Connective Tissue Growth Factor Expression in Glaucomatous Human TM Cells and Aqueous Humor and Upregulation by TGFbeta2

Abstract

Abstract: : Purpose: Connective Tissue Growth Factor (CTGF) is a secretory protein involved in a variety of pathological disorders leading to increased extracellular matrix production. We examined the expression of CTGF in glaucomatous vs. normal TM cell lines and tissue, determined the CTGF expression profile in ocular tissue, and examined its mode of regulation in TM cells. Methods: GeneFilter (ResGen) and cDNA subtraction (Clontech) analyses were performed on pooled normal vs. glaucomatous TM cell mRNA. Aqueous humor and TM cell lysates and media were examined for CTGF expression by Western blot analysis using a commercially available polyclonal anti-CTGF antibody. Quantitative PCR analysis was used to measure CTGF mRNA levels. Results: CTGF expression was elevated in glaucomatous TM cells by GeneFilter and cDNA subtraction analyses. Ocular tissue profiling showed CTGF mRNA expression levels in TM=RPE=Ciliary Body>Iris>ONH>Retina with cultured TM cells expressing amounts similar to HUVEC, HMVEC-L, HAEC, utMVEC-myo, and HASMC cells. We stably transfected CHO cells with a CMV promoter-driven CTGF expression construct. Media from these cells, TM cells, and primate aqueous humor was analyzed for CTGF expression by Western blot analysis. A specific protein of the expected size for CTGF (38-kDa) was detected. Treatment of cultured TM cells with TGFß1 or TGFß2 resulted in significant elevation of CTGF expression, whereas treatment with Dexamethasone resulted in variable CTGF expression. Conclusions: CTGF mRNA is elevated in glaucomatous TM cells and is present in aqueous humor. TGFß2, shown by several groups to be elevated in glaucomatous AH, consistently up-regulated CTGF expression in TM cells. CTGF overexpression may be a consequence of elevated TGFß2 in glaucomatous AH and may play a key role in the remodeling of the extracellular matrix resulting in compromised AH filtering in the glaucomatous eye.

Keywords: growth factors/growth factor receptors • trabecular meshwork • aqueous