Browsing by Subject "Follistatin"
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Item Characterization and Function of Follistatin in Human Trabecular Meshwork Cell and Tissues(2013-05-01) Fitzgerald, Ashley M.; Robert WordingerPrimary Open Angle Glaucoma (POAG) is a leading cause of blindness affecting over 70 million people worldwide. The most important risk factor for developing POAG is elevated intraocular pressure (IOP), which results from increased resistance of aqueous humor (AH) through the trabecular meshwork (TM) outflow pathway. Transforming growth factor- beta II (TGF-β2) is elevated in the AH and TM of glaucoma patients. Recent evidence indicate an extracellular BMP antagonist, gremlin, regulates BMP signaling and TGF-β2 activity. Follistatin (FST), another secreted BMP antagonist is recognized for its ability to bind BMPs and their type I receptor, sequestering BMP signaling. The purpose is to evaluate the presence and relevant activity of follistatin in TM tissues and cells. We hypothesize expression of follistatin in human trabecular meshwork cells alters the expression of extracellular matrix (ECM) deposition seen in the pathogenesis of glaucoma. First, we examined differential FST expression in human trabecular meshwork cells and tissues. We observed a significant increase in expression of FST in glaucomatous as compared to normal protein and mRNA expression. Next, we determined if FST could be induced upon treatment of exogenous TGF-ß2 protein in human TM cells. Studies showed TGF-ß2 up-regulated FST mRNA transcript in a time dependent manner. FST protein secretion was increased in a time and does dependent manner. Third, we assessed FST effects on induction or inhibition of ECM proteins in human TM cells. ECM protein and mRNA expression was time dependent; nevertheless the response of ECM protein to FST treatment is different depending on isoform presence. Additional studies will be done to further elucidate these findings. Lastly, we evaluated FST-288 and FST-315 inhibition of BMP4 attenuation of TGF-ß2 induced ECM expression. Data suggest FST-315 to suppress BMP-4 effects on TGF-ß2 induced ECM and FST-288 enhanced BMP-4 effects on TGF-ß2 induced ECM. The goal is to evaluate additional factors that contribute to the pathogenesis of POAG and assess how these factors can provide possible therapeutic mechanisms for the treatment of glaucoma.Item EFFECTS OF TGF-BETA2, FOLLISTATIN AND ACTIVIN A ON EXTRACELLULAR MATRIX IN NORMAL HUMAN TRABECULAR MESHWORK CELLS AND TISSUES.(2013-04-12) Fisher, AndrewPurpose: Primary open angle glaucoma (POAG) is characterized as a group of eye diseases resulting in optic nerve head damage and irreversible blindness. A major risk factor for developing POAG is increased intraocular pressure (IOP) leading to decreased outflow of aqueous humor (AH) through the trabecular meshwork (TM). Transforming growth factor-beta2 (TGF-β2) is increased in the AH of glaucoma patients, and causes increased extracellular matrix (ECM) protein synthesis in the TM. Bone morphogenetic protein-4 (BMP-4) has been shown to inhibit TGF-β2 actions. Follistatin (FST) is an antagonist of BMP-4 and is elevated in the glaucomatous TM. Elevated levels of FST in the TM may block BMP-4 ability to attenuate TGF-β2 induction of ECM proteins. FST may also have a direct role in regulating ECM protein expression in human TM (HTM) cells. HTM cells also express Activin A (Act A). The purpose of this study was to assess the role of FST and Act A in HTM cells as related to TGF-β2/BMP-4 signaling. Understanding these interactions may provide possible new therapeutic targets for the treatment of glaucoma. Methods: Normal HTM cell lines were cultured and treated with TGF-β2 (5ng/ml), FST-315, FST-288, or Act A (each at 50ng/ml) alone and/or simultaneously for 24 and 48 hrs. Western blot analysis was used to evaluate the effects of FST-315/288, Act A, and TGF-β2 on ECM protein synthesis including fibronectin (FN), PAI-1, and collagen1A. Results: TGF-β2 induced expression of PAI-1 and FN.. ACT-A mildly induced PAI-1 and FN proteins as compared to TGF-β2. TGF-β2 and Act A treatment appeared to have a synergistic effect on the expression of PAI-1 and FN protein as compared to individual treatment (Tx) of TGF-β2 or Act A. FST 288 does not seem to change the Act A + TGF-β2 synergism. FST 315 inhibits the synergism of Act A + TGF-β2 showing a decrease in PAI-1 and FN protein. Conclusions: FST-315 decreased the induction of ECM proteins by TGF-β2 and Act-A. FST-288 increased induction of ECM proteins in cells treated with TGF-β2 and Act A. FST-288 treatment for 24 hours induced increased ECM proteins PAI-1 and FN, but there was no induction at 48 hours. FST-315 treatment for 24 hours slightly induced ECM PAI-1 and FN, but there was no induction at 48 hours. Act A treatment for 24 and 48 hours increased induction of PAI-1 or FN. These results further our knowledge of the potential role of BMP antagonists in the human TM and their potential roles in the pathogenesis of glaucoma.