Regulation of matrix contraction in chronic venous disease

Abstract

Objective: The role of TGF-beta(1) in venous ulcer healing and the signalling cascades regulating dermal fibroblast function are poorly understood. To elucidate these processes, we hypothesized that TGF-beta(1) facilitates wound healing by increasing chronic venous insufficiency (CVI) induced matrix contraction via intracellular cross-talk between TGF-beta(1) and the ERK-1/2 MAP kinase signalling cascades.

Methods: Fibroblasts isolated from calf biopsies (LC) of patients with different severity of CVI (CEAP, Clinical Etiological Anatomical Pathological classes) were seeded into 200 microl collagen gels under isometric conditions. Fibroblasts from neonatal foreskins (HS68), non-CVI patients (NC), and the ipsilateral normal thigh of each CVI patient (LT) served as controls. Thirteen patients with CVI (class 2, n=5; class 4, n=5; class 6, n=3) and 2 non-CVI controls (NC, n=2) were included in the study. All experimental conditions were determined by dose-response and time-course experiments. Gels were cultured with/without 0.1 ng/ml TGF-beta(1) and with/without 50 microM PD98059 (MEK and downstream-MAPK inhibitor). Additional patient fibroblasts were transfected with constitutively active Ras (pCMV-Ras) or an empty vector (pCMV-beta) with/without 0.1 ng/ml TGF-beta(1) and with/without 50 microm PD98059. The collagen gels were released after 4 days and the percent contraction was determined by area measurements using image analysis. Differences in alpha-smooth muscle actin (alpha-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 of MAPK and/or stimulation with TGF-beta(1) increased the contraction of LC gels compared to unstimulated controls. Agonist induced gel contraction correlated with CVI disease severity. alpha-SMA protein expression in LC fibroblasts increased with MAPK inhibition with/without TGF-beta(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-beta(1). Transfection with the pCMV-beta empty vector had no effect on gel contraction.

Conclusions: TGF-beta1 stimulation of CVI patient fibroblasts grown in 3D collagen gels results in conversion to a contractile phenotype through upregulation of alpha-SMA, and in enhanced gel contraction. Inhibition of MAPK further increases gel contraction, while Ras activation of ERK-1/2 inhibits TGF-beta1-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-beta(1) signalling cascades. These data identify potentially clinically relevant therapeutic molecular targets that could enhance matrix contraction and thereby improve venous ulcer wound healing.

Figure 1

Patient fibroblast response to TGF-β1 stimulation. 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). After stimulation with 0.1 ng/ml of TGF-β1, gels were released and percent contraction was compared with untreated controls for each CVI class respectively (a, p<0.001; b, p<0.001; c, p<0.01; d, p<0.001; e, p<0.001, f, p<0.001; g, p<0.01).

Figure 2. Contraction-response of fibroblast-seeded collagen gels to MEK inhibition

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).

Figure 3. ERK-1/2 phosphorylation in response to release of fibroblast-seeded collagen gels

3a. Time-course of ERK-1/2 response to gel-release. Representative western blot and graph demonstrating relative band intensities of protein expression for total and phosphorylated (pERK-1/2) ERK-1/2 after the release of gels seeded with HS68 fibroblasts (a, p<0.001). Total ERK-1/2 expression remained unchanged. 3b. pERK-1/2 response to TGF-β1 stimulation and MEK blockade (PD98059). Representative western blots and graphs demonstrating relative band intensities of protein expression for pERK-1/2 10 minutes after the release of gels seeded with fibroblasts derived from calves (LC) and thighs (LT) of classes 2, 4 and 6 CVI patients. Gels were stimulated with 0.01 ng/ml TGF-β1 prior to release. Percent contraction of LC gels (first graph) compared to the respective untreated controls in each group (a, p<0.001; b, p<0.05; c, p<0.001; d, p<0.001; e, p<0.01; f, p<0.05). Expression could not be restored with simultaneous stimulation with TGF-β1 (for each, compared to their respective controls). Percent contraction of LT gels (second graph). A similar response was observed when gels seeded with fibroblasts from CVI patient thighs (LT) were tested with TGF-β1 (a, p<0.01; b, p<0.05; c, p<0.01; d, p<0.05; e, p<0.001; f, p<0.05). 3c. Total ERK-1/2 response to TGF-β1 stimulation and MEK blockade (PD98059). Representative western blots and graphs demonstrating that no change in total ERK-1/2 expression was observed when gels seeded with CVI patient fibroblasts were treated with TGF-β1, PD98059, or a combination of TGF-β1+PD98059.

Figure 3. ERK-1/2 phosphorylation in response to release of fibroblast-seeded collagen gels

3a. Time-course of ERK-1/2 response to gel-release. Representative western blot and graph demonstrating relative band intensities of protein expression for total and phosphorylated (pERK-1/2) ERK-1/2 after the release of gels seeded with HS68 fibroblasts (a, p<0.001). Total ERK-1/2 expression remained unchanged. 3b. pERK-1/2 response to TGF-β1 stimulation and MEK blockade (PD98059). Representative western blots and graphs demonstrating relative band intensities of protein expression for pERK-1/2 10 minutes after the release of gels seeded with fibroblasts derived from calves (LC) and thighs (LT) of classes 2, 4 and 6 CVI patients. Gels were stimulated with 0.01 ng/ml TGF-β1 prior to release. Percent contraction of LC gels (first graph) compared to the respective untreated controls in each group (a, p<0.001; b, p<0.05; c, p<0.001; d, p<0.001; e, p<0.01; f, p<0.05). Expression could not be restored with simultaneous stimulation with TGF-β1 (for each, compared to their respective controls). Percent contraction of LT gels (second graph). A similar response was observed when gels seeded with fibroblasts from CVI patient thighs (LT) were tested with TGF-β1 (a, p<0.01; b, p<0.05; c, p<0.01; d, p<0.05; e, p<0.001; f, p<0.05). 3c. Total ERK-1/2 response to TGF-β1 stimulation and MEK blockade (PD98059). Representative western blots and graphs demonstrating that no change in total ERK-1/2 expression was observed when gels seeded with CVI patient fibroblasts were treated with TGF-β1, PD98059, or a combination of TGF-β1+PD98059.

Figure 4. Contraction of gels seeded with Ras-transfected CVI patient fibroblasts

4a. Transfection with Ras enhances expression of Ras and activates ERK-1/2. (Top panel) Fibroblasts derived from the lower calf (LC) and thigh (LT) of a patient with class 2 chronic venous insufficiency were transfected with an active mutant of Ras (pCMV-Ras) or empty vector (pCMV-β) and Ras expression was compared to controls by western blotting. (Lower Panel) Fibroblasts derived from the lower calf (LC) of the same patient underwent similar transfections and pERK-1/2 expression was compared with controls. 4b. ERK-1/2 activation by Ras transfection reduces TGF-β1-induced matrix contraction in class 2 CVI. Fibroblasts derived from the calves (LC) and thighs (LT) of class 2 CVI patients were transfected with (pCMV-Ras) or empty vector (pCMV-β), seeded into collagen gels and treated with TGF-β1. Percent contraction for each group was compared with the response of fibroblasts from normal patient controls (a, p<0.05; b, p<0.001; c, p<0.05; d, p<0.05; e, p<0.001). 4c. ERK-1/2 activation by Ras transfection reduces TGF-β1-induced matrix contraction in class 4 CVI Gel contraction after similar transfections of fibroblasts from the calves (LC) and thighs (LT) of class 4 CVI patients that underwent similar treatment (a, p<0.01; b, p<0.001; c, p<0.001; d, p<0.001; e, p<0.001). 4d. ERK-1/2 activation by Ras transfection reduces TGF-β1-induced matrix contraction in class 6 CVI Gel contraction after similar transfections of fibroblasts from the calves (LC) and thighs (LT) of class 6 CVI patients that underwent similar treatment (a, p<0.001; b, p<0.001; c, p<0.01; d, p<0.01).

Figure 5. Expression of α-SMA in CVI patient fibroblasts in response to TGF-β1 stimulation and MAPK inhibition

5a. Representative western blots for α-SMA expression in fibroblasts from class 2, 4 and 6 CVI patients. Cells were seeded in collagen gels, treated with TGF-β1 and/or PD98059, and protein measured prior to release. 5b. Graphs demonstrating relative band intensities of protein expression for α-SMA before release of gels seeded with fibroblasts derived from calves (LC) and thighs (LT) of classes 2 CVI patients. Gels were treated with 0.01 ng/ml TGF-β1 and/or 50 μM PD98059 (a, p<0.01; b, p<0.001 vs. untreated cells). 5c. Graph demonstrating results of similar experiments with fibroblasts derived from classes 4 CVI (a, p<0.05; b, p<0.01; c, p<0.001 vs. untreated cells). 5d. Graph demonstrating results of similar experiments with fibroblasts derived from classes 6 CVI (a, p<0.05; b, p<0.01; c, p<0.05; d, p<0.001 vs. untreated cells).