Arterioscler Thromb Vasc Biol cAMP Involvement in the Expression of MMP-2 and MT-MMP1 Metalloproteinases in Human Endothelial Cells by Francesca Peracchia, Antonio Tamburro, Cesaria Prontera, Barbara Mariani, and Domenico Rotilio Arterioscler Thromb Vasc Biol Volume 17(11):3185-3190 November 1, 1997 Copyright © American Heart Association, Inc. All rights reserved.

Gelatin zymogram of MMP-2 in the CM of HUVECs after treatment for 12 hours with FK. HUVECs were stimulated with 100 and 25 μmol/l FK(lanes 2 and 3, respectively) vs control (lane 1) and control ethanol (lane 4), representing the vehicle in which FK was dissolved. Gelatin zymogram of MMP-2 in the CM of HUVECs after treatment for 12 hours with FK. HUVECs were stimulated with 100 and 25 μmol/l FK(lanes 2 and 3, respectively) vs control (lane 1) and control ethanol (lane 4), representing the vehicle in which FK was dissolved. Double lanes are representative of samples harvested from two different culture dishes treated similarly. Results are representative data of three separate experiments. Francesca Peracchia et al. Arterioscler Thromb Vasc Biol. 1997;17:3185-3190 Copyright © American Heart Association, Inc. All rights reserved.

Effect of FK on cAMP levels in HUVECs Effect of FK on cAMP levels in HUVECs. HUVECs were treated for 1 hour with 100 μmol/L FK. Then cAMP was extracted by ethanol in the presence of 0.5 mmol/L 3-isobutyl-1-methylxanthine, and cAMP levels were measured by radioimmunoassay. Effect of FK on cAMP levels in HUVECs. HUVECs were treated for 1 hour with 100 μmol/L FK. Then cAMP was extracted by ethanol in the presence of 0.5 mmol/L 3-isobutyl-1-methylxanthine, and cAMP levels were measured by radioimmunoassay. Results are mean±SD of quadruplicate wells. Francesca Peracchia et al. Arterioscler Thromb Vasc Biol. 1997;17:3185-3190 Copyright © American Heart Association, Inc. All rights reserved.

Gelatin zymogram of MMP-2 in the CM of HUVECs treated for 12 hours with 0.5 mmol/L dibutyryl-cAMP (lane 2) vs control (lane 1) and control ethanol (lane 3). Gelatin zymogram of MMP-2 in the CM of HUVECs treated for 12 hours with 0.5 mmol/L dibutyryl-cAMP (lane 2) vs control (lane 1) and control ethanol (lane 3). Each single lane (two for control and control ethanol; four for treated cells) is representative of samples harvested from different culture dishes treated similarly. Results are representative data of two separate experiments. Francesca Peracchia et al. Arterioscler Thromb Vasc Biol. 1997;17:3185-3190 Copyright © American Heart Association, Inc. All rights reserved.

Effects of FK on MMP-2 mRNA expression. Effects of FK on MMP-2 mRNA expression. HUVECs were treated for 6 hours with 100 μmol/l FK (lane 2) or 25 μmol/l FK (lane 3) vs control (lane 1). Total RNA was prepared and run in formaldehyde-agarose gel, blotted on nytran membrane, and hybridized with MMP-2 cDNA probe, 32P-labeled by the random priming method, and GAPDH cDNA probe, 32P-labeled by the nick translation method. Similar results were obtained in three different experiments. Radioactivity quantitation was performed by Instant Imager. Densitometric data are mean±SD normalized for control value of three separate experiments. Student’s t test was used, and the difference was considered statistically significant when P<.05. Ethidium bromide stained 28 S and 18 S after transfer to nytran membrane, demonstrating the application of approximately equal amounts of total RNA. Francesca Peracchia et al. Arterioscler Thromb Vasc Biol. 1997;17:3185-3190 Copyright © American Heart Association, Inc. All rights reserved.

Effect of FK on MT-MMP1 and TIMP-2 mRNA expression. Effect of FK on MT-MMP1 and TIMP-2 mRNA expression. HUVECs were treated for 6 hours with 100 μmol/l FK (lane 2) or 25 μmol/l FK (lane 3) vs control (lane1). Total mRNA was run in formaldehyde-agarose gel, blotted on nytran membrane, and hybridized with MT-MMP1 and TIMP-2 cDNA probes, 32P-labeled by random priming. Representative results of three separate experiments are shown. Densitometric data are mean±SD of three separate experiments normalized for control value. Student’s t test was used for statistical analysis. *P<.001. Ethidium bromide stained 28 S and 18 S after transfer to nytran membrane, demonstrating the application of approximately equal amounts of total RNA. Francesca Peracchia et al. Arterioscler Thromb Vasc Biol. 1997;17:3185-3190 Copyright © American Heart Association, Inc. All rights reserved.

Activation of MMP-2 gelatinase from CM of HUVECs by plasma membranes of cells treated for 12 hours with FK. Membranes of HUVECs were obtained as described in “Methods”: 4 mg of plasma membrane extracts was incubated with control CM in 10-mL final volume of 25 mmol/L HEPES/KOH (pH 7.5) containing 0.1 mmol/L CaCl2. Activation of MMP-2 gelatinase from CM of HUVECs by plasma membranes of cells treated for 12 hours with FK. Membranes of HUVECs were obtained as described in “Methods”: 4 mg of plasma membrane extracts was incubated with control CM in 10-mL final volume of 25 mmol/L HEPES/KOH (pH 7.5) containing 0.1 mmol/L CaCl2. The reaction was incubated at 37°C for 4 hours, terminated by addition of sample buffer, and subjected to gelatin zymogram analysis. Lane 1, control CM; lane 2, CM incubated with membrane extract of control HUVECs; and lane 3, CM incubated with membrane extract of HUVECs treated with 100 μmol/l FK. The zymogram is representative of two separate experiments. Francesca Peracchia et al. Arterioscler Thromb Vasc Biol. 1997;17:3185-3190 Copyright © American Heart Association, Inc. All rights reserved.

Western blot analysis of MT-MMP1 of cell extract from HUVECs after 12 hours of treatment, using rabbit polyclonal antibodies to MT-MMP1. Western blot analysis of MT-MMP1 of cell extract from HUVECs after 12 hours of treatment, using rabbit polyclonal antibodies to MT-MMP1. Lanes 1 to 2 contain 45 mg of protein/lane of cell extract from control (lane 1) and 100 μmol/L FK (lane 2). Cell extracts were prepared by 1% SDS treatment of cell layers. SDS-polyacrylamide gel electrophoresis was followed by immunoblotting. Antibodies were visualized using the ECL detection system. Francesca Peracchia et al. Arterioscler Thromb Vasc Biol. 1997;17:3185-3190 Copyright © American Heart Association, Inc. All rights reserved.