The Effects Of Transforming Growth Factor-Beta On Cultured ...



The Effects Of Transforming Growth Factor-Beta On Cultured Dupuytren’s Fibroblasts In A Collagen Matrix

Raymond Tse, MD, Jeffrey Howard PhD, Bing Siang Gan, MD, PhD

INTRODUCTION: In search of the underlying etiology of Dupuytren’s disease, recent evidence suggests that transforming growth factor-β2 (TGF-β2) may play a key role in the underlying pathogenic mechanism1. The purpose of this study was to further explore this hypothesis by examining the effects of TGF-β2 on primary cell cultures derived from patient-matched disease and normal palmar fascia tissue using a three-dimensional collagen contraction assay.

METHOD: Surgical samples of diseased and non-diseased (control) palmar fascia were taken from the same patients and studied in fibroblast-populated collagen lattices (FPCL). Contraction assays were used to define baseline cellular properties as well as to compare responses to exogenous TGF-β2 and anti-TGF-β2 antibodies.

RESULTS: Basal collagen contraction of the disease cells occurred at a greater rate and to a greater degree than control cells demonstrating a true biologic difference in the tissues sampled from the palmar fascia of the same patients (Fig. 1).

A dose response curve to exogenously added TGF-β2 was developed to establish a maximal response at 1 ng/mL (Fig. 2). An enhanced rate and total collagen contraction for both control and disease cells in response to this dose of TGF-β2 was clearly demonstrated (Fig. 3). The ligand-dependent nature of this response was confirmed by the addition of neutralizing anti-TGF-β2 antibodies that inhibited the increased collagen contraction induced by exogenous TGF-β2 in a dose-dependent manner (Fig. 4).

When neutralizing anti-TGF-β2 antibodies alone were added to the cells there was no change in the basal levels of collagen contraction for either control or disease cell cultures (Fig. 5). Since more localized interactions between endogenously produced TGF-β2 and its cell-surface receptors may account, in part, for the observed increase in disease FPCL contraction, we also pre-incubated with neutralizing anti-TGF-β2 antibodies prior to mixing with collagen. Regardless of the way the neutralizing antibodies were added, no difference in basal contraction was observed suggesting that endogenous activity of TGF-β2 does not contribute to the observed in vitro collagen contraction events.

CONCLUSION: Although exogenous TGF-β2 enhances the contractile properties of both disease and control fibroblasts, ligand-directed antibodies do not alter their basal contractile properties. The increased collagen contraction activity in vitro of Dupuytren’s fibroblasts in three-dimensional culture is, therefore, most likely not mediated by endogenous activity of TGF-β2.

REFERENCES:

1. Kloen P. New insights in the development of Dupuytren’s contracture: a review. British Journal of Plastic Surgery 52:629-635, 1999.

2. Kuhn MA, Payne WG, Kierney PC, Pu LL, Smith PD, Siegler K, Ko F, Wang X, Robson MC. Cytokine manipulation of explanted Dupuytren’s affected human palmar fascia. International Journal of Surgical Investigation 2:443-456, 2001.

Figure 1 Collagen contraction of primary control and disease FPCL cultures: Patient-matched fibroblast explant cultures derived from uninvolved palmar fascia (control) and areas of Dupuytren’s contracture (disease). FPCL contraction was quantified with the aid of Image J software, with the average mean FPCL surface area (mm2) plotted against the indicated time points. Each data point represents mean +/- SD of three independent patient-matched cell lines with experiments repeated in quadruplicate.

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Figure 2 TGF-β2 dose response: The dose response curve presented here is plotted as the percent FPCL contraction 5 days after being mechanically released. The degree of TGF-β2 stimulated contraction was measured over a large concentration range (>5 orders of magnitude). The figure shows a clear dose-response relationship, with the maximal response elicited by 1ng/mL of TGF-β2.

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Figure 3 Collagen contraction of primary control and disease FPCL cultures in response to exogenous TGF-β2: The extent of FPCL contraction in response to exogenous TGF-β2 (1ng/ml) was measured and quantified using Image J software, and plotted against the indicated time points (hours) for the control and disease fibroblast cultures. Each data point represents the average mean surface area ± SD of FPCL measured from three independent patient-matched primary cell cultures. Experiments were repeated in quadruplicate.

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Figure 4a Neutralizing anti- TGF-β2 antibodies block TGF-β2 stimulated FPCL contraction. Control fibroblasts were treated with 1ng/mL TGF-β2 in the presence or absence of the indicated concentrations of neutralizing anti-TGF-β2 antibodies. Measurements of FPCL contraction for both disease and control cultures were plotted as the average mean surface area ± SD for quadruplicate cultures per treatment.

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Figure 4b Neutralizing anti- TGF-β2 antibodies block TGF-β2 stimulated FPCL contraction: Disease fibroblasts were treated with 1ng/mL TGF-β2 in the presence or absence of the indicated concentrations of neutralizing anti-TGF-β2 antibodies. Measurements of FPCL contraction for both disease and control cultures were plotted as the average mean surface area ± SD for quadruplicate cultures per treatment.

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Figure 5 – Neutralizing anti- TGF-β2 antibodies do not alter the basal collagen contraction of control and disease FPCL cultures:

Disease and control FPCL cultures were incubated in the absence or presence of the indicated concentrations of neutralizing anti-TGF-β2 antibodies. Measurements of the contracting FPCL were plotted as the average mean surface area ± SDM for quadruplicate cultures per treatment.

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