Effects of TIMP-3 peptides on VEGF signaling in endothelial cells.

<p>(a) TIMP-3 loop 6 and tail inhibit VEGF-mediated phosphorylation of VEGFR-2 in PAE/KDR cells in a concentration dependent manner. Prior to treatment with indicated concentrations of TIMP-3 peptides for 30 min, the serum-starved cells were stimulated or not with 50 ng/ml VEGF for 10 min in the presence or absence of the same concentration of peptide. Cell lysates were separated by SDS-PAGE (10%) and analyzed by immunoprecipitation with an anti-KDR anti-body followed by immunoblotting with an anti-phosphotyrosine antibody, 4G10 (top panel). KDR protein was analyzed by immunoprecipitation and immunoblotting of cell lysates using anti-KDR antibody (bottom panel). (b) Densitometric quantitation of 210-kDa bands for phospho- or total mature form of KDR. (c) TIMP-3 loop 6 and tail but not loop 5 and N-peptide inhibit VEGF-mediated phosphorylation of KDR in PAE/KDR cells. (d) TIMP-3 loop 6 and tail inhibit ERK phosphorylation. Phosphorylation of ERK1 and ERK2 in response to VEGF was detected by immunoblotting with phosphospecific MAP kinase antibodies (top panel). Total protein levels of ERK was determined by immunoblotting with anti- MAP kinase antibodies (bottom panel). (e) TIMP-3 loop 6 and tail but not loop 5 nor N-peptide inhibit VEGF-mediated phosphorylation of ERK in PAE/KDR cells.(f) Densitometric quantitation of pERK (top panel of d) and ERK bands (bottom panel of d) for phospho- or total ERK proteins. **significantly different from control (without peptides+VEGF), p<0.01 (Student’s t test).