Effects of PDGF-BB or IGF-1 on proliferation, migration, and MMP activities of wild type and <i>HtrA1</i>^<i>-/-</i> mouse VSMCs.

<p><b>(A)</b> Effects of PDGF-BB or IGF-1 on VSMC proliferation. Wild type (WT) and <i>HtrA1</i>^<i>-/-</i> (KO) VSMCs were cultured in medium containing 0.5% FBS with or without 20 ng/ml PDGF-BB or 10 ng/ml IGF-1, as indicated. Cell proliferation was assayed at the indicated time points (0 h was set at 15 h after plating). Points represent means ± SD (n = 3–4). The experiments were carried out independently for three different batches of WT and <i>HtrA1</i>^<i>-/-</i> VSMCs at the matching passage. The data shown are a representative result from cells at passage 7. Statistical significance was determined by Student’s <i>t</i>-test. *; p <0.05. **; p < 0.01. <b>(B-C)</b> Effects of PDGF-BB or IGF-1 on VSMC migration. <b>(B)</b> Cell migration was analyzed by the wound-healing assay. WT and <i>HtrA1</i>^<i>-/-</i> VSMCs were cultured in medium containing 0.5% FBS with or without 20 ng/ml PDGF-BB or 10 ng/ml IGF-1, as indicated. Photographs were taken at the indicated time points after wounding. Dotted white lines indicate the borders of the initial wounded area. The experiment was repeated three times and representative results are shown. Cells at passage 13 were used. <b>(C)</b> Cell migration was analyzed by the modified Boyden chamber assay. Cells were cultured in the assay chamber in medium containing 0.5% FBS with or without 20 ng/ml PDGF-BB or 10 ng/ml IGF-1, as indicated, for 24 h. The number of cells that migrated to the bottom side of the chamber was counted after DAPI staining. Data shown are means ± SD (n = 3). The experiment was repeated twice and representative results are shown. Cells at passage 9 were used. <b>(D)</b> Effects of PDGF-BB or IGF-1 on MMP9 activity of VSMCs. WT and <i>HtrA1</i>^<i>-/-</i> VSMCs were cultured for 72 h in medium containing 0.5% FBS without (- or Control) or with 20 ng/ml PDGF-BB (PDGF) or 10 ng/ml IGF-1, as indicated. The culture supernatants were recovered and applied to the zymography gel in volumes adjusted for the tubulin content in the cell lysates. Bands of MMP9 were analyzed by densitometer and are presented in the graph in the right panel. The experiment was repeated twice and representative results are shown. Cells at passage 12 were used.