Modulation of Migration and Ca²⁺ Signaling in Retinal Pigment Epithelium Cells by Recombinant Human CTGF

Abstract

Purpose: The migration of retinal pigment epithelium (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). We investigated the expression of connective tissue growth factor (CTGF) in an in vitro model of wound healing and effects of recombinant human CTGF (rhCTGF) on modulating migration and Ca²⁺ signaling in RPE cells.

Methods: Cultured human RPE monolayers were used to establish a wound-healing model. Western blot and in situ hybridization were used to detect the CTGF expression in RPE cells. Migration of RPE cells was measured under the stimulation of rhCTGF alone or in combination with dexamethasone (DEX) or 8-Br-cAMP. To determine the concentration of cytoplasmic-free Ca²⁺ ([Ca²⁺]i) responding to CTGF, the fluo-3/AM-loaded RPE cells were observed with a laser scanning confocal microscope.

Results: The CTGF expression first increased after being wounded in RPE cells, then reached a peak and maintained at a high level. The positive expression was mainly at the edge of scrape and in motile RPE cells. rhCTGF-stimulated RPE cells migrated in a dose-dependent manner, and both DEX and 8-Br-cAMP could significantly inhibit the CTGF-induced migrations. CTGF induced a (Ca²⁺)i elevation in RPE cells in a concentration-dependent manner. Moreover, stimulation of RPE cells with CTGF and DEX or 8-Br-cAMP counteracted the elevation of (Ca²⁺)i induced by CTGF.

Conclusions: The CTGF expression could be induced by an in vitro model of scrape wounding. rhCTGF stimulated the migration and Ca²⁺ signal pathway in RPE cells in a dose-dependent manner, and DEX and 8-Br-cAMP suppressed this effect. Our results indicate that CTGF is involved in the wound-healing process and plays an important role in the pathogenesis of intraocular proliferative diseases.

KEYWORDS:

• Ca2+ signaling
• cell migration
• Connective tissue growth factor
• Proliferative vitreoretinopathy
• Retinal pigment epithelial cell
• Wound healing

ACKNOWLEDGMENTS

We would like to thank Professor Qihan Li of the Department of Viral Immunology, Institute of Medical Biology, Chinese Academy of Medical Science, for his generous gift of the recombinant CTGF and mouse anti-CTGF antibody. The project was supported partly by the equipment donation from the Alexander von Humboldt Foundation in Germany (to Y. S. Wang, V-8151/02085), by the Chinese National Nature Science Foundation (to Y. N. Hui, No. 39970780; to Q. H. Han, No. 30200311), and by the Scientific and Technological Project in Shaanxi Province (to C. M. Guo, No. 2008 K14-04).

Delcaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.