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RC Tripathi, J Chen, BJ Tripathi, D Ehrich, S Gotsis; Effect of Growth Factors on Expression of Fibronectin and MMP-3 by Trabecular Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1044.
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Purpose: To determine the mRNA expression of fibronectin and matrix metalloproteinase (MMP)-3, a protease that degrades fibronectin, by trabecular cells exposed to growth factors (GFs) present in primary and secondary aqueous humor. Methods: First-passage porcine trabecular cells were maintained in serum-free medium (SFM) for 48 hrs prior to incubation in SFM that contained a mixture of GFs at concentrations either present in primary aqueous humor (1.36 ng/ml bFGF, 1.74 ng/ml IGF-1, 14.2 mg/ml transferrin; 0.57 ng/ml TGF-ß1 and 2.47 ng/ml TGF-ß2) or those in secondary aqueous humor (0.91 ng/ml bFGF, 79.4 ng/ml IGF-1, 23.1 mg/ml transferrin, 3.86 ng/ml TGF-ß1, and 3.46 ng/ml TGF-ß2), or in SFM alone for 48 hrs or for 7 days. After extraction of total RNA, we performed RT-PCR using primer pairs specific for fibronectin and MMP-3. We quantified and normalized the products to mRNA expression of GAPDH as the internal control. Western blot analysis was performed to detect fibronectin protein levels in the cellular extracts. Results: Compared to control cells in SFM, fibronectin mRNA expression in trabecular cells incubated for 48 hrs in medium containing GFs present in primary or in secondary aqueous humor was up-regulated 60% and 50%, respectively. Fibronectin mRNA increased 3-fold and the amount of fibronectin protein increased 6-fold after 7 days. After incubation in primary or in secondary aqueous humor GFs, the mRNA level for MMP-3 expressed by trabecular cells decreased 20% and 70%, respectively, but this effect was not time-dependent. Conclusion: The combination and concentration of GFs in primary or secondary aqueous humor induced a similar expression of fibronectin mRNA. However, GFs in secondary aqueous humor had a greater effect in decreasing mRNA levels of MMP-3 than did GFs in primary aqueous humor. The down-regulation of MMP-3 mRNA expression by trabecular cells exposed to GFs present in secondary aqueous humor augments the up-regulation of fibronectin gene expression and contributes to the accumulation of this glycoprotein in the trabecular meshwork of glaucomatous eyes.
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