ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

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Research Article

ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor Junko Inagakia, Katsuyuki Takahashia, Hiroko Ogawaa, Keiichi Asanoa, Omer Faruk Hatipoglua, Mehmet Zeynel Cileka, Masanari Obikaa, Takashi Ohtsukia, Matthias Hofmannd, Shozo Kusachib, Yoshifumi Ninomiyaa, Satoshi Hirohataa,c,n a

Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan b Department of Medical Technology, Okayama University Graduate School of Health Sciences, Okayama, Japan c International Center, Okayama University, Okayama, Japan d Department of Dermatology, Venereology and Allergology, Goethe University, Frankfurt, Germany

article information

abstract

Article Chronology:

Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and

Received 8 October 2013

metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angio-

Received in revised form

genesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether

6 February 2014

ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell

Accepted 1 March 2014

migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/

Keywords:

VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube

ADAMTS1

formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-

Lymphangiogenesis

transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene

VEGFC

therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with

VEGFR-3

VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data

Lymphatic endothelial cell

highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. & 2014 Elsevier Inc. All rights reserved.

Introduction A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family is a recently identified group of multifunctional zinc

metalloproteinases that are composed of propeptide, metalloproteinase, disintegrin-like, and spacer-region domains and a carboxylterminal region containing a variable number of thrombospondin (TSP) type Ι motifs [1–5]. At least 19 members have been identified to

n Corresponding author at: Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama 700-8558, Japan. Fax: þ81 86 222 7768. E-mail address: [email protected] (S. Hirohata).

http://dx.doi.org/10.1016/j.yexcr.2014.03.002 0014-4827 & 2014 Elsevier Inc. All rights reserved.

Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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date and have proteolytic activities against various extracellular matrix (ECM) proteins, particularly proteoglycans such as aggrecan, brevican, and versican, which are key components of the ECM and their degradation is regulated by these metalloproteinases [2–5]. These members have been implicated in diverse physiological and pathological processes including embryogenesis [6], wound healing [7], osteoarthritis [8], inflammation [1,9,10], ovulation [11–13], and cancer growth and metastasis [14,15]. ADAMTS1 was first identified among this family as a mediator of inflammation [1] and its function has been characterized in physiological and pathological conditions, such as organogenesis [6,16–18], ovarian folliculogenesis [19], ovulation [20,21], ovarian lymph vessel formation [19,22], inflammation [1,23,24], angiogenesis [7,25–27], and tumor invasion and metastasis [28–31]. Since ADAMTS1 was implicated in ECM remodeling through proteolytic degradation, this molecule has been shown to contribute to tumor progression [32] and tumor invasion and metastasis [15]. Interestingly, ADAMTS1 is also known to have anti-angiogenic activities and may, therefore, have an anti-tumorigenic role [25,33]. Previous studies reported that ADAMTS1 inhibited endothelial cell proliferation in vitro [33], directly bound to VEGF165, one of the most specific mediators of angiogenesis, dampened VEGFR2 phosphorylation and VEGFR2-associated downstream signaling, and then suppressed endothelial cell proliferation [26]. In addition, ADAMTS1 has been shown to cleave both thrombospondin-1 (TSP1) and TSP2 and mediate the release of anti-angiogenic polypeptides from the trimeric structure of both molecules [27]. Moreover, we recently demonstrated that ADAMTS1 induced endothelial cell apoptosis and inhibited tumor angiogenesis, resulting in the suppression of tumor growth, and that the induction of endothelial cell apoptosis by ADAMTS1 occurred independently of protease activity [34]. Previous studies demonstrated that ADAMTS1 was essential for the hormonally-regulated remodeling of normal ovarian lymphatic vasculature [19,22]. Although ADAMTS1 is known to be required during ovarian lymphangiogenesis, few reports have examined the direct effect of ADAMTS1 on lymphangiogenesis in vitro. Therefore, in the present study, we investigated the effect of ADAMTS1 on lymphangiogenesis in vitro using normal HMVEC-dLy transduced with or without the adenoviral human ADAMTS1 gene. We then examined the effect on the vascular endothelial growth factor-C (VEGFC)stimulated phosphorylation of VEGFR3 and associated downstream signaling molecules in ADAMTS1-transduced HMVEC-dLy.

Materials and methods Cell lines and cultures HMVEC-dLy obtained from Lonza Inc. (Walkersville, MD, USA) were maintained in EGM-2MV BulletKit. HEK-293 cells were purchased from ECACC (Dainippon Sumitomo Pharmaceutical Co., Osaka, Japan) and MDA-MB-231 human breast adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). HEK-293 and MDA-MB231 were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were cultured at 37 1C under 5% CO2 and 20% O2 in a humidified chamber. HMVEC-dLy were used at passage 3–6 for all experiments.

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ADAMTS1 expression vectors and VEGFC expression vector The ADAMTS1 plasmid, designed to add v5 epitope tag sequences to the COOH-terminal end of human full-length ADAMTS1 using the expression vector (pcDNA/v5-D-TOPO; Invitrogen, Carlsbad, CA, USA), was constructed as previously described [34]. The recombinant adenovirus vectors carrying the human fulllength ADAMTS1 gene (Ad-ADAMTS1) or LacZ reporter gene as a negative control (Ad-LacZ) were generated using the Adenovirus Expression Vector Kit (Dual Version) (TaKaRa Bio Inc., Kyoto, Japan) according to the manufacturer's instructions. The recombinant adenoviruses contained the human full ADAMTS1 or LacZ genes under the control of the cytomegalovirus (CMV) immediate-early enhancer/ promoter. This adenovirus vector (pAxCAwtit2) was replication deficient because it lacked the E1A and E3 regions. These adenovirus particles were then amplified in HEK 293 cells, purified by cesium chloride density gradient centrifugation, and titered using a standard plaque-forming assay. For VEGFC expressing vector, we used HaloTags system (Promega, Madison, WI, USA). The VEGFC carrying halotagged vector was purchased (FHC09559, Promega) and used for the transient transfection.

Adenovirus infection HMVEC-dLy were plated at 1.0  105 cells in a 6-well plate. After overnight incubation, these cells were infected with Ad-ADAMTS1 or Ad-Lac Z at a multiplicity of infection (MOI) from 0 to 100 in a minimal volume of serum-free culture medium for 1 h at 37 1C. The necessary amount of the medium was then added and cells were incubated for 1 h. The medium was replaced with 5% FBScontaining complete medium and further incubated overnight at 37 1C. The negative control was treated equally, but did not receive any virus load.

Plasmid electroporation and transfection Electroporation of HMVEC-dLy was performed using a Microporator (BMS-MP-100; Microporator, Seoul, Korea), in accordance with the manufacturer's instructions [35]. Briefly, cells were removed from the plate with trypsin, centrifuged, and dissolved in a serum-free medium. The cells and plasmids were mixed into the buffer at room temperature, after exposure to electricity (1000 V 30 mS, 2-pulse). Cells were plated at a density of 1.5  105 cells in 24-well dishes with 500 μL culture medium without antibiotics. After seeding, cells were allowed to recover for 24 h. The medium was then changed and cells were used for the analysis. In another experiments, lipofection was used for the transient transfection as we previously described [34,36] with slight modification. Lipofectamine LTX & PLUS (Life technologies Corp., CA, USA) was used for co-transfection into HEK293 cells with expression constructs containing inserts encoding full ADAMTS1-V5 and VEGFC-HaloTag according to the manufacturer's protocol. Empty vector was transfected as a negative control. HMVEC-dLy were plated at 2.5  105 cells in a 6-well plate. After overnight incubation, we added the diluted DNA and Lipofectamine LTX reagent with OptiMEM medium to the cells and incubated for 48 h.

ADAMTS1 overexpression analyses HMVEC-dLy infected with Ad-ADAMTS1 or Ad-LacZ at MOIs of 10, 30, 50, and 100 for 2 h were incubated in fresh medium and were

Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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collected 24 h post-infection. The cell lysates and conditioned media were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Advantech, Tokyo, Japan), immunoblotted with the anti-v5-tag antibody (1:1,000; Medical & Biological Laboratories Co., ltd, Nagoya, Japan) or anti-ADAMTS1 antibody (AF5867, 1:200; R&D systems, Inc., Minneapolis, MN, USA), and probed with an appropriate secondary antibody (HRP conjugated goat anti-mouse IgG, 1:5,000; or HRP conjugated donkey anti-sheep IgG, 1:2500; R&D systems, Inc.). Immunoreactive protein bands were visualized using the enhanced chemiluminescence detection system (ECL; Amersham Bioscience, NJ, USA). HMVEC-dLy were plated at a concentration of 1.5  105 cells/mL in 8-well LAB-TEK II Chamber Slide (Thermo Fisher Scientific Inc., NY, USA) coated with 0.1% gelatin for immunofluorescence analysis. HMVEC-dLy were infected with Ad-ADAMTS1 at 50 MOI for 2 h, the medium was replaced with fresh medium containing 5% FBS, and incubated for 24 h. After incubation, the cells were washed, fixed with 4% PFA/PBS at room temperature for 15 min, and then blocked with 2% BSA/PBS, treated with the anti-V5-tag antibody (1:1000) and antiVEGFR3 antibody (C-20, 1:50; Santa Cruz Biotechnology, Inc., CA, USA) at room temperature for 1 h. These slides were subsequently incubated with secondary antibodies (anti-rabbit IgG-Cy3 conjugate at 1:1500; Sigma-Aldrich or anti-mouse IgG-Alexa Fluor 488 conjugate at 1:1500; Life Technologies Corp.) in blocking buffer at room temperature for 1 h. Fluorescence images were observed on Zeiss Confocal Laser Scanning Microscope Model LSM510 (Carl Zeiss, Göttingen, Germany).

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expressed as the percentage of proliferation compared with control empty adenovirus-infected cells.

Co-culture system and cell migration assay HMVEC-dLy cell migration to Ad-ADAMTS1- or Ad-Lac Z-transduced MDA-MB-231 cells was evaluated by a BD Biocoat™ Angiogenesis System: Endothelial Cell Migration using Falcon FluoroBlok™ 24Multiwell Insert Plate (3 μm pore size) coated with Human fibronectin (Becton Dickinson). In the Transwell co-culture system, MDA-MB231 cells (1.0  105/well) were seeded in the lower chambers overnight, followed by the transduction of Ad-ADAMTS1 or Ad-Lac Z at 100 MOI for 2 h. The medium was replaced with 5% FBS-containing complete medium, incubated overnight, and then replaced with serum-free medium. After a further overnight incubation, the bottom well was replaced with the culture medium supplemented with 50 ng/mL recombinant human VEGFC (rVEGFC) (R&D systems, Inc.). HMVEC-dLy (1.0  105/well) suspended in serum-free medium were seeded onto Transwell inserts fitted with a polyethylene terephthalate (PET) insert filter, the insert wells were put in the bottom wells in which MDA-MB-231 cells were placed, and then inserts were incubated for 22 h. Migrated endothelial cells were post-labeled with BD Calcein AM Fluorescent Dye, and were quantified by a MultiDetection Microplate Reader (a bottom-reading fluorescence plate reader) (PowerScan HT, Dainippon Sumitomo Pharmaceutical, Japan), which only detected fluorescently-labeled cells in the chamber below the insert (i.e., migrating cells). Experiments were performed in triplicate and repeated three times.

Immunoprecipitation and phosphorylation of VEGFR3, Akt, and ERK1/2 Endothelial tube formation assay The endothelial tube formation assay was performed as previously described. HMVEC-dLy infected with Ad-ADAMTS1 or AdLac Z at 0, 50, or 100 MOI for 24 h were plated on a Matrigel surface and incubated for 14 h. Briefly, 100 μL of Matrigel Matrix (Becton Dickinson, Franklin Lakes, NJ, USA) was applied to 96-well plates, and a suspension of 1.0  104 of Ad-ADAMTS1-transduced or Ad-Lac Z-transduced HMVEC-dLy in 100 μL complete medium was seeded on each well. All assays were performed in triplicate and the experiments were repeated at least three times. Cells were incubated at 37 1C for 16 h and tube formation was evaluated by measuring the number of tube-like structures per well (magnification at 40  ) using an IX71 inverted microscope (Olympus Japan, Tokyo, Japan). Three random areas of triplicate wells were imaged, the total tube number was measured using Image J software (W. Rasband, Research Services Branch, NIMH, National Institutes of Health, Bethesda, MD, USA), and the recorded number was averaged.

Cell proliferation assays After adenovirus infection, HMVEC-dLy (5.0  103 cells/well) were seeded on 96-well tissue culture plates and incubated for 72 h. Cell proliferation was examined using a CellTiter AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) according to the manufacturer's instructions. OD was measured at 490 nm. All assays were performed at least three times. Cell proliferation was

HMVEC-dLy were plated at 1.0  105 cells in 6-well plates. After overnight incubation, cells were infected with Ad-ADAMTS1 or Ad-Lac Z at 50 MOI for 2 h. The medium was replaced with 5% FBS-containing complete medium and incubated overnight, and cells were then serum-starved overnight in the presence of 0.1% BSA (Sigma-Aldrich). After starvation, cells were treated with 0, 50, 100, or 200 ng/mL rVEGFC for 10 min. After washing with icecold PBS, cells were lysed with immunoprecipitation assay buffer (20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40, 0.5% Triton X-100, 1.0 mM EDTA, 0.5% sodium deoxycholate, 10% glycerol, and 10 mM NaF) supplemented with Complete, Mini protease inhibitor cocktail Tablets (Roche Applied Science, Mannheim, Germany) and Phosphatase inhibitor Cocktail 3 (SigmaAldrich). Cell lysates were centrifuged at 15,000 rpm for 15 min, and total protein concentrations were quantified using a Bio-Rad DCTM Protein Assay (Bio-Rad Laboratories, CA, USA). Cell lysates were used immediately for immunoprecipitation experiments or stored at  80 1C. Immunoprecipitation was performed using the ImmunoCruz™ IP/WB Optima B System (Santa Cruz Biotechnology), according to the manufacturer's instructions. Cell lysates were precleared by incubation with IP Matrix B at 4 1C for 1 h. After removal of the Matrix by centrifugation, the precleared cell lysates were incubated with IP Matrix B along with 3 μg of the anti-VEGFR3 antibody (C-20, Santa Cruz Biotechnology) on a rotor wheel at 4 1C overnight. Protein-Matrix B complexes were washed four times with wash buffer (20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1.0% Nonidet P-40, 1.0 mM EDTA) and the precipitated

Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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immunocomplexes were eluted in 50 μl of 2  reducing sample buffer by boiling for 5 min. The eluted samples were separated by 10% SDS-PAGE, transferred onto PVDF membranes, immunoblotted with phospho-tyrosine antibodies (4G10, 1:1,000; Cell Signaling Technology, MA, USA), probed with HRP-conjugated Exacta Cruz™ reagent B (1:1000), and immunoreactive protein bands were visualized using the ECL. Phosphorylated VEGFR3 levels were quantified using Image J software. Western blots of the treated lysates were also performed with antiphospho-Erk1/2 (#4370, 1:1000; Cell Signaling Technology) or antiphospho-Akt antibodies (#4060, 1:1000; Cell Signaling Technology) to detect the phosphorylation levels of Akt and Erk1/2 as previously described [37]. To show that equal amounts of protein have been loaded, total ERk1/2 or Akt protein levels were also detected with anti-ERK1/2 (#4695, 1:1000; Cell Signaling Technology) or anti-Akt (#4691, 1:1000; Cell Signaling Technology) antibodies.

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RNA extraction and quantitative RT-PCR Total RNA was extracted as we previously described [38,39]. Briefly, cellular RNAs were extracted using TRIzols (Life technologies Corp.) and subsequently quantified by measuring absorbance at 260 nm and then stored at 80 1C until assay. The extracted RNA (2 μg) was subjected to a reverse transcription reaction using ReverTra Aces at 42 1C for 50 min and at 70 1C for 10 min [40,41,42]. The cDNA was diluted five-fold prior to quantitative PCR amplification. Subsequently, quantitative real-time PCR was performed using Taqman Fast Advanced Master Mix (Life technologies Corp.). The code number for each primer and probe set ((Life technologies Corp.) used for the analysis are following: ADAMTS1, hs00199608_m1; COX, hs00153133_m1; GAPDH, hs02758991_g1. The quantification was performed by comparative Ct method.

Statistical analysis Co-immunoprecipitation For co-immunoprecipitation, co-transfected HEK-293 cells were washed with ice-cold PBS and lysed with immunoprecipitation assay buffer. Co-immunoprecipitation was performed using anti-v5-tag antibody, anti-HaloTags polyclonal antibody (Promega), or normal mouse IgG (Santa Cruz Biotechnology) and ImmunoCruz™ IP/WB Optima B or C System (Santa Cruz Biotechnology), as described above. The precipitated immunocomplexes were confirmed by Western blot analysis with either monoclonal anti-v5 antibody or polyclonal antiHalo antibody. To detect the immunoprecipitated protein, we performed Western blotting using anti-v5-tag antibody and antiHaloTags polyclonal antibody, and immunoreactive protein bands were visualized using the ECL.

Dot blot analysis BSA and rVEGFC (R&D systems, Inc.) were diluted to 50, 100, and 250 μg/mL in TBS for dot blot assay. Two μl of each diluted protein were spotted onto the PVDF membrane and allowed to air dry for 30 min. After extensive drying of the samples, the membrane was blocked with 5% (w/v) skim milk in TBS containing 0.025% Tween 20 (TBST) for 1 h. The conditioned medium of Ad-ADAMTS1transduced (or Ad-Lac Z-transduced) HMVEC-dLy at 50 MOI was diluted two times in the blocking buffer and the membrane was incubated with the diluted conditioned medium for 2 h at room temperature. After incubation, the membrane was incubated with the anti-v5-tag antibody (1:1000) at room temperature for 1 h. The membrane was subsequently incubated with a secondary antibody (HRP conjugated goat anti-mouse IgG, 1:5000; MP Biomedicals, LLC., CA, USA) in blocking buffer at room temperature for 1 h. Following the last wash, dot blots were visualized using ECL.

Titration assay of Ad-DAMTS1-transduced conditioned medium To titrate the Ad-ADAMTS1-containing conditioned medium, HMVEC-dLy were plated at 1.0  105 cells in a 6-well plate. After 48 h incubation, the medium was replaced with the 50 MOI AdADAMTS1-conditioned medium with the dilution at 1/2 and 1/10. The phosphorylation of VEGFR3, Akt and Erk1/2 were examined as described above.

Statistical analyses were performed using the Student's t-test for nonparametric correlations. A po0.05 was considered to be significant.

Results ADAMTS1 protein overexpression in HMVEC-dLy Since the expression of ADAMTS1 protein was slightly detected under normal conditions in HMVEC-dLy (data not shown) and high-level transgene expression was crucial for local gene therapy, we constructed recombinant adenovirus vectors carrying the human fulllength ADAMTS1 gene (Ad-ADAMTS1) and LacZ gene (Ad-LacZ) and examined the effects of ADAMTS1 on lymphangiogenesis in vitro. We used Western blot and immunofluorescence analyses to observe the efficiency of adeno-virus-mediated ADAMTS1 gene expression in HMVEC-dLy. The protein bands of full-length ADAMTS1 with a C-terminal v5-tag were visualized by Western blot analysis (Fig. 1A). Three major bands were detected in conditioned media and cell lysates that consisted of the unprocessed form (110 kDa), mature form (88 kDa), and a proteolytic C-terminal fragment (25 kDa) of ADAMTS1, based on a previous reported (Fig. 1A) [43]. This expression was also confirmed by the anti-ADAMTS1 antibody (AF5867) (Fig. 1B). ADAMTS1 expression levels in the conditioned medium and cell lysates increased in a dose (MOI)-dependent manner (Fig. 1A and B), which indicated that ADAMTS1 proteins were efficiently expressed and secreted into the conditioned supernatants of transduced HMVEC-dLy. Immunofluorescence analysis with the anti-V5-tag antibody and anti-VEGFR3 antibody revealed that ADAMTS1 proteins were efficiently expressed in the cytoplasm of transduced HMVEC-dLy and secreted to the ECM of the cells (Fig. 1C).

ADAMTS1 transduced via adenoviral vectors inhibited tube formation by HMVEC-dLy To examine the effects of ADAMTS1 on tube formation by HMVECdLy, cells infected with Ad-ADAMTS1 or Ad-Lac Z at 0, 50, or 100 MOI were plated on a Matrigel surface and incubated for 14 h. As shown in Fig. 2A, uninfected HMVEC-dLy controls (0 MOI of Ad-ADAMTS1 or Ad-Lac Z) efficiently formed capillary-like structures. Although tube

Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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Fig. 1 – Human ADAMTS1 expression in HMVEC-dLy. (A) HMVEC-dLy were infected with Ad-ADAMTS1 at MOIs of 10, 30, 50, and 100, or with Ad-Lac Z at MOIs of 50 and 100, or uninfected controls. Cells were harvested at 24 h post-infection, and Western blot analysis for the ADAMTS1 protein was performed using an anti-V5-tag antibody. Locations of the proforms (110 kDa), mature forms (87 kDa), and Cterminal fragments of ADAMTS1 (22 kDa) are indicated. (B) After stripping the membrane (A), ADAMTS1 levels were assessed using the antibody AF5867. (C) Immunofluorescence analysis of ADAMTS1 expression in HMVEC-dLy. Cells were infected with Ad-ADAMTS1 at 50 MOI and incubated for 24 h. Immunofluorescence analysis was performed using an anti-V5-tag antibody (green staining) and using an anti-VEGFR3 antibody for lymphatic EC (red staining). Scale bars are 100 μm.

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formation by HMVEC-dLy transfected with Ad-Lac Z at 100 MOI was approximately 20% less than that of uninfected control cells, transfection with ADAMTS1 significantly inhibited tube formation in a dosedependent manner (Fig. 2A and B). Tube formation was quantified by measuring the number of tubes that formed (Fig. 2B). Three random areas of triplicate wells were imaged and the total number of tubes was calculated using Image J software. The mean and SEM values from each condition were shown relative to HMVEC-dLy transfected with Ad-Lac Z. Tube formation by HMVEC-dLy transfected with AdADAMTS1 was inhibited by approximately 99% at 100 MOI (Fig. 2B). These results showed that the ADAMTS1 proteins secreted by transduced HMVEC-dLy were capable of inhibiting tube formation in vitro.

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ADAMTS1 inhibited proliferation of HMVEC-dLy Cell proliferation was assessed 72 h after infection at different MOIs of Ad-ADAMTS1 or control empty adenovirus (Ad-Lac Z) (Fig. 3A). Cell proliferation was expressed as a percentage of proliferation compared to that without infection (0 MOI of AdADAMTS1 or Ad-Lac Z). Although the negative control viral vector alone (Ad-Lac Z) inhibited cell proliferation by approximately 20.0% at MOI of 50 and 100, the proliferation of HMVEC-dLy infected with Ad-ADAMTS1 was significantly less at both 50 and 100 MOI than that of the negative controls. These results indicated that the ADAMTS1 transfectants secreted by transduced

Fig. 2 – Inhibition of capillary-like tube formation by ADAMTS1. (A) Uninfected (0 MOI) and Ad-ADAMTS1-transduced (Ad-ADAMTS1) or Ad-Lac Z-transduced (Ad-Lac Z) HMVEC-dLy at 50 and 100 MOI were plated on a Matrigel surface and incubated for 14 h. (40x magnification). (B) Quantification of capillary-like tube formation. Tube formation was quantified by counting the number of tubes that formed per field. Three random areas of triplicate wells were imaged, the total tube number was measured using Image J software, and the recorded number was averaged. The results shown are the mean7SD of three independent experiments. Asterisks indicate significant differences from control values (*, po0.0001). Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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cells were bioactive and attenuated the proliferation of HMVECdLy in vitro. We further examined the effect of ADAMTS1 transduced by electroporation on the proliferation of HMVECdLy. As shown in Fig. 3B, HMVEC-dLy proliferation with the ADAMTS1 overexpression was also significantly less than that of control cells treated with the empty vector after 72 and 96 h incubation.

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Inhibition of HMVEC-dLy migration to MDA-MB-231 cells infected with Ad-ADAMTS1 As shown in Fig. 4A, a HMVEC-dLy-MDA-MB-231 cell Transwell co-culture system was established using the BD Biocoat™ Angiogenesis System to examine the effect of HMVEC-dLy cell migration on Ad-ADAMTS1- or Ad-Lac Z-transduced MDA-MB-231 cells. After HMVEC-dLy were co-cultured with Ad-ADAMTS1-transfected MDAMB-231 cells for 22 h, HMVEC-dLy migration to Ad-ADAMTS1transfected MDA-MB-231 cells was significantly less than that of control and Ad-Lac Z-transfected MDA-MB-231 cells with or without VEGFC (po0.005, po0.05) (Fig. 4B and C).

Inhibitory effect of ADAMTS1 on VEGFC-stimulated phosphorylation of VEGFR3 and associated downstream signaling molecules in HMVEC-dLy Cell proliferation, migration, and tube formation was significantly less with transduced ADAMTS1 than with the control (nontransduced HMVEC-dLy). Therefore, we examined the effect on the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1transduced HMVEC-dLy. VEGFC-stimulated phosphorylation of VEGFR3 was significantly inhibited by ADAMTS1 gene transduction in HMVEC-dLy (Fig. 5A and B). Since signaling by VEGFR3 promotes lymphatic endothelial cell proliferation, migration, and survival via activation of the Erk 1/2, Akt, and Jnk1/2 pathway, we next examined whether transduced ADAMTS1 also inhibited Erk1/2 and Akt phosphorylation. The phosphorylation of Erk1/2 and Akt was less in ADAMTS1-transduced HMVEC-dLy than in uninfected and Ad-Lac Z-infected control cells, and phosphorylation decreased in a dose-dependent manner (Fig. 6A and B).

ADAMTS1 bound to VEGFC

Fig. 3 – Inhibition of ADAMTS1-transduced HMVEC-dLy proliferation. (A) After adenovirus infection for 2 h, HMVEC-dLy were seeded on 96-well tissue culture plates and incubated for 72 h. Cell proliferation was assessed by the MTS assay. Cell proliferation was expressed as a percentage of proliferation relative to that of the control empty adenovirus (Ad-Lac Z)infected cells. The mean7SD of quadruplicates are shown. Asterisks indicate significant differences from control values (*, po0.005, **, po0.001). B: Inhibition of HMVEC-dLy proliferation by transfected-ADAMTS1 using electroporation. After electroporation, cells were seeded on 24-well tissue culture plates with 500 μL of culture medium without antibiotics and incubated for 24 h. The medium was then changed and cells were incubated for 24, 48, 72, and 96 h. Cell proliferation was assessed and compared with that of empty vector by the MTS assay. Relative cell proliferation was normalized to untreated controls (0 h incubation). The mean7SD of quadruplicates are shown. Asterisks indicate significant differences from control values (*, po0.005, **, po0.001).

ADAMTS1 was previously shown to exhibit anti-angiogenic properties by interacting with VEGF165 and inhibiting VEGF165-stimulated VEGFR2 phosphorylation [26]. In view of the inhibition of lymphatic endothelial cell proliferation, migration, tube formation, and VEGFCstimulated VEGFR3 phosphorylation by transduced ADAMTS1 (shown above), we investigated whether ADAMTS1 could also directly bind to rVEGFC using dot blot assays. As shown in Fig. 7A, transduced ADAMTS1 of the conditioned medium interacted with rVEGFC, but not with BSA in a dose-dependent manner. Furthermore, the interaction of ADAMTS1 with rVEGFC was also evaluated by co-immunoprecipitation assay. The recombinant full ADAMTS1-V5 and VEGFC-HaloTag proteins were extracted and served for the immunoprecipitation. As shown in Fig. 7B, we detected VEGFC-HaloTag protein immunoprecipitated by anti-V5 antibody. We also detected the ADAMTS1 protein precipitated with anti-HaloTag antibody (Fig. 7C). No band of VEGFC or ADAMTS1 was detected in the immunoprecipitated complexes in the transfectants with CMV control vector (lane 2 in Fig. 7B and C) and the immunocomplexes with normal mouse IgG (lane 3 in Fig. 7B and C). The target protein expression was confirmed by Western blot and shown as a positive control (lane 4 in Fig. 7B and C).

Titration of Ad-ADAMTS1 transduced conditioned medium As shown in Fig. 8A, the inhibitory effect of VEGFR3 phosphorylation by ADAMTS1 was attenuated by the dilution with serum-free

Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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medium. The phosphorylation of Akt and Erk1/2 were also recovered by diluting Ad-ADAMTS1-conditiomed medium with serum-free conditioned medium (Fig. 8B).

Effect of Ad-ADAMTS1 on COX2 expression in HMVEC-dLy We next examined the effect of Ad-ADAMTS1 on COX2 mRNA expression in the lymphatic endothelial cells. Interestingly, AdADAMTS1 attenuated COX2 mRNA expression levels (Fig. 9).

Discussion The purpose of this study was to evaluate the possibility of using an adenoviral gene transduction approach to induce exogenous ADAMTS1 in lymphatic endothelial cells and assess the in vitro effect on lymphangiogenesis. We herein report that adenoviral gene

Fig. 4 – ADAMTS1 inhibited VEGFC-induced HMVEC-dLy migration to MDA-MB-231. (A) Schematic diagram of the co-culture system of HMVEC-dLy and ADAMTS1-transduced MDA-MB-231. Ad-ADA MTS1-, Ad-Lac Z-transduced, or uninfected MDA-MB-231 cells were seeded on the lower chambers, followed by a replacement with 5% FBS-containing complete medium overnight. After further incubation under the starved condition, the bottom well was replaced with the culture medium supplemented with 50 ng/mL VEGFC. HMVEC-dLy were seeded onto Transwell inserts, the insert wells were put in the bottom wells in which MDA-MB-231 cells was placed, and then incubated for 22 h. (B) The migrated endothelial cells were post-labeled with BD Calcein AM Fluorescent Dye, and then photographed under a fluorescence microscopy at 40  magnification. (C) Calcein AM-labeled migrated endothelial cells were quantified by a Multi-Detection Microplate Reader, and data was expressed as the mean7SD from three independent experiments. Asterisks indicate significant differences from control values (*, po0.05, **, po0.005).

Fig. 5 – Inhibitory effect of ADAMTS1 on the VEGFC-stimulated phosphorylation of VEGFR3. (A) uninfected, Ad-ADAMTS1- or Ad-Lac Z-transduced HMVEC-dLy were serum-starved overnight in the presence of 0.1% BSA, and then stimulated with 100 or 200 ng/mL rVEGFC for 10 min. Cell lysates prepared from treated and non-treated cells were subjected to immunoprecipitation (IP) with the VEGFR3-specific antibody before separation by 10% SDS-PAGE, and Western blotting was then performed with the phospho-tyrosine antibody (top), followed by detection with the VEGFR3-specific antibody (bottom). The two main isoforms (250, 150 kDa) of VEGFR3 were phosphorylated on tyrosine residues. (B) Data of VEGFR3 phosphorylation were quantified using Image J software and normalized to the signal intensity of the VEGFR3 protein (n¼ 34). Asterisks indicate significant differences from control values (*, po0.05).

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transduction into lymphatic endothelial cells resulted in the efficient expression of ADAMTS1 in cell lysates and conditioned media. We demonstrated that adenovirus-transduced ADAMTS1 inhibited lymphangiogenesis in vitro. Furthermore, ADAMTS1 directly bound to VEGFC and dampened VEGFC-stimulated VEGFR3 phosphorylation,

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thereby suppressing lymphatic endothelial cell proliferation, migration, and tube formation. We used adenoviral transduction system in this study. Although the cell damage induced by the adenovirus should be considered, it was relatively small as indicated by the control adenovirus

Fig. 6 – ADAMTS1 decreased the phosphorylation of VEGFR3-associated downstream signals (Erk1/2 and Akt). Uninfected, Ad-ADAMTS1- or Ad-Lac Z-transduced HMVEC-dLy were treated with 50 or 100 ng/mL rVEGFC, harvested, and subjected to western blot analysis with the indicated antibodies. β-actin was used as an internal control. A dose-dependent decrease in the phosphorylation of Erk1/2 and Akt was less in ADAMTS1-transduced HMVEC-dLy than in uninfected and Ad-Lac Z-infected control cells, and phosphorylation decreased in a dose-dependent manner. (B) Data of Erk1/2 and Akt phosphorylation were quantified using Image J software and normalized to the signal intensity of Erk1/2 and Akt protein, respectively (n¼ 3–4). Asterisks indicate significant differences from control values (*, po0.05) and n.s. indicated no significant difference. Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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Fig. 7 – Binding of ADAMTS1 and VEGFC. (A) Dot blot analysis of a serial dilution of rVEGFC using the conditioned medium of Ad-ADAMTS1-transduced HMVEC-dLy and the V5-tag antibody. BSA was used as a negative control. Diluted BSA and rVEGFC (0.1, 0.2, and 0.5 μg) were blotted onto the PVDF membrane and incubated at a dilution of 1/2 with the conditioned medium of Ad-ADAMTS1-transduced HMVEC-dLy at 50 MOI for 2 h at room temperature. The binding of ADAMTS1 and rVEGFC was detected via the V5-tag antibody, followed by the HRP-conjugated anti-mouse IgG antibody. (B) Co-immunoprecipitation assay with anti-V5 antibody was performed. Lane 1: HEK293 cell lysates co-transfected with full ADAMTS1-v5 and VEGFC-HaloTag were probed with anti-HaloTag antibody. The band indicated the VEGFC-HaloTag protein pull-down by anti-V5 antibody. Lane 2: HEK293 cell lysate transfected with CMV (control vector) and probed with anti-HaloTag antibody. Lane 3: HEK293 cell lysate co-transfected with full ADAMTS1-v5 and VEGFC-HaloTag were immunoprecipitated with normal mouse IgG and probed with anti-HaloTag antibody. Lane 4: HEK293 cell lysate co-transfected with full ADAMTS1-v5 and VEGFC-HaloTag before immunoprecipitation was loaded and probed with anti-HaloTag antibody. (C) Co-immunoprecipitation assay with antiHaloTag antibody was performed. Lane 1: HEK293 cell lysates co-transfected with both constructs were probed with anti-V5 antibody. The band indicated the full ADAMTS1-v5 protein pull-down by anti-HaloTag antibody. Lane 2: HEK293 cell lysate transfected with CMV (control vector) and probed with anti-V5 antibody. Lane 3: HEK293 cell lysate co-transfected with full ADAMTS1-v5 and VEGFC-HaloTag were immunoprecipitated with normal mouse IgG and probed with anti-V5 antibody. Lane 4: HEK293 cell lysate co-transfected with full ADAMTS1-v5 and VEGFC-HaloTag before immunoprecipitation was loaded and probed with anti-V5 antibody. Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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Fig. 9 – Effect of Ad-ADAMTS1 on COX2 mRNA expression. The COX2 mRNA expression was examined by qRT-PCR. Control indicated the RNA extracted from uninfected cells and the COX2 mRNA expression in control RNA was indicated as 1 and relative expressions levels were shown. Asterisks indicate significant differences from control values (n, po0.05).

Fig. 8 – Recovery of the phosphorylation of VEGFR3 by titrating the Ad-ADAMTS1-containing conditioned medium. AdADAMTS1-conditioned medium were diluted 1/2 and 1/10 and compared the phosphorylation of VEGFR3, Akt, Erk1/2. Control; serum-free conditioned medium. Note that the inhibitory effect of phosphorylation was decreased by titration of the Ad-ADAMTS1-containing conditioned medium.

(adeno-LacZ) used in this study. We also confirmed that other methods (i.e., electroporation) had similar inhibitory effects on lymphatic endothelial cell proliferation. Accordingly, the inhibitory effect of adenovirus-transduced ADAMTS1 was not a non-specific effect caused by the adenovirus, but was caused by the exogenous ADAMTS1 itself. Lymphangiogenesis play roles for tissue development and homeostasis. Disrupted lymphangiogenesis in ovaries from postnatal day 10 and 21 in ADAMTS1-null homozygotes mice has been reported, indicating that ADAMTS1 is essential for lymphangiogenesis in ovary at this stage [19]. However, LYVE-1 positive lymphatic vessels were observed in extra-ovarian fat tissue at day 10 and 21. Interestingly, LYVE-1 positive lymphatic vessels were observed in ovaries of adult cycling ADAMTS1 null homozygotes and immature mice after treatment on day21 with pregnant mare serum gonadotropin (PSMG). Furthermore, LYVE-1 protein expression was similar in the liver at day 10 mice between ADAMTS1 null homozygotes and heterozygotes and there was no developmental defect in the dermal lymphatic vessel formation in back and ear skin of day 10 and 21 mice in ADAMTS1 null homozygotes. Brown et al. investigation indicated that lymphatic vessel formation in the immature ovary

may be hormonally regulated [19]. Such hormonal regulation of ADAMTS1 was also reported, indicating that ADAMTS1 may play an important role for lymphangiogenesis and follicle growth. Our results were not matched with their data because lymphangiogenesis is regulated by a complex mechanism. Thus, it is not fully clarified how ADAMTS1 interacts with VEGFC in lymphangiogenesis in developing mouse organs and this interesting theme remains to be elucidated. Lymphangiogenesis are also important process that disseminates tumor cells through the lymphatic system to distant organs as well as regional lymph nodes. Metastatic tumor cells and tumor-associated macrophages can actively induce lymphangiogenesis via VEGFC, VEGFD, and VEGFA production and further promote tumor metastasis. These pro-lymphangiogenic factors interact with lymphatic endothelial receptor VEGFR3 and VEGFR2 and result in the activation of lymphatic endothelial cell proliferation, survival, and migratory signaling [44,45]. We herein showed that ADAMTS1-transduced MDA-MB231 cells inhibited HMVEC-dLy migration. Tube formation assay revealed that ADAMTS1 inhibited tube formation in HMVECdLy. MTS assay also demonstrated that ADAMTS1 inhibited HMVECdLy proliferation. Together with these data, ADAMTS1 is likely to show an inhibitory effect on lymphangiogenesis. VEGFC/VEGFR-3 signaling is essential for the development of lymphatic vessels and tumor dissemination via the lymphatics. Our immunoprecipitation study demonstrated that ADAMTS1 bound to the VEGFC. Luque et al., showed that recombinant ADAMTS1 directly bound to VEGF165, affected the subsequent phosphorylation of VEGFR2, and consequently inhibited VEGF-induced angiogenesis [26]. Our results indicated that ADAMTS1 was able to bind VEGFC as well as VEGFA, which subsequently attenuates further lymphangiogenesis signals. Western blot data on phosphorylation showed the attenuation of VEGFR3 phosphorylation induced by VEGFC, Akt and Erk1/2 signaling. Akt and Erk1/2 signals play essential roles in lymphangiogenesis. VEGFC is known to activate Akt and Erk1/2 signaling [46] and these signals are also known to induced VEGFC expression [47]. Our titration data of Ad-ADAMTS1 transduced conditioned medium indicated that secreted ADAMTS1 contained the inhibitory effect of Akt and Erk1/2 phosphorylation. Interestingly,

Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002

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the inhibitory effect on VEGFR3, Akt and Erk1/2 phosphorylation by ADAMTS1 was reduced by the addition of excess VEGFC recombinant protein, which indicated that this action was reversible (data not shown). To the best of our knowledge, this is the first study to clarify inhibition of the signaling pathway by ADAMTS1 in lymphatic endothelial cells. Thus, dampening VEGFC/VEGFR-3 signaling by ADAMTS1 may function as a new therapeutic tool. Cursiefen C et al. reported that the endogenous angiogenesis inhibitor TSP-1 can inhibit inflammatory lymphangiogenesis. This inhibitory action driven by TSP-1 was considered to be due to the down-regulation of VEGFC expression in macrophages via CD36, a receptor for TSP-1 on macrophages. The ligation of TSP-1 to CD36 on macrophages was shown to prevent their expression of VEGFC, and thus explains the indirect inhibitory effect of TSP-1 under inflammatory conditions on corneal lymphangiogenesis [48]. Since ADAMTS1 can cleave ECM-bound TSP-1 homotrimers to release active anti-angiogenic fragments and generate a pool of these fragments [27], ADAMTS1 may also be able to indirectly attenuate lymphangiogenesis in vivo using cleaved TSP-1 fragments via its CD36 ligation on macrophages. However, previous reports have demonstrated that ADAMTS1 expression levels in tumor xenografts inversely correlated with TSP-1 and VEGFA [49,50]. In addition, the anti-angiogenic activity of TSP-1 was found to be differentially regulated by ADAMTS1 in different tissue microenvironments [51]. Therefore, the effect of ADAMTS1 on TSP-1 activity in regard to lymphangiogenesis remains to be elucidated. In conclusion, we demonstrated that ADAMTS1 produced by adenovirus-infected lymphatic endothelial cells inhibited VEGFR3 signaling under VEGFC stimulation, and that it also inhibited cell migration, proliferation, and tube formation induced by VEGFC. In particular, we showed that the inhibitory effect of ADAMTS1 was caused by direct binding to VEGFC. The present study provides the first evidence that ADAMTS1 has an anti-lymphangiogenic function in addition to its anti-angiogenic function. Therefore, ADAMTS1 is considered to be a potential dual inhibitor for tumor angiogenesis and lymphangiogenesis.

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Acknowledgments [18]

The authors are grateful to Drs. Toshitaka Oohashi, Tomoko Yonezawa, and Aiji Ohtsuka and other members of our department for their stimulating discussions and suggestions. This work was supported in part by a Grant-in-Aid (23612004 to J.I. and 23390366 to S.H.) for Scientific Research and an Invitation Fellowship (S13731 to M.H.) from the Japan Society for the Promotion of Science.

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Please cite this article as: J. Inagaki, et al., ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.002