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

Abstract

Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two‐hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity‐binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 ± 11 nM and 125 ± 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type‐1 repeat (TSP‐1) domain of CTGF binds to the exon 7‐coded region of VEGF165 and that the COOH‐terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full‐length CTGF and the deletion mutant possessing the TSP‐1 domain inhibited VEGF165‐induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein‐to‐protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.

Anti-Vascular Endothelial Growth Factor Gene Therapy Attenuates Lung Injury and Fibrosis in Mice

“…We found an association between angiogenesis and inflammation in this model, which is earlier than that found in the study by Fehrenbach et al (1). This may be the reason why we found an association between VEGF expression and von Willebrand factor in this model in contrast to the results of Fehrenbach et al Recently, Inoki et al (22) demonstrated that connective tissue growth factor (CTGF) binds to VEGF and inhibits VEGF-induced angiogenesis. CTGF and VEGF are downstream effectors of TGF-␤, and these growth factors are known to be up-regulated in this model (23).…”

The VEGF angiogenic switch of fibroblasts is regulated by MMP-7 from cancer cells

“…Previous reports have shown that some secreted proteins, including soluble VEGF receptor 1, platelet factor 4, thrombospondin, ADAMTS1 and CTGF, bind to VEGF and inhibit angiogenic activity (Kendall and Thomas, 1993;Gengrinovitch et al, 1995;Gupta et al, 1999;Luque et al, 2003;Inoki et al, 2002;Jang et al, 2004). As one of these proteins, CTGF is typically expressed in fibroblasts (Brigstock, 1999), we considered the possibility that CTGF might function as an inhibitor of VEGF from fibroblasts.…”

“…However, the mechanism by which vascular quiescence is maintained in normal tissues, wherein VEGF is widely expressed (Berse et al, 1992;Hanahan and Folkman, 1996), remains unclear. Several secreted proteins have been reported to bind and sequester VEGF (Kendall and Thomas, 1993;Gengrinovitch et al, 1995;Gupta et al, 1999;Inoki et al, 2002;Luque et al, 2003). This type of regulation could account for the vascular quiescence in the presence of VEGF, although in most tissues except the cornea (Ambati et al, 2006), the relevance of these proteins to vascular control has yet to be demonstrated.…”

First Structural Glimpse of CCN3 and CCN5 Multifunctional Signaling Regulators Elucidated by Small Angle X-ray Scattering

“…These include the integrins, which are considered to be the "functional receptors of the CCN family"; the small cysteine knot growth factors (vascular endothelial growth factor (VEGF), transforming growth factor (TGF), and bone morphogenic protein (BMP) 2 ); extracellular matrix proteins (collagen, fibulin, and Notch); heparin sulfated proteoglycans; LDL receptor protein-1; and insulin growth factor (11,12,19) In some cases, the module that these receptors bind is known. VEGF isoforms bind to domains 3 and 4 of CCN2 (20) and domain 2 of CCN3 binds BMP2 (21). Aggrecan binds to the N terminus of CCN2 (either domain 1 or domain 2) (22), and domain 3 and domain 4 facilitate binding to heparin sulfated proteoglycans (23) and fibulin (16).…”

“…Given the high degree of sequence identity between the proteins, it is likely that domain-domain interactions may be one of the factors that control the subtle differences in behavior and binding of these proteins (1,2). In the case of CCN2, domain cooperativity has already been seen to influence ligand binding with domain 4 binding to VEGF 121 and both domain 3 and domain 4 being required to bind the longer VEGF1 165 (20). Experiments done by Kubota et al (24) also demonstrated that certain biological effects were only seen when multiple domains were involved.…”