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Ann Thorac Surg 1997;64:1204-1211
© 1997 The Society of Thoracic Surgeons

Current review

Angiogenesis in the Pathobiology and Treatment of Vascular and Malignant Diseases

David S. Winlaw, MD, BS

Department of Vascular Surgery and Molecular Genetics Unit, Kolling Institute for Medical Research, Royal North Shore Hospital, Sydney, Australia


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Occlusive Vascular Disease and...
 Malignant Disease
 Summary and Future Directions
 Acknowledgments
 References
 
Cardiovascular disease and cancer account for the majority of adult disease in the developed world. This review focuses on current concepts in the study of angiogenesis (new vessel formation) as related to these conditions and highlights the role of vascular endothelial growth factor. Developments in therapeutic angiogenesis have raised the possibility that pharmacologic or gene-directed interventions, based on the ability of vascular endothelial growth factor to promote new vessel formation, may soon gain clinical application for the treatment of occlusive vascular disease. Similarly, the future treatment of malignant disease is likely to involve antiangiogenic agents that, in preliminary animal work, have demonstrated an efficacy that is not limited by adverse affects. Aside from these potential applications, current investigations have enhanced our understanding of mechanisms involved in the development of atherosclerotic and malignant disease.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Occlusive Vascular Disease and...
 Malignant Disease
 Summary and Future Directions
 Acknowledgments
 References
 
Physiologic new vessel formation occurs in response to metabolic need, and is observed in circumstances such as wound repair, ischemia, and cellular proliferation. In these and other conditions, tissue hypoxia is a potent stimulus for neovascularization, inducing expression of vascular endothelial growth factor (VEGF) in cardiac myocytes [1], vascular smooth muscle cells [2], and malignant cells [3]. Hypoxic induction of gene expression occurs for VEGF as well as erythropoietin, platelet-derived growth factor-ß-chain, endothelin, and angiotensin converting enzyme, possibly through a hypoxia-specific transcription factor [4]. The intracellular events underlying this effect involve NF-{kappa}B and other transcription factors [5] (Fig 1Go). Hypoxia has been shown to stabilize VEGF mRNA, and this is thought to be a major factor regulating VEGF mRNA levels in vivo [6]. The effects of VEGF are further regulated by the expression of various VEGF receptors, and receptor expression itself is enhanced by hypoxia through augmentation of receptor protein synthesis [7].



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  		Fig 1. . Schematic representation of cellular processes involved in vascular endothelial growth factor (VEGF) production, and expression of receptors mediating effects of VEGF. Hypoxia upregulates transcription of VEGF and VEGF receptors, and stabilizes receptor messenger RNA (mRNA). Specific cytokines also regulate this process. Endothelial cells respond to VEGF through specific receptors but do not produce VEGF, whereas vascular smooth muscle cells express VEGF receptors and produce VEGF. Many neoplastic cells produce VEGF, express VEGF receptors, and proliferate in response to exogenous VEGF, suggesting paracrine or even autocrine pathways promoting cell growth and proliferation.

 
Vascular endothelial growth factor and basic fibroblast growth factor (bFGF) target receptors present on endothelial cells, and binding sets in motion a number of mitogenic and enzymatic events that culminate in the growth of new blood vessels. Basic FGF is capable of this effect in its own right, and although bFGF expression may not be enhanced by hypoxia [8], it potently synergizes the expression and angiogenic effects of VEGF [9].

Neovascularization begins with increased permeability, or vascular leakage through endothelial cells, which is associated with enrichment of interstitial fluid with plasma components. Vascular endothelial growth factor achieves this by increasing the activity of microvascular vesicular-vacuolar organelles that transiently fenestrate endothelial cells. In this favorable environment, proliferating endothelial cells form cordlike structures in target tissue that later canalize to form functional vessels. Vascular endothelial growth factor is chemotactic and mitogenic to endothelial cells and enhances the expression of plasminogen activators and collagenases, which aid the growth of new vessels into target areas. New vessel formation is a crucial part of embryonic development, as well as the response to disease. Mice with both heterozygous [10] and homozygous [11] inactivation of the VEGF gene die early in utero, with impaired circulatory development, indicating the importance of VEGF in vasculogenesis (the growth of new vessels de novo) as well as angiogenesis (development of new blood vessels from existing networks).

The human VEGF gene is located on chromosome 6 and encodes a dimeric glycoprotein comprising four possible monomers as a result of differential splicing of eight exons that make up the gene product. The four VEGF subtypes are 121-, 165-, 189-, and 206-amino acids in length. The smaller forms are secreted, whereas VEGF189 and VEGF206 are retained close to the membrane of producing cells bound to heparan proteoglycans. The longer splice variants can be mobilized by heparanases and proteases, and in this way, alternative splicing may control release of VEGF. In the last year, two additional forms of VEGF, VEGF-B [12] (or VEGF related peptide [13]) and VEGF-C [14], have been described with affinities for muscular and lymphatic tissues, respectively, but are yet to be fully characterized.

There are several receptors for VEGF, all of which have receptor tyrosine kinase activity. Ligand binding induces dimerization, which triggers signal transduction by phosphorylation of downstream transduction proteins. Receptors for vascular endothelial growth factor, VEGF-R1 (previously known as flt-1) and VEGF-R2 (flk/KDR), bind VEGF, whereas VEGF-R3 (flt-4) appears to be specific for VEGF-C [14]. The expression of VEGF-R2 is confined to endothelial cells, accounting for the selective nature of VEGF-induced mitogenesis.

Cytokines and other growth factors are involved in the regulation of angiogenesis, providing a balance between proangiogenic and antiangiogenic forces. Individual cytokines such as transforming growth factor-ß may have differing effects depending on their local concentration and the presence of other cytokines. Platelet-derived growth factor promotes angiogenesis by enhancing VEGF expression [15], as does interleukin-1ß in vascular smooth muscle [16]. Expression of VEGF in vivo is downregulated by cytokines including interleukin-12 and thrombospondin, a naturally occurring and widely distributed heparin-binding glycoprotein [17].


   Occlusive Vascular Disease and Vascular Injury
Top
 Footnotes
 Abstract
 Introduction
 Occlusive Vascular Disease and...
Malignant Disease
 Summary and Future Directions
 Acknowledgments
 References
 
New vessel formation is an integral part of the circulatory response to large vessel disease caused by atherosclerosis. Vascular endothelial growth factor and bFGF mediate the development of new collateral vessels that supplement existing networks in ischemic tissue. Paradoxically, production of VEGF by vascular smooth muscle cells (VSMCs) may actually facilitate the neovascularization of stable fibrous plaques causing complicated lesions. The following section integrates the study of classical atherosclerosis, restenosis after balloon angioplasty of atherosclerotic lesions, and the response of normal vessels to balloon injury to provide a better understanding of the biologic roles of angiogenic peptides.

Plaque Dynamics
Atherosclerotic lesions arise as a consequence of the inflammatory response to oxidized low-density lipoproteins incorporated by macrophages and deposited in the intima. This is associated with the accumulation of mesenchymal cells, traditionally thought to be derived from the media, in the neointima. In the stable plaque, a fibrous cap excludes material contained within the lesion from the circulation. Several events may lead to breakdown of this cap, such as plaque fissuring or rupture, causing distal embolization or vessel occlusion. Candidate processes that may weaken the fibrous cap include heightened immunologic activity and plaque neovascularization.

Carotid artery plaque has been extensively studied, because of well-characterized clinical syndromes associated with embolization of plaque material or thrombi and cerebral hypoperfusion leading to cerebral infarction. Furthermore, removal of human plaque at endarterectomy allows direct study of the lesion and correlation with clinical behavior. Activated immune cells such as macrophages have been observed within carotid artery plaque, adjacent to and within the fibrous cap. Liberation of metalloproteinases (collagenase, gelatinase, and stromelysin) and hydrogen peroxide by these cells may be responsible for cap thinning and breakdown [18]. Indirect evidence of this inflammatory process is provided by increased temperature within plaque from individuals with significant disease [19]. Plaque infiltrating lymphocytes produce bFGF and other cytokines that regulate expression of VEGF [20], which is itself capable of promoting migration of monocytes from surrounding tissues through increased expression of VEGF-R1 [21].

Neovascularization of plaque is associated with symptomatic carotid artery disease [22]. The dilated microvessels frequently observed have thin walls and may be susceptible to rupture by physical forces such as wall stress and pulsatile flow as the plaque enlarges. The stimulus for neovascularization is unclear although it has been observed that homogenates of explanted human carotid artery plaque have been shown to stimulate new vessel formation in vitro [23], suggesting expression of an angiogenic factor. One likely mediator is platelet-derived growth factor through a direct effect, and indirectly by promoting VEGF expression. Histologic examination of coronary artery plaque specimens obtained by atherectomy from patients with restenotic lesions yields microvessel counts above that of primary lesions [24], possibly reflecting a more complex and intense cytokine response.

In normal circumstances, the media is dependent on diffusion of oxygen from the lumen and from vessels of the vasa vasorum. Wall thickening associated with injury or atherosclerosis impairs oxygen delivery from the lumen, requiring a compensatory increase from the vasa vasorum [25]. Hypoxic induction of VEGF expression by VSMCs could explain neovascularization of the vasa vasorum and the thickened intima. Growth factors such as platelet-derived growth factor and transforming growth factor-ß implicated in the pathogenesis of atherosclerosis and restenosis, are known to synergize the effect of hypoxia on VEGF expression [9, 15]. The concept that intraplaque hypoxia causes an increase in expression of angiogenic peptides by mesenchymal cells is reinforced by the finding that human microvascular endothelial cells cultured in the presence of VSMC-conditioned medium demonstrate marked proliferation, an effect that is abolished by anti-VEGF monoclonal antibodies [26].

In advanced atherosclerosis, there is a pronounced increase in vascularity of the adventitia; however, neither the cause nor the significance of this finding is known. As suggested earlier, expression of VEGF stimulated by hypoxia and cytokines within the plaque may contribute. An alternative explanation is that the adventitia may play a primary role in the response to physical vessel wall injury. Interference with the vasa vasorum of normal vessels causes intimal thickening and changes characteristic of early atherosclerosis [27]. Expression of bFGF is upregulated in the adventitia of arteries affected by atherosclerosis compared with normals, with relatively less expression in the media [28]. Interestingly, the translocation and phenotypic modulation of adventitial fibroblasts into neointimal myofibroblasts after balloon injury involves the expression of growth factors by these cells [29], with angiogenic potential.

Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor in the Response to Vessel Injury
Vascular endothelial growth factor is generally viewed as a selective endothelial cell mitogen, whereas bFGF is also a mitogen for VSMCs. The significance of this finding has been explored in detail because of the potentially detrimental effect of VSMC proliferation within plaque. Nabel and colleagues [30] demonstrated that a recombinant homologue of bFGF expressed in porcine arteries promoted intimal hyperplasia and neovascularization. Edelman and colleagues [31] demonstrated that bFGF administered into the periadventitial space of rat carotid artery increased the intimal hyperplastic response to balloon denudation in proportion to the amount of damage produced. There was no intimal hyperplasia present in bFGF-treated but uninjured arteries. Furthermore, the angiogenic effect of bFGF on the vasa vasorum was linearly related to the amount of VSMC proliferation. The authors speculate that injury causes bFGF-mediated VSMC proliferation and induction of angiogenesis. It is possible that enhanced capillary permeability caused by bFGF and VEGF may create a favorable environment for proliferation, not just for endothelial cells but also for VSMCs.

Reendothelialization After Intimal Injury and Intimal Hyperplasia
Because bFGF and VEGF are mitogenic for endothelial cells, their ability to reconstitute an endothelial lining after injury, such as that caused by balloon angioplasty, has been assessed. The importance of a functional endothelial lining is underscored by the protective role of constitutively expressed nitric oxide synthase by these cells. Nitric oxide (NO) produced in this fashion has a tonic dilator effect in those vessels capable of relaxation and an inhibitory effect on platelet aggregation and activation [32]. The antiplatelet effect is important not just in terms of thrombosis, but also because platelet-derived growth factor is an important mediator of restenosis [33], and NO itself has a weak inhibitory effect on VSMC proliferation [34]. Systemic administration of bFGF increases the rate of reendothelialization after balloon injury of the iliac artery in rabbits, and this corresponds with partial restoration of endothelium (NO) -dependent relaxation [35]. Similarly, systemic administration of VEGF has also been shown to enhance reendothelialization after balloon injury, and intraarterial administration of VEGF attenuates intimal thickening after injury [36]. An approach using molecular techniques, in which the gene for VEGF is incorporated by host cells that then produce VEGF, may speed reendothelialization and reduce the proliferative complications of clinical balloon angioplasty, and similar technology may be applicable to endothelial seeding of vascular prostheses [37].

Strategies to Increase Collateral Formation
In most vascular beds, the circulation adapts to high-grade stenoses of major vessels by forming collateral vessels, essentially bypassing the lesion. Although these vessels do not have the same ability to dilate quickly in response to metabolic need, they are often capable of maintaining end-organ function and viability. In occlusive disease of the superficial femoral artery, the profunda femoris and geniculate arteries compensate by increasing in size and provide a natural bypass of the blockage. An exercise program can increase the distance walked before onset of ischemic leg pain, presumably by recruitment and enlargement of existing collaterals, possibly the result of local increase in the pressure gradient across capillary beds, as well as increasing the size and number of new collateral pathways in response to distal tissue hypoxia. In this way, the development of collateral vessels probably involves a combination of vasculogenesis and angiogenesis.

The therapeutic effect of increased VEGF availability has been studied in animal models and humans with limb ischemia, and a large body of this work has been reported by Dr Jeffrey Isner and colleagues in Boston. This group demonstrated that in rabbit hindlimbs rendered chronically ischemic by surgical excision of external iliac and femoral arteries, an increase in collateral vessel development occurred in response to intramuscular administration of VEGF into the affected limb, which was associated with an increase in capillary density and calf systolic blood pressure compared with controls. It was subsequently shown that local intraarterial and peripheral intravenous administration of VEGF had comparable effects [38]. A clinical trial using VEGF gene therapy in patients with ischemic limbs not amenable to surgical revascularization is currently being undertaken. An early report documented the successful transfection of DNA encoding human VEGF165 within a plasmid into a disease-free segment of popliteal artery through a balloon angioplasty device. Preliminary results suggested that enough VEGF was expressed to cause an increase in collateral formation within 4 weeks of the procedure. Leg swelling, consistent with a VEGF-mediated increase in capillary permeability, and cutaneous angiomas were associated with this treatment, and there was no intimal thickening evident at the site of transfection [39].

In this approach, a cytomegalovirus promoter was used to drive VEGF expression by cells that incorporated the plasmid, presumably endothelial and VSMCs. No viral vector was used in this experiment—the naked DNA was physically transferred on the surface of an angioplasty balloon, and significant expression was achieved by increasing the dose of plasmid DNA that was transfected. Expression can be expected to have lasted for up to 3 weeks and is terminated by destruction of the plasmid DNA. High doses have been associated with resolution of ischemic pain in a limited number of patients, and higher doses are currently being tried. It is probable that similar results can be obtained by intramuscular administration of naked DNA directly into ischemic muscle, using the capacity of postischemic regenerative skeletal myocytes to easily incorporate foreign DNA [40].

Vascular endothelial growth factor-dependent neovascularization and collateral formation compensates for occlusive disease affecting the coronary arteries. Ischemia and hypoxia enhance expression of angiogenic peptides in chronically ischemic myocardial tissue [41]. Furthermore, in a rat model of myocardial infarction, Li and colleagues [42] demonstrated an initial dramatic rise in VEGF expression, paralleled by increased expression of VEGF-R1 and R2, throughout the myocardium. Subsequently, expression of VEGF and its receptors was limited to areas around the infarct, a response that lasted for up to 6 weeks after infarction. Extraluminal administration of VEGF has been shown to improve coronary flow and myocardial function in a porcine model of gradual circumflex coronary arterial occlusion [43]. In a similar model, direct coronary injection of VEGF also improved myocardial blood flow, but was associated with profound—and sometimes fatal—hypotension [44]. This fall in blood pressure was associated with a significant decrease in peripheral resistance, and has been shown by several groups to be caused by endothelium-dependent relaxation. Competitive inhibitors of NO production such as NG-monomethyl-L-arginine prevent these effects, which occur both in chronically ischemic and normal myocardium [44, 45]. Intracoronary injection of bFGF does not have a discernible haemodynamic effect [46].

It remains to be seen whether continuous release of VEGF or bFGF, possibly using gene therapy, is more or less efficacious than intraarterial or systemic administration. Mild marrow and renal toxicity are observed with high-dose intraarterial administration of bFGF [47, 48]. Lazarous and associates [49] demonstrated mild anemia and thrombocytopenia in dogs treated with bFGF for 9 weeks, but noted no adverse effects attributable to VEGF. At the present time, the cost of recombinant peptides suitable for use in humans is prohibitive and would preclude their use in clinical practice.

Interactions Between Nitric Oxide and VEGF
Nitric oxide directly influences angiogenesis and its effects are related to the amount generated as well as the context in which it is produced. Nitric oxide stimulates migration and proliferation of coronary venular endothelial cells, and the mitogenic effect of VEGF on endothelial cells occurs partly by this mechanism [50]. Constitutive (basal) NO production is enhanced by shear stress, and Morbidelli and colleagues [50] suggest that NO may couple local physical effects of low-flow situations with VEGF-mediated angiogenesis. This coupling may work in the opposite direction, as demonstrated by the sudden increase in NO production and resulting hypotension during administration of intracoronary VEGF as discussed in the previous section. Reactive oxygen intermediates also cause an increase in VEGF expression [51], and these agents enhance VEGF production during reperfusion after ischemia. Nitric oxide may facilitate angiogenesis by increasing vascular permeability during an inflammatory response, yet higher concentrations of NO, produced by the inducible isoform of NO synthase after stimulation with endotoxin, actually modulate VEGF gene expression [52]. It is not known to what extent VEGF-mediated vasodilator activity in existing vessels explains improvement in limb perfusion or coronary flow seen with supplemental VEGF, compared with improvements derived from new vessel formation.


   Malignant Disease
Top
 Footnotes
 Abstract
 Introduction
 Occlusive Vascular Disease and...
 Malignant Disease
Summary and Future Directions
 Acknowledgments
 References
 
A significant increase in cell number in vivo depends on a commensurate increase in blood supply. Many tumor cell lines have been demonstrated to produce VEGF during normal growth in vitro, and this may facilitate tumor vascularization in vivo allowing further growth. In the following section the effects of tumor-derived VEGF, regulation of expression, and prognostic implications are reviewed. Interactions between the angiogenic potential of primary and secondary tumor deposits are discussed, as well as basic research examining the effects of modulating angiogenesis on tumorigenicity.

Production of Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor by Tumors
Vascular endothelial growth factor and bFGF production is a characteristic of many human tumors, including carcinoma of the lung [53–55]. Production is detectable by direct measurement in urine and plasma and tumor-specific expression has been confirmed by Northern analysis of explanted tumors.

Angiogenesis involves a net balance of positive versus negative regulators of vessel growth. There is evidence that this process is, in part, genetically mediated. Mutant ras and raf protooncogenes upregulate VEGF expression [56], and oncogenes such as Src enhance VEGF expression, whereas tumor suppressor genes such as p53 inhibit expression [57], probably by enhancing production of thrombospondin, an inhibitor of angiogenesis [58]. Mazure and associates [59] recently suggested that the ras component of the signal transduction pathway, which is often activated during malignancy, enhances hypoxic induction of VEGF expression occurring in response to changes in the tumor microenvironment.

Tumors do not outgrow their blood supply as was previously thought. Areas of central necrosis evident in advanced tumors actually arise because of excess VEGF production. The VEGF-mediated increase in permeability of existing and immature vessels is responsible for the development of intersitital edema that eventually arrests flow in the microcirculation because of extrinsic compression [17].

The Role of Angiogenesis in Tumor Dissemination and the Development of Metastatic Deposits
Blood vessels within primary tumors deliver oxygen and nutrients required for tumor growth, and probably also provide a route through which cells metastasize. Vascular endothelial growth factor may promote dissemination by increasing vessel permeability and allowing egress of cells into the circulation, and this mechanism is independent of primary tumor growth [60]. Whereas dissemination of tumor cells may occur long before the onset of clinically evident metastatic disease, peripheral deposits only become biologically significant when they develop a blood supply. Small tumor masses, perhaps as small as 1 mm3, may lie dormant, relying on diffusion of oxygen and nutrients for survival. The histologic correlate of this period is carcinoma in situ as occurs in breast and other malignancies. Folkman [17] suggests that neovascularization is the principal event in the development of these so-called prevascular deposits, involving a switch to an angiogenic phenotype. Cell-mediated immunity, hormones, and elaboration of antiangiogenic cytokines by lymphocytes may perpetuate this prevascular phase. Somewhat paradoxically, a primary tumor may also prolong dormancy by producing inhibitors of angiogenesis, such as angiostatin [61]. Angiogenic cytokines may also alter the immune response by modulating the expression of adhesion molecules on tumor endothelium [62].

Vascular Endothelial Growth Factor and Malignant Cell Growth
Vascular endothelial growth factor promotes growth of malignant cells in several ways: by enhancing delivery of oxygen and nutrients through new vessel formation; by enriching the extracellular matrix through increased vessel permeability; and, in some circumstances, by a direct receptor-mediated effect. Intuitively, a generous blood supply is likely to facilitate growth of a tumor, and a correlation between vascularity and poor clinical prognosis is evident for a number of tumors including carcinoma of the lung [63].

In vitro, nearly 80% of non–small cell lung carcinoma lines express VEGF [54]. Ohta and colleagues [53] described similar levels of expression in human lung tumors of all histologic types and a dramatic decrease in median survival for patients with high-level expression of VEGF121, with a 5-year survival of 17% versus 78% for those with low-level expression following curative resection for stage I disease. A recent study examining pulmonary adenocarcinoma also suggests that VEGF and bFGF expression is of prognostic significance [55]. Reinforcing the relevance of this finding, melanoma cells transfected with a VEGF complementary DNA shown to overexpress VEGF are seen to promote tumor vascularization and cause an increase in tumor size and the number of lung metastases. In the same cells transfected with vector alone, these changes were not observed [64].

The second proposed means by which VEGF enhances malignant cell growth is through the enrichment of tumor extracellular matrix, caused by increased vessel permeability. This is reviewed in detail by Senger and colleagues [65], who describe the expression of VEGF close to and within hyperpermeable blood vessels. By contrast, little expression of VEGF and its receptors is observed in nonmalignant tissues surrounding a tumor [3]. Unlike the content of plasma exudate enriching the extracellular space during normal angiogenesis, fibrin deposition features prominently in tumor stroma. This provides a structural framework for the development of new tissue and is noted on histology of malignant tumors [65]. The development of a matrix to facilitate tumor growth is analogous to the way in which wound healing takes place, and VEGF is a key mediator of both processes.

Tumors may respond directly to VEGF though expression of VEGF receptors. Supraphysiologic concentrations of VEGF (2 ng/mL) have been shown to enhance proliferation of cultured choriocarcinoma cells, an effect that was blocked by VEGF-neutralizing monoclonal antibodies. The choriocarcinoma cells used in this experiment expressed VEGF-R1 and -R2, and the growth-promoting effect of VEGF was associated with phosphorylation of several proteins in the receptor tyrosine kinase cascade, including MAP kinase [66]. Vascular endothelial growth factor (10 ng/mL) added to cultures of melanoma cells bearing VEGF-R2 receptors has also been shown to increase the rate of cell replication. Because these melanoma cells were also shown to produce VEGF there is the likelihood that VEGF is involved in paracrine, and probably autocrine, stimulation of melanoma growth in vitro [67].

The upregulation of angiogenesis associated with cell proliferation may be associated with a slower rate of programmed cell death, possibly through a direct effect of angiogenic peptides such as VEGF. Inhibitors of angiogenesis are thought to control growth of metastatic deposits by enhancing apoptosis in tumor cells [68]. Conversely, expression of VEGF and its receptors may decrease the rate of apoptosis and confer a survival advantage to tumor cells [69].

Inhibition of Angiogenesis
Inhibition of angiogenic activity is a logical therapeutic target and may provide a means of shrinking primary tumors and diminishing metastatic spread. Antiangiogenic agents are being tested in trials for treatment of childhood hemangiomas and benign conditions such as diabetic ocular neovascularization, arthritis, psoriasis, and duodenal ulceration [17]. Research into neoplasia-driven angiogenesis is directed toward establishing mechanisms and significance of VEGF expression. Widely used tools include neutralizing monoclonal antibodies and transfection of VEGF and VEGF receptor antisense constructs, inhibiting the transcription of DNA to RNA.

In 1993 it was reported that monoclonal antibodies to VEGF inhibited the growth of several tumor cell lines in vivo [70] but have no effect on cell growth in vitro. Although increased expression of VEGF sometimes acts directly to cause growth promotion, neutralization of VEGF using monoclonal antibodies appears to have no discernible effect in vitro. This effect may relate to differences in the amount of constitutive VEGF expression, as different cell lines were used and anti-VEGF monoclonal antibodies can be expected to have little effect where there is minimal constitutive expression. Alternatively, the disparity may indicate that VEGF mediates growth promotion predominantly through its angiogenic activity (not evident in isolated cell cultures) with little direct effect on malignant cell proliferation. Tumor dissemination, as well as the growth of primary lesions, are inhibited by anti-VEGF monoclonal antibodies [71].

Antisense VEGF constructs also alter the angiogenicity and tumorigenicity of human tumors. Glioblastoma cells expressing the construct produce less VEGF, are unable to induce proliferation of endothelial cells, and do not proliferate in vivo [72]. Similar results were obtained using C6 glioma cells expressing an antisense construct in vivo [73]. Expression of a construct that modifies VEGF-R1 to effect loss of the kinase domain, thereby preventing signal transmission, also inhibits growth [74], and other antireceptor strategies are being investigated for use in cancer treatment [75].

The utility of thrombospondin and angiostatin, naturally occurring antiangiogenic substances, is currently being investigated. Thrombospondin is produced by normal cells but synthesis is massively downregulated during tumor development [76]. Gene therapy aimed at transfection of a thrombospondin cDNA into a human breast carcinoma cell line has demonstrated a reduction in angiogenesis, primary tumor growth, and development of metastases in an animal model [77]. Angiostatin arrests the development of new blood vessels by blocking endothelial proliferation, and is a naturally occurring substance, sometimes produced by primary tumors. Treatment of tumor-bearing mice with this agent can arrest the growth of well-vascularized lesions and cause regression to the point of dormancy, in which the rate of proliferation is balanced by the rate of apoptosis [78]. Initial experiments included human colon, breast, and prostate cancers implanted into immunodeficient mice, and large deposits were reduced to microscopic foci with no apparent side effects. Interestingly, angiostatin disappears from the circulation after resection of human tumors, raising the prospect that it may be largely responsible for suppression of metastatic deposits during primary tumor growth [79]. Supplemental angiostatin may be of use in preventing the progression of tumor deposits from dormancy to vascularized tumors after surgical removal of the primary lesion. Other antiangiogenic agents such as the synthetic analogue of fumagillin derivative AGM-470 (TNP-470), thalidomide, and interleukin-12 are currently being investigated in human trials [17].


   Summary and Future Directions
Top
 Footnotes
 Abstract
 Introduction
 Occlusive Vascular Disease and...
 Malignant Disease
 Summary and Future Directions
Acknowledgments
 References
 
The development of collateral vessels supplying tissue beyond stenosed arterial segments is enhanced by angiogenic agents, and delivery strategies for molecules such as VEGF, including transfection of naked DNA, are nearing clinical application. The functional capacity of new vessels formed by angiogenic agents is yet to be fully defined, and a compound that enhances vasculogenesis (de novo formation) as well as angiogenesis (new vessels from existing capillary beds) is likely to be of greater benefit in coronary and peripheral vasculature affected by occlusive disease. Vascular endothelial growth factor therapy may also be used to restore a functional endothelial monolayer after interventions such as balloon angioplasty and endoluminal stenting, and this is likely to have significant benefit. Concerns exist over the potential pro-proliferative effect of bFGF, and to a lesser extent VEGF, on other cell types that may accelerate intimal thickening because of VSMC proliferation. The clinical relevance of plaque neovascularization in the natural history of atherosclerosis is highlighted by current angiogenesis research. Debate regarding the significance of adventitial neovascularity in diseased vessels may be clarified by ongoing investigations pointing toward a primary role for adventitial cells in the elaboration of growth factors and development of intimal thickening.

Although new vessel formation is sought to combat occlusive vascular disease, it is also recognized that an adequate blood supply is a fundamental requirement for tumor growth, raising the prospect that antiangiogenic therapy may slow disease progression. The regulation of new vessel formation is a dynamic equilibrium between metabolic requirements of the tumor mass, apoptosis induced by various inhibitors of tumor cell growth, and immune-mediated events. Expression of factors such as VEGF may facilitate tumor growth, not just by inducing vessel formation, but through paracrine and autocrine mechanisms. The integration of antiangiogenic treatments into contemporary clinical oncology is likely in the near future, with the evolution of therapies causing regression by selective apoptosis and maintenance of metastatic deposits in a dormant phase. Should treatments of this nature prove effective in human hosts, they are likely to complement existing surgical practice by inhibiting the development of metastatic disease.


   Acknowledgments
Top
 Footnotes
 Abstract
 Introduction
 Occlusive Vascular Disease and...
 Malignant Disease
 Summary and Future Directions
 Acknowledgments
References
 
I thank Professor Michael Appleberg (Vascular Surgery), Professor Bruce Robinson, and Dr Jan Zedenius (Molecular Genetics) for their critical review; Mr Jeremy Hogan (Molecular Genetics) for assistance in preparation of the manuscript; and Dr Andrew Wilks (Ludwig Institute for Cancer Research, Melbourne) and Dr Mary Saleh (Department of Surgery, Royal Melbourne Hospital, Melbourne, Australia) for useful discussion.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Occlusive Vascular Disease and...
 Malignant Disease
 Summary and Future Directions
 Acknowledgments
 References
 
Address reprint requests to Dr Winlaw, Department of Cardiothoracic Surgery, St Vincent's Hospital, Victoria St, Darlinghurst, NSW 2010, Australia (e-mail:dwinlaw{at}wr.com.au).


   References
Top
 Footnotes
 Abstract
 Introduction
 Occlusive Vascular Disease and...
 Malignant Disease
 Summary and Future Directions
 Acknowledgments
 References
 

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