Similar to other investigators, we have reported a loss of growth factor responsiveness in fibroblasts isolated from venous ulcer patients indicating that cellular senescence may be involved in CVI dermal pathology 15. were cultured with/without 0.1 ng/ml TGF-1 and with/without ITI214 50 M PD98059 (MEK and downstream MAPK inhibitor). Additional patient fibroblasts were transfected with constitutively active Ras (pCMV-Ras) or an empty vector (pCMV-) with/without 0.1 ng/ml TGF-1 and with/without 50 m PD98059. The collagen gels were released after 4 days and the percent contraction was determined by area measurements using image analysis. Differences in -smooth muscle actin (-SMA) and ERK-1/2 MAPK (phosphorylated and total) protein levels were analyzed with western blotting. Results Gels seeded with CVI fibroblasts contracted more than HS68, NC and LT fibroblasts. Inhibition Rabbit Polyclonal to OR5K1 of MAPK and/or stimulation with TGF-1 increased the contraction of LC gels compared to un-stimulated controls. Agonist induced gel contraction correlated with CVI ITI214 disease severity. -SMA protein expression in LC fibroblasts increased with MAPK inhibition with/without TGF-1 stimulation, and correlated with the degree of gel contraction. Transfection with pCMV-Ras (activator of ERK-1/2) inhibited gel contraction; this inhibition was not reversed by addition of TGF-1. Transfection with the pCMV- empty vector had no effect on gel contraction. Conclusions TGF-1-stimulation of CVI patient fibroblasts grown in 3D collagen gels results in conversion to a contractile phenotype through upregulation of -SMA, and in enhanced gel contraction. Inhibition of MAPK further increases gel contraction, while Ras activation of ERK-1/2 inhibits TGF-1-induced gel contraction. These responses correlate with increasing CEAP severity. CVI fibroblast mediated gel contraction is therefore regulated through cross-talk between the ERK-1/2 MAPK and TGF-1 signaling cascades. These data identify potentially cliniclly relevant therapeutic molecular targets that could ITI214 enhanc matrix contraction and thereby improve venous ulcer wound healing. gels seeded with calf fibroblasts also demonstrated progressively increasing contraction with CVI severity (p 0.05 for both, LC2 vs. LC4, and LC4 vs. LC6). This was not seen when the responses of untreated thigh fibroblasts were compared (p=not significant for LT2 vs. LT4 and LT4 vs. LT6). Stimulation with TGF-1 resulted in a further increase of gel contraction above that observed with unstimulated gels. Interestingly, TGF-1-treated thigh cells of class 6 patients contracted more than those of class 2 patients (LT2 vs. LT6, p ITI214 0.01) indicating that molecular defects in patient fibroblasts extended well beyond the clinically affected part of the lower extremity. These data may reflect effects related to pressure as LC fibroblasts are exposed to higher levels of venous hypertension compared to LT fibroblasts in-vivo. ERK inhibition enhances TGF1-induced matrix contraction by CVI patient fibroblasts Inhibition of MAPK with 50 (p 0.01) and 100 (p 0.01) M PD98059 enhanced gel contraction caused by HS68 fibroblasts, compared to untreated cells (Fig 2a). When gels containing HS68 cells were simultaneously incubated with 0.1 ng/mL of TGF-1 and 50 (p 0.001) or 100 (p 0.05) M PD98059, an even greater contraction ITI214 was observed compared to PD98059 alone (Fig 2a). Open in a separate window Figure 2 Contraction-response of fibroblast-seeded collagen gels to MEK inhibition2a. 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 M concentrations. Contraction was further enhanced when 0.01 ng/ml TGF-1 was added to PD98059 50 M (c, p 0.001 vs. 50 M PD alone) or to 100 mM (d, p 0.05 vs. 100 M PD98059 alone and p 0.05 vs. combined TGF-1+50 M 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-1, and percent contraction for each group was measured. Gels treated with PD98059 alone did not demonstrate increased contraction. Gels treated with TGF-1 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;.