Vascular endothelial growth factor (VEGF), a potent angiogenic mitogen, plays a crucial role in angiogenesis under various pathophysiological conditions. We have recently demonstrated that VEGF(165), one of the VEGF isoforms, binds connective tissue growth factor (CTGF) and that its angiogenic activity is inhibited in the VEGF(165).CTGF complex form (Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H., Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K. and Okada, Y. (2002) FASEB J. 16, 219-221). In the present study, we further examined the susceptibility of the VEGF(165).CTGF complex to matrix metalloproteinases (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4 (aggrecanase-1), and serine proteinases, and evaluated the recovery of the angiogenic activity of VEGF(165) after the treatment. Among the MMPs, MMP-1, -3, -7, and -13 processed CTGF of the complex into the major NH(2)- and COOH-terminal fragments, whereas VEGF(165) was completely resistant to the MMPs. On the other hand, elastase and plasmin cleaved both CTGF and VEGF(165) of the complex, but they were completely resistant to ADAMTS4. By digestion of the immobilized VEGF(165).CTGF complex with MMP-3 or MMP-7, both NH(2)- and COOH-terminal fragments of CTGF were dissociated and released from the complex into the liquid phase. The in vitro angiogenic activity of VEGF(165) blocked in the VEGF(165).CTGF complex was reactivated to original levels after CTGF digestion of the complex with MMP-1, -3, and -13. Recovery of angiogenic activity was further confirmed by in vivo angiogenesis assay using a Matrigel injection model in mice. These results demonstrate for the first time that CTGF is a substrate of MMPs and that the angiogenic activity of VEGF(165) suppressed by the complex formation with CTGF is recovered through the selective degradation of CTGF by MMPs. MMPs may play a novel role through CTGF degradation in VEGF-induced angiogenesis during embryonic development, tissue maintenance, and/or pathological processes of various diseases.