum starvation-induced endothelial apoptosis, induced sprouting angiogenesis, and stabilized HUVEC network organization (Koblizek et al., 1998; Korpelainen and Alitalo, 1998; Kwak et al., 1999). When combined with other angiogenic factors such as VEGF or growth factor supplements containing FGF-1, the survival of both endothelial cells and vascular networks increased even more (Kwak et al., 1999; Papapetropoulos et al., 1999). Although being chemotactic for endothelial cells and Tie2-transfected fibroblasts, no mitogenic responses of endothelial cells to Ang-1 could be observed (Koblizek et al., 1998; Witzenbichler et al., 1998).

In various animal models, the effects of either plasmid DNA encoding angiogenic factors or their respective protein have been studied. In pigs, gradual narrowing of the coronary artery leading to complete blood vessel occlusion was induced by use of an ameroid constructor. In this model, the continuous perivascular administration of VEGF via an osmotic pump, or FGF-2 containing heparin-alginate microspheres, led to improved resting and stress-induced collateral blood flow values. Although the number of (vWF positive) blood vessels in nonischemic heart tissue was unchanged, the vessel density significantly increased in the ischemic areas (Lopez and Simons, 1996).

In a mouse model of hindlimb ischemia, created by femoral artery ligation, the question of whether diabetes leads to impaired neovascularization was addressed, because diabetes is a major risk factor for artery diseases. It was shown, that nonobese diabetic mice exerted a lower angiogenic response in ischemic tissue compared with normal mice. This impaired response could be reduced by intramuscular (i.m.) gene therapy with recombinant adenovirus expressing murine VEGF cDNA (Rivard et al., 1999). Local administration of a plasmid encoding the 165-amino acid isoform of human VEGF₁₆₅ (phVEGF₁₆₅) during balloon cathetherization of the femoral arteries of rabbits resulted in an increased rate of re-endothelialization. This effect was also observed in the contralateral femoral artery that underwent simultaneous balloon injury but was not transfected. As a result of the increased re-endothelialization, the intimal thickening was diminished in both limbs, thrombotic occlusions were less frequent, and recovery of the endothelial cell-dependent vasomotor reactivity was accelerated (Asahara et al., 1996).

For reconstructive surgery and organ transplantation procedures, hypoxia or ischemia of the organs will negatively influence organ viability and function. The local administration of VEGF cDNA or FGF-2 protein into ischemic experimental skin flaps in rats and rabbits, respectively, significantly enhanced survival time of isolated skin flaps after 1 week (Hickey et al., 1998; Taub et al., 1998). In the case of a solid support system exploited for the grafting of, e.g., pancreatic islets as a means of bioartificial organ development, incorporation of FGF-1 led to ingrowth of blood vessels 4 weeks after implantation under the liver. Engrafting of pancreatic islets into these FGF-1 prevascularized solid support systems resulted in a better survival of the graft compared with islets engrafted without a solid support, although islet function was somewhat less than in normal rats (de Vos et al., 1997). This study indicates that therapeutic angiogenesis may also have a potential in organ transplantation and bioartificial organ development. It should be realized, however, that in the case of allotransplantation, immune cell activation will occur. Increased neovascularization may facilitate immune cell infiltration by virtue of the fact that more blood vessels allow better access to the graft. On the other hand, endothelium under the influence of proangiogenic factors may exhibit impaired leukocyte recruitment functions (see Section IV.A).

After i.m. administration of a plasmid encoding the human Ang-1 gene in a rabbit ischemic hindlimb model, human Ang-1 mRNA could be detected 3 to 14 days after gene transfer. No mRNA was found in sites distant from the ischemic hindlimb. Both the angiographic score and the capillary density were increased in the hindlimb 40 days after Ang-1 encoding plasmid administration (Shyu et al., 1998).

The increase in re-endothelialization of balloon injured vessels in the femoral artery that was not treated with the phVEGF₁₆₅ cDNA (Asahara et al., 1996), poses the question whether angiogenic therapy for a specific purpose may affect other sites in the body as well. For example, the question comes to mind whether therapeutic angiogenesis in a patient with myocardial ischemia is able to induce angiogenesis in an otherwise dormant, undetected, tumor nodule. Until now, however, laboratory studies did not demonstrate that stimulation of angiogenesis alone was sufficient for malignant growth (Isner et al., 1996).

So far, results are available from several pilot studies on the clinical application of therapeutic angiogenesis. In all three studies described, naked plasmid DNA encoding human phVEGF₁₆₅ under the cytomegalovirus promoter/enhancer was administered. In the case of ischemic limbs in patients with critical limb ischemia or thromboangiitis obliterans, the DNA was i.m. injected in the ischemic limb (Baumgartner et al., 1998; Isner et al., 1998). The DNA was administered directly in the myocardium of patients suffering from myocardial ischemia. In patients suffering from myocardial ischemia, the DNA was administered directly in the myocardium (Losordo et al., 1998).

Using contrast angiography, newly formed collateral blood vessels could be visualized in critical limb ischemia and thromboangiitis obliterans patients treated with phVEGF₁₆₅. Ischemic ulcers markedly improved or healed, resulting in successful limb salvage in several patients. Documented adverse effects were transient ankle or calf edema in some limbs. Patients suffering from myocardial ischemia had significant reduction in angina and reduced ischemia after phVEGF₁₆₅ treatment.

VEGF was also administered as a protein to patients with angina. Although the 120-day follow-up showed promising results, the 60-day follow-up showed no difference in exercise time or improvement of angina compared with placebo. An unexpected improvement in the placebo group may be the reason for this result. Furthermore, a clinical study on the applicability of FGF-2 in a similar patient group has started. Results are expected to be presented early 2000 (anonymous, 1999a).

It was concluded from these preliminary studies that therapeutic angiogenesis was able to induce neovascularization, and if instituted at the proper time, it may improve ischemic disease conditions in humans. The finding that endothelial progenitors can be isolated from human peripheral blood opens another possibility to augment collateral vessel growth to ischemic tissue (Asahara et al., 1997). The homing potential of these progenitors to foci of angiogenesis may be exploited for their application as autologous vectors for gene therapy with, e.g., cDNA encoding VEGF after angiogenesis induction with nontargeted plasmid DNA.

	III. Angiogenesis Inhibition

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Virchow was among the first to demonstrate the high vascularization in tumors in his publication Die Krankhaften Geschwülste published in 1863. He suggested that this was associated with the disorganized nature of tumor cells. The origin of the observed blood vessels was uncertain by then, it either developed from the transformed tumor cells or, alternatively, from normal cells that had been derived from the neighboring benign tissues. In a later period it was suggested that the trigger for enhanced blood vessel growth in tumors emanated from the invading malignant cells. It was proposed that the ability to attract new vasculature from the host was a characteristic feature of tumor cells. Recently, two paradigms were added to the vascular processes thought to prevail in tumor-induced neovascularization. During vessel co-option, tumors will initially exploit the host vasculature for survival, which coincides with host vasculature regression. Ongoing tumor cell growth will subsequently lead to initiation of angiogenesis (Holash et al., 1999). Furthermore, circulating endothelial progenitor cells can form an additional source for postnatal vasculogenesis in tumor growth (Asahara et al., 1999).

Since then many researchers have studied angiogenesis in a variety of test systems. Only after it was recognized that new vessels at the tumor site were absolutely required for solid tumor expansion beyond the size of approximately 1 to 2 mm in diameter, it was suggested that the process of angiogenesis might be a target for therapy. Recent studies have demonstrated that lymphoproliferative diseases, such as leukemia and lymphoma, are dependent on angiogenesis as well. Elevated expression of FGF and VEGF has been observed in acute myeloid leukemia, acute lymphoblastoid leukemia, and lymphomas (Fiedler et al., 1997; Foss et al., 1997; PerezAtayde et al., 1997). These studies indicate, therefore, that angiogenesis might also be a therapeutical target for hematologic tumors.

The first molecule identified as an angiogenic factor was described in 1984 (Shing et al., 1984) after which a large number of angiogenic factors followed. These factors can be produced by the tumor cells themselves, cells present in the tumor stroma such as fibroblasts, smooth muscle cells, or by infiltrating immune cells. To complicate matters, these cells are all able to produce angiogenesis inhibitors as well. More recent attention has been paid to the isolation and characterization of these angiogenesis inhibitors because they may have potential as therapeutic agents. Most of them have been studied for their applicability in cancer therapy but may also be suitable for the therapy of chronic inflammatory conditions (see Section IV.C).

Angiogenesis can be qualitatively and quantitatively measured in a large variety of in vitro and in vivo model systems. As discussed in Section I.C, the angiogenic cascade can be dissected in different sequential steps so that can each be studied separately in vitro. Research has mainly focused on proliferation and migration of endothelial cells. For this research, different endothelial cell sources can be applied. For human research most laboratories make use of HUVECs. This is the best available source of human endothelium, but the major drawback of these cells is their macrovascular origin, which makes them less suitable for studies on angiogenesis, a microvascular process. Although more laborious, human microvascular endothelial cells can be isolated from other organs such as foreskin or adipose tissue. For all primary isolates, the number of in vitro passages (4-5, and in the presence of growth factors, approximately 10) is, however, limited, which poses a major problem for their application. The required regular isolations furthermore introduce significant donor variation. To circumvent these drawbacks, one can use immortalized endothelial cell lines, such as HMEC-1 (Ades et al., 1992), EA.hy926 (Edgell et al., 1983), or ECL4n (Griffioen et al., 1996b). ECV304, a spontaneous immortalized endothelial cell line, has been applied for in vitro angiogenesis studies, although recently it has become apparent that this cell line contains a nonendothelial background as well. Endothelial cells from other species are also available, e.g., bovine capillary endothelial cells or cell lines from mouse and rat origin. It should be kept in mind that the effects of angiogenesis-inducing or -inhibiting factors can be different in the different species.

Assays to study proliferation of endothelial cells are based on cell counting or radiolabeled thymidine incorporation, or on colorimetric systems for measurement of mitochondrial activity [cell counting kit-8 (CCK-8) or dimethylthiazol diphenyl tetrazolium bromide (MTT)]. Alternatively, proliferation of endothelial cells can be analyzed by DNA profiling or determination of cell cycle-dependent expression of molecules such as proliferating cell nuclear antigen or Ki-67. Also, detection of cell death is a commonly used approach to average cell growth; e.g., apoptosis induction can be studied by detection of subdiploid cells or analysis of DNA degradation profiles, cell morphology or nick-end labeling by terminal dUTP nick-end labeling analysis.

For analysis of migration of endothelial cells, Boyden chambers are primarily used. An easier method is the wound assay. This assay system is based on wounding of a confluent monolayer of endothelial cells and measurement of the wound width in time.

Although the advantage of these in vitro assays is clearly the control over the few parameters present, the angiogenic cascade consists of multiple steps. This as a more extended process, can be studied in vitro, too. Most of these assays studying more complex processes of angiogenesis are based on tube formation of long-term cultured endothelial cells in a 3-dimensional seminatural matrix microenvironment. The most commonly used assay system to measure tube formation is the growth factor-induced sprouting of capillary-like structures from a confluent monolayer of endothelial cells grown on a thick gel. These gels can either be composed of a seminatural matrix with or without growth factors (e.g., Matrigel), or be based on collagen (Barendsz-Janson et al., 1998) or fibrin (Koolwijk et al., 1996). The demonstration of lumina in these endothelial cell sprouts is regarded as a criterion for vessel growth, in contrast to just migration of endothelial cells in the matrix or just rearrangement of endothelial cells on the gel. An elegant method to measure capillary formation has been described where endothelial cells grown on gelatin-coated cytodex-3 microcarrier beads were cultured in a fibrin gel (Nehls and Drenckhahn, 1995; Trochon et al., 1998). Quantification of sprouting can subsequently be performed by either measurement of maximal sprouting distance or by computer-based determination of total vessel length. Assays based on the sprouting of capillaries out of fresh tissue embedded in matrix gels more closely reflect the in vivo situation. This has been described for rat aortic rings (Nicosia and Ottinetti, 1990; Malinda et al., 1999) and human placenta tissue (Brown et al., 1996). This procedure is not applicable for all tissues because it has been described that, e.g., tumor biopsies often produce too much proteases digesting the matrix and thereby prevent endothelial cell sprouting (Barendsz-Janson et al., 1998). Figure 3 shows some examples of in vitro angiogenesis assays. A recently published novel way of measurement of angiogenesis in vitro, which is even more close to the in vivo situation, is the use of embryoid bodies (Wartenberg et al., 1998). In vitro cultured mouse blastocyst cells (Evans and Kaufman, 1981) recapitulate several steps of murine embryogenesis, including vasculogenesis and angiogenesis (Risau et al., 1988). There is a complete blood vessel development in these embryoid bodies (Vittet et al., 1996) making this system suitable for the study of angiogenesis modulators.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   In vitro angiogenesis assays. Tube formation of human umbilical vein endothelial cells on the seminatural matrix, Matrigel, 2 h (panel A) and 16 h (panel B) after seeding. Growth factor induced (20 ng/ml bFGF) sprouting of bovine microvascular endothelial cells grown as a monolayer on a collagen-based gel (panel C) can be inhibited by the angiogenesis inhibitor platelet factor-4 (panel D). Similar regulation is shown in panels E and F in a tube formation/migration assay of bovine endothelial cells grown on cytodex-3 beads and embedded in a fibrin gel. Panel G represents the rat aortic ring assay under control conditions and after exposure for 5 days with 200 µg/ml endothelial cell growth supplement (H). Panels A, B,G, and H were kindly provided by Dr. H. Kleinman (Bethesda, MD).

Besides the advantages that in vitro angiogenesis assays clearly have, the major drawback of all these assays is that they require the endothelial cells to be removed from their natural microenvironment, which may alter their physiologic properties. To study angiogenesis in vivo, the most frequently used assay systems are the chicken chorioallantoic membrane assay (Nguyen et al., 1994), the corneal pocket (Conrad et al., 1994), transparent chamber preparations such as the dorsal skin-fold chamber (Algire, 1943; Lichtenbeld et al., 1998), the cheek pouch window (Shubik et al., 1976), and the polymer matrix implants (Mahadevan et al., 1989; Plunkett and Hailey, 1990). However, in vivo assays also have several disadvantages: the pharmacokinetic properties of the compounds tested, necessary for proper interpretation of results, are often not known and the host will respond nonspecifically to the implantation. In this review, these assays will not be discussed in more detail because recently an elegant review on this issue appeared, discussing the pro's and con's of in vivo quantitative angiogenesis assays (Jain et al., 1997).

1. Intervention with Endothelial Cell Growth. The most successful approach to modulate angiogenesis, to date, is the use of agents that specifically inhibit the growth of the endothelial cells. One of the first compounds identified to exhibit inhibitory effects on cell growth with specificity for endothelial cells was O-chloroacetylcarbamoyl fumagillol or AGM-1470/TNP-470, an analog of the fungus-derived antibiotic fumagillin (Ingber et al., 1990; Kusaka et al., 1991). The mechanism of action of this compound was found to be prevention of endothelial cells to enter G₁ phase of the cell cycle, resulting in a decrease in proliferation (reviewed in Castronovo and Belotti, 1996). In later years, several endogenous molecules with angiostatic activity were described. Among these molecules are thrombospondin-1 (Rastinejad et al., 1989; Good et al., 1990; Grossfeld et al., 1997), platelet factor-4 (Gupta et al., 1995; Kolber et al., 1995), and interferon-inducible protein-10 (Luster et al., 1995). Two other members of this class of endogenously produced antiangiogenic proteins are angiostatin (O'Reilly et al., 1994) and endostatin (O'Reilly et al., 1997). Angiostatin is an internal fragment of plasminogen with multiple antiangiogenic activities in vitro and in vivo. Endostatin is a proteolytic fragment of collagen XVIII that affects endothelial cell survival via the induction of an imbalance between the antiapoptotic proteins Bcl-2 and Bcl-X_L and the proapoptotic protein Bax (Dhanabal et al., 1999). Both induced tumor regression, not only growth inhibition, in tumor-bearing mice, an effect that was most pronounced with endostatin and demonstrates the potential of these proteins. Direct inhibition of endothelial cell growth was also obtained with two other recently described endogenously produced angiostatic proteins, namely vasostatin (Pike et al., 1998) and restin (Ramchandran et al., 1999). Detailed mechanisms of action have not been described yet for these angiogenesis inhibitors.

2. Intervention with Endothelial Cell Adhesion and Migration. Because the process of angiogenesis also depends on endothelial cell adhesion events to, and migration of cells through, the extracellular matrix, effort is put in the search for modulators of these interactions. The first identified member of this group of compounds is the endogenously produced cytokine interferon. Antiendothelial activity was recognized by the observation that interferon could inhibit the migration of capillary endothelial cells (Brouty and Zetter, 1980). Subsequently, both interferons  and  were shown to have in vivo antiangiogenic activity (Sidky and Borden, 1987). Although interferons are probably not sufficiently active for treatment of all tumors, benign tumors predominantly comprised of endothelial cells are particularly sensitive to treatment with interferon (Ezekowitz et al., 1992). When it was found that activated endothelial cells up-regulate receptors for extracellular matrix components (Re et al., 1994; Frisch et al., 1996; Griffioen et al., 1997), interaction of endothelial cells with the matrix was chosen as a target for inhibition of angiogenesis. This proved to be a relevant approach by the demonstration that _v3 integrin molecules, the biological function of which is binding of vitronectin and other RGD-containing matrix components, are overexpressed in angiogenically stimulated blood vessels. Ligation of these receptors with an antibody called LM609 interferes with endothelial cell growth leading to inhibition of subsequent tumor growth (Brooks et al., 1994a). In addition, the exposure of endothelial cells to anti-_v3 antibodies resulted in the induction of apoptosis in these cells via loss of cell anchorage to the extracellular matrix (Brooks et al., 1994b). This is most likely the mechanism by which proliferation of endothelial cells and angiogenesis in vivo is blocked by _v3-directed antibodies.

3. Intervention with Metalloproteinases. Another mechanism of angiogenesis inhibition, related to the inhibition of endothelial cell adhesion and migration, is the use of specific inhibitors of proteinases that dissolve the connective tissue, thereby facilitating endothelial cell migration and subsequent vessel formation. Matrix metalloproteinases are a homologous family of enzymes that are involved in tissue remodeling and morphogenesis. Collectively, these enzymes are capable of degrading all components of the extracellular matrix (Rasmussen and McCann, 1997). Increased activity of these enzymes has been observed in tumor formation, and therefore inhibitors of MMPs represent an attractive approach to treat cancer. MMP inhibitors can be divided in synthetic protease inhibitors and naturally occurring MMP inhibitors, the tissue inhibitors of metalloproteinase. Belonging to the former group, batimastat, marimastat, and prinomastat/AG3340 are potent broad-spectrum inhibitors of the major MMPs and can prevent or reduce spread and growth of several different malignant tumors in numerous animal models (Brown and Giavazzi, 1995; Shalinsky et al., 1999). Cell adhesion and proteolytic mechanisms are functionally associated, as recently demonstrated by the observation that the collagenase MMP-2 can bind to integrin _v3 on angiogenic blood vessels. Most interestingly, it was found that a naturally occurring MMP-2 breakdown product, called PEX, can inhibit cell-associated collagenolytic activity. It is suggested that this breakdown product is an important regulator of protease activity during angiogenesis and vasculogenesis. A recombinant form of PEX was useful in blocking angiogenesis and tumor growth in vivo, providing a novel therapeutic approach for angiogenesis inhibition at this level (Brooks et al., 1998).

The pivotal role of angiogenesis in tumor progression and metastasis has urged researchers to test newly developed angiogenesis inhibitors in a broad variety of animal tumor growth models. Studies with one of the first angiogenesis inhibitors, AGM-1470/TNP-470, were performed in the early 1990s. Although in vitro the sensitivity for TNP-470 was not completely restricted to endothelial cells, doses of the compound had to be 10 to 100 times higher for comparable inhibition of tumor cell lines. Treatment of tumor-bearing mice resulted in a significantly increased survival time of 260% over untreated control animals. Daily treatment was not necessary; optimal treatment regimens were s.c. or i.v. administration once every three days. Oral administration had weaker effects on tumor growth, likely a result of lower bioavailability. The fact that sensitivity of tumor cells in vitro and effect on tumor growth in vivo did not correlate is seen as evidence for antitumor effects through the tumor vasculature (Ingber et al., 1990; Yamaoka et al., 1993). Angiogenesis inhibitors have also been expected to be efficient metastasis inhibitors based on the concept that tumors require new vasculature for spreading and outgrowth at a secondary site. TNP-470 reduced both the number and size of metastases of the B16BL6 melanoma and the M5076 reticulum cell sarcoma cell line in mice by 80 to 90% (Yamaoka et al., 1993). Also in rats, inhibition of tumor growth and metastasis was observed (Futami et al., 1996).

The next breakthrough in the search for novel antiangiogenic compounds occurred when the hypothesis that a primary tumor, whereas capable of stimulating angiogenesis for its own blood supply, can produce angiogenesis inhibitors that suppress the outgrowth of distant metastases, was proven to hold true. This hypothesis came from the observation that the removal of primary tumors could lead to the accelerated growth of metastases (Sugarbaker et al., 1977). To test this hypothesis, the Lewis lung carcinoma mouse model was used in which the primary tumor completely suppresses the growth of its metastases. From the urine of these mice a cleavage fragment of plasminogen called angiostatin was purified, which completely replaced the inhibitory activity of the primary tumor (O'Reilly et al., 1994). Treatment of tumor-bearing mice with angiostatin almost completely prevented metastasis formation in the lung. Using a similar strategy, endostatin was discovered (O'Reilly et al., 1997). Treatment of mice carrying different syngeneic malignant tumors with endostatin led to a rapid regression of tumors. As with angiostatin, there was no sign of toxicity, and continued endostatin therapy maintained the tumors in a state of dormancy. Discontinuation of treatment led to renewed growth at the primary site, which would eventually lead to death of the animals. However, when therapy was restarted tumors regressed again for nearly 100%. Subsequent intermittent cycles of treatment were able to maintain the tumors in a state of dormancy and showed no sign of drug-induced resistance (Boehm et al., 1997). When angiostatin and endostatin therapy were combined for 25 days, both at relatively high doses (20 mg/kg/day), tumors regressed completely yielding tumor-free survival of up to 11 months after start of therapy.

The nature of this dormancy is still obscure since these tumors have the capacity of renewed outgrowth when transplanted to the other flank or to another animal. This makes the involvement of the immune system, i.e., the development of a specific antitumor immune response in these mice, unlikely. It has been suggested that the sequential accumulation of endostatin during these cycles of growth and regression can locally lead to a high concentration, and hence keep the tumor dormant (Black and Agner, 1998). The data on endostatin suggest that the endothelial cell compartment is directly involved in the dormancy of the tumor cells, demonstrating the powerful control exerted by the vascular endothelial cell population over the tumor cell population.

The discovery of overexpression of _v3 integrin in tumor vessels and the dependence of angiogenesis on this matrix receptor (Brooks