Trabecular cell expression of fibronectin and MMP-3 is modulated by aqueous humor growth factors

• Published in: Experimental eye research Vol. 78; no. 3; pp. 653 - 660
• Main Authors: Tripathi, Brenda J., Tripathi, Ramesh C., Chen, Jianguang, Gotsis, Stelios, Li, Junping
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.03.2004
• Subjects: Animals; Aqueous Humor - chemistry; Cells, Cultured; Culture Media, Serum-Free; Fibronectins - genetics; Fibronectins - metabolism; Gene Expression Regulation - drug effects; gene therapy; glaucoma; Growth Substances - pharmacology; Matrix Metalloproteinase 3 - genetics; Matrix Metalloproteinase 3 - metabolism; primary aqueous humor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; secondary aqueous humor; Swine; trabecular meshwork; Trabecular Meshwork - drug effects; Trabecular Meshwork - metabolism; western blot; secondary aqueous humor; gene therapy; primary aqueous humor; western blot; glaucoma; trabecular meshwork
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2003.09.011

We investigated the mRNA and protein expression of fibronectin and stromelysin-1 (matrix metalloproteinase-3, MMP-3) by trabecular cells treated with growth factors present in primary and secondary aqueous humors. Serum-deprived trabecular cells were incubated for 48 hr or 7 days in medium containing either primary or secondary aqueous humor growth factors or in serum-free medium. We extracted total RNA, performed reverse transcription-polymerase chain reaction using primer pairs for fibronectin, stromelysin-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and quantified the products. We utilized Western blotting to detect and quantify fibronectin and stromelysin-1 protein. Compared to controls, expression of fibronectin mRNA by trabecular cells was increased by 50 and 100% after incubation in primary aqueous humor growth factors for 48 hr or 7 days, respectively, and 50 and 130% after incubation in secondary aqueous humor growth factors. Stromelysin-1 mRNA expression was decreased by 25 and 50% after incubation in primary aqueous humor growth factors for 48 hr or 7 days, respectively, and 80 and 85% after incubation for 48 hr or 7 days, respectively, in secondary aqueous humor growth factors. Fibronectin protein increased 3·5-fold and 6-fold after incubation for 48 hr with primary or secondary aqueous humor growth factors, respectively; after 7 days, the level increased 4- and 7-folds, respectively. Stromelysin-1 protein was not detectable by western blotting. The up-regulation of fibronectin mRNA by trabecular cells exposed to growth factors present in secondary aqueous humor augmented by the down-regulation of stromelysin-1 mRNA contributed to the accumulation of fibronectin. Our findings open the possibility that induction of stromelysin-1 gene expression in the trabecular meshwork of glaucomatous eyes could effectively reduce buildup of fibronectin in the aqueous outflow pathway to decrease outflow resistance in glaucomatous states of the eye.