[CANCER RESEARCH 61, 1207–1213, February 1, 2001]

Involvement of Flt-1 Tyrosine Kinase (Vascular Endothelial Growth FactorReceptor-1) in Pathological Angiogenesis1

Sachie Hiratsuka, Yoshiro Maru, Akiko Okada, Motoharu Seiki, Tetsuo Noda, and Masabumi Shibuya2

Departments of Genetics [S. H., Y. M., M. Sh.] and Cancer Cell Research [A. O., M. Se.], Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, andCancer Institute, Tokyo 170-0012 [T. N.], Japan

ABSTRACT

Vascular endothelial growth factor (VEGF) and its two receptors,Fms-like tyrosine kinase 1 (Flt-1) (VEGFR-1) and KDR/Flk-1 (VEGFR-2),have been demonstrated to be an essential regulatory system for bloodvessel formation in mammals. KDR is a major positive signal transducerfor angiogenesis through its strong tyrosine kinase activity. Flt-1 has aunique biochemical activity, 10-fold higher affinity to VEGF, whereasmuch weaker tyrosine kinase activity compared with KDR. Recently, weand others have shown that Flt-1 has a negative regulatory function forphysiological angiogenesis in the embryo, possibly with its strong VEGF-trapping activity. However, it is still open to question whether the tyrosinekinase of Flt-1 has any positive role in angiogenesis at adult stages. In thisstudy, we examined whether Flt-1 could be a positive signal transducerunder certain pathological conditions, such as angiogenesis with tumorsoverexpressing a Flt-1-specific, VEGF-related ligand. Our results showclearly that murine Lewis lung carcinoma cells overexpressing placentagrowth factor-2, an Flt-1-specific ligand, grew in wild-type mice muchfaster than in Flt-1 tyrosine kinase domain-deficient mice. Blood vesselformation in tumor tissue was higher in wild-type mice than in Flt-1tyrosine kinase-deficient mice. On the other hand, the same carcinomacells overexpressing VEGF showed no clear difference in the tumorgrowth rate between these two genotypes of mice. These results indicatethat Flt-1 is a positive regulator using its tyrosine kinase under patholog-ical conditions when the Flt-1-specific ligand is abnormally highly ex-pressed. Thus, Flt-1 has a dual function in angiogenesis, acting in apositive or negative manner in different biological conditions.

INTRODUCTION

Angiogenesis is an essential process in normal embryogenic devel-opment and in a variety of pathological conditions, such as diabeticretinopathy and tumor growthin vivo (1). VEGF3 and its receptor(VEGFR) family, including Flt-1 (VEGFR1), KDR/Flk-1 (VEGFR2),and Flt-4 (VEGFR3), are well known to be a crucial regulatory systemfor normal and pathological angiogenesis (2–8). Flt-1 as well as KDRand Flt-4 is structurally related to the Fms/Kit/platelet-derived growthfactor receptor family and contains an extracellular domain carryingseven immunoglobulin-like sequences and a cytoplasmic TK domainwith a long kinase insert.

Flt-1 is expressed as a full-length tyrosine kinase receptor and insome cases as a soluble form, which carries only the extracellulardomain (9–13). Biochemically, Flt-1 shows an affinity to VEGF thatis at least 10-fold higher than that of KDR/Flk-1, but its TK activityis ;10-fold weaker than that of KDR/Flk-1 (2–4, 14–19), suggesting

a function different from KDR/Flk-1. Flt-1 and KDR/Flk-1 are spe-cifically expressed on vascular endothelial cells (20–24), and as anexception, theflt-1 mRNA has been shown to be expressed in mono-cyte/macrophages (25, 26).

Recently, gene targeting studies were carried out onflt-1 andKDR/flk-1 in mice (7, 8). TheKDR/flk-1 (2/2) homozygous micedied at embryonic day 8.5 (E8.5) because of a severe deficiency invascular formation associated with a strong hematopoietic impairment(7). The flt-1 (2/2) homozygous mice also showed embryonic le-thality at almost the same stage (E8.5–9.0); however, the phenotypewas quite different. The blood vessels inflt-1 (2/2) mice weredisorganized, and the endothelial-like abnormal cells were overgrow-ing within the vascular lumens (8). These results suggest that Flt-1 hasa negative role in the early angiogenesis in embryo. More recently, wehave shown that the Flt-1 TK domain-deficient [flt-1 TK (2/2)] micedeveloped basically normal blood vessels and survived with an im-pairment of macrophage migration toward VEGF (27). Thus, Flt-1 isconsidered to play a negative function in embryogenesis by trappingthe endogenous VEGF and adjusting the levels of VEGF to anappropriate range.

Although one important function of Flt-1 was found to be a VEGF-absorbing activity, it is still not clear whether the Flt-1 has a positiverole in pathological angiogenesis, such as tumor angiogenesis. Toanswer this question, we introduced an Flt-1-specific ligand into atumor cell line and examined the tumor growth and tumor angiogen-esis in both the wild-typeflt-1 TK (1/1) and flt-1 TK (2/2) mice.Our results indicate clearly that the TK of Flt-1, although it does nothave strong enzymatic activity, is important for angiogenesis undercertain pathological conditions.

MATERIALS AND METHODS

Animals. F1 mice of flt-1 TK (1/2) heterozygotes were intercrossed, andamong offspring the F2 littermates offlt-1 TK (1/1) andflt-1 TK (2/2) micewere selected for analysis. Seven to 10flt-1 TK (1/1) andflt-1 TK (2/2) micederived from several litters were used for each assay.

Cells Overexpressing Flt-1 Ligand.Human PlGF2 or human VEGF165

cDNA was inserted into murine pSRaMSVtk-neo retroviral vector, and thehigh titer retroviruses were used to infect murine endothelial cell-derived F2

(kindly provided by Dr. K. Toda, Department of Dermatology, Kyoto Univer-sity, Kyoto, Japan; Ref. 28) or LLC cell line by the Polybrene method.G418-resistant cells, designated as F2-PlGF, LLC-PlGF, or LLC-VEGF, wereharvested as a mixed population, and the production of human PlGF2 orVEGF165 from these cells into culture medium was examined by Westernblotting with antibody against PlGF or VEGF.

Injection of Ligand-containing Matrigels and Ligand-overexpressedLLC Cells into Mice. F2-PlGF (53 106 cells) or 200 ng/ml of PlGF purifiedfrom F2-PlGF culture medium using HiTrap Heparin Affinity Column (1 ml;Pharmacia, Uppsala, Sweden) as described previously (27) were mixed with 1ml of Matrigel (Becton Dickinson Laboratory) and s.c. injected into the backof anesthetized mice. Mouse VEGF164 and antimouse VEGF164 neutralizingantibody were purchased from R&D Systems (Minneapolis, MN). ParentalLLC, LLC-PlGF, or LLC-VEGF cells were collected by centrifugation, resus-pended in DMEM at 53 106 cells/ml, and injected into the backs of mice.

Preparation of Proteins and Western and Northern Blot Analyses.LLCs were lysed in an ice-cold buffer containing 150 mM NaCl, 50 mM

HEPES (pH 7.4), 10 mM EDTA, 1% Triton X-100, 10% glycerol, 2% apro-

Received 4/24/00; accepted 11/30/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant-in-Aid for Special Project Research on Cancer-Bioscience 04253204 from the Ministry of Education, Science, Sports and Culture inJapan and by research grants for the program “Research for the Future” of the JapanSociety for Promotion of Science.

2 To whom requests for reprints should be addressed, at Department of Genetics,Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 Japan.Phone: 81-3-5449-5550; Fax: 81-3-5449-5425.

3 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR,vascular endothelial growth factor receptor; Flt-1, Fms-like tyrosine kinase 1; PlGF,placenta growth factor; TK, tyrosine kinase; vWF, von Willebrand factor; MMP, matrixmetalloproteinase; LLC, Lewis lung carcinoma.

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tinin, and 1 mM phenylmethylsulfonyl fluoride and incubated with heparinbeads for 2 h. The protein-bound beads were collected by centrifugation,washed in PBS, and analyzed by SDS-PAGE and Western blotting. ForNorthern blot analysis, 0.2 kb of mouse PlGF cDNA and 0.5 kb of mouseVEGF-B cDNA were used for hybridization probes.

Immunohistochemistry. Tumor tissue sections were immunohistochemi-cally stained with an antiserum against human vWF (Dako, Carpinteria, CA)as an endothelial cell-specific marker or antiserum against mouse F4/80antibody (BMA) as a macrophage marker. Tissue sections (8-mm thick) werefixed with acetone at220°C for 5 min and rehydrated in PBS. For inhibitionof endogenous peroxidase, sections were incubated in 0.1% H2O2 methanolsolution for 10 min at room temperature and washed three times with PBS.

Nonspecific binding of antibody was blocked by incubation with 10%normal goat serum-containing PBS for 30 min. Subsequently, sections wereincubated with primary antibody for 2 h, washed three times with PBS,incubated with biotinylated goat antirabbit IgG (Vector, Burlingame, CA) forvWF, washed again with PBS, and then incubated with ABC kit (Vector).Horseradish peroxidase-conjugated sheep antirabbit IgG was used for F4/80.After washing with PBS, vWF and F4/80 were detected by 3-amino-9-ethylcarbazole (Vector). The smooth muscle cells in the thoracic aorta and tumortissues were stained with a FITC-conjugated anti-a-smooth muscle actinantibody (Sigma) at 4°C overnight.

RESULTS

Murine F2 Cells Overexpressing PlGF-2 Induce an AngiogenicResponse inflt-1 TK (1/1) Mice but not in flt-1 TK (2/2) Mice

To examine whether the TK domain of Flt-1 is important for anypathological angiogenesisin vivo, we took advantage of the pair ofFlt-1 TK domain (1/1) and (2/2) mice. We have shown already thatthe flt-1 TK (2/2) mice develop essentially normal physiologicalangiogenesis except for an impaired response of macrophages toVEGF (27).

To continuously stimulate the Flt-1 TK, at the first step, we usedF2-PlGF cells, a murine endothelial-derived cell line (28) that ex-presses relatively low levels of endogenous VEGF but high levels ofexogenously introduced Flt-1-specific ligand, PlGF-2 (Refs. 13, 15,and 29–32; see “Materials and Methods”). F2-PlGF cells were mixed

with Matrigel and s.c. injected intoflt-1 TK (1/1) or flt-1 TK (2/2)mice (Fig. 1).

After a 4-day incubation, we found that the s.c. tissues attached tothe Matrigel/F2-PlGF mixture and the surface of the Matrigel hadbecome reddish with dilated fine blood vessels inflt-1 TK (1/1) micebut not inflt-1 TK (2/2) mice (Fig. 1,C andD). Microscopically, theMatrigel/F2-PlGF mixture inflt-1 TK (1/1) mice showed a clearangiogenic response with several dilated vessels but not inflt-1 TK(2/2) mice. The original F2 cells without the exogenous PlGFexpression showed no detectable angiogenic response either inflt-1TK (1/1) or in TK (2/2) mice (Fig. 1,A andB).

During this incubation period, most of the F2 or F2-PlGF cells diedwithin the Matrigel because of its weak transforming potential (Fig.1). However, these results strongly suggest that Flt-1 TK is directlyinvolved in angiogenesis under certain conditions,i.e., when theFlt-1-specific ligand is highly expressed.

Purified PlGF-2 Induces an Angiogenic Response inflt-1 TK(1/1) Mice

To avoid any effects produced by F2 cells on the angiogenesisassay, we next used the PlGF-2 purified from the culture medium ofcells overexpressing PlGF-2 (see “Materials and Methods”) andmixed it with Matrigel for in vivo analysis. The same amounts (200ng) of PlGF-2 or VEGF164 mixed with Matrigel were inoculated s.c.for 4 days. The Matrigel containing PlGF-2 showed a mild angiogenicresponse with dilated blood vessels inflt-1 TK (1/1) mice but not inflt-1 TK (2/2) mice (Fig. 2,A andB). On the other hand, the Matrigelcontaining VEGF instead of PlGF induced small new blood vessels toform both in flt-1 TK (1/1) andTK (2/2) mice (Fig. 2,C andD).

In some areas of the PlGF-Matrigel inflt-1 TK (1/1) mice, weobserved an infiltration of macrophage-like, F4/80-positive cells. Be-cause macrophages were reported to secrete VEGF to some extent, wetested whether macrophage-mediated VEGF participates in this an-giogenic response by using antimouse-VEGF neutralizing antibody.The Matrigel containing both PlGF and antimouse-VEGF neutralizingantibody still induced an angiogenic response inflt-1 TK (1/1) mice

Fig. 1. Angiogenesis induced by F2-PlGF cellsin flt-1 TK (1/1) or flt-1 TK (2/2) mice. Matri-gels mixed with parental F2 cells (Aand B) orF2-PlGF cells (CandD) were s.c. injected intoflt-1TK (1/1) (A andC) or flt-1 TK (2/2) (B andD)mice (see “Materials and Methods”). After a 4-dayincubation, these Matrigels were recovered, fixed,and stained with H&E.3100.Bar, 50 mm.

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(Fig. 2E), indicating that the major angiogenic factor in this system isPlGF itself. In total, however, the responses to purified ligands weremild, even in the wild-type mice, possibly because of a rapid inacti-vation and degradation of ligands in Matrigels.

Comparison of Tumor Angiogenesis inflt-1 TK (1/1) Mice andin flt-1 TK (2/2) Mice

Expression of PlGF-2 or VEGF165 in LLC Cells Does NotModify the in Vitro Cell Growth. Because F2 or F2-PlGF cellscould not make tumorsin vivo, we used the murine LLC cell line foran in vivo tumorigenicity assay. Before introducing human PlGF orVEGF cDNA to LLCs, we examined the endogenous VEGF-relatedligands in LLCs. We found that after overnight culture, LLCs secreted;40 ng/ml of VEGF164 (Fig. 3A), less than the ascites-generatingsarcoma cell lines (170 to 850 ng/ml; Ref. 33). LLCs also expressedVEGF-B mRNA (34–36) at levels similar to a melanoma but higherthan normal placenta and lung (Fig. 3C). LLCs did not expressdetectable levels of PlGF mRNA (Fig. 3B).

After infecting LLCs with retrovirus expression vector for PlGF orVEGF cDNA, we harvested the infected cells as a mixed populationto avoid selection of specific clones. LLC-PlGF thus obtained secreted;200 ng/ml of PlGF, and LLC-VEGF secreted;40 ng/ml of exog-enous VEGF165(total, 80 ng/ml) per 13 107 cells in overnight culture(data not shown). The growth rate of these LLCs overexpressingeither PlGF or VEGF165 was essentially the same as that of theparental LLCs inin vitro culture (Fig. 4).

LLC-PlGF-induced Tumors in flt-1 TK (1/1) Mice GrowFaster Than Those in flt-1 TK (2/2) Mice, with Higher Angio-genesis.Cells (53 106) of LLC-PlGF or LLC-VEGF were injectedinto the back offlt-1 TK (1/1) or flt-1 TK (2/2) mice, and the

growth rate of tumors was monitored for 1 month (Fig. 5). PlGFprotein secreted from LLC-PlGF could be detected in tumor tissues 3weeks after inoculation (data not shown).

Eight days after inoculation, the tumor growth of LLC-PlGF wasfaster in flt-1 TK (1/1) mice thanflt-1 TK (2/2) mice. After 4weeks, the tumor mass inflt-1 TK (1/1) mice was;300% that inflt-1 TK (2/2) mice (Fig. 5A).

On the other hand, LLC-VEGF cells induced tumors of almost thesame size in the two genotypes of mice (Fig. 5C). This is to beexpected because the overexpressed VEGF could stimulate KDR/Flk-1 in these genotypes, which carries potent TK activity and medi-ates a strong angiogenic signal.

The tumor growth rate of the original LLCs was basically low butslightly better inflt-1 TK (1/1) mice when compared withflt-1 TK(2/2) mice (Fig. 5B). This minor difference could be attributable to anendogenous VEGF-B expression in LLCs. The LLC-PlGF-induced tu-mors showed a clear increase in tumor vessel density, particularly small-to middle-sized blood vessels, as indicated in Table 1 and Fig. 6.

Morphological Differences in the Blood Vessels between LLC-PlGF- and LLC-VEGF-induced Tumor Tissues. The blood vesselsinduced by LLC-PlGF were morphologically different from those in-duced by LLC-VEGF. In LLC-PlGF, relatively large vessels.100mmin diameter were often observed (Fig. 6,E andG). However, LLC-VEGFcells induced many small vessels not.50 mm in diameter (Fig. 6,I andJ; Table 1). The large vessels induced by LLC-PlGF, as well as smallvessels induced by LLC-VEGF, carried a single layer of vWF-positiveendothelial cells (Fig. 7,upper), but smooth muscle cells positive fora-smooth muscle actin staining were almost undetectable (Fig. 7,lower).These results suggest that the processes of angiogenesis stimulated byPlGF and by VEGF are not identical.

Fig. 2. Angiogenic response induced by PlGF-containing Matrigel inflt-1 TK (1/1) mice. Matrigelscontaining purified human PlGF2 (200 ng;A and B),mouse VEGF164 (200 ng; C and D), PlGF (200 ng)mixed with antimouse VEGF neutralizing antibody (E),or mouse VEGF (200 ng) mixed with antimouse VEGFneutralizing antibody (F) were s.c. injected intoflt-1TK (1/1) (A, C, E,andF) or flt-1 TK (2/2) (B andD)mice. The Matrigels were recovered after 4 days, fixed,and analyzed by H&E staining.Arrowheads,bloodvessels.3100 (A, B, rightin C, andD–F), 3200 (leftin C and D), 3400 (lower right in C and D). Bar,50 mm.

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Number of Macrophages within the LLC-PlGF-induced TumorTissues.Macrophages are known to be partly involved in the processof angiogenesis by secreting MMPs and angiogenic factors. To ex-amine whether macrophage-like cells participate in the LLC-PlGF-

induced tumor angiogenesis, we counted the number of cells posi-tively stained with anti-F4/80 antibody, which was specific formacrophages. The numbers of the F4/80-positive macrophages at day8 were almost the same between the tumors inflt-1 TK (1/1) and inflt-1 TK (2/2) mice, probably because of a nonspecific infiltration.At day 16, the number of these macrophages in the tumors weredecreasing in both cases but 3-fold larger inflt-1 TK (1/1) mice thanthat in flt-1 TK (2/2) mice (Table 2). Because LLC-PlGF tumors aregrowing faster even at day 8 inflt-1 TK (1/1) mice when comparedwith those inflt-1 TK (2/2) mice, we do not think that the macro-phages infiltrated into tumors play a major role in LLC-PlGF-inducedtumor angiogenesis.

Fig. 3. Endogenous expression of several ligands for Flt-1 in LLC cells.A, productionof mouse VEGF in LLC. Aliquots (1, 10, and 100 ng) of purified mouse VEGF (Lanes1–3) and VEGF in LLC-cultured medium (Lane 4) were analyzed by Western blotting.The LLC-culture medium and pure mouse VEGF in the same volume of basal mediumwere concentrated to 20ml with heparin beads in the same procedure and separated bySDS-PAGE. Western blots were probed with antimouse VEGF antibody.B and C,Northern blot analysis of PlGF (B) and VEGF-B (C) mRNAs in LLC cells. Twentymg/lane of total RNA obtained from placenta (Lane 1), lung (Lane 2), melanoma (Lane 3in C), and LLC (Lane 3in B; Lane 4in C) were hybridized with mouse PlGF (B) or mouseVEGF-B (C) cDNA probe. Amounts of 18S and 28S rRNA were monitored by ethidiumbromide staining.

Fig. 4. No major differences in thein vitro cell proliferation among LLC, LLC-PlGF,and LLC-VEGF. The cells (13 104) were cultured in the 96 wells of rat collagen I-coatedplates for 3 days. 3-(4,5-Dimethylthizol-2-yl)-5-(8-carboxymethoxy-phenyl)-2-(4-sulfo-nyl)-2H- tetrazolium solution was added, and the absorbance at 490 nm was examined byusing a 96-well plate reader. The assay was performed with triplicate samples.Bars,SD.

Fig. 5. Tumorigenicity of LLC-PlGF, LLC, and LLC-VEGF inflt-1 TK (1/1) or flt-1TK (2/2) mice. Growth rates of LLC-PlGF (A), LLC (B), and LLC-VEGF (C) during 4weeks after s.c. implantation are indicated. For each experiment, seven 8–10-week-oldflt-1 TK (1/1) andflt-1 TK (2/2) mice of the same litter (total, three to four litters) wereused.Bars,SD.

Table 1 The number of blood vessels in LLC-PlGF- and LLC-VEGF-induced tumortissues derived from flt-1 TK (1/1) and flt-1 TK (2/2) micea

50–100mm 100–150mm 150–200mm 200–300mm

LLC-PlGF8-day flt-1 TK (1/1) 406 25b 0 0 08-day flt-1 TK (2/2) 206 12 0 0 016-dayflt-1 TK (1/1) 16.56 7.1 36 3 0.8 6 1.4 016-dayflt-1 TK (2/2) 5.66 3.7 0.36 0.5 0 0

LLC-VEGF16-dayflt-1 TK (1/1) 156 2.3 16 1.4 0 016-dayflt-1 TK (2/2) 136 1.7 16 0.8 0 0

a Average numbers of three sagittal sections of three tumor tissues were counted. Thediameters of blood vessels are shown at 8 and 16 days (numbers/cm2) after intradermalimplantation.

b Mean6 SD.

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DISCUSSION

Mechanisms for the Tumor Angiogenesis Induced by the Flt-1TK Receptor. The TK activity of Flt-1 has been shown to be oneorder of magnitude weaker than that of KDR/Flk-1. We reportedpreviously that PlGF-2, which binds both Flt-1 and neuropilin-1 (37),a subtype-specific receptor for VEGF, showed a weak stimulatoryactivity for proliferation of endothelial cellsin vitro (2–4, 14–19).PlGF-1, which binds Flt-1 but not neuropilin-1, was found to induceangiogenesis using a highly sensitive systemin vivo (38). However,generally the biological activity of PlGF is not strong; thus, it is stillnot clear whether the Flt-1 tyrosine kinase induces pathological an-giogenesis, such as tumor angiogenesisin vivo, because of the un-availability of a sensitive enough assay.

In this study, using LLC-PlGF and both wild-typeflt-1 TK (1/1)and flt-1 TK (2/2) mice, we have shown that the tumor growth was;3-fold higher in the wild-type compared withflt-1 TK (2/2) mice.The increase in the tumor growth in the wild-type mice correlated wellwith the increase in tumor angiogenesis. These results strongly sug-gest that the TK domain of Flt-1 is required for a certain pathological

angiogenesis, such as Flt-1-specific, ligand-induced tumor angiogen-esisin vivo.

Because Flt-1 bears weak stimulatory activity for endothelial cellproliferation and because angiogenesis is a multistep process involv-ing matrix proteolysis, migration, proliferation, and tube formation,several steps other than direct cell proliferation are also considered tobe stimulated by Flt-1.

Recently, our preliminary results suggested that MMP-2 was acti-vated in blood vessel-enriched regions in tumor tissues in wild-typebut not in theflt-1 TK (2/2) mice.4 MMP is known to be importantfor tumor growth because MMP-2 null mutation in mice resulted inthe reduction of tumor growth via a decrease in angiogenesis (39–41).In addition, batimastat (BB94), an inhibitor for MMPs, disturbed thegrowth of a transplanted LLC-induced tumor by 25% (42).

Precursor forms of MMP-2 and MMP-1, as well as tissue inhibitorof MMP-1 and tissue inhibitor of MMP-2 are known to be producedin endothelial cells (39). Therefore, signaling via the TK domain of

4 S. Hiratsuka and M. Shibuya, unpublished observation.

Fig. 6. Tumor angiogenesis induced by LLC-PlGF and LLC-VEGF inflt-1 TK (1/1) or flt-1 TK(-/-) mice. LLC-PlGF-induced tumor tissues at day8 (A and B) and day 16 (C–H) and LLC-VEGF-induced tumor tissues at day 16 (IandJ) after s.c.injection were analyzed with H&E staining. Histo-logical samples were obtained from tumor tissuesin flt-1 TK (1/1) (A, C, E, G,andI) and inflt-1 TK(2/2) (B, D, F, H,andJ) mice.3100.Bar, 50mm.

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Flt-1 appears to activate MMP-2, leading to a proteolytic digestion ofmatrix in tumor tissue to facilitate migration and tube formation ofendothelial cells.

It is also possible that some angiogenic factors, such as VEGFsecreted from the macrophages that migrated in tumor tissues, induceangiogenesis. However, this seems unlikely, because approximatelythe same number of macrophages have migrated into tumor tissues atday 8, when a difference in tumor angiogenesis between the twostrains of mice was already observed (Tables 1 and 2). Moreover, apartially purified PlGF-induced angiogenesis in wild-type mice, evenin the presence of anti-VEGF neutralizing antibody, suggested aminor contribution of VEGF secreted from infiltrating inflammatorycells (Fig. 2E).

Taken together, a simple explanation for the rapid growth of LLC-PlGF in wild-type mice might be direct stimulation of both theactivation of proteases and the proliferation of endothelial cells via apositive signal from the Flt-1 tyrosine kinase.

Possible Mechanisms for Morphological Differences in BloodVessels Induced by PlGF and VEGF.We found a difference in theformation of blood vessels between LLC-PlGF- and LLC-VEGF-induced angiogenesis. The vessels induced by LLC-PlGF were oftenlarger in diameter than those induced by LLC-VEGF. In our prelim-inary experiments, the proteolysis surrounding the newly formedblood vessels was increased in LLC-PlGF-induced tumor tissuescompared with LLC-VEGF-induced tumor.4 Dvorak’s group reportedpreviously that, in addition to the sprouting mechanism, angiogenesisoccurred via an increase in the diameter of blood vessels because ofa proliferation of endothelial cells, followed by segregation of anenlarged vessel to several small ones (43). Therefore, the morpholog-ical difference induced by PlGF and by VEGF might be attributable toa difference in the remodeling process of angiogenesis. PlGF mayinduce less sprouting-type vessels but rather large-diameter-type ves-sels.

Another difference between PlGF- and VEGF-induced angiogene-sis may be in the cells surrounding endothelial cells, such as smoothmuscle cells and pericytes. However, this seems unlikely because novessels induced by either PlGF or VEGF were stained with anti-a-smooth muscle actin antibody, except for preexisting arteries, indi-cating that in both cases the association of smooth muscle cells withvessels was equally low.

Differential Roles of Flt-1 during Embryonic and PathologicalAngiogenesis.We have shown previously that the extracellular do-main of Flt-1 plays a negative role in embryogenesis. Although nullmutation of theflt-1 gene results in embryonic lethality because of anovergrowth of endothelial-like cells, theflt-1 TK (2/2) mice devel-oped an almost normal vascular system and could be mated aswild-type mice. Because the loss of a single allele of theVEGFgenein mice strongly disturbed angiogenesis and was lethal between thestage of E11–E12, a tight regulation of VEGF levelsin vivo is thoughtto be necessary for normal angiogenesis in embryogenesis. Thus, theextracellular domain of Flt-1 may function as a ligand-trapping mol-ecule and negatively regulate the levels of VEGF, decreasing thesignals from KDR/Flk-1.

Recently, elevations of humanPlGF mRNA in renal cell carcinomaas well as some brain tumors (44, 45) and an increase in humanVEGF-BmRNA in melanoma, lung carcinoma, and colon cancer havebeen reported (46). These observations, as well as the results shownhere, suggest that not only KDR/Flk-1 but also Flt-1 is involved intumor angiogenesis in a positive manner. Therefore, in addition to theblocking of VEGF and KDR/Flk-1, suppression of the Flt-1-PlGF/VEGF-B pathway may be required to decrease certain types of path-ological angiogenesisin vivo.

On the basis of these findings, Flt-1 is considered to have dual

Fig. 7. Staining of blood vessels in Matrigel and tumor tissues with anti-vWF oranti-a-SM actin antibody.Upper panels(A–H), vWF-positive endothelial cells in F2-PlGF-, PlGF-LLC-, and VEGF-LLC-induced angiogenesis. Endothelial cells in the Ma-trigels containing F2-PlGF (AandB), PlGF-LLC-induced tumor tissues (CandD), andVEGF-LLC-induced tumor tissues (E–H) were stained with anti-vWF antibody (A, C, E,andG). Samples were obtained fromflt-1 TK (1/1) (A–F) or flt-1 TK(2/2) (G andH)mice. B, D, F, and H, negative control staining without antibody.Lower panels(I–L),staining of blood vessels with anti-a-SM actin antibody as a smooth muscle cell marker.The wall of the thoracic aorta (leftin A) and blood vessels in normal s.c. tissues (rightinA) were stained with anti-a-SM actin antibody as a positive control. Essentially no vesselsin LLC-VEGF-induced (leftin C) or LLC-PlGF-induced tumor tissues (rightin C) werestained with anti-a-SM actin antibody (IandK). J andL, negative control staining withoutantibody.3200.Bar, 50 mm.

Table 2 The numbers of anti-F4/80 antibody-positive macrophage-like cells in LLC-PlGF- and LLC-VEGF-induced tumor tissues derived fromflt-1 TK (1/1) and

flt-1 TK (2/2) micea

F4/80-positivemacrophage-like cells (3102/cm2)

LLC-PlGF 8-day flt-1 TK (1/1) 92 6 56b

8-day flt-1 TK (2/2) 1086 7616-dayflt-1 TK (1/1) 40 6 2916-dayflt-1 TK (2/2) 11 6 10

LLC-VEGF 16-dayflt-1 TK (1/1) 50 6 2416-dayflt-1 TK (2/2) 48 6 30

a The cells were counted at random in 10 fields for one tumor section. Three sectionsat 8 and 16 days were used for the analysis.

b Mean6 SD.

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INVOLVEMENT OF Flt-1 (VEGFR-1) IN TUMOR ANGIOGENESIS

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functions in endothelial cells, either negative or positive, dependingon the biological conditions. Because Flt-1 can bind VEGF, PlGF, andVEGF-B with different affinity and because the gene regulation ofeach of these three ligands is different, further characterization isrequired to elucidate the network of the VEGF ligand family and Flt-1in each pathological condition.

ACKNOWLEDGMENTS

We thank Drs. P. Brown and K. Toda for supplying BB94 and F2 cells,respectively. We are also grateful to Dr. Hiroaki Kinou for helpful discussionsand technical assistance.

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INVOLVEMENT OF Flt-1 (VEGFR-1) IN TUMOR ANGIOGENESIS

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2001;61:1207-1213. Cancer Res   Sachie Hiratsuka, Yoshiro Maru, Akiko Okada, et al.   Growth Factor Receptor-1) in Pathological AngiogenesisInvolvement of Flt-1 Tyrosine Kinase (Vascular Endothelial

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