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• Methods - (Adobe Acrobat file) (51 KB).
• Figure I - (Adobe Acrobat file) (86 KB). Carotid injury induces consistent endothelial denudation. A, Adult, C57Bl/6J mice were subjected to mechanical injury of the left carotid artery. Two hours after injury, six mice were infused with Evans blue dye prior to perfusion-fixation in situ with buffered formalin. A representative non-injured, right carotid artery and injured, left carotid artery is shown. The arteries were pinned by the en face method and images were collected with a digital camera. B, Adult, C57Bl/6J mice were subjected to carotid injury as described in “A”. Two hours after surgery, three mice underwent perfusion-fixation in situ with buffered formalin. Then, carotid cross-sections were isolated and stained for von Willebrand factor. Micrographs were collected with a 63x objective. Two, representative non-injured, right and two representative injured, left carotid arteries are shown. Red arrows point at the vascular endothelium (VE, vascular endothelium; M, media).
• Figure II - (Adobe Acrobat file) (22 KB). SB202190 blocks PDGF-induced cell proliferation and reduces DNA replication in RAOSMCs. A, Evaluation of PDGF-stimulated cell proliferation after SB202190 treatment. Serum-deprived RAOSMCs were pre-treated with SB202190 (10 μM) or vehicle (DMSO) for 1 hour. Then, in triplicate wells, the monolayers were treated with PDGF-BB (5 ng/ml) or 0.1% BSA, as a vehicle control for PDGF-BB, in the continued presence of SB202190 or DMSO. After 40 hours cell number was determined. HPF, 400x high power field (DMSO, + PDGF-BB vs. DMSO, - PDGF-BB, *P<0.05; DMSO, + PDGF-BB vs. SB202190, + PDGF-BB, **P<0.05). B, Examination of PDGF-induced DNA replication after SB202190 treatment. Serum-deprived RAOSMCs were pre-treated with SB202190 (10 μM) or DMSO as in “A”. Then, the monolayers were treated with PDGF-BB (5 ng/ml) or 0.1% BSA for 15 hours in a tissue culture incubator in the continued presence of SB202190 or DMSO. Next, each well was pulsed with 6-3H-thymidine (1 μCi/ml) for 2.5 hours and thymidine incorporation was determined; r.u., relative units (DMSO, + PDGF-BB vs. DMSO, - PDGF-BB, *P<0.05; DMSO, + PDGF-BB vs. SB202190, + PDGF-BB, **P<0.05).
• Figure III - (Adobe Acrobat file) (69 KB). SB202190 blocks PDGF-induced Rb hyper-phosphorylation in RAOSMCs cells. Analysis of PDGF-induced Rb phosphorylation after SB202190 treatment. Serum-deprived RAOSMCs were pre-treated with SB202190 (10 μM) or vehicle (DMSO) for 1 hour, and then monolayers were treated with PDGF-BB (5 ng/ml) or 0.1% BSA in the continued presence of SB202190 or DMSO. After 20 hours, cells were lysed and immunoblotting was performed with an anti-phospho (Ser 807/811) Rb antibody. Re-probing was conducted with anti-phospho (Thr180/Tyr182) p38 MAPK and anti-actin antibodies (SB, SB202190). Densitometric analysis of the Western blot is shown in parentheses; r.u., mean, relative units of phosho Rb signal for each group normalized to actin.