Expression and Function of Connective Tissue Growth Factor in Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose:To determine the expression and function of connective tissue growth factor (CTGF) in retinal pigment epithelial cells (RPE). Methods:Fetal human RPE were isolated and used at passages 3-5. mRNA expression was determined using RT-PCR and Northern blot. Protein expression was determined by Western blot and ELISA. Tritiated thymidine uptake and modified Boyden chamber assay were used to measure proliferation and chemotactic migration. Attachment to fibronectin substrate was determined using a fluorescent label. Immunohistochemistry was used to localize CTGF in membranes removed at vitrectomy from patients with proliferative vitreoretinopathy (PVR). Results:RPE demonstrate a specific CTGF band on RT-PCR and a single transcript on Northern blot; expression is increased by stimulation with TGF-beta. Western blot reveals a doublet (38-40 kDa) whose expression level is increased by stimulation with TGF-beta. ELISA of RPE supernatants (24 hrs) reveals a CTGF concentration of 9 ng/ml that increases to 40 ng/ml after TGF-beta (10 ng/ml) stimulation. CTGF induces a moderate proliferative response in RPE that is maximal at 100 ng/ml, and a strong chemotactic migration response similar to that induced by platelet derived growth factor. Attachment to fibronectin was significantly increased by CTGF. All PVR membranes showed some immunoreactivity for CTGF; double staining revealed colocalization with cytokeratin-positive RPE. Conclusion:The expression of CTGF in RPE and its stimulatory effects on RPE proliferation, migration and attachment to fibronectin suggest that CTGF may play an important role in the pathogenesis of PVR.

Keywords: 423 growth factors/growth factor receptors • 524 proliferative vitreoretinopathy • 567 retinal pigment epithelium