Figure 1.
Figure 1. MT1-MMP induces morphogenic differentiation and endothelial cell migration in cooperation with serum factors. (A) BAECs were transfected with empty vector (pcDNA3.1) or with an MT1-MMP construct and allowed to recover for 40 hours. (i) Transfected cells were plated on Matrigel in serum-free media and were either left unstimulated or were stimulated with either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, 50 ng/mL VEGF, 20 ng/mL bFGF, 20 ng/mL IGF-1, 20 ng/mL TGF-β2, 20 ng/mL EGF, 20 ng/mL PDGF, or 1 U/mL thrombin. Original magnification, × 50. (ii) Formation of capillary-like structures was recorded after 6 hours and quantified using a computer-based program. Results are expressed as x-fold induction ± SD of nonstimulated control (Ctl) and are the means of 3 different experiments. (B) Transfected BAECs were subjected to migration assay for 3 hours in serum-free media containing either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, 50 ng/mL VEGF, 20 ng/mL bFGF, 20 ng/mL IGF-1, 20 ng/mL TGF-β2, 20 ng/mL EGF, 20 ng/mL PDGF, or 1 U/mL thrombin. Cell migration was quantified using a computer-based program. Data are expressed as x-fold induction ± SD of nonstimulated control (Ctl). Results are the means of 3 independent experiments. (C) HUVECs were pretreated for 2 hours with BB94 (5 μM) or dimethyl sulfoxide (DMSO). Cells were then harvested and plated on Matrigel in serum-free media containing either 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. A representative of 2 independent experiments is shown. Original magnification, × 50. (D) HUVECs were treated with human MT1-MMP antisense (MT1-MMP AS) or control oligonucleotides (Ctl AS) for 72 hours. Cells were plated on Matrigel in serum-free media containing 1 μM S1P. A representative of 2 independent experiments is shown. Original magnification, × 50. The inhibition of MT1-MMP expression after 72 hours of antisense oligonucleotide treatment was monitored by Western blotting. TD indicates immunodetection. (E) (Left) HUVECs were pretreated for 2 hours with BB94 (5 μM) or DMSO and allowed to attach to filters. The medium in the lower chambers was then replaced with serum-free media containing either 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF and were incubated for 3 hours. (Right) HUVECs were treated with human MT1-MMP antisense (MT1-MMP AS) or control oligonucleotides (Ctl AS) for 72 hours and submitted to migration assay for 3 hours in the presence of 1 μM S1P. Cell migration was quantified as described and data are expressed as x-fold induction ± SD of nonstimulated control (Ctl). Results are the means of 2 independent experiments.

MT1-MMP induces morphogenic differentiation and endothelial cell migration in cooperation with serum factors. (A) BAECs were transfected with empty vector (pcDNA3.1) or with an MT1-MMP construct and allowed to recover for 40 hours. (i) Transfected cells were plated on Matrigel in serum-free media and were either left unstimulated or were stimulated with either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, 50 ng/mL VEGF, 20 ng/mL bFGF, 20 ng/mL IGF-1, 20 ng/mL TGF-β2, 20 ng/mL EGF, 20 ng/mL PDGF, or 1 U/mL thrombin. Original magnification, × 50. (ii) Formation of capillary-like structures was recorded after 6 hours and quantified using a computer-based program. Results are expressed as x-fold induction ± SD of nonstimulated control (Ctl) and are the means of 3 different experiments. (B) Transfected BAECs were subjected to migration assay for 3 hours in serum-free media containing either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, 50 ng/mL VEGF, 20 ng/mL bFGF, 20 ng/mL IGF-1, 20 ng/mL TGF-β2, 20 ng/mL EGF, 20 ng/mL PDGF, or 1 U/mL thrombin. Cell migration was quantified using a computer-based program. Data are expressed as x-fold induction ± SD of nonstimulated control (Ctl). Results are the means of 3 independent experiments. (C) HUVECs were pretreated for 2 hours with BB94 (5 μM) or dimethyl sulfoxide (DMSO). Cells were then harvested and plated on Matrigel in serum-free media containing either 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. A representative of 2 independent experiments is shown. Original magnification, × 50. (D) HUVECs were treated with human MT1-MMP antisense (MT1-MMP AS) or control oligonucleotides (Ctl AS) for 72 hours. Cells were plated on Matrigel in serum-free media containing 1 μM S1P. A representative of 2 independent experiments is shown. Original magnification, × 50. The inhibition of MT1-MMP expression after 72 hours of antisense oligonucleotide treatment was monitored by Western blotting. TD indicates immunodetection. (E) (Left) HUVECs were pretreated for 2 hours with BB94 (5 μM) or DMSO and allowed to attach to filters. The medium in the lower chambers was then replaced with serum-free media containing either 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF and were incubated for 3 hours. (Right) HUVECs were treated with human MT1-MMP antisense (MT1-MMP AS) or control oligonucleotides (Ctl AS) for 72 hours and submitted to migration assay for 3 hours in the presence of 1 μM S1P. Cell migration was quantified as described and data are expressed as x-fold induction ± SD of nonstimulated control (Ctl). Results are the means of 2 independent experiments.

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