The effect of dexamethasone on synthesis of collagen, fibronectin and α-smooth muscle actin in cultured human trabecular meshwork cells

Purpose: Steroids cause increased extracellular matrix (ECM) deposition in trabecular meshwork (TM) leading to increased aqueous outflow resistance. Since, the same is also observed in primary open angle glaucoma, steroid-treated TM cells are commonly used as in vitro model of glaucoma. However, the time-dependent effects of steroids on ECM production by TM cells have not been described. Hence, we investigated the time-dependent effects of dexamethasone on collagen type I (COLI), collagen type III (COLIII), collagen type IV (COLIV), fibronectin and α-smooth muscle actin (SMA) production by cultured human TM cells. Methods: TM cells were cultured in DMEM with or without dexamethasone (100 nM) for 7, 14 and 21 days. At the end of each time point, cells morphology was observed and cell viability was assessed by MTS assay. Cells and media were collected to measure the protein expression of COLI, COLIII, COLIV, fibronectin and SMA using Elisa and immunocytochemistry. Results: Secretion of all 3 collagen subtypes and fibronectin in the dexamethasone-treated cells were significantly greater (p<0.001) than untreated cells. Similarly, SMA protein expression was increased about 2-fold compared to untreated cells. Additionally, COLI, COLIII and COLIV secretion was significantly greater on days 14 and 21 compared to day 7 (p<0.05). Conclusion: Dexamethasone significantly increased synthesis of COLI, COLIII, COLIV, fibronectin and SMA by TM cells. The effect of dexamethasoneon the synthesis of COL I, III and IV, in particular, was found to be time-dependent. Indian J Physiol Pharmaco 2016; 60(4) : 352–363 *Corresponding author : Dr. Renu Agarwal, Associate Professor, Department of Pharmacology, Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Sungai Buloh, 47000 Selangor, Malaysia; Email: renuag02@gmail.com (Received on April 12, 2015) Indian J Physiol Pharmacol 2016; 60(4) Experimental Studies on The Role of Nitric Oxide (NO) 353 pathological changes in TM including the ECM changes (Wilson et al. 1993; Wordinger et al. 1999; Tawara et al. 2008; Razali et al. 2015). Several s tud ies have shown tha t t rea tment w i th glucocorticoids such as dexamethasone results in excessive ECM deposition in TM (Lo et al. 2003; Rozsa et al. 2006; Razali et al. 2016). Hence, glucocorticoid-treated TM cells are considered a good representative in vitro model for glaucoma related research. Although, glucocorticoid-induced changes in ECM deposition have widely been investigated, most studies describe the effects of only a shortterm treatment with glucocorticoids and the timedependent effects of glucocorticoids on human TM cells have not been studied. Since, glucocorticoid treated TM cells are commonly used as in vitro model of glaucoma, it is extremely important to elucidate how secretion of ECM is affected by glucocorticoids in a t ime-dependent manner. Time-dependent pathological ef fects are of great s igni f icance particularly when evaluating extent of change in the pathology in response to change in experimental condition, experimental substances or other stimuli over a period of time. Hence, we carried out this study to demonstrate the time-dependent effects of glucocorticoids on the production of COLI, COLIII, COLIV, fibronectin and α-smooth muscle actin by TM cells over a prolonged period of 21 days.