Development of a novel, anti-angiogenic peptide derived from the C-terminal of MMP-inhibitor, TIMP-3
Year: 2008
Session type: Poster / e-Poster / Silent Theatre session
University of Sheffield, Sheffield/South Yorkshire, UK
Abstract
Tissue inhibitor of metalloproteinases-3 (TIMP-3), is one of a family of 4 inhibitors of the matrix metalloproteinases (MMPs). While TIMPs are known to regulate angiogenesis by inhibiting the function of MMPs, TIMP-3 has recently been shown to exert a unique anti-angiogenic activity by binding to and inhibiting VEGF-R2.
The aim of this project is to identify the portion of the TIMP-3 molecule that interacts with this receptor and inhibits its activation by VEGF. Our initial solid phase assays using biotinylated VEGF indicated that the C-terminal domain of TIMP-3 interacts with VEGF-R2. Indeed, transfection of human endothelial cells (ECs) with wild-type TIMP-3, a mutant form of TIMP-3 which lacks most of the C-terminal domain of the protein, and a TIMP-2/TIMP-3 chimera confirmed that the C-terminal domain of TIMP-3 is involved in the interaction of TIMP-3 with VEGFR-2. We then synthesised a 16 amino acid peptide derived from the C-terminal domain of TIMP-3 (which we called ‘p700’) and showed in a series of in vitro angiogenesis assays that it was able to inhibit VEGF-induced proliferation, migration and differentiation by ECs in vitro. These data are supported by immunoblotting studies showing that the peptide also markedly reduced VEGF-induced pERK1/2 and PI3K pathways in human ECs. Moreover, p700 was able to inhibit EC tubule formation induced by VEGF121. As this form of VEGF lacks a heparin binding site it suggests that the latter is not involved in the interaction between p700 (and thus possibly TIMP-3) and VEGFR-2.
Taken together our studies show that a novel peptide derived from the C-terminal domain of TIMP-3 may have therapeutic potential as an angiogenesis inhibitor. Further work is now under way in our laboratory to confirm the receptor specificity of this TIMP-3 derived peptide.
Tissue inhibitor of metalloproteinases-3 (TIMP-3), is one of a family of 4 inhibitors of the matrix metalloproteinases (MMPs). While TIMPs are known to regulate angiogenesis by inhibiting the function of MMPs, TIMP-3 has recently been shown to exert a unique anti-angiogenic activity by binding to and inhibiting VEGF-R2.
The aim of this project is to identify the portion of the TIMP-3 molecule that interacts with this receptor and inhibits its activation by VEGF. Our initial solid phase assays using biotinylated VEGF indicated that the C-terminal domain of TIMP-3 interacts with VEGF-R2. Indeed, transfection of human endothelial cells (ECs) with wild-type TIMP-3, a mutant form of TIMP-3 which lacks most of the C-terminal domain of the protein, and a TIMP-2/TIMP-3 chimera confirmed that the C-terminal domain of TIMP-3 is involved in the interaction of TIMP-3 with VEGFR-2. We then synthesised a 16 amino acid peptide derived from the C-terminal domain of TIMP-3 (which we called ‘p700’) and showed in a series of in vitro angiogenesis assays that it was able to inhibit VEGF-induced proliferation, migration and differentiation by ECs in vitro. These data are supported by immunoblotting studies showing that the peptide also markedly reduced VEGF-induced pERK1/2 and PI3K pathways in human ECs. Moreover, p700 was able to inhibit EC tubule formation induced by VEGF121. As this form of VEGF lacks a heparin binding site it suggests that the latter is not involved in the interaction between p700 (and thus possibly TIMP-3) and VEGFR-2.
Taken together our studies show that a novel peptide derived from the C-terminal domain of TIMP-3 may have therapeutic potential as an angiogenesis inhibitor. Further work is now under way in our laboratory to confirm the receptor specificity of this TIMP-3 derived peptide.
