Transforming growth factor β2-induced myofibroblastic differentiation of human retinal pigment epithelial cells: Regulation by extracellular matrix proteins and hepatocyte growth factor

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Abstract

Retinal pigment epithelial (RPE) cells possess the potential to transdifferentiate into myofibroblasts after stimulation with transforming growth factor beta (TGFβ) and are implicated in the pathogenesis of proliferative vitreoretinopathy. In this study we evaluated how TGFβ2 and various extracellular matrix (ECM) proteins modulate the transdifferentiation of human fetal retinal pigment epithelial cells (RPE) cells into myofibroblast-like cells. Furthermore, we investigated whether hepatocyte growth factor (HGF) can suppress this transdifferentiation. RPE cells were cultured on ECM coated or uncoated surfaces in the presence or absence of TGFβ2. HGF was added to certain cultures only once or on a daily basis during the treatment. Transdifferentiation of RPE cells into myofibroblasts was assessed by the quantitation of α-smooth muscle actin (α-SMA) using immunocytochemistry, flow cytometry, real-time PCR and Western blotting. TGFβ2 induced a significant increase of α-SMA expression in a dose-dependent manner. Compared with growth on uncoated surfaces, RPE cultured on fibronectin (FN)-coated surfaces and stimulated with TGFβ2 showed a significantly higher α-SMA expression than untreated cells. This upregulation of α-SMA could be markedly reduced by daily treatment with HGF; however, a single HGF administration did not significantly reduce α-SMA. These findings are important for further understanding the interaction of cytokines, RPE cells and their environment in mesenchymal transformation as well as its possible modulation. Continuous or long-term treatment with HGF should be further investigated for its potential to prevent mesenchymal transdifferentiation of RPE cells, and ultimately, PVR in vivo.

Introduction

Proliferative vitreoretinopathy (PVR) is a common sequel of untreated rhegmatogenous retinal detachment, but also a common cause for failure of retinal detachment surgery (Walshe et al., 1992, Wiedemann and Weller, 1988). It is characterized by the formation of contractile membranes in the vitreous cavity and on the surface of the retina (Wiedemann and Weller, 1988). Although the exact pathologic mechanisms involved in PVR are not completely clarified, it has been suggested that PVR is actually an excessive wound healing reaction (Weller et al., 1990). Studies in other fields have shown that wound healing involves the transdifferentiation of fibroblasts into myofibroblasts, which typically express high levels of alpha smooth muscle actin (α-SMA) (Desmouliere et al., 1993, Fan et al., 1999, Jester et al., 1999, Ronnov-Jessen and Petersen, 1993). These myofibroblasts form tight adhesions to the extracellular matrix (ECM) and in the eye are the main factor in the process of membrane contraction (Hiscott et al., 1999, Singer et al., 1984). Among the cellular components involved in PVR reaction, RPE cells are key players, but the cells in the membranes differ phenotypically from the normal retinal pigment epithelium (RPE) on Bruch's membrane: the RPE cells in PVR membranes look and behave like fibroblasts or macrophages, which are usually found in wound healing processes (Glaser et al., 1987, Hiscott et al., 1999, Newsome et al., 1981, Vinores et al., 1990). In vitro studies confirm that RPE cells can transdifferentiate from epithelial cells into myofibroblasts-like cells (Casaroli-Marano et al., 1999, Grisanti and Guidry, 1995, Hiscott et al., 1999). Guidry et al. (2002) as well as Lopez et al. (1996), were also able to show that RPE can transdifferentiate with upregulation of α-SMA expression in other ocular disorders, such as age-related macular degeneration and formation of choroidal neovascularization.

Transforming growth factor beta (TGFβ) is considered to be the main inducer of the myofibroblastic phenotype: it upregulates α-SMA as well as ECM protein expression (e.g. collagen type I (C-I) and fibronectin (FN)) in fibroblasts (Arora et al., 1999, Ignotz and Massague, 1986, Xu et al., 2001). TGFβ is also able to control cell adhesion and migration by modulating adhesion molecules on the cell surface (Gailit et al., 1994, Riikonen et al., 1995, Wang et al., 1993). Furthermore, ECM has been shown to regulate the expression of the TGFβ1 gene, suggesting a feedback loop in vivo (Streuli et al., 1993).

In the eye, TGFβ2 is the main isoform of TGFβ (Connor et al., 1989). It has been shown to inhibit proliferation (Lee et al., 1999, Lee et al., 2001a, Lee et al., 2001b), but to promote migration of cultured RPE cells (Mitsuhiro et al., 2003). In PVR, vitreous TGFβ levels increase in proportion to disease severity (Connor et al., 1989). Bochaton-Piallat et al. (2000) reported that TGFβ1 and its receptor TGFβRII are always present in PVR membranes, concomitant with the expression of ED-A fibronectin. ED-A containing polymerized fibronectin even seems to be necessary for the TGFβ induced myofibroblastic transformation of cells (Serini et al., 1998). Kurosaka et al. (1996) confirmed that TGFβ2 significantly induced expression of α-SMA in cultured bovine RPE cells, and this was further confirmed for porcine RPE cells (Lee et al., 2001a, Lee et al., 2001b).

Hepatocyte growth factor (HGF), a cytokine produced mainly by mesenchymal cells, acts on epithelial cells through its membrane-spanning tyrosine kinase receptor c-met (Matsumoto and Nakamura, 1996). It promotes proliferation, dissociation, motility and invasiveness of epithelial and endothelial cells (Matsumoto and Nakamura, 1996, Miura et al., 2003) and is believed to be involved in the invasion and metastasis of tumor cells (Hiscox and Jiang, 1999). It has widespread effects on embryogenesis and regeneration by acting on angiogenesis and branching morphogenesis of epithelial cells (Crepaldi et al., 1994, Zarnegar and Michalopoulos, 1995). These unique but widespread biological effects and the pattern of HGF and c-met expression in different cells have led to the postulate that the HGF/c-met system is critical for epithelial–mesenchymal interactions (Zarnegar, 1995). RPE cells, however, have been shown to differ from the normal epithelial pattern and to express not only c-met, but also HGF itself, suggesting an autocrine loop for retinal development, angiogenesis and wound healing (He et al., 1998, Van Aken et al., 2003).

The above-mentioned investigations have been conducted mostly in fibroblasts, mesangial cells and established cell lines with little relevance to ophthalmologic pathologies such as PVR. We investigated the effects of TGFβ2, ECM proteins and HGF in early passages of human RPE cells, in order to obtain conditions that would better mimic in vivo condition. In this paper, we describe the changes of human RPE cells during mesenchymal transdifferentiation, stimulated by TGFβ2 and modulated by ECM proteins. Furthermore, we show that HGF can inhibit TGFβ2-induced upregulation of α-SMA-expression, an important characteristic of mesenchymal transdifferentiation.

Section snippets

Cell culture

Primary human fetal RPE cells were isolated from eyes obtained from the Anatomic Gift Foundation (Woodbine, GA). The Institutional Review Board of the University of Southern California approved our use of cultured human RPE cells. The eyes were cut circumferentially, the vitreous removed and the retina gently detached from the RPE cell layer. The choroid/RPE layer was placed in 2% Dispase (Gibco, Madison, WI) in Hanks’ balanced salt solution (HBSS) (Irvine Scientific, Santa Ana, CA) for 25 min

ECM proteins upregulate α-SMA expression in RPE in a dose-dependent manner

Plates or slides were precoated with FN, C-I, C-IV, LN and TSP in three different concentrations. In flow cytometric analyses, each one of the tested ECM proteins had a dose dependent effect on the expression of α-SMA in the RPE cells. Yet, there only was a statistically significant increase in α-SMA expression between control (uncoated) and FN- and TSP-coated plates (P < 0.05), when the coating was performed with ECM proteins at medium or high concentration. FN-coated plates displayed the

Discussion

Myofibroblastic transformation of RPE cells induced by TGFβ2 includes morphologic changes as well as changes in protein expression of the mesenchymal marker, α-SMA. The results of our study demonstrate that this effect of TGFβ2 on RPE cells is dose-dependent and can be further augmented by growing the cells on ECM coated surfaces. This transformation can be blocked by TGFβ neutralizing antibody, indicating that this effect is TGFβ-specific. Furthermore, HGF can reduce α-SMA expression at both

Acknowledgements

The authors thank Ernesto Barron for technical assistance, Laurie LaBree for statistical analysis and Dr. Thomas E. Ogden for the kind revision of the manuscript. This project was supported by following grants: NIH EY02061, NIH EY03040, Research to Prevent Blindness, and Arnold and Mabel Beckman Foundation; M.A.G. was supported by a grant from the Deutsche Akademie der Naturforscher Leopoldina, Halle/Saale, Germany, BMBF-LPD 9901/8-52.

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