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Deborah Wallace, Abbot Clark, Noelynn Oliver, John Crean, Colm O'Brien; Connective Tissue Growth Factor Induction of Lysl Oxidase (LOX) Enzyme Expression in Human Trabecular Meshwork Cells is reduced by FG-3019. Invest. Ophthalmol. Vis. Sci. 2013;54(15):763.
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The matricellular protein Connective Tissue Growth Factor (CTGF) has been shown to play an integral role in extracellular matrix (ECM) deposition in trabecular meshwork (TM) cells in glaucoma. Furthermore, we have previously described a role for anti-CTGF immunotherapy (FG-3019, FibroGen Inc. USA) in combating glaucoma-associated fibrosis in in vitro models. LOX enzymes are an important class of ECM cross-linking enzymes involved in the pathogenesis of glaucoma with identified single nucleotide polymorphisms (SNP) in LOXL-1 associated with an increased risk of developing pseudoexfoliation glaucoma. The purpose of this study was to firstly investigate if CTGF induced expression of LOX enzymes in human TM cells and whether pre-treatment with anti-CTGF (FG-3019) could decrease LOX expression and therefore be an attractive therapeutic approach for the treatment of glaucoma.
TM cells from a normal donor (NTM5) were grown to confluence and placed in serum free media for 24 hours prior to treatment with a recombinant fragment of CTGF (25ng/ml for 24hours) (Sigma). Levels of LOX and LOXL genes (1-4) were determined by quantitative PCR using gene specific exon-exon spanning primers and / or specific probes. NTM5 cells were then cultured in the presence or absence of the therapeutic, humanized monoclonal anti-CTGF antibody FG-3019 (10μg/ml) prior to treatment with CTGF as described above. The modulatory effect of FG-3019 (10μg/ml) was assessed and IgG (10μg/ml) was included as a control. LOX and LOXL genes (1-4) expression were determined by quantitative PCR using gene specific exon-exon spanning primers and / or specific probes. The equation 2ΔΔcT was used to derive a fold difference for gene expression. Results are representative of three independent experiments.
Treatment of NTM cells with CTGF induces a significant (P<0.05) increase in the expression of LOX & LOXL 1-4. Pre-treatment with anti-CTGF (FG-3019) significantly reduces (P<0.01) the CTGF-induced expression of all LOX enzymes.
CTGF can drive the expression of the ECM cross-linking LOX enzymes in NTM cells. The anti-CTGF antibody FG-3019 is effective in reducing LOX production. These observed anti-fibrotic effects support a pathologically significant role for the use of anti-CTGF immunotherapy as a possible approach for treatment of glaucoma.
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