NFAT and fibrosis in the trabecular meshwork

NFAT 和小梁网纤维化

• 批准号: 10630268
• 负责人: Donna M Peters
• 金额: $ 41.6万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630268
• 关键词: Adenovirus Vector; Anterior; Aqueous Humor; Binding; Biological Process; Blindness; Calcineurin; Cannulations; Cells; Chelating Agents; Chronic; Clinical; Combined Modality Therapy; Deposition; Development; Dexamethasone; Etiology; Extracellular Matrix; Extracellular Matrix Proteins; Feedback; Fibronectins; Fibrosis; Genetic Transcription; Glaucoma; Glucocorticoids; Goals; Grant; Human; Immunofluorescence Microscopy; In Vitro; Injections; Integrin Signaling Pathway; Integrin alphaVbeta3; Integrins; Ionomycin; Ionophores; Laboratories; LoxP-flanked allele; Luciferases; Measures; Mechanics; Monkeys; Mus; Pathway interactions; Patients; Phosphoric Monoester Hydrolases; Physiologic Intraocular Pressure; Primary Open Angle Glaucoma; Property; Protein Isoforms; Proteins; Reporter; Role; Signal Pathway; Signal Transduction; Stretching; Testing; Tissues; Trabecular meshwork structure; Transcriptional Activation; Transforming Growth Factor beta; Transgenes; Western Blotting; Work; age related; anterior chamber; cell type; cellular transduction; connective tissue growth factor; early onset; in vivo; mechanical force; myocilin; novel; nuclear factors of activated T-cells; optic nerve disorder; overexpression; pressure; prevent; small hairpin RNA; transcription factor; vector

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

小梁网（TM）中TGFβ2水平的水平升高，这是主要原因 增加限制水性幽默出口（AHO）的细胞外基质（ECM）的沉积增加并导致 眼内压（IOP）的高升高（POAG）。然而路径（S） POAG患者中TGFβ2表达的增加尚不清楚。我们假设协调的激活 转录因子NFATC1和AαVβ3整合素信号通路形成正反馈回路 这驱动了POAG中TGFβ2的升高水平。为了支持这一假设，我们的研究表明 αVβ3整联蛋白信号传导的激活触发了人小梁中TGFβ2表达的增加 网格工作（HTM）细胞，并且αVβ3整合素表达受NFATC1的转录活性控制。 了解NFATC1和αVβ3整合素活性如何共同控制TGFβ2水平很重要，因为它很重要 可以展示新颖的方法（使用组合疗法）来控制POAG。 AIM＃1将测试 假设NFATC1的激活参与确定TGFβ2和ECM的表达 HTM细胞和C57BL/6J小鼠中的蛋白质。腺病毒（AD5）向量表达始终如一的活性 （CA）-NFATC1将用于激活HTM细胞和C57BL/6J小鼠中的NFATC1。 lenti-nfatc1 shrnas和 用AD5-CRE载体转移的NFATC1FLOX/FLOX小鼠将在体外沉默NFATC1表达，并且 分别在体内。地塞米松或Ca2+离子酚离子霉素（已知的NFATC1激活剂）将是 用于确认NFATC1活性及其在调节HTM细胞中的TGFβ2和ECM表达中的作用 老鼠。 TGFβ2和ECM表达的变化将使用免疫荧光显微镜测量， 蛋白质印迹和PCR。 IOP和AHO将使用传动计和前室插管测量 AIM＃2将检验αVβ3整合素活性驱动TGFβ2和ECM的假设 通过生成由NFATC1协调的Ca2+依赖性反馈回路的表达。 AD5矢量 表达CA-αVβ3整联蛋白或非活性（D119Y）αVβ3整合素将用于改变 HTM细胞和C57BL/6J小鼠体内的αVβ3整联蛋白。与 AD5-αVβ3整合素或AD5-BioActiveTGFβ2转化将用于检测αVβ3整合素和 TGFβ2在体内NFATC1活性上。 Ca2+ chelator（BAPTA-AM）将用于抑制Ca2+信号传导。 TGFβ2和ECM表达的变化将如AIM＃1所述测量。 AIM＃3将测试 假设与IOP升高相关的机械力使Ca2+的高度永久存在 这增加了TM中NFATC1和αVβ3整合素活性。前体内猴子和人类前部 将承受升高的压力。 Ca2+离子载体离子霉素将用于激活NFATC1，而 CA2+螯合剂（BAPTA-AM）将用于阻止它。 Ca2+指示器，Fura2-Am将用于测量Ca2+ 水平。 TGFβ2，αVβ3整合素和ECM表达的变化将如AIM＃1所述测量