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. 2000 Nov;84(11):1306–1311. doi: 10.1136/bjo.84.11.1306

Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells by platelet derived growth factor

A Ando 1, M Ueda 1, M Uyama 1, Y Masu 1, S Ito 1
PMCID: PMC1723316  PMID: 11049960

Abstract

AIMS—To clarify factor(s) involved in morphological dedifferentiation of retinal pigment epithelial (RPE) cells in vitro from mitotically quiescent hexagonal cells to flattened cells that lack epithelial characteristics and concurrent myoid differentiation.
METHODS—RPE cells which retained their differentiated hexagonal morphology were isolated from bovine eyes by mechanical pipetting. Dedifferentiation and myoid differentiation of RPE cells were examined by microscopic observation and immunohistochemical analysis using antibodies against cytokeratin, an epithelial marker, and α smooth muscle actin, a marker of myoid differentiation. The contractile ability of RPE cells was evaluated by collagen gel contraction assay.
RESULTS—Platelet derived growth factor (PDGF) enhanced morphological changes in the RPE from hexagonal-shaped cells to flattened cells. Coincident with this morphological alteration, the expression of cytokeratin in RPE cells decreased and expression of α smooth muscle actin began and was increased in a time dependent manner. These alterations were completely blocked by collagen synthesis inhibitors. Interleukin 1β, transforming growth factor β1, insulin-like growth factor I, and basic fibroblast growth factor had little or no effect on the dedifferentiation. PDGF also potentiated the RPE induced collagen gel contraction.
CONCLUSIONS—These results demonstrate that PDGF enhanced the dedifferentiation of RPE cells, the initial step of proliferative vitreoretinopathy (PVR), as well as myoid differentiation and collagen gel contraction. PDGF may have a versatile role in the pathogenesis of PVR involving collagen synthesis.



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Figure 1  .

Figure 1  

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Photomicroscopy of retinal pigment epithelial (RPE) cells under bright field illumination (A, B), and immunofluorescent staining for cytokeratin (C, D) and α smooth muscle actin (α-SMA) (E, F) on day 0 (A, C, E) and day 14 of incubation with 100 ng/ml platelet derived growth factor (PDGF) (B, D, F). Freshly isolated RPE cells were hexagonal-shaped and mostly pigmented (A). RPE cells partly became flattened and lost their pigment granules (B). They exhibited a decline in immunoreactivity for cytokeratin (D) and an increase in α-SMA immunoreactivity (F). Original magnification, ×150.

Figure 2  .

Figure 2  

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Effect of platelet derived growth factor (PDGF) and protein kinase inhibitors on dedifferentiation of hexagonal-shaped retinal pigment epithelial (RPE) cells. Freshly isolated RPE cells (5 × 104 cells/well) were cultured in DMEM-5 in the absence or presence of the indicated agents for 7 days. Dedifferentiation was evaluated as described under "Materials and methods". Data (mean (SE), n=12) represent the percentage of the number of positive wells where dedifferentiated cells were observed.

Figure 3  .

Figure 3  

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Western blotting for cytokeratin (upper) and α-smooth muscle actin (α-SMA) (lower) on day 0 (lane 1), day 7 (lane 2), day 10 (lane 3), and day 14 (lane 4). Positions of rainbow protein molecular weight markers (Amersham) are indicated in kDa on the right.

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