American Society of Hematology

Figure 2.

$Figure 2. S1P-, LPA-, and VEGF-induced MT1-MMP–dependent morphogenic differentiation and endothelial cell migration involve the catalytic activity and cytoplasmic domain of the protein. (A) BAECs were transfected with empty vector (pcDNA3.1) or with the various MT1-MMP constructs and allowed to recover for 40 hours. (i) Transfected cells were plated on Matrigel in media and stimulated with either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. Original magnification, × 50. (ii) Formation of capillary-like structure was recorded after 6 hours and quantified using a computer-based program. Results are expressed as x-fold induction ± SD of control (pcDNA3.1) and are the means of 2 independent experiments. (B) (i) Transfected BAECs were allowed to migrate for 3 hours in media containing either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. Cell migration was quantified as described. Data are expressed as x-fold induction ± SD of control (pcDNA3.1). Results are the means of 2 independent experiments. (ii) Forty hours after transfection, crude membrane fractions were isolated and the expression levels of MT1-MMP constructs were monitored by Western blotting using anti–MT1-MMP polyclonal antibodies. NS indicates not specific. The ability of MT1-MMP constructs to induce proMMP-2 activation was monitored by gelatin zymography.$

S1P-, LPA-, and VEGF-induced MT1-MMP–dependent morphogenic differentiation and endothelial cell migration involve the catalytic activity and cytoplasmic domain of the protein. (A) BAECs were transfected with empty vector (pcDNA3.1) or with the various MT1-MMP constructs and allowed to recover for 40 hours. (i) Transfected cells were plated on Matrigel in media and stimulated with either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. Original magnification, × 50. (ii) Formation of capillary-like structure was recorded after 6 hours and quantified using a computer-based program. Results are expressed as x-fold induction ± SD of control (pcDNA3.1) and are the means of 2 independent experiments. (B) (i) Transfected BAECs were allowed to migrate for 3 hours in media containing either 10% serum (BCSi), 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. Cell migration was quantified as described. Data are expressed as x-fold induction ± SD of control (pcDNA3.1). Results are the means of 2 independent experiments. (ii) Forty hours after transfection, crude membrane fractions were isolated and the expression levels of MT1-MMP constructs were monitored by Western blotting using anti–MT1-MMP polyclonal antibodies. NS indicates not specific. The ability of MT1-MMP constructs to induce proMMP-2 activation was monitored by gelatin zymography.

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