--…… REVIEW
`
`Promoting the formation of new collateral vessels in ischemic tissues using angiogenic growth factors (therapeutic
`angiogenesis) is a an exciting frontier of cardiovascular medicine. Conversely, inhibition of the action of key
`regulators of angiogenesis, such as VEGF, constitutes a promising approach for the treatment of solid tumors and
`intraocular neovascular. syndromes. These concepts are being tested now in clinical trials.
`
`Clinical applications of angiogenic growth factors
`and their inhibitors
`
`NAPOLEON£ FERRARA1 &
`KARI ALITAL02
`
`In embryos, blood vessels form through
`two distinct processes, vasculogenesis
`in(cid:173)
`and angiogenesis. Vasculogenesis
`valves the de novo differentiation of en-
`in
`from mesodermal precursors, whereas
`dothelial cells
`angiogenesis new vessels are generated from pre-existing ones1.
`Vasculogenesis takes place only during embryonic develop(cid:173)
`men t and leads to the formation of a primary vascular plexus.
`Later these rather uniformly sized endothelial channels are re(cid:173)
`modeled into a mature system consisting of a tree-like hierar(cid:173)
`chy of large and small vessels . New capillaries then form
`I through angiogenesis, either by sprouting or by splitting (in(cid:173)
`I tus susc빡ion) from their vessels of origin. In adults, angiogen(cid:173)
`esis is essential for the female reproductive cycle, and for repair,
`remodeling and regeneration of tissues, for exa~ple during
`wound healing2. Neovascularization is also important in patho(cid:173)
`logical processes such as tumor growth and metastasis2.
`The known endothelial cell specific growth factors and theu
`receptors can be classified into vascular endothelial growth fac(cid:173)
`tor (VEGF) and angiopoietin (Ang) families3 (Fig. 1).Among the
`various angiogenic factors, VEGF is probably the most essen(cid:173)
`tial for the development and differentiation of the vascular
`system4. Loss of a single VEGF allele results in embryonic
`leth ality5·6 (Fig. 2) . Even selective inactivation of the heparin(cid:173)
`binding isoforms of VEGF, leaving one functional isoform
`(VEGF120), is insufficient for the proper development of the
`cardiovascular system and results in myocardial ischemia and
`perinatal or early postnatal lethality7. Also, other angiogenic
`fa ctors, such as FGFs may work more indirectly, some of them
`through the VEGFs and their receptors8, so that a thorough
`knowledge of the signal transduction pathways of VEGFs and
`angiopoietins is essential for their use in therapeutic settings.
`
`Therapeutic angiogenesis and inhibition of arterial restenosis
`An exciting frontier of cardiovascular medicine is therapeutic
`angiogenesis. Promoting the formation of new collateral ves(cid:173)
`sels on the ischemic myocardium, leg muscles and other tissues
`would have an important effect on the treatment of disorders
`for which pharmacological intervention has been ineffective in
`controlled trials and for which therapy is now limited to surgi(cid:173)
`cal revascularization or endovascular interventional therapy9 .
`Several angiogenic molecules have been tested in animal
`models, including bFGF, aFGF, FGF-5, VEGF isoforms, VEGF(cid:173)
`C, HGF/SF and Ang-l/Ang-2. The factors tested most exten(cid:173)
`sively are VEGF and bFGF . In some cases, the recombinant
`protein was tested. In others, gene transfer using naked DNA
`or adenoviral vectors was used. A single intra-arterial adminis(cid:173)
`tration of 500-1000 µg of rhVEGF165 augmented perfusion and
`in a rabbit model of
`development of collateral vessels
`hindlimb ischemia in which the femoral artery was surgically
`removed10. Similar results were obtained in the same model
`
`NATURE MEI기 CINE • VOLUME 5 • NUMBER 12 • DECEMBER 1999
`
`with intramuscular or intra-arterial ad(cid:173)
`ministration of aFGF, bFGF, HGF/SF and
`VEGF-C (refs. 11-14). VEGF administra-
`tion after removal of the femoral artery
`not only resulted in increased vascularization but also led to
`recovery of the normal endothelial reactivity to various media(cid:173)
`tors15. Arterial gene transfer with cDNA encoding VEGF iso(cid:173)
`forms also led to revascularization to an extent comparable to
`that achieved with the recombinant protein 16. Moreover, ad(cid:173)
`ministration of a VEGF165 adenovirus vector shortly after com(cid:173)
`mon iliac artery ligation in the rat was capable of stimulating
`an angiogenic response that protects against subsequent oc(cid:173)
`clusion of the femoral artery, indicating that gene transfer of
`VEGF might be useful in the prophylaxis of advancing arterial
`occlusive disease1 7. As little as 2 µg rhVEGF delivered over 4
`weeks periadventitially, distal to the occlusion, resulted in a
`significant increase in coronary blood flow and functional im(cid:173)
`provement in a pig model of chronic myocardial ischemia18.
`Very similar results were obtained using bFGF (ref. 19).
`Unexpectedly, even a single intracoronary administration of
`VEGF (or bFGF) was efficacious in this model to an extent
`comparable to that of 4-week infusion, despite the fact that
`only a small fraction of protein localizes to the ischemic area20.
`Given such results, it is conceivable that young and otherwise
`healthy animals are very responsive to exogenous growth fac(cid:173)
`tors in the context of ischemia. At least some of this respon(cid:173)
`siveness may be due to the upregulation of VEGF receptors in
`the endothelia of ischemic tissues21 . Adenovirus-mediated
`gene transfer of VEGF121 (ref. 22) or FGF-5 (ref. 23) also resulted
`in collateral vessel growth and functional improvement in
`porcine models of myocardial ischemia.
`These encouraging animal studies led to clinical trials using
`recombinant VEGF165, aFGF, bFGF or gene therapy with plas(cid:173)
`mid or with adenoviral vectors. There is considerable debate
`whether gene therapy or administration of recombinant pro(cid:173)
`tein would be preferable. Delivery of angiogenic proteins by
`gene therapy might not only minimize their systemic side ef(cid:173)
`fects, such as hypotension (VEGF) or nephrotoxicity (bFGF),
`but also provide a slow release of the encoded factor for 1-2
`lasting angiogenic response.
`to a more
`leading
`weeks,
`However, slow release of the recombinant protein, using mi(cid:173)
`crospheres or heparin-alginate formulations, might achieve
`the same results, without the potential risks associated with
`the use of viral vectors.
`Arterial gene transfer of naked plasmid DNA encoding
`VEGF1 65 in a patient with severe limb ischemia produced angio(cid:173)
`graphic and histologic evidence of angiogenesis in the knee,
`mid-tibial and ankle levels 4 weeks after the transfer24 . In a sub(cid:173)
`sequent study, the VEGF165 plasmid cDNA was injected intra(cid:173)
`muscularly25. Gene transfer was done in ten limbs of nine
`patients with nonhealing ischemic ulcers and/or rest pain due
`
`1359
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`wall was not very efficient35, but secreted proteins such as VEGF
`could be used for therapeutic gene transfer trials using infu(cid:173)
`sion-perfusion catheters36 or histamine-induced increase of en(cid:173)
`dothelial permeability37. Because VEGF and VEGF-C share one
`receptor (VEGFR-2) but differ in the other receptor, VEGF-C
`and VEGF1 65 might have overlapping but distinct effects in the
`vessel wall. However, VEGF-C gene transfer inhibits intimal
`thickening early, and the protective effect is at least equal to
`that seen with VEGF165 gene transfer38.
`
`inhibition of vascular endothelial growth factor
`
`Therapeuti[
`Tumors
`The growth of tumor xenografts in transparent chambers in
`mice is preceded by an increase in vascular density, indicating
`that the rapid growth of tumors depends on the development
`of a neovascular supply39. In 1971, inhibition of angiogen esis
`was proposed as a valid strategy for the treatment of solid tu(cid:173)
`mors and the search for the mediator(s) of tumor angiogen esi s
`was begun얘
`Although inhibition of bFGF (ref. 41) or angiopoietin/Tie2
`(refs. 42,43) may inhibit tumor growth, so far VEGF and its re(cid:173)
`ceptors constitute the most extensively investigated system in
`tumor angiogenesis and are now a main target of anti-cancer
`strategies. VEGF mRNA is substantially upregulated in most
`human tumors4. Although tumor cells represent the main
`source of VEGF, tumor-associated stroma is also an important
`site of VEGF production44. There is a correlation between VEGF
`expression and microvessel density in primary breast cancer
`sections45. A similar correlation has been described in several
`other malignancies,
`including
`gastric
`carcinoma46.
`Furthermore, there are increases in plasma levels of VEGF in
`tumor patients compared with tumor-free individuals, and
`high VEGF levels before chemotherapy are associated with a
`poor outcome'‘7
`Direct evidence for involvement of VEGF in tumorigenesis
`was first demonstrated using monoclonal antibodies against
`
`따 따
`
`다 /
`
`·%
`
`VE-
`Cadhenn
`
`따
`
`따
`
`νk
`
`따 順
`
`때
`
`Pll 14L
`
`Mig 「ation, permeability, DNA synthesis, survival
`
`∼、 ι- 「~r
`
`ang1ogenes1s
`
`Lymphang1ogenes1s
`
`REVIEW······
`
`to peripheral arterial disease. Improvement in the ankle(cid:173)
`brachial index and distal flow in eight limbs were reported25.
`Additional small trials by the same group have also shown that
`local injection of the VEGF1 65 plasmid DNA resulted in clinical
`improvement in patients affected by myocardial ischemia26 or
`Burger's disease (thromboangiitis obliterans)27. However, none
`of these studies were placebo-controlled. Clinical trials using
`VEGF-C naked DNA or adenovirus mediated gene transfer of
`VEGF1 21 in myocardial ischemia patients are now in phase I.
`Femoral angiograms from a patient with limb ischemia, before
`and 3 months after transfection of a VEGF1 65 plasmid/liposome
`expression vector, show increased vascular density after the
`treatment (Fig. 3). However, the trial is ongoing and some cau(cid:173)
`tion should be used in interpreting such data, until more pa(cid:173)
`tients and the effect of placebo are more extensively evaluated.
`Clinical trials using recombinant VEGF1 65 and bFGF are also
`ongoing. In a phase I study in patients with coronary ischemia
`in which rhVEGF1 65 was administered by intracoronary infu(cid:173)
`sion, the molecule was safely tolerated at all doses tested28.
`There was evidence of improvement in perfµsion in seven of
`fifteen subjects and improved collateralization in five of seven
`who underwent follow-up coronary angiography. However, a
`subsequent placebo-controlled phase II study,
`in which
`rhVEGF was delivered as a single intracoronary infusion, fol(cid:173)
`lowed by three intravenous infusions, has not demonstrated
`clinical benefit29. The treatment was not better than placebo in
`treadmill time and pain relief, at least at 60 days29. Brief expo(cid:173)
`sures to rh VEGF1 65, such as those achieved in this trial, may be
`insufficient to trigger and maintain a therapeutically meaning(cid:173)
`ful angiogenic response, especially in the context of extensive
`atherosclerotic disease. Also, systemic administration of
`rhVEGF1 65 or other factor may fail to generate an appropriate
`angiogenic concentration gradient from ischemic to non-is(cid:173)
`chemic areas, a requisite aspect of angiogenesis in a variety of
`physiological and path이ogical circumstances1. Moreover, the
`placebo effect is probably greater than initially suspected, and
`even patients with very compromised myocar-
`dial function may show a substantial improve(cid:173)
`ment with placebo. A phase II study with bFGF
`for coronary ischemia is now ongoing.
`Local gene transfer into the vascular wall of(cid:173)
`fers a promising alternative for the treatment
`of the complication of restenosis after percuta(cid:173)
`neous transcoronary angioplasty (PTCA) and
`coronary stenting. Restenosis occurs in many
`treated patients in 6 months, leading to ob-
`struction in 20-35% of the patients30. The
`pathogenesis of restenosis depends on en-
`dothelial damage, which also predisposes ar-
`teries to other pathological conditions, such as
`spasms or thrombosis. Prophylaxis of resteno-
`sis could therefore be based on strategies for
`endothelial protection or enhancement of en-
`dothelial repair and endothelial growth factors
`or vascular gene transfer could be used for
`this31. Re-endothelization in balloon-injured
`rat carotid artery was accelerated by a single
`dose of recombinant VEGF injected into the
`bloodstream or locally32·33. Vessel status was
`also improved by injection of VEGF plasmid
`into adventitial surface of rabbit carotid arter(cid:173)
`ies34. Intravascular gene transfer in the arterial
`
`g 잉R 淑
`
`Fig. 1 VEGFs, thei 「 「eceptors and some of their endothelial effects in cells and tissues.
`Ligand binding induces recepto 「 dime「ization and subsequent auto/transphosphorylation,
`activates various signal transduction pathways and leads to differential cellular responses.
`sVEGFR-1, soluble VEGFR-1; HSPG, heparan sulphate proteoglycan; NP-1, neuropilin-1 ; avbi,
`integrin avb3 (reported to make a molecular complex with activated VEGFR-2; ref. 95). VE(cid:173)
`cadherin is also able to form a complex with VEGFR-2, a requirement for VEGF-dependen~
`anti-apoptotic signals involving the Pl3-kinase/Akt pathway96. Pl l 14L, point mutation 01
`VEGFR-3 affecting patients in a family with lymphoedema97
`
`1360
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`,.:---
`
`Fig. 2 Yolk sac of El 0.5 VEGF+i+ and VEGF +/- mouse embryos5. There is
`an apparent absence of vasculature in the yolk sac of the heterozygous,
`wh ich die around Ell. This is probably the only example among verte(cid:173)
`brates of lethality after inactivation of a single allele of a gene that is not
`maternally imprinted
`
`VEGF in human xenografts in nude mice48. These initial studies
`: showed that several tumor cell lines can be substantially
`I growth-inhibited by this treatment샌 These findings were ex(cid:173)
`tended to a broad variety of tumor cell lines, including carcino(cid:173)
`mas, sarcomas and gliomas4. Intravital videomicroscopy
`techniques have augmented our understanding of VEGF in tu(cid:173)
`imaging of the vasculature
`morigenesis49·50. Non-invasive
`dem onstrated a nearly complete suppression of tumor-associ(cid:173)
`ated angiogenesis in animals treated with monoclonal antibod(cid:173)
`ies against VEGF compared with controls, providing a direct
`verification that inhibition of angiogenesis is the mechanism
`of tumor suppression after anti-VEGF treatment49. Intravital
`microscopy techniques have also been used to investigate the
`effects of VEGF on the permeability and other properties of
`tumor vessels50. Treatment with antibodies against VEGF re(cid:173)
`sulted in time-dependent reductions in vascular permeability,
`in the diameter and tortuosity and eventually to a regression of
`tumor blood vessels; thus, VEGF is also an essential survival fac(cid:173)
`tor for tumor endothelial cells50. Further evidence that VEGF
`action is required for tumor angiogenesis has been provided by
`the finding that retrovirus-mediated expression of a dominant
`negative VEGFR-2 mutant, which inhibits signal transduction
`through wild-type VEGFR-2 receptor, suppresses the growth of
`glioblastoma multiforme as well as other tumor cell lines in
`vivo51. Furthermore, high local expression of the soluble extra(cid:173)
`cellular domain of VEGFR-1 or VEGFR-2, achieved by adminis(cid:173)
`tration of the recombinant proteins, adenoviral-mediated gene
`transfer or by stable transfection of tumor cells, may signifi(cid:173)
`cantly inhibit tumor growth, metastasis and mortality rate in
`nude mice52·53.
`Several strategies have been used to generate VEGF inhibitors
`suitable for clinical trials. One approach involves the ‘human(cid:173)
`ization’ of mouse monoclonal antibodies. A chief advantage of
`‘humanized' antibodies is a high degree of specificity, com(cid:173)
`bin ed with a long half-life and little or no immunogenicity. A
`1humanized’ high-affinity monoclonal antibody against VEGF
`(rhuMAb VEGF) with the same affinity and biological proper-
`ties as the original murine antibody has been described54.
`Toxicological studies in primates have shown that the effects of
`rh uMAb VEGF are limited to inhibition of angiogenesis in the
`female reproductive tract and in the epiphyseal growth plate in
`
`NATURE MEI기〔 INE • VOLUME 5 • NUMBER 12 • DECEMBER 1999
`
`·····REVIEW
`
`sexually immature animals that have not completed statural
`growth55. rhuMAb VEGF is now in phase II clinical trials for the
`treatment of non-small cell lung carcinoma and colorectal car(cid:173)
`cinoma in conjunction with standard chemotherapy and for
`breast and renal cell carcinoma as a single agent. In addition,
`small molecules that inhibit VEGFR-2 signal transduction are
`in cancer patients56.
`trials
`undergoing phase II clinical
`Furthermore, monoclonal antibodies against VEGFR-2 are en(cid:173)
`tering clinical trials.
`
`Retinal ischemia and other conditions
`Diabetes mellitus, occlusion of the central retinal vein or pre(cid:173)
`maturity with subsequent exposure to oxygen can all be associ(cid:173)
`intraocular neovascularization57. A common
`ated with
`denominator among these conditions is retinal ischemia57. The
`new blood vessels may lead to vitreous hemorrhage, retinal de(cid:173)
`tachment, neovascular glaucoma, and eventual blindness.
`Diabetic retinopathy is the leading cause of blindness in the
`working population. The hypothesis that ischemia-induced
`VEGF may be pathogenic in these conditions was initially
`tested by measuring VEGF levels in the eye fluids of patients. In
`a large series with 165 patients, a strong correlation was found
`between concentrations of VEGF in both aqueous and vitreous
`and active proliferative retinopathy associated with diabetes,
`occlusion of central retinal vein or prematurity58. Direct evi(cid:173)
`dence for the role of VEGF as a mediator of intraocular neovas(cid:173)
`cularization has been generated in several animal models,
`including a primate model of iris neovascularization and a
`mouse model of retinopathy of prematurity. In the former, in(cid:173)
`traocular administration of monoclonal antibodies against
`VEGF substantially inhibits the neovascularization that follows
`the occlusion of central retinal veins59. Likewise, soluble
`VEGFR-1 or VEGFR-2 extracellular domains fused to the im(cid:173)
`munoglobulin y Fc domain suppress retinal angiogenesis in the
`that growth
`also evidence
`is
`mouse model60. There
`hormone/insulin-like growth factor-1 is involved in ischemia(cid:173)
`induced retinal neovascularization61.
`Neovascularization is a principal cause of visual loss also in
`the wet form of age-related macular degeneration (AMD), the
`overall leading cause of blindness62. Several studies have docu(cid:173)
`mented the immunohistochemical localization of VEGF in sur(cid:173)
`gically resected choroidal neovascular membranes from AMD
`patients63. These findings suggest involvement of VEGF in the
`progression of AMD-related choroidal neovascularization.
`Anti-VEGF strategies for AMD are now being explored in clini(cid:173)
`cal trials. One approach consists in the intravitreal administra(cid:173)
`tion of a recombinant humanized anti-VEGF Fab antibody
`fragment. Another strategy involves the injection of 2’-fluo(cid:173)
`ropyrimidine RNA 이igonucleotide ligands (aptamers)64.
`VEGF inhibition may also have therapeutic value for the
`treatment of ischemic-reperfusion related brain edema and in(cid:173)
`jury. VEGF antagonism has shown beneficial effects in a mouse
`model of cortical ischemia65; reducing acutely the volume of
`edematous tissue and resulting in a significant sparing of corti(cid:173)
`cal tissue.
`VEGF is important in angiogenesis in the female reproduc(cid:173)
`tive tract. VEGF inhibition results in suppression of corpus lu(cid:173)
`teum angiogenesis in rodents66 and primates55. VEGF inhibitors
`might be used to treat conditions characterized by ovarianhy(cid:173)
`perplasia and hypervascularity, such as the polycystic ovary
`syndrome66. VEGF-dependent angiogenesis may also be impor(cid:173)
`tant pathogenically in endometriosis. Furthermore, VEGF is a
`
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`REVIEW
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`...... ..
`
`mediator of the ovarian growth and increased vascular perme(cid:173)
`ability of ovarian hyperstimulation syndrome, a potentially
`fatal condition characterized by massive ovarian enlargement
`that may follow medical induction of ovulation with go(cid:173)
`nadotropins67.
`
`Perspectives
`VEGF165 binds to neuropilin-1, which functions as a ligand
`binding subunit of putative transmembrane receptors mediat(cid:173)
`ing specific signals for different semaphorins, the molecules
`mediating the collapse of axonal growth cones68. Neuropilin is
`expressed in endothelial cells and enhances the mitogenic ef(cid:173)
`fects of VEGFR-2 upon VEGF165 stimulation. Thus, there may be
`an as-yet ill-defined cross-regulation of cellular signals between
`these two families of factors. These findings lead to the intrigu(cid:173)
`ing conclusion that the processes of axon guidance and devel(cid:173)
`opment of a network of capillary tubes share at least some
`common molecular mechanisms. In addition, the angiopoietin
`receptor/Tie and ephrin families of endothelial tyrosine kinases
`have important functions in the formation and maintenance
`of the vascular system69.71. Endothelial cell-specific members of
`the TGF-~ receptor and Notch families have also been de(cid:173)
`scribed72·73. Given this complexity of vascular endothelial sig(cid:173)
`naling, therapies using VEGF alone or any other single
`angiogenic factor may produce incompletely functioning or
`unstable endothelial channels with defective arteriovenous
`and pericellular differentiation, characteristic of many tu(cid:173)
`mors74. Combinations of growth factors may be preferable in
`future therapies directed to neovascularization of tissues, with
`an adequate investment of the formed vessels with perien(cid:173)
`dothelial matrix and pericyte/smooth muscle cells. In fact, a
`more heterogenous set of genes coordinating angiogenic func(cid:173)
`tions may be provided by active ongoing research of hypoxia(cid:173)
`regulated gene expression in mammalian cells75. Also, some
`virus-encoded proteins, such as the VEGFR-2 activating HIV
`Tat protein76, Kaposi sarcoma herpesvirus-associated G-protein(cid:173)
`coupled receptor77 or Orf virus encoded VEGF-£78-80 may offer
`new insights into the mechanism of regulation of angiogenesis.
`Although recent research has focused on the combination of
`VEGF and Ang-1 as being especially promising, it is not known
`now which growth factor combinations will prove to be the
`most effective therapeutically. VEGF and bFGF have a very
`synergistic effect in the induction of angiogenesis, both in vitro
`and in vivo4. The interaction between VEGF and HGF/SF is also
`being actively investigated. Although transgenic expression of
`Ang-1 in the skin epidermis under the keratin (K)14 promoter
`has been associated with neovascularization81’ other studies,
`using defined amounts of the recombinant protein in a model
`of adult neovascularization, have failed to demonstrate strong
`angiogenic responses to Ang-1, unless it is used in combina(cid:173)
`tion with VEGF (refs. 71,82). This discrepancy may be ex(cid:173)
`plained by the fact that the expression of the K14 promoter is
`initiated already at midgestation, and thus the results may re(cid:173)
`flect persistence of the fetal neovascularization. It is possible,
`however, that Ang-1 may provide a co-factor for combination
`therapies. A further unresolved issue is the correct dosage of
`growth factor(s) . This seems particularly important for a mole(cid:173)
`cule like VEGF, which has several isoforms and such a tight
`dose-response effect that a 50% reduction in expression re(cid:173)
`sults in lethality during embryonic life5·6. Conversely, continu(cid:173)
`ous
`local overexpression of VEGF may
`result
`in a
`hemangioma-like vasculature and thus can be deleterious83.
`
`Also, it is unknown whether an angiogenic treatment m ay be
`sufficient to induce functional blood vessels for prolonged pe(cid:173)
`riods or will need to be re-administered periodically in order to
`maintain such vessels.
`A K14-driven VEGF-C transgene induced lymphangiogenesis
`but no angiogenesis in mouse skin84, and recombinant VEGF-C
`also stimulated lymphatic vessel hyperplasia in mature chick
`chorioallantoic membrane85. Thus, besides angiogenesis, it may
`also become possible to direct therapeutic lymphangiogenesis
`in patients, such as after evacuation of axillary lymph nodes in
`breast carcinoma surgery.
`Despite the potential redundancy of tumor angiogenesis fac(cid:173)
`tors, inhibition of VEGF alone seems sufficient to achieve con(cid:173)
`siderable tumor growth suppression in a wide variety of
`models. However, it remains to be established whether tumors
`are able to activate, after prolonged therapy, alternative angio(cid:173)
`genic pathways that might confer resistance to the treatment.
`These issues should be addressed in the current clinical trials
`with various VEGF inhibitors. A challenge now in anti-VEGF
`(and anti-angiogenic) therapy is devising appropriate and reli(cid:173)
`able markers to monitor tumor progression. There is consider(cid:173)
`able debate whether blood vessel count
`in biopsy
`specimens45·46 may provide a reliable indicator of response to
`the treatment. There are also efforts to identify surrogate end(cid:173)
`points, applying non-invasive approaches, such as magnetic
`resonance imaging86 .
`VEGF is not only a mitogen but also a potential survival fac(cid:173)
`tor for endothelial cells4. Such a 'maintenance’ function seems
`to be developmentally regulated, as it is very dependent on the
`age of the animal87. VEGF inactivation during early postnatal
`life, achieved by Cre-loxP-mediated inducible gene targeting of
`by administration of a soluble VEGFR-1 chimeric protein, re(cid:173)
`sults in regression of the vasculature, kidney failure and lethal(cid:173)
`ity87. However, in adult animals a similar treatment h as no
`effects on the existing vasculature. Therefore, a process of mat(cid:173)
`uration occurs in endothelial cells such that VEGF eventually is
`not essential for survival. This switch seems to take place in the
`mouse around the fourth postnatal week. Absence of pericyte
`
`Fig. 3 Angiography of the lower extremity of a patient with limb 15·
`chemia before (PRE) and 3 months afte「 (3 MO) the transfection of a
`VEG Fl 65 plasmid/liposome expression vector, showing strongly increaseu
`vascula 「 density after the treatment. Courtesy H. Manninen, P. Matsi, "·
`Makinen, M. Hilpelainen, M. Laitinen, E. Alhava and S. Yla-Herttuala, A. I.
`Virtanen Institute and Kuopio University Hospital (Kuopio, Finland).
`
`1362
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`… ·REVIEW
`
`~:erage in im
`dependence on VEGF (ref. 88). However, other evidence sug(cid:173)
`gests that the molecular/intracelullar nature of this switch may
`be more complex and mostly still to be determined87. In juve(cid:173)
`nile animals, VEGF is essential for endochondral bone forma(cid:173)
`tion and longitudinal growth89'55. In the fully developed
`anim al, VEGF may be required mainly for active angiogenic
`processes such as corpus luteum development or wound heal(cid:173)
`ing. Neverthless, VEGF may be important for endothelial
`I homeostasis in the adult in certain circumstances; for example
`during disease states. Indeed, prolonged VEGF inhibition failed
`to induce glomerular damage in normal primates55 or ro(cid:173)
`dents87·90, despite the strong constitutive expression of the
`VEGF mRNA in podocytes and other cell types in the adult kid(cid:173)
`ney4. However, administration of VEGF inhibitors to rats with
`mesangiopr이iterative nephritis results in impaired glomerular
`endothelial regeneration and increased endothelial cell death90.
`Some CD34+ hematopoietic progenitor cells mobilized by
`GM-CSF from human peripheral blood, bone marrow, fetal
`liver or umbilical cord blood were shown to express VEGFR-2
`on their surface91, and VEGFR-2 is expressed on human
`hematopoietic stem cells92. Endothelial progenitor cells expand
`and differentiate into endothelial cells after addition of bFGF
`and VEGF to the cultures, and they can thus be considered to
`provide endothelial progenitor cells91 93. The endothelial prog(cid:173)
`enitor cells from bone marrow may be mobilized using the stro(cid:173)
`mal-derived factor 1 chemokine, the GM-CSF cytokine or tissue
`hypoxia94. As these cells may be capable of participating in ac(cid:173)
`tive angiogenesis after entry into the circulatory system94, they
`provide an interesting possibility for the delivery of cellular or
`gene therapy to sites of neovascularization.
`Finally, the first placebo-controlled clinical study with
`rhVEGF may have brought a more realistic assessment of the
`potential of therapeutic angiogenesis and raised a number of
`questions. For example, how can one explain the discrepancy
`between the considerable efficacy observed even with very
`small amounts of growth factors in animal models of coronary
`or limb ischemia and the rather disappointing clinical results?
`An essential difference may lie in the fact that young and oth(cid:173)
`erwise healthy animals are able to mount an effective endoge(cid:173)
`nous angiogenic response that can be maximized by an
`additional stimulus provided by a recombinant protein or gene
`therapy. In contrast, patients with extensive atherosclerotic
`disease may have poor responses. It is possible, however, that a
`more persistent exposure to an individual growth factor or to a
`combination of growth factors may be effective. Clinical trials
`now ongoing should answer at least some of these questions
`over the next 2-3 years.
`
`1. Risau, W. Mechanisms of angiogenesis. Nature 386, 671 674 (1997).
`2. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other diseases.
`Nature Med. 1, 27-31 (1995).
`3. Korpelainen, E.I. & Alitalo, K. Signaling angiogenesis and lymphang1ogenes1s
`Curr. Opin. Cell Biol. 10, 159 164 (1998).
`4. Fe rrara, N . & Davis-Smyth, T. The biology of vascular endothelial growth factor.
`Endocr. Rev. 18, 4 25 (1997).
`5. Ferrara, N . et al. Heter。zygous embryonic lethality induced by targeted inact1va(cid:173)
`tion of the VEGF gene. Nature 380, 439-442 (1996) .
`6. Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryos
`lacking a single VEGF allele. Nature 380, 435-439 (1996).
`7. Carmeliet, P. et al. Impaired myocardial angiogenesis and ischemic cardiomy(cid:173)
`opathy in mice lacking the vascula 「 endothelila growth factor isofo 「ms VEGF164
`and VEG Fl 88. Nature Med. 5, 495-502 (1999).
`8. Seghezzi, G. et al. Fibroblast growth factor-2 (FGF-2) induces vascular endothe(cid:173)
`li al growth factor (VEGF) expression in the endothelial cells of forming capillar(cid:173)
`ies: an autocrine mechanism contributing to angiogenesis. }. Cell Biol. 171,
`1659-1673 (1998)
`
`9. Rivard, A. & Isner」 .M . Angiogenesis and vascul 。genesi s in t「eatment of card10-
`vascular disease. Mo/. Med. 4, 429-440 (1998) .
`10. Takeshita, S. et al. Therapeutic angiogenesis. A single intra-arterial bolus of vas(cid:173)
`cul a 「 endothelial growth fact。r augments revascularizati 。 n in a rabbit ischemic
`hind limb model.}. Clin. Invest. 93, 662-670 (1994)
`11. Pu, L.Q. et al. Enhanced revascularization of the ischemic limb by ang1 。genic
`therapy. Circulation 88, 208-215 (1993) .
`12. Asahara, T. et al. Synergistic effect of vascular endothelial growth factor and
`basic fibroblast growth factor on angiogenesis in vivo. Circulation 92, 11365-11371
`(1995).
`1 3. Van Belle, E. et al. Potentiated angiogenic effect of scatter factor hepatocyte
`growth factor via induction of vascular endothelial growth factor: the case for
`paracrine amplification of angiogenesis. Circulation 97, 381-390 (1998).
`14 Witzenbichler, B. et al. Vascular endothelial 9 「owth factor-C (VEGF-C/VEGF-2)
`promotes angiogenesis in the setting 。f tissue ischemia. Am. }. Patho/‘ 153,
`381-394 (1998) .
`15. Bauters, C. et al. Physi이ogical assessment of augmented vascularity induced by
`VEGF in ischemic