NFAT and fibrosis in the trabecular meshwork

小梁网(TM)中转化生长因子β2水平升高被认为是导致 细胞外基质(ECM)沉积增加，限制房水流出(Aho)并导致 原发性开角型青光眼的眼压升高。然而，这条道路(S) 转化生长因子β-2在POAG患者中的表达增加尚不清楚。我们假设，协同激活 转录因子NFATc1和αvβ3整合素信号通路形成正反馈环 这导致了POAG患者转化生长因子β-2水平升高。为了支持这一假设，我们的研究表明 α-v-β-3整合素信号通路激活促进人眼小梁细胞转化生长因子β-2表达 αvβ3整合素的表达受NFATc1转录活性的调控。 了解NFATc1和αvβ3整合素活性如何共同作用来控制转化生长因子β2水平是很重要的，因为它 可以展示控制POAG的新方法(使用组合疗法)。目标1号将测试 假设NFATc1参与调节转化生长因子β-2和细胞外基质的表达 在HTM细胞和C57BL/6J小鼠中的蛋白质。腺病毒(Ad5)载体表达A组分活性 (CA)-NFATc1将用于在HTM细胞和C57BL/6J小鼠中激活NFATc1。Lenti-NFATc1 shRNA和 转导Ad5-cre载体的NFATc1FLOX/FLOX小鼠将在体外沉默NFATc1的表达 分别在活体内。已知的NFATc1激活剂地塞米松或钙离子载体离子霉素将被 用于确定NFATc1的活性及其在调节转化生长因子β-2和细胞外基质表达中的作用 老鼠。用免疫荧光显微镜检测转化生长因子β-2和细胞外基质表达的变化。 蛋白质印迹和聚合酶链式反应。使用眼压计和前房插管测量眼压和AHO， 分别进行了分析。Aim#2将验证α与β3整合素活性驱动转化生长因子β2和细胞外基质的假设 通过产生一个由NFATc1协调的钙依赖反馈环来表达。Ad5向量 表达CA-αvβ3整合素或失活(D119Y)αvβ3整合素将用于改变 αvβ3整合素在人巨噬细胞瘤细胞和C57BL/6J小鼠体内的表达转导NFAT-荧光素酶报告基因小鼠 Ad5-αvβ3整合素或Ad5生物活性转化生长因子β2转基因将用于检测αvβ3整合素和 转化生长因子β-2对体内NFATc1活性的影响钙离子螯合剂(BAPTA-AM)将用于抑制钙信号转导。 转化生长因子β2和细胞外基质表达的变化将按照目标1中的描述进行测量。目标3将测试 与眼压升高相关的机械力使钙离子升高永久化的假说 这增加了TM中NFATc1和αvβ3整合素的活性。离体猴和人的眼前节 将承受更高的压力。钙离子载体离子霉素将用于激活NFATc1，而 用钙离子螯合剂(BAPTA-AM)阻断。将使用钙离子指示剂FURA2-AM来测量钙离子 级别。转化生长因子β2、αvβ3整合素和细胞外基质表达的变化将按照目标1中的描述进行测量

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