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Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis ¹

ISAO INOKI^*,, TAKAYUKI SHIOMI^*, GAKUJI HASHIMOTO^*, HIROYUKI ENOMOTO^*, HIROYUKI NAKAMURA^*, KEN-ICHI MAKINO^*, EIJI IKEDA^*, SHIGEO TAKATA, KEN-ICHI KOBAYASHI and YASUNORI OKADA^*2

^* Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-0016, Japan; and
First Department of Internal Medicine, School of Medicine, Kanazawa University, 13–1 Takaramachi, Kanazawa 920-8640, Japan

²Correspondence: Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku Tokyo 160-0016, Japan. E-mail: okada{at}sc.itc.keio.ac.jp

SPECIFIC AIMS

In this study we screened the binding inhibitors against vascular endothelial growth factor (VEGF) in a human chondrocyte cDNA library by a yeast two-hybrid system using VEGF₁₆₅ as a bait and identified connective tissue growth factor (CTGF) as a candidate. We further studied the binding mechanism of these two molecules and the inhibitory activity of CTGF to VEGF₁₆₅-induced angiogenesis both in vitro and in vivo.

PRINCIPAL FINDINGS

1. CTGF binds to VEGF₁₆₅ through specific interaction between the TSP-1 domain of CTGF and the exon 7-coded region of VEGF₁₆₅
Of 120 clones isolated by yeast two-hybrid system and sequenced, six clones were human CTGF. The complex formation of VEGF₁₆₅ with CTGF was first established by immunoprecipitation from the 293T cells overexpressing both binding partners. A competitive binding assay and inhibition of the binding by anti-CTGF antibodies demonstrated that CTGF binds specifically to VEGF₁₆₅. Since CTGF consists of four distinct domains, i.e., the insulin-like growth factor binding protein (IGFBP, I), von Willebrand factor type C (vWFC, II), thrombospondin type 1 repeat (TSP-1, III), and COOH-terminal (CT, IV) domains, we determined the domains involved in the binding to VEGF₁₆₅ using radioiodinated full-length CTGF(I.II.III.IV.) and its deletion mutants. Full-length CTGF(I.II.III.IV.) could bind to both VEGF₁₆₅ and VEGF₁₂₁. Scatchard analyses of the data indicated the presence of two classes of binding sites of CTGF(I.II.III.IV.) to VEGF₁₆₅ with K_d values of 26 ± 11 nM and 125 ± 38 nM, whereas only one class of binding site with a K_d value of 167 ± 36 nM was obtained with VEGF₁₂₁. In contrast, CTGF(I.II.) lacking both TSP-1 and CT domains bound to neither VEGF₁₆₅ nor VEGF₁₂₁. CTGF(I.II.III.) without the CT domain showed affinity only to VEGF₁₆₅, whereas CTGF(I.II.IV.) lacking the TSP-1 domain interacted with both VEGF₁₆₅ and VEGF₁₂₁. These data indicate that the third TSP-1 domain is involved in the specific binding to the exon 7-coded region of VEGF₁₆₅ and that the CT domain interacts with both VEGF₁₆₅ and VEGF₁₂₁.

2. CTGF inhibits the binding of VEGF₁₆₅ to human umbilical vein endothelial cells (HUVEC) or to a recombinant VEGF receptor 2 (VEGFR-2), KDR/IgG Fc
The binding of VEGF₁₆₅ to HUVEC was specifically inhibited by full-length CTGF(I.II.III.IV.) up to the level of 30% of the maximal binding capacity, whereas CTGF did not affect the binding of VEGF₁₂₁. CTGF also attenuated the binding of VEGF₁₆₅ to immobilized KDR/IgG Fc up to 30% of the total binding, but the binding of VEGF₁₂₁ was not altered with CTGF. In contrast, CTGF showed no effects on the binding of both VEGF isoforms to immobilized Flt-1/IgG Fc, a recombinant VEGFR-1.

3. Full-length CTGF and its deletion mutant containing TSP-1 domain efficiently inhibit VEGF₁₆₅-induced in vitro tube formation of bovine aortic endothelial cells (BAEC)
The in vitro tube formation of BAEC in type I collagen gel was stimulated in the presence of CTGF(I.II.III.IV.) up to 2.5-fold compared with that of negative control. On the other hand, VEGF₁₂₁ (40 ng/ml) and VEGF₁₆₅ (20 ng/ml) enhanced by sixfold the tube formation compared with that of the control. The stimulation of tube formation by VEGF₁₆₅ was significantly inhibited < 45% of the maximal effect by low concentrations of CTGF (10 and 20 ng/ml) (Fig. 1 A, B), whereas inhibition of the VEGF₁₂₁-induced tube formation by CTGF was less effective and required higher concentrations of 500 and 1000 ng/ml CTGF. Addition of anti-CTGF polyclonal antibodies to the VEGF₁₆₅/CTGF(I.II.III.IV.) complex completely reversed the inhibition of the VEGF₁₆₅-induced tube formation. To identify which domains of CTGF are involved in the inhibition of the VEGF-induced angiogenesis, we further studied the effects of two deletion mutants of CTGF, i.e., CTGF(I.II.III.) and CTGF(I.II.IV.). Although CTGF(I.II.III.) neither stimulated the tube formation nor inhibited the VEGF₁₂₁-dependent angiogenesis, the VEGF₁₆₅-dependent tube formation was remarkably inhibited with CTGF(I.II.III.) in a pattern similar to that with CTGF(I.II.III.IV.). CTGF(I.II.IV.) did not greatly inhibit the VEGF₁₆₅-dependent tube formation, although a high concentration of 1000 ng/ml showed significant inhibition of VEGF₁₂₁- and VEGF₁₆₅-dependent angiogenesis.

View larger version (52K): [in this window] [in a new window]   Figure 1. Effect of full-length CTGF(I.II.III.IV.) on VEGF165-induced tube formation. A) Representative micrographs of the VEGF165-induced tube formation of BAEC in the presence of CTGF(I.II.III.IV.). BAEC were stimulated for 3 days with VEGF165 in the presence of 0 (1), 10 (2), 20 (3), 100 (4), 500 (5) or 1000 ng/ml CTGF(I.II.III.IV.) (6), 20 ng/ml CTGF(I.II.III.IV.) and 500 ng/ml anti-CTGF IgG (7), or 20 ng/ml CTGF(I.II.III.IV.) and 500 ng/ml nonimmune rabbit IgG (8) and photographed. B) Tubular length was measured in 5 different areas of 0.25 mm2 and expressed as mm/mm2. *P < 0.05.

4. CTGF inhibits VEGF₁₆₅-induced in vivo angiogenesis in the Matrigel model
To determine the effects of full-length CTGF on VEGF₁₆₅-induced angiogenesis, an in vivo angiogenesis assay using the Matrigel injection model in mice was carried out. Whereas the Matrigel plugs without any growth factors showed only a few spindle-shaped cells and few blood vessels (Fig. 2 A), VEGF₁₆₅ markedly stimulated the migration of the cells and blood vessel formation (Fig. 2B ). The angiogenic reaction by VEGF₁₆₅ was remarkably inhibited by CTGF (Fig. 2C ), whereas CTGF itself showed only a weak angiogenic reaction (Fig. 2D ). Most of the spindle-shaped cells and the vessel-forming cells within the plugs were immunostained with the vWF antibody (Fig. 2E –G). The number of vWF-positive cells and blood vessels in the plugs was significantly 3.3-fold and 6.4-fold increased with VEGF₁₆₅, respectively. CTGF inhibited the number of the vWF-positive cells and blood vessels stimulated by VEGF₁₆₅ up to 37% and 30% of the maximal activity, respectively (Fig. 2I , 2J ). The stimulatory effect of CTGF itself was negligible in the assay.

View larger version (61K): [in this window] [in a new window]   Figure 2. Antiangiogenic effect of CTGF(I.II.III.IV.) against VEGF165-induced angiogenesis in vivo. Matrigel containing phosphate buffered saline (PBS) (A, E), VEGF165 (50 ng/ml) (B, F, H), VEGF165 (50 ng/ml) mixed with CTGF (100 ng/ml) (C, G), or CTGF (100 ng/ml) (D) was injected subcutaneously (s.c.) into C57BL/6 mice. Skin tissues with Matrigel plugs were removed 5 days after the injection, fixed, and embedded in paraffin. The sections were stained with hematoxylin and eosin (A–D) or immunostained with anti-vWF antibody (E–G) or nonimmune rabbit serum (H). Bars, 50 µm. The degree of angiogenesis was evaluated by counting the number of the vWF-immunostained spindle cells (I) and blood vessels (J) in the Matrigel plugs with PBS, VEGF165, VEGF165 mixed with CTGF, or CTGF. The bars represent mean ± SD of six mice. *P < 0.05.

CONCLUSIONS

VEGF is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. However, the in vivo angiogenic activity of secreted VEGF may be regulated by inhibitors in the extracellular milieu, since it is also produced in avascular tissues such as cartilage. To find the binding inhibitors against VEGF, we screened a chondrocyte cDNA library by a yeast two-hybrid system using VEGF₁₆₅ as a bait and identified CTGF as a candidate. The specific interaction of CTGF with VEGF₁₆₅ was demonstrated by immunoprecipitation, competitive affinity binding assay, and an inhibition study with anti-CTGF polyclonal antibodies. The data on the binding of VEGF₁₆₅ and VEGF₁₂₁ to CTGF deletion mutants indicate that of the four functional domains of CTGF, the TSP-1 domain is responsible for the specific interaction with the exon 7-coded region of VEGF₁₆₅ and that the CT domain interacts with some domain other than the exon 7-coded region of VEGF (Fig. 3 ).

View larger version (48K): [in this window] [in a new window]   Figure 3. Schematic diagram of VEGF165-induced angiogenesis and its inhibition by CTGF(I.II.III.IV.). Left column: VEGF165 binds to VEGFR-2 (KDR) of endothelial cells and the exon 7-coded region (Exon 7) interacts with NRP-1 and HSPG to promote angiogenesis. Right column: TSP-1 domain of CTGF (TSP-1) binds to Exon 7 and COOH-terminal domain (CT) binds to some region of VEGF165 except for Exon 7. Complex formation of VEGF165 with CTGF may interrupt the binding of VEGF165 to KDR, NRP-1, and HSPG of endothelial cells and inhibits VEGF165-induced angiogenesis.

The most interesting finding of our study is that CTGF remarkably inhibits VEGF₁₆₅-induced angiogenesis by forming a complex. This inhibition by CTGF was demonstrated by the in vitro tube formation assay and further confirmed by the in vivo angiogenesis assay. Since full-length CTGF specifically inhibited the binding of VEGF₁₆₅ to HUVEC and the CTGF deletion mutant lacking the CT domain exhibited inhibition of VEGF₁₆₅-induced angiogenesis without any effects on VEGF₁₂₁-mediated angiogenesis, it is reasonable to think that the interaction between the TSP-1 domain of CTGF and the exon 7-coded region of VEGF₁₆₅ causes the antiangiogenic effect of CTGF to VEGF₁₆₅. As the present data demonstrate that CTGF inhibits only the binding of VEGF₁₆₅ to KDR/IgG Fc, the complex formation of VEGF₁₆₅ with CTGF may directly interrupt the binding of VEGF₁₆₅ to its major receptor, i.e., VEGFR-2 (Fig. 3) . However, since the exon 7-coded region of VEGF₁₆₅ binds to neuropilin 1 (NRP-1), a coreceptor of VEGF₁₆₅, the complex with the TSP-1 domain of CTGF may cause VEGF₁₆₅ to be unable to interact with NRP-1 (Fig. 3) . In addition, the exon 7-coded region of VEGF₁₆₅ has binding activity to cell surface heparan sulfate proteoglycan (HSPG). Thus, the interaction of VEGF₁₆₅ with HSPG may be modulated by CTGF (Fig. 3) .

The gene expression of CTGF is up-regulated in various fibrotic conditions such as hepatic fibrosis, atherosclerosis, and myocardial fibrosis. These fibrotic lesions are often accompanied by neovascularization especially in the early stages and followed by progressive fibrosis. Although VEGF and CTGF are downstream effectors of transforming growth factor ß (TGF-ß), CTGF can be induced by VEGF. Thus, the present finding of the antiangiogenic activity of CTGF against VEGF indicates the possibility that CTGF plays a dual role in completing the fibrotic processes by diminishing VEGF-induced blood vessel formation and enhancing fibroblast proliferation to produce collagens, the latter of which is a well-known activity of CTGF.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0332fje; to cite this article, use FASEB J. (December 14, 2001) 10.1096/fj.01-0332fje

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This Article Full Text (PDF) All Versions of this Article: 16/2/219 01-0332fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by INOKI, I. Articles by OKADA, Y. Search for Related Content PubMed PubMed Citation Articles by INOKI, I. Articles by OKADA, Y.