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doi: 10.1242/10.1242/jcs.00119

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Thrombospondin-1 differentially induces chemotaxis and DNA synthesis of human venous smooth muscle cells at the receptor-binding level

Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology & Medicine, QEQM Wing, St Mary's Hospital, Paddington, London W2 1NY, UK

View larger version (15K): [in a new window]   Fig. 1. Effect of RGD peptide on cellular responses. (A) Histogram shows the effect of the RGD and ESP peptides (0.1 mM) on TSP-induced chemotaxis of quiescent (serum-deprived) cells. Each bar represents the mean±s.e.m. of three separate cell strains. Data are expressed as a percentage of the stimulated response (449±112 cells), which was taken to be 100%. (B) Histogram shows the effect of the RGD peptide (0.1 mM) on TSP-1 and PDGF-induced chemotaxis of cells through collagen-coated filters. Each bar represents the mean±s.e.m. of 3-5 separate cell strains. Data are expressed as a percentage of the stimulated response (TSP-1, 343±123 cells; PDGF-BB, 767±98 cells n=3), which was taken to be 100%.

View larger version (19K): [in a new window]   Fig. 2. Effect of integrin antibodies on cellular responses. (A) Histogram shows the dose-dependent effect of the function-blocking antibody to vß3 (LM609, 20 µg/ml and 50 µg/ml) on TSP-1 and PDGF-BB-induced chemotaxis on collagen-coated filters. Each bar represents the mean±s.e.m. of six separate cell strains. Data are expressed as a percentage of the stimulated response (TSP-1, 606±107 cells; PDGF-BB, 775±302 cells) which was taken to be 100%. (B) Histogram shows the effect of 20 µg/ml of the function-blocking antibodies LM609 and CD29 (ß1 integrin subunit) on TSP-1 and PDGF-BB-induced DNA synthesis. Each bar represents the mean±s.e.m of 6-9 separate cell strains. Data are expressed as a percenatage of the stimulated response (TSP-1, 884±112 cpm; PDGF-BB, 1660±410 cpm) which was taken to be 100%. (C) Histogram shows the effect of function-blocking antibodies to 5ß1, 3ß1 and 2ß1 on TSP-1-induced DNA synthesis. All antibodies were used at a final concentration of 20 µg/ml. Each bar represents the mean±s.e.m. of six separate cell strains. Data are expressed as a percentage of the stimulated response (TSP-1, 1041±255 cpm), which was taken to be 100%.

View larger version (17K): [in a new window]   Fig. 3. Role of IAP and PTX in regulating TSP-1-induced cell responses. (A) Histogram shows the effect of a function-blocking IAP antibody (B6H12) on both TSP-1-induced cell chemotaxis (20 and 50 µg/ml) and DNA synthesis (50 µg/ml). Each bar represents the mean±s.e.m. of four (chemotaxis) or six (DNA synthesis) separate cell strains. Data are expressed as a percentage of the TSP-1-stimulated response (chemotaxis, 248±60 cells; DNA synthesis, 404±64 cpm), which was taken to be 100%. (B) Histogram shows the effect of pre-treatment of vascular smooth muscle cells with pertussis toxin (0.5 µg/ml for 18 hours) on both TSP-1-induced cell chemotaxis and DNA synthesis. Each bar represents the mean±s.e.m. of six (chemotaxis) or four (DNA synthesis) separate cell strains. Data are expressed as a percentage of the TSP-1-stimulated response (chemotaxis, 229±34 cells; DNA synthesis, 456±101 cpm), which was taken to be 100%.

View larger version (29K): [in a new window]   Fig. 4. Effect of RGD peptides on TSP-1-induced cellular signals. (A) Histograms show the effect of RGD peptide (0.1 mM) on the TSP-1-induced tyrosine phosphorylation of signalling molecules as determined by densitometric analysis of western blots. Each bar represents the mean±s.e.m. of four separate cell strains. Data are expressed as a percentage of the TSP-1-stimulated response, which was taken to be 100% Representative western blots depicting tyrosine phosphorylation of FAK, p85PI3K and Erk2 are shown above the relevant bar on the histogram.

View larger version (26K): [in a new window]   Fig. 5. TSP-1-induced activation of Erk2. (A) Western blot showing the effect of TSP-1 alone and in the presence of RGD peptide (0.1 mM), function-blocking ß1- integrin antibody (50 µg/ml), heparin (100 µg/ml), function-blocking vß3 antibody (50 µg/ml) and function-blocking 3ß1 antibody (20 µg/ml) on the activity of Erk. Total Erk2 was immunoprecipitated from whole-cell lysates prior to western blotting with a monoclonal anti-phospho Erk1/2 antibody (0.5 µg/ml, 1 hour). The western blot shown is representative of n=3-10 separate cell strains. (B) The histogram shows the mean activation of Erk2 as determined by densitometric analysis of the dual-phosphorylated kinase. Dual phosphorylation of Erk2 in the TSP-1-stimulated cells was taken to be 100% and other data are normalised with respect to this value. Data represent the mean±s.e.m. of all 3-10 cell strains.

View larger version (16K): [in a new window]   Fig. 6. Effect of MEK inhibitor PD98059 on TSP-1-induced cell responses. Histogram shows the effect of a MEK inhibitor on both TSP-1-induced cell chemotaxis and DNA synthesis. Cells were preincubated with or without 10 µM PD98059 for 1 hour at 37°C prior to stimulation with TSP-1 and their cellular response in terms of DNA synthesis and chemotaxis was measured. Each bar represents the mean±s.e.m. of five (DNA synthesis) or six (chemotaxis) separate cell strains. The cellular response to TSP-1 (DNA synthesis, 584±265 cpm; chemotaxis, 680±201 cells) was taken to be 100% and the effect of PD98059 was calculated with respect to this.