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First published online 18 May 2004
doi: 10.1242/jcs.01112

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Anti-angiogenic action of the C-terminal domain of tenomodulin that shares homology with chondromodulin-I

¹ Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
² Department of Ophthalmology, Osaka University Medical School, Suita 565-0871, Japan
³ Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Suita 565-0871, Japan

View larger version (23K): [in a new window]   Fig. 1. Structural features of human tenomodulin (TeM) and the human chondromodulin-I (ChM-I) precursor proteins deduced from their cDNA sequences. Analysis of primary amino acid sequences reveals several structural features in the putative TeM protein. These include a type II transmembrane domain at the N terminus, a BRICHOS domain and a cysteine-rich domain (Sanchez-Pulido et al., 2002). The TeM protein contains two N-glycosylation sites within the BRICHOS domain. The highest homology with the ChM-I precursor was scored in the cysteine-rich domain at the C terminus (Sachdev et al., 2001; Shukunami et al., 2001).

View larger version (92K): [in a new window]   Fig. 2. Characterization of the TeM protein. (A) Detection of human TeM and ChM-I proteins expressed in COS7 cells. COS7 cells were transfected with empty vector (mock), N-terminal FLAG-tagged (FLAG-hTeM) or C-terminal FLAG-tagged (hTeM-FLAG) full-length hTeM (wild), N-terminal FLAG-tagged secreted human TeM (Glu202-Val317) (FLAG-shTeM) and N-terminal FLAG-tagged secreted human ChM-I precursor (Glu215-Val334) (FLAG-shChM-I). Cell lysates and culture media were analyzed by western blotting using anti-FLAG monoclonal antibodies. (B) Identification of human TeM as a transmembrane protein by cell surface biotinylation. COS7 cells transfected with empty vector (mock), FLAG-hTeM cDNA or hTeM-FLAG cDNA were biotinylated on their cell surfaces. The expressed protein was immunoprecipitated with anti-FLAG antibody and detected by streptavidin binding and anti-TeM antibody, respectively. (C,E,G) COS7 cells expressing TeM tagged with either FLAG or empty vector were incubated with FITC-conjugated anti-FLAG M2 antibody for 2 hours at 4°C under nonpermeabi lizing conditions and fixed with PBS containing 4% paraformaldehyde. (D,F,H) Permeabilized COS7 cells were incubated with FITC-conjugated anti-FLAG M2 antibody. Cells expressing hTeM-FLAG are shown in C and D, FLAG-hTeM in E and F and empty vector in G and H, respectively. Scale bars: 20 µm.

View larger version (47K): [in a new window]   Fig. 3. Detection of TeM protein in mouse skeletal muscle. Tissue extracts were prepared from muscle and liver of 4-week-old DDY mice. Western blotting was carried out with anti-TeM antibodies. Immunoreactive bands were detected in muscle but not in liver extracts.

View larger version (46K): [in a new window]   Fig. 4. Effects of conditioned media derived from COS7 cells expressing hTeM, shTeM or shChM-I on HUVEC tube formation. (A) HUVECs were suspended in a mixture of culture media from COS7 cells transfected with the indicated cDNAs and fresh EBM medium at a ratio of 1:1. The cells were then plated on growth factor-reduced Matrigel in the presence of VEGF (20 ng/ml). Representative phase-contrast microscopic images after 6 hours incubation are shown. HUVECs suspended in fresh medium derived from wild-type untransfected COS7 cells and in medium collected from either mock- or hTeM-transfected cultures organized themselves into tubular networks. By contrast, HUVECs suspended in media derived from either shTeM- or shChM-I-transfected COS7 cells did not form tubular structures. Scale bar: 100 µm. (B) Total tube length per field was measured using image processing and analysis software. The data represent the mean±s.d. from three independent experiments. *P<0.01 versus HUVECs suspended in the EBM medium.

View larger version (38K): [in a new window]   Fig. 5. Construction and expression of Ad-shTeM, Ad-shChM-I and Ad-hTeM in adenoviral vectors. (A) The structure of the pALC3 cosmid vector with an expression cassette containing either a shTeM, shChM-I or hTeM insert. The 34-kb adenoviral genome is indicated as a closed ring. The 7-kb cosmid vector backbone is flanked by loxP sites and includes a cos site, a bacterial ori site and the ampicillin resistance gene (Amp). The expression cassette consisting of the cytomegalovirus immediate-early enhancer-chicken ß-actin hybrid (CAG) promoter, the preprotrypsin secretion signal (ss), the indicated FLAG inserts, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) cDNA followed by a polyadenylation signal (pA) was cloned into the SwaI site of the pALC3 vector, resulting in the pALC3-shChM-I, pALC3-shTeM or pALC3-hTeM cosmid. Recombinant adenoviruses were generated upon cotransfection of a Cre-expression plasmid with pALC3-shChM-I, pALC3-shTeM or pALC3-hTeM cosmids into 293 cells and are designated as Ad-shChM-I, Ad-shTeM or Ad-hTeM. Empty vector expressing EGFP alone was designated as Ad-EGFP. (B) Phase-contrast microscopic image (upper panel) indicating the cobblestone-like morphology of HUVECs 24 hours after adenoviral infection. The fluorescence microscopic image (lower panel) shows efficient adenoviral gene transduction in HUVECs. Scale bar: 40 µm. (C) Fluorescence microscope images of uninfected HUVECs (upper panel) or HUVECs transduced with Ad-EGFP. Dil-AcLDL uptake is observed as red spots regardless of adenoviral transduction. Cell nuclei were stained blue-violet with Hoechst dye 33342 (upper panel). Green fluorescence is observed in the HUVECs expressing EGFP (lower panel). Scale bar: 40 µm. (D) Cell lysates from uninfected HUVECs (wild) or the HUVECs infected with Ad-hTeM were directly harvested with 1x SDS sample buffer and then subjected to western blotting (left panel). Secreted proteins were concentrated by a Butyl-Toyopearl column from conditioned medium derived from wild type HUVECs (wild) or HUVECs transduced by Ad-EGFP, Ad-shChM-I or Ad-shTeM, and subjected to western blot analysis by anti-FLAG monoclonal antibodies.

View larger version (38K): [in a new window]   Fig. 6. Inhibition of HUVEC tube formation (A) HUVECs were transduced with Ad-EGFP, Ad-shChM-I or Ad-shTeM and then plated on growth factor-reduced Matrigel in the presence of VEGF (20 ng/ml). Representative phase-contrast micrographs (left panels) and fluorescence micrographs (right panels) after 6 hours incubation at 37°C are shown. Wild-type HUVECs (wild) or HUVECs transduced with either Ad-EGFP or Ad-hTeM organized into tubular networks, whereas HUVECs transduced with either Ad-shChM-I or Ad-shTeM were inhibited from doing so. Scale bar: 100 µm. (B) Total tube length per field was measured by image processing and analysis software. The data represent the means±s.d. from four independent experiments. *P<0.01 versus wild-type HUVECs.

View larger version (18K): [in a new window]   Fig. 7. Inhibition of HUVEC proliferation following transduction with either Ad-shChM-I or Ad-shTeM. HUVECs transduced with either Ad-EGFP, Ad-shChM-I or Ad-shTeM were plated into 96-well plates. The cells were starved in 0.5% FBS-containing MEM for 6 hours and then incubated with VEGF (20 ng/ml) for 12 hours. The rate of DNA synthesis was evaluated by BrdU incorporation followed by measurement of BrdU ELISA chemiluminescence. A MAP kinase inhibitor, PD98059 (50 µM), was used as a negative control. Data are mean±s.d. of triplicate measurements.

View larger version (70K): [in a new window]   Fig. 8. Inhibition of migration in HUVECs transduced with Ad-shChM-I or Ad-shTeM. (A) Cell migration was evaluated using a modified Boyden chamber assay with vitronectin-coated filters. Representative images of HUVECs migrating to the lower side of the membranes in the presence of VEGF (20 ng/ml) are shown. Bar, 50 µm. (B) The number of cells migrating to the lower side of the membranes was measured. Data are mean±s.d. of triplicate measurements. *P<0.01 versus wild-type HUVECs.

View larger version (38K): [in a new window]   Fig. 9. Inhibition of cell adhesion and spreading via vitronectin in HUVECs transduced with Ad-shChM-I or Ad-shTeM. (A) Both adenovirus-transduced and wild-type HUVECs were harvested with trypsin/EDTA and suspended in culture medium for 20 minutes. The cells were re-suspended in MEM containing 0.1% BSA at a density of 5x105 cells/ml and seeded on 96-well culture plates coated with type I collagen, fibronectin or vitronectin. After incubation for 1 hour at 37°C, unattached cells were removed by gentle washing with PBS. Cellular morphologies of adherent cells were observed and photographed by fluorescent microscopy. Bar, 100 µm. (B) For quantification of adhesion, the adherent cells were stained with crystal violet. The crystal violet stain was eluted with 0.1 M sodium citrate (pH 4.2) in 50% ethanol and the number of adherent cells in each well was evaluated by the measurement of absorbance at 596 nm. Data are means±s.d. of triplicate assays.

View larger version (81K): [in a new window]   Fig. 10. Suppression of both tumor growth and tumor-induced angiogenesis by transduction of Ad-shChM-I or Ad-shTeM. (A) C57BL/6 mice at 21 days following implantation of melanoma cells (BL-6). Red circles indicate implanted tumors. Scale bar: 1.2 cm. (B) Images of subcutaneously injected melanoma tumor cells, transduced with or without Ad-shChM-I, Ad-shTeM and Ad-EGFP, were photographed also on day 21. (C) Time course of increase in tumor weight following implantation. At 7, 14 and 21 days after subcutaneous implantation of both transduced and non-transduced melanoma cells, the tumor volume was determined in Ad-shChM-I (open boxes), Ad-shTeM (open circles)-, Ad-EGFP (filled boxes)-transfected groups or in the wild-type group. The plots represent the mean values of tumor volumes from four animals in each group. Bars indicate±s.d. (D) Reduced angiogenesis in Ad-shChM-I or Ad-shTeM transduced tumors is apparent. Vasculariza tion in tumors on day 21 following implantation is visualized by staining of PECAM-1 with monoclonal antibodies. Cell nuclei are stained blue-violet with Hoechst dye 33342 as. Scale bar: 400 µm. (E) Microvessel numbers were counted from four different random fields of both adenovirus transduced or non-transduced melanoma cells. Data are represented as mean values (±s.d.) of microvascular densities per field (x40) from sections of Ad-shChM-I-, Ad-shTeM-, Ad-EGFP-transduced or non-transduced tumors (P<0.005).