2a. Dose response to MEK inhibition (PD98059). Gels seeded with neonatal fibroblasts (HS68) were treated with increasing concentrations of PD98059 and percent contraction for each group was compared to the untreated control (a, p<0.001; b, p<0.01). Responses were not different between 50 and 100 μM concentrations. Contraction was further enhanced when 0.01 ng/ml TGF-β1 was added to PD98059 50 μM (c, p<0.001 vs. 50 μM PD alone) or to 100 mM (d, p<0.05 vs. 100 μM PD98059 alone and p<0.05 vs. combined TGF-β1+50 μM PD 98059).
2b. Patient fibroblast response to MEK inhibition (PD98059). Gels were seeded with fibroblasts derived from the thighs of normal controls (NC) and CVI classes 2, 4 & 6 (LT2-6), and from the calves of CVI classes 2, 4 & 6 (LC2-6). Gels were treated with PD98059 and/or TGF-β1, and percent contraction for each group was measured. Gels treated with PD98059 alone did not demonstrate increased contraction. Gels treated with TGF-β1 alone or in combination with PD98059 demonstrated increased contraction compared to their respective untreated controls, that varied with the severity of disease (a, p<0.001; b, p<0.001; d, p<0.01; e, p<0.001; g, p<0.001; i, p<0.001; j, p<0.001). Combined treatment with TGF-β1+PD98059 resulted in more contraction than TGF-β1 alone in normal controls, class 2 and in class 4 patients (c, p<0.05; f, p<0.001; h, p<0.05).