NFAT and fibrosis in the trabecular meshwork

Elevated levels of TGFβ2 levels in the trabecular meshwork (TM) are thought to the major cause for the increased deposition of extracellular matrix (ECM) that restricts aqueous humor outflow (AHO) and causes an elevation in intraocular pressure (IOP) in primary open angle glaucoma (POAG). Yet the pathway(s) that increase TGFβ2 expression in POAG patients is unclear. We hypothesize that the coordinated activation of the transcription factor, NFATc1 and a αvβ3 integrin signaling pathway forms a positive feedback loop that drives the elevated levels of TGFβ2 in POAG. In support of this hypothesis, our studies have shown that activation of αvβ3 integrin signaling triggers an increase in TGFβ2 expression in human trabecular meshwork (HTM) cells and that αvβ3 integrin expression is controlled by the transcriptional activity of NFATc1. Understanding how NFATc1 and αvβ3 integrin activity work together to control TGFβ2 levels is important as it could demonstrate novel ways (using combinational therapies) to control POAG. Aim#1 will test the hypothesis that activation of NFATc1 is involved in regulating the expression of TGFβ2 and ECM proteins in HTM cells and in C57BL/6J mice. Adenoviral (Ad5) vectors expressing a constitutively active (CA)-NFATc1 will be used to activate NFATc1 in HTM cells and in C57BL/6J mice. Lenti-NFATc1 shRNAs and a NFATc1flox/flox mouse transduced with Ad5-cre vector will be used to silence NFATc1 expression in vitro and in vivo, respectively. Dexamethasone or the Ca2+ ionophore ionomycin, known activators of NFATc1, will be used to confirm NFATc1 activity and its role in regulating TGFβ2 and ECM expression in HTM cells and in mice. Changes in TGFβ2 and ECM expression will be measured using immunofluorescence microscopy, western blots, and PCR. IOP and AHO will be measured using a tonometer, and anterior chamber cannulation, respectively. Aim#2 will test the hypothesis that αvβ3 integrin activity drives TGFβ2 and ECM expression by generating a Ca2+-dependent feedback loop coordinated by NFATc1. Ad5 vectors expressing a CA-αvβ3 integrin or inactive (D119Y) αvβ3 integrin will be used to alter the expression/activity of αvβ3 integrin in HTM cells and in C57BL/6J mice in vivo. A NFAT-luciferase reporter mouse transduced with Ad5-αvβ3 integrin or an Ad5-bioactive TGFβ2 transgene will be used to detect the effect of αvβ3 integrin and TGFβ2 on NFATc1 activity in vivo. The Ca2+-chelator (BAPTA-AM) will be used to inhibit Ca2+ signaling. Changes in TGFβ2 and ECM expression will be measured as described in aim#1. Aim#3 will test the hypothesis that mechanical forces associated with an elevated IOP perpetuate the elevation in Ca2+ that increases NFATc1 and αvβ3 integrin activity in the TM. Ex vivo monkey and human anterior segments will be subjected to elevated pressure. The Ca2+ ionophore ionomycin will be used to activate NFATc1 while a Ca2+ chelator (BAPTA-AM) will be used to block it. A Ca2+ indicator, Fura2-AM will be used to measure Ca2+ levels. Changes in TGFβ2, αvβ3 integrin and ECM expression will be measured as described in aim#1