Connective Tissue Growth Factor Modulates Human Choroidal Endothelial Cell Expression of TGF–ß and VEGF Receptors, and Increases Human Retinal Endothelial Cell Permeability

Abstract

Abstract: : Purpose: Whilst there is increasing evidence that Connective Tissue Growth Factor (CTGF) may play a role in ocular angiogenesis, its effect on retinal vascular permeability has not yet been reported. We used in vitro models to study the effect of CTGF on human retinal capillary endothelial monolayer permeability. Since VEGF and TGF–ß have been shown to mediate increased permeability of the retinal vasculature, we also studied the expression of transforming growth factor ß (TGF–ß) receptors I and II, and vascular endothelial growth factor (VEGF) receptors flk–1 and flt–1, and the production of VEGF, and TGF–ß1, ß2 and ß3 by human choroidal endothelial cells (HCEC). Methods:Human retinal capillary endothelial cells (HREC) and human choroidal endothelial cells (HCEC) isolated from post–mortem human eyes were exposed to rhCTGF(0–250ng/ml). The expression of TGF–ß RI and RII, flk–1 and flt–1 on HCEC was quantitated by flow cytometry and confirmed by immunocytochemistry. The permeability of HREC monolayers was studied by measuring transendothelial electrical resistance across monolayers grown on Millipore filters coated with laminin, fibronectin and type IV collagen in Transwell inserts. The production of VEGF and TGF–ß1, ß2 and ß3 from HCEC was assessed by ELISA. Results: CTGF significantly upregulated TGF–ß RI, RII, and flk–1 expression on HCEC, determined by both flow cytometry and immunocytochemical observations. CTGF increased HREC monolayer permeability more than two fold 24–48 hours after stimulation as reflected by a significant reduction in the transmonolayer electrical resistance of treated cells. CTGF treatment did not result in significant changes in the secretion of VEGF or TGF isoforms by HCEC Conclusions: CTGF increases human retinal capillary endothelial cell monolayer permeability and upregulates VEGF and TGF–ß receptors on human choroidal endothelial cells without a discernible effect on secretion of these cytokines, at least by vascular endothelial cells. These data indicate that CTGF may play a role in the pathogenesis of retinal edema, possibly through its regulation of other key retinal growth factor signaling pathways.

Keywords: growth factors/growth factor receptors • retinal culture • choroid