MCP-1 mediates TGF-β effect on VSMC recruitment onto endothelial cells. (A) The conditioned medium (CM) of ECs treated with TGF-β1 stimulated VSMCs migration via MCP-1. The CM from HUVECs treated with none (i), anti–TGF-β antibody (ii), or with TGF-β1 (iii) for 24 hours was added into VSMCs in wound healing assay. Aliquots of the CM from the TGF-β1–treated ECs were added into VSMCs together with anti–MCP-1 antibodies (iv) or anti–TGF-β antibody (v). A representative was shown (bar represents 200 μm) and the migrated distance of the wound edge quantitated (n = 3) (bottom panel). (B) Blockage of MCP-1 activity impaired VSMC recruitment induced by TGF-β–treated ECs. HUVECs were first seeded to the lower wells of transwell chambers and pretreated with mock (i-iii) or TGF-β1 (iv-vi) for 24 hours. Then anti–TGF-β antibody (ii,vi), SB431542 (10 μM) (iii), or anti–MCP-1 antibody (v) was added into the lower wells 1 hour before VSMCs were seeded in the upper wells. After 20 hours, the cells migrating to the lower surface of the membrane were fixed, stained, and examined (original magnification ×100). Five different areas of migrated cells were counted for each data point. Asterisks indicate a significant difference compared with the control (*P < .05, ***P < .001; n = 5). Error bars indicate SD.