Figure 4.

TIMP-2 α3β1-binding peptides retain anti-mitogenic activity in vitro and anti-angiogenic activity in vivo. a) VEGF-A-stimulated (10 ng/mL) hMVECs demonstrate greater than two-fold increase in cell number after 24 h. TIMP-2 α3β1-binding peptides, peptide 8, peptide 9 and peptide 8–9 at 1 µM concentrations are essentially equipotent with 100 nM Ala+TIMP at inhibition of the VEGF-A-induced mitogenic response in hMVEC in vitro. Statistical analysis: ** denotes p<0.001 and * denotes p≤0.01. b) FGF-2-stimulated mitogenic response of hMVECs was also inhibited by TIMP-2 as previously reported. In addition, mitogenic stimulation of hMVECs was also inhibited by N-TIMP-2, and by the peptide containing the eight C-terminal amino acid residues of peptide 9, peptide 9–13, but loss of this peptide 9 sequence (peptide 11–13) or disruption of the primary sequence of peptide 9 (peptide 9–13 SCR) sequence resulted in significant loss of anti-mitogenic activity. Statistical analysis: ** denotes p<0.0005; * denotes p<0.005. c) The α3β1-binding TIMP-2 peptides retain anti-angiogenic activity in vivo. The α3β1-binding peptides, peptide 8, peptide 8–9, peptide 9, as well as peptide 9–13 and 9–13SCR were tested for anti-angiogenic activity using the directed in vivo angiogenesis assay [14, 28, 29]. The results demonstrate that at 0.5 µM local concentration of peptides 8, 8–9 and 9 showed statistically significant inhibition of angiogenic activity in vivo. The angio-inhibitory activity of peptide 9–13 was somewhat less (~ 50 %) but remained statistically significant. However, scrambling peptide 9–13 sequence destroyed this angio-inhibitory activity. Statistical analysis: *** denotes p<0.0001, ** denotes p<0.0005 and * denotes p<0.05.