Tie2 Vascular Endothelial Receptor Expression andFunction in Hepatocellular Carcinoma

Shinji Tanaka,1 Keishi Sugimachi,1 Yo-ichi Yamashita,1 Takefumi Ohga,1 Ken Shirabe,1 Mitsuo Shimada,1

Jack R. Wands,3 and Keizo Sugimachi1,2

Hepatocellular carcinoma (HCC) is generally characterized as a hypervascular tumor ofrapid growth. We have previously reported that angiopoietin (Ang), a ligand for Tie2vascular endothelial-specific receptor tyrosine kinase, may play a role in the progression ofhuman HCC (J Clin Invest 1999;103:341-345) and matrix proteinase expression (CancerRes 2001;61:2145-2153). However, the role of Tie2 receptor in hepatic oncogenesis isunknown. The Tie2 receptor protein was overexpressed in the neovascular endothelium of31 of 39 (80%) human HCC tumors by immunohistochemical analysis with significantcorrelation to cell dedifferentiation and tumor size (P < .05). In vitro expression of adominant-negative construct, containing a soluble Tie2 ectodomain (sTie2), led to Angprotein interaction, inhibition of endogenous Tie2 phosphorylation in vascular endothelialcells and matrix metalloproteinase 9 (MMP-9) suppression. In conclusion, tumorigenicitywith neovascularization was suppressed by in vivo gene transfer and sTie2 expression in amurine HCC model, suggesting a possible role for Tie2 expression in the induction of HCCneovascularization and disease progression. Inhibition of the Ang/Tie2 signal transductioncascade is a promising approach for tumor treatment. (HEPATOLOGY 2002;35:861-867.)

Hepatocellular carcinoma (HCC) is one of themost prevalent malignancies worldwide, and itsincidence continues to increase in the United

States primarily because of chronic hepatitis B virus(HBV) and hepatitis C virus (HCV) infection.1 BecauseHCC tumors are hypervascular, angiogenesis is impli-

cated in survival and growth.2,3 Two growth factors (en-dothelial cell restricted), identified as specific ligands forreceptor tyrosine kinases, are divided into subfamilies.4

The first contains vascular endothelial growth factor(VEGF) (designated VEGF-A through VEGF-D) thatbinds and activates various VEGF receptors (VEGFR-1[Flt-1], VEGFR-2 [KDR/Flk-1], and VEGFR-3 [Flt-4]);the second was recently identified as vascular endothe-lium-specific (angiopoietin [Ang]), composed of Ang1through Ang4.6

We have shown that Ang2 plays a critical physiologicand pathologic role in clinical HCC in vivo murine tumormodels7 and matrix proteinase expression.8 Angs are vas-cular endothelial specific as a result of Tie2 (tyrosine ki-nases with immunoglobulin and epidermal growth factorhomology domains 2)-restricted cellular distribution.9,10

Tie2 expression in embryonic endothelium transcription-ally activates and maintains endothelial precursors; unlikethe VEGF receptor system, it is not required for vasculargenesis, but is essential to the more mature endothelium.6

Tie2 knockout mice lack proper hierarchical vasculature,culminating in death during embryogenesis.11 Althoughpostnatally Tie2 expression is down-regulated, it persistsin quiescent adult endothelial cells; in normal vasculature,tyrosyl-phosphorylation suggests an active role in bloodvessel maintenance.12 Additionally, Tie2 is up-regulatedin capillaries as a function of neovascularization during

Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV,hepatitis C virus; VEGF, vascular endothelial growth factor; Ang, angiopoietin;sTie2, murine-soluble Tie2 ectodomain; HUVEC, human umbilical vein endothe-lial cell; FBS, fetal bovine serum; EDTA, ethylenediaminetetraacetic acid; cDNA,complementary DNA; HA, hemagglutinin; EGTA, ethylene glycol-bis(-amino-ethyl ether)-N,N-tetraacetic acid; DNase, deoxyribonulease; RT, reverse transcrip-tase; PCR, polymerase chain reaction; mRNA, messenger RNA.

From the 1Department of Surgery and Science, Graduate School of MedicalSciences, and 2Station for Collaborative Research, Kyushu University, Fukuoka,Japan; 3Liver Research Center, Rhode Island Hospital, Brown University School ofMedicine, Providence, RI.

Received August 9, 2001; accepted January 24, 2002.Supported by Fukuoka Cancer Association; Ichiro Kanehara Foundation; Ue-

hara Memorial Foundation; Mochida Memorial Foundation; Kaibara MorikazuResearch Fund; Kanae Foundation; a Grant-in-Aid from Ministry of Education,Science, Sports, and Culture of Japan; and CA 35711 from the National Institutesof Health, Bethesda, MD. S.T. is a recipient of the Japan Cancer Society IncitementAward.

Address reprint requests to: Shinji Tanaka, M.D., Ph.D., Department of Surgeryand Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Mai-dashi, Fukuoka 812-8582, Japan. E-mail: shinjit@surg2.med.kyushu-u.ac.jp; fax:(81) 92-642-5482.

Copyright 2002 by the American Association for the Study of Liver Diseases.0270-9139/02/3504-0016$35.00/0doi:10.1053/jhep.2002.32535

861

wound healing12 and appears involved in breast tumorgrowth.13

Because the specific role of Tie2 in human HCC tis-sues is unknown, Tie2 expression was analyzed with cor-relation made to cell differentiation and tumor size;moreover, in vivo gene transfer of a dominant-negativeTie2 ectodomain (sTie2) into murine HCC tumors alsowas performed to evaluate tumor growth. Our studiessuggest a role for endothelial receptor tyrosine kinase inHCC development, and we present a novel approach totherapy of this devastating disease.

Materials and Methods

Clinical Samples. We studied 39 (31 male/8 female;63.2 years mean age) primary surgical HCC tumors (22with cirrhosis, 17 with chronic hepatitis without cirrho-sis) for Tie2 expression. The HBV- and HCV-associatedviral causes were as follows: HCV () HBV (): n 6;HCV () HBV (): n 26; HCV () HBV (): n4; HCV () HBV (): n 3. Thus, 25.6% (10 cases)were HBV-related (including HBV/HCV-positive cases),and 76.9% (30 cases) were HCV related (including HBV/HCV-positive cases). HCC tumors were histologicallyclassified as follows: 6 well-differentiated, 27 moderatelydifferentiated, and 6 poorly differentiated. Histologic dif-ferentiation of the tumors was evaluated based on classi-fication of the Liver Cancer Study Group of Japan.33

Immunohistochemical Analysis for Tie2 Expres-sion. Formalin-fixed, paraffin-embedded tissue sectionswere sectioned (4 mm) and mounted on silanated slidesfor Tie2 receptor protein expression analysis. After depar-affinization in xylene and rehydration in graded ethanol,endogenous peroxidase activity was blocked with metha-nol containing 0.3% H2O2 for 30 minutes. Sections wereincubated in trypsin (1 mg/mL) (Sigma Chemical, St.Louis, MO) at 37C for 30 minutes for antigen enhance-ment. Nonspecific antibody binding was blocked by in-cubation with 10% normal serum for 20 minutes.Sections were incubated with the primary polyclonal an-tibody against Tie2 (Santa Cruz Biotechnology; diluted1:250) and CD31 (DAKO A/S, Glostrup, Denmark; di-luted 1;20) overnight at 4C. Staining of the protein wasvisualized using streptavidin-biotin-peroxidase kits (di-aminobenzidine tetrahydrochloride as the chromogen[Nichirei, Tokyo, Japan]); sections were lightly counter-stained with hematoxylin. The immunohistochemicalstaining was evaluated under a light microscope by 3 in-dependent pathologists. The blood vessels in the tumorexpressing Tie2 and CD31 were determined respectively,as described by Peters et al.32 The most highly vascular-ized areas of each tissue were identified by scanning the

CD31-stained sections at a low-power field ( 40). Thenthe positive vessels were counted in 3 different high-power fields ( 200) for Tie2 and CD31 to evaluate theratio of Tie2 to CD31.

Cell Cultures. Human umbilical vein endothelialcells (HUVECs) were isolated by collagenase treatment ofhuman umbilical cord veins14; the cells were characterizedby acetylated low-density lipoprotein uptake, factor VIIIexpression, and cobblestone morphology. Cell cultureswere maintained in medium 199 with Earles salts(Sigma) supplemented with 10% heat-inactivated fetalbovine serum (FBS) (Sigma), 1% antibiotic-antimycoticsolution (100 stock) (Sigma), 2 mmol/L L-glutamine,120 mg/mL heparin (Sigma), and 100 mg/mL endothe-lial cell growth supplement (Collaborative Biomedical,Bedford, MA). Cells, grown in tissue culture flasks (Corn-ing, Corning, NY), were coated with 0.1% porcine gela-tin (Sigma) in medium 199; monolayers were split 1:4using 0.05% trypsin ethylenediaminetetraacetic acid(EDTA) (Sigma), and grown to confluency. Vascular en-dothelial-specific receptor Tie2 was detected by indirectimmunofluorescent technique using a primary polyclonalantibody against Tie2 (Santa Cruz Biotechnology) andanti-immunoglobulin preparation conjugated with fluo-rescence (CAPPEL Research Products, Durham, NC), asdescribed previously.15 A mouse HCC cell line MH134,derived from a carbon tetrachloridetreated C3H liver,was maintained in RPMI-1640 medium (Nipro, Osaka,Japan), supplemented with 10% (vol/vol) FBS and a1.0% penicillin/streptomycin solution (Sigma).

Construction and Protein Expression of Plasmids.Human Ang1 complementary DNA (cDNA) and Ang2cDNA were isolated from HCC tumors, modified atamino acid 245 of Ang1,10 and subcloned into a pcDNA3mammalian expression vector (pcDNA3-Ang1 andpcDNA3-Ang2) (Invitrogen, Carlsbad, CA).7 The cDNAof murine soluble Tie2 ectodomain (sTie2) was generatedby introduction of hemagglutinin (HA)-tagged epitopewith a stop codon just before the transmembrane regionof Tie2 cDNA.16 After sequence certification, sTie2cDNA was subcloned into a pcDNA3 vector (pcDNA3-sTie2). The biochemical effects of Ang1 or Ang2 wereassessed by DNA transfection, immunoprecipitation, andimmunoblot analysis on HUVEC cell lysates.15,17 Briefly,50% confluent HUVECs were transfected with the ex-pression vector using Superfect reagent (QIAGEN, SantaClarita, CA).

Cells, prepared from transfectants, were lysed in coldTriton buffer (50 mmol/L Tris-HCl, pH 7.5, containing1% Triton, 2 mmol/L ethylene glycol-bis(-aminoethylether)-N,N-tetraacetic acid (EGTA), 10 mmol/L EDTA,100 mmol/L NaF, 1 mmol/L Na4P2O7, 2 mmol/L

862 TANAKA ET AL. HEPATOLOGY, April 2002

NaVO4, 1 mmol/L phenylmethylsulfonyl fluoride, 25mg/mL aprotinin, 3.5 mg/mL pepstatin A, and 25mg/mL leupeptin). Lysates (10 mg) were subjected to gelelectrophoresis, blotted with specific antibody, and de-tected by the ECL system (Amersham Co., ArlingtonHeights, IL). Cell lysates (500 mg) were incubated withprotein A-agarose for immunoprecipitation analysis byimmunoblot. In addition, 0.5 mL of culture medium wasemployed for the sTie2/Ang analysis. For immunologicdetection, we used anti-Ang1, anti-Ang2, anti-Tie2 (C-terminus) antibodies (Santa Cruz Biotechnology), anti-phosphotyrosine antibody conjugated with horseradishperoxidase (PY20H; Transduction Laboratories), anti-HA, anti-survivin, and antiMMP-9 antibodies (Onco-gene Research Products, Cambridge, MA). Recombinanthuman VEGF 165 was purchased from R&D Systems,Inc. (Minneapolis, MN).

A Mouse Model of HCC Tumorigenicity and InVivo Gene Expression. The mouse HCC cell line,MH134, derived from a carbon tetrachloridetreatedC3H liver, was employed. In brief, 2.5 104 MSH134cells suspended in 0.5 mL phosphate buffered saline wereinoculated subcutaneously in the right flank of 6-week-old female C3H mice. Subcutaneous tumors (0.5 mm3)developed 7 days after inoculation. After saline injection(100 L) with 100 g DNA of sTie2 expression plasmidand mock empty plasmid (control) into 5 murine tumors,a pair of electrode needles was inserted (5 mm depth) toencompass the injection sites. The growth of MH134HCC cells implanted on the contralateral side also wasassessed simultaneously. Then 50-millisecond electricpulses (1 time/sec) at 150 V were delivered with an elec-tric pulse generator (CUY-21, BEX Co, Tokyo, Japan).18

The pulse remained as a square wave (i.e., constant voltagefor the duration of the pulse). Electrodes consisted of apair of tungsten needles (10 mm in length and 0.4 mm

diameter). At the rate of 1 pulse/sec, pulses of the oppositepolarity were administered to each DNA injection site.Genetic expression of transferred sTie2 was analyzed us-ing the specific PCR primers for Tie2 and HA sequences(5-TCAGCTCAGCCGCCGCCTCATCC-3 and 5-ATCAGCTTCCCCTTCTTGTTCAT-3). For thisprocedure, total RNA was extracted from transfected tu-mors, followed by thorough treatment of RNA withdeoxyribonuclease (DNase) to remove contaminatingplasmid DNA, and then reverse transcription (RT) and25 cycles of PCR were conducted.14 The integrity of themessenger RNA (mRNA) per sample was certified usingRT-PCR for glyceraldehyde-3-phosphate dehydrogenase(5-TTCCAGTATGACTCCACTCA-3 and 5-AT-CACGCCACAGCTTTCCAG-3. The sTie2 proteinwas determined on serum samples obtained from mice atday 14 following gene transfer. Tumor size was observeddaily, and mice were sacrificed on day 14 for histologicexamination of tumor tissue. Results were obtained from3 independent experiments.

Results

Tie2 Expression in HCC. Immunohistochemicalanalysis was performed to assess Tie2 levels in 39 humanHCC samples; Tie2 immunostaining was prominent inthe vascular endothelium, but not in normal liver tissue.The immunoreactive vessels were counted in 3 differentfields, and greater than 10% as the positive. Strong Tie2immunoreactivity was evident in 31 (80%) of 39 humanHCC samples. Tie2 expression was present in the endo-thelium of moderate or poorly differentiated HCC (Fig.1). Significant Tie 2 expression in HCC was observed in24 of 27 moderately differentiated (89%) and in 4 of 6poorly differentiated (67%) tumors; however, only 1 of 6well-differentiated tumors (17%) showed positive immu-

Fig. 1. Immunohistochemical anal-ysis of CD31 protein expression (up-per) and Tie2 receptor tyrosine kinaseprotein expression (lower) in humanliver samples (original magnification100). Moderately differentiatedHCC (right), well-differentiated HCC(center), and noncancerous liver(left) (original magnification 100).Moderately differentiated HCC showshigh-level expression of Tie2 proteincompared with uninvolved liver tissueand well-differentiated HCC.

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noreactivity (P .0019; Fishers exact test). Tie2 expres-sion correlated with tumor size: 4.2 2.1 cm of Tie2-positive versus 2.1 0.9 cm of Tie2-negative tumors(P .0034; t test) (Fig. 2). These results suggest a closeassociation between Tie2 expression, cellular dedifferen-tiation, and tumor size. All of the Tie2 positive vesselswere revealed to express CD31 protein. Whereas theTie2/CD31 ratio was 0.39 0.12 in well-differentiatedHCC, the ratio was increased to 0.60 0.18 in moder-ately and poorly differentiated HCC (P .05). TheTie2/CD31 ratio tended to increase with histologicdedifferentiation.

Ectopic Expression of Ang and Tie2 Activation inHUVECs. Tie2 is a transmembrane protein composed ofextracellular and intracellular domain(s), containing 2 ty-rosine kinase regions (Fig. 3B). The expression vector ofsTie2) was generated by inserting a stop codon just beforethe Tie2 cDNA transmembrane region.16 To explore theeffects of sTie2 on Ang signaling, Ang cDNA, sTie2cDNA, or both were transiently transfected to determineeffects on HUVECs that endogenously expressed theTie2 receptor (Fig. 3A); 5 independent experiments usinga green fluorescent protein (GFP) expression vector re-vealed that HUVEC transfection efficiency was 9.6 0.9%. Ectopic expression of Ang1- as well as Ang2-in-duced tyrosyl phosphorylation of the Tie2 receptor pro-tein (Fig. 3C; Tie2/pY). The product of sTie2 cDNAbound to Ang proteins (Fig. 3C; HA/Ang1 and HA/Ang2). Phosphorylation of endogenous Tie2 was de-creased after sTie2 cotransfection in Ang1- or Ang2-expressing HUVECs (Fig. 3C; Tie2/pY).

In addition, previous studies have shown that Ang-induced Tie2 phosphorylation resulted in enhanced ex-pression of survivin, an anti-apoptotic protein.19 Survivinis a member of the inhibitor of apoptosis protein (IAPfamily), and suppresses apoptosis through direct inhibi-tion of caspase and procaspase(s) (primarily caspase 3 and7) and modulates nuclear factor B transcription factorexpression. Ang1 and Ang2 stimulation induced expres-sion of survivin in HUVECs; this induction was substan-tially reduced by sTie2 expression after transfection (Fig.3C; survivin). It is noteworthy that Ang1 and a high con-centration of Ang2 may act as a survival factor for endo-thelial cells after serum deprivation to induce apoptosis.20

Thus, sTie2 may function as an inhibitor of vascular en-dothelial cell survival. Tyrosine phosphorylation of Tie2has been known to lead to proMMP-9 expression,34 andwe recently reported that MMP-9 expression was specif-ically induced by Ang2 in VEGF-stimulated HUVECs.8

As shown in Fig. 3D, Ang2 induced the active form ofMMP-9 (80 kd), and this response was inhibited by sTie2transfection. The soluble form sTie2 bound to Ang2 pro-tein, and thus antagonized Ang2 function for MMP-9expression. Expression of sTie2 appears to have a domi-nant-negative effect on Ang-induced cell survivin andMMP-9 expression, and may inhibit vascular progressionby endothelial cells in HCC tumors.

Electroporation-Mediated Gene Expression ofsTie2 in a Mouse Model. Additional studies have re-vealed that sTie2 was a potent inhibitor of in vivo vascu-larization of melanoma and mammary tumors.21 Tofurther explore this phenomenon in HCC progression,the electroporation-mediated gene transfer method wasused with C3H-derived MH134 HCC cells to investigatethe in vivo effects of sTie2 on the tumor growth rate.18

Mock genetransferred MH134 cells grew rapidly (12-fold increase) in the subcutaneously inoculated flank ofC3H mice after 14 days (Fig. 4A). In contrast, sTie2 genetransfer and expression significantly inhibited MH134HCC growth in vivo (P .001) (Fig. 4A). The inhibitoryeffects of sTie2 were recognized also on the contralaterallyimplanted tumors during the 8 days after the gene trans-fer. Expression of sTie2 gene was shown in several repre-sentative tumors by RT-PCR analysis, as shown in Fig.4B. There was no contaminating plasmid DNA, as con-firmed by PCR on the samples without the RT reaction.Protein expression of sTie2 was observed also in serumsamples of the mice at day 14 after gene transfer (Fig. 4C).It was difficult to determine the exact localization of sTie2protein in the tumor tissues. Immunohistologic analysisof HCC tumors revealed that the vascular endothelium,expressing CD31, was substantially reduced at day 14after sTie2 gene transfer (Fig. 4D).18 Additionally, areas

Fig. 2. Relationship between Tie2 expression, cellular differentiation,and tumor size. () Tie2 positive; (E) Tie2 negative.

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Fig. 3. Transfection of Ang1/Ang2 and sTie2 into HUVECs. (A) Demonstration of endogenous Tie2 expression in HUVECs by immunofluorescence.(B) Schematic representation of wild-type and soluble forms of Tie2 protein tagged with a HA-epitope. (C) HUVECs cultures were transfected () withAng1/Ang2 or together with the sTie2 expression vector. Ectopic expression of Ang1/Ang2 was detected by immunoblot analysis using anti-Ang1 oranti-Ang2 antibody (Ang1, Ang2). Expressions of endogenous Tie2 and survivin were detected using anti-Tie2 and survivin antibodies (survivin),respectively. Expression of HA-tagged sTie2 was detected by immunoblot analysis with culture medium derived from transfected cells. Tyrosylphosphorylation of Tie2 (Tie2/PY) and sTie2 interaction with Ang1/Ang2 (HA/Ang1, HA/Ang2) were detected by immunoprecipitation analysis. (D)Expression of MMP-9 in HUVECs transfected with or without Ang2 in HUVECs after administration of 50 ng/mL of VEGF. Ang2-induced expressionof the latent form of MMP-9 (92 kd) as well as the active form (80 kd) was inhibited by sTie2 transfection. Molecular weight: **131 kd, *75 kd.

Fig. 4. Inhibition of HCC growth after gene transfer and expression of sTie2. (A) Effect of cDNA electroporation-mediated gene transfer of mockempty plasmid (mock) as the control or sTie2 expression plasmid (sTie2) on tumor growth in a mouse tumor model using C3H-derived MH134 HCCcells. The volume of MH134 tumors was determined every 2 days after gene transfer. There were 10 animals each in the 2 groups. (B) Gene expressionof sTie2 was analyzed by RT-PCR in representative tumors transferred with mock (lanes 1-5) or sTie2 cDNA (lanes 6-10). Equal expression ofglyceraldehyde-3-phosphate dehydrogenase showed the integrity of the mRNA in each sample. M, 100-bp DNA marker. (C) Protein expression ofsTie2 was analyzed by anti-HA immunoblotting in the serum samples of mice transferred with mock (lanes 1-5) or sTie2 cDNA (lanes 6-10). Molecularweight, *75 kd. (D) Expression of CD31 protein in the endothelium of vessels surrounding and penetrating a mouse HCC tumor. Sections of a MH134tumor tissue at 14 days after gene transfer were analyzed by immunohistochemistry using the anti-CD31 antibody (original magnification 400).Significant CD31 staining was observed in vessels penetrating the highly vascular HCC tumors after mock gene transfer (mock). Note the lack ofneovascularization in HCC tumors after sTie2 gene transfer. Necrotic tissue was often apparent in sTie2-transfected HCC tumors as illustrated by theright area of the lower panel.

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of necrotic tissue were present and prominent in sTie2-treated tumors probably as the result of poor neovascular-ization. These observations suggest a stimulatory role forTie2 expression in promoting the growth of HCC tu-mors. More important, inhibition of this process is possi-ble with a dominant-negative mutant of the Tie2 receptorprotein, because there was substantial reduction of tumorsize in this experimental animal model system.

DiscussionFormation of de novo blood vessels is absolutely re-

quired in certain pathologic conditions involving tumorgrowth and subsequent metastases. It is also important inseveral physiologic conditions, including wound healing,and in female reproductive system regeneration.3 Angio-genesis represents the formation of new blood vesselsthrough sprouting and branching of endothelial cells.4

Endothelial receptor tyrosine kinases play a central role invascular establishment, maintenance, and adaptation.22,23

Our study shows significant Tie2 endothelial receptor ty-rosine kinase up-regulation in tumor vessels within HCCtissues possibly contributing to human disease progres-sion. The Tie2 receptor for Angs is pivotal in the angio-genic process because it involves remolding of the primaryvascular plexus into large/small vessels and the recruit-ment of periendothelial cells into the vascular wall.3,6

The induction of Tie2 expression in human endothe-lial cells is dependent on hypoxia, a frequent event inrapidly growing tumors.24 Several characteristics of theresponse to hypoxia have been described that allow for theaccumulation of a family of hypoxia-inducing factors. Inthis regard, Ang2 expression is up-regulated in humanHCC and may be hypoxia induced; further studies will berequired to substantiate this hypothesis. Although Ang2was originally identified as an Ang1 antagonist, wepresent evidence that Ang2 expression activates the Tie2receptor as an agonist. Ang1/Ang2 may function as a sur-vival factor required for vascular stable growth.18 Takentogether, Ang2 may play the dual role of initiating andpromoting vascular remodeling. The local balance ofAng1/Ang2 may disrupt endothelial cell interaction andmicroenvironment, thereby making such cells responsiveto an angiogenic initiator.25 More important, sTie2 mayact as an inhibitor of this process.

Endothelial cells interact with components of the un-derlying basement membrane to promote angiogenesis.Indeed, the activity of MMPs, that can cleave basementmembranes and interstitial matrix molecules has beenshown to be required for the angiogenic process.25 Re-cently, Hanahan et al.26 revealed MMP-9 is a trigger ofthe angiogenic switch that occurs during carcinogenesis.In our studies, MMP-9 was induced by Ang2 and VEGF

stimulation in HUVECs (Fig. 3D).8 The ligand stimu-lates its receptor tyrosine kinase to transmit intracellularsignals. For example, the activated Tie2 interacts withadaptor proteins Grb2, Grb7, and Grb14.27 In this con-text, we have previously isolated and characterized a hu-man Grb7 cDNA sequence,28,29 and showed that it servesas a substrate of focal adhesion kinase during the cellularinvasion process.30 This finding raises the possibility thatGrb7 participates in Tie2-mediated endothelial cell mi-gration. Further analysis of Ang/Tie2 signaling cascademay contribute to the understanding of the functionalsignificance of this pathway. It is a formal possibility thatAng1 and Ang2 activate Tie2 in fundamentally differentways by stimulation of signaling pathways, which result invaried biologic responses.

Human HCC is a devastating malignancy and is on theincrease worldwide.1 The major risk factors for the devel-opment of HCC are now well recognized, and detailsregarding the pathogenesis are emerging.31 Our study re-veals a role of Tie2 receptor tyrosine kinase in HCC pro-gression that involves the neovascularization process.Establishment of angiogenesis dependence of HCC sug-gests that tumor progression may be inhibited and leads tolong-term disease control. Indeed, we have shown thatsTie2 blocks Ang2-mediated Tie2 signaling required forthe growth and assembly of neovessels, but not for themaintenance of existing vessels. Finally, realizing the fulltherapeutic potential of modulation of the Ang2/Tie2pathway will require a greater understanding of the bio-logic effect of this signaling cascade in the context ofHCC tumor progression.

Acknowledgment: The authors thank Dr. S. A.Stacker (Ludwig Institute for Cancer Research, Mel-bourne, Australia) for providing Tie2 cDNA, Dr. I.Masaki for preparation of HUVEC cultures, and Ms. M.Hirata for technical assistance. The authors acknowledgeMrs. Sharon Chiott for editorial review.

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