Figure 3.

Uremic serum increases proliferation of vascular smooth muscle cells (VSMC) but has not effect on apoptosis. (A) VSMC were plated on 96 well plates (3×103³ cells/well) in growth media. Cells were subsequently serum starved for 72 hours and then treated with basal media (Ctrl 0.1%FBS), growth media GM (5%FBS), normal serum NS (10%) or dialysis patient serum DS (10%) for another 72 hours. Proliferation was measured using the Cyquant reagent. For the assay, the media was removed from the cells, dye binding solution was added to each well and nucleic acid was measured using a fluorescence plate reader using a 485/535nm filter set. (B) VSMC were plated on 96 well plates 5×10³ (cells/well) after adhesion cells were serum starved for 48 hours and then treated for 24 hours in either, basal media (ctrl), normal human serum (NS) or dialysis patient serum (DS). Cells were then washed in HBSS and exposed to 0.5M staurosporine for 4 hours at which time supernatant was removed and 100ul of Caspase3/7 substrate was added and further incubated for 1 hour at room temperature. Enzymatic activity was detected using a luminometer. Results shown are mean ± SD of 3-5 independent experiments. (C) Expression of Cleaved Caspase 3 was measured by Western blot analysis in VSMC lysates of cells that were serum starved (ctrl), treated with 10% normal healthy serum (NS), or treated with dialysis patient serum (DS) and then treated with staurosporine for 6 hours.