Abstract
Purpose: :
Primary open angle glaucoma (POAG) is a blinding ocular disease affecting 70 million people worldwide. A major risk factor for POAG is elevated intraocular pressure resulting from increased resistance of aqueous humor outflow through the trabecular meshwork (TM). We have previously reported that BMP-4 attenuates TGF-β2 induced extracellular matrix (ECM) deposition in TM cells, and that gremlin, a BMP antagonist, blocks this inhibitory effect. Follistatin (FST) has also been reported to inhibit BMPs as well as activins (Act). Three isoforms of FST (288, 315, 303) have been described. FST and Act expression and function in normal (NTM) and glaucomatous (GTM) TM cells and tissues are unknown. The purpose of this study was to determine if: (a) FST isoforms and activin A are differentially expressed in NTM vs. GTM cells and tissues, and (b) exogenous FST or activin A regulate ECM protein production in TM cells.
Methods: :
QRT-PCR and RT-PCR were used to determine mRNA expression of FST isoforms and Act A in TM cells (N=10). Immunohistochemistry (IHC) was used to demonstrate FST isoforms and Act A in NTM (N=3) and GTM (N=3) tissues. TM cells were cultured with FST or Act A, at previously reported concentrations, for 6, 24, and 48 hrs. Western immunoblot analysis was used to evaluate FST and Act A effects on ECM proteins fibronectin (FN), PAI-1, and collagen1A.
Results: :
mRNA for FST isoforms was detected in TM cells, but with significantly higher expression in GTM cells (p<0.05). Using IHC, a greater amount of FST was also seen in GTM tissues compared to NTM tissues. Act A mRNA was detected in TM cells and proteins were detect in TM tissues with significantly lower protein expression in GTM tissues (p<0.05). Exogenous FST and activin A increased FN, and PAI-1 expression in a dose-dependent manner.
Conclusions: :
This is the first report in differences of FST and Act A mRNA and protein expression in NTM vs. GTM cells and tissues. GTM cells and tissues had significantly higher expression of FST while NTM tissues had significantly lower expression of Act A. This is also the first documentation of FST and Act A function in TM cells. Both exogenous FST and Act A increased ECM proteins in TM cells. These results further our knowledge of the potential role of BMP antagonists in the human TM and their potential pathogenic roles in the pathogenesis of glaucoma.
Keywords: extracellular matrix • trabecular meshwork • protein structure/function