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水平的升高被认为是小梁切除术后的主要原因。 细胞外基质（ECM）沉积增加，限制了房水流出（AHO），并导致 原发性开角型青光眼（POAG）的眼内压（IOP）升高。而这条路， TGFβ2在POAG患者中表达增加尚不清楚。我们假设，协调激活 转录因子NFATc 1和αvβ3整合素信号通路形成正反馈环 导致POAG中TGFβ2水平升高。为了支持这一假设，我们的研究表明， αvβ3整合素信号的激活触发了人小梁细胞中TGFβ2表达的增加， 在HTM细胞中，αvβ3整合素的表达受NFATc 1的转录活性控制。 了解NFATc 1和αvβ3整合素活性如何共同控制TGFβ2水平是重要的，因为它 可以展示新的方法（使用组合疗法）来控制POAG。目标1将测试 假设NFATc 1的激活参与调节TGFβ2和ECM的表达 HTM细胞和C57 BL/6 J小鼠中的蛋白质。腺病毒（Ad 5）载体表达组成型活性的 （CA）-NFATc 1将用于激活HTM细胞和C57 BL/6 J小鼠中的NFATc 1。Lenti-NFATc 1 shRNA和 用Ad 5-cre载体转导的NFATc 1 flox/flox小鼠将用于在体外沉默NFATc 1表达， 在体内，分别。地塞米松或Ca 2+离子载体离子霉素，已知的NFATc 1激活剂，将被 用于证实NFATc 1活性及其在调节HTM细胞中TGFβ2和ECM表达中的作用，以及在 小鼠将使用免疫荧光显微镜测量TGFβ2和ECM表达的变化， 蛋白质印迹和PCR。将使用眼压计和前房插管测量IOP和AHO， 分别目标2将检验αvβ3整联蛋白活性驱动TGFβ2和ECM的假设 通过产生由NFATc 1协调的Ca 2+依赖性反馈环来表达。ad 5载体 表达CA-αvβ3整联蛋白或无活性（D119 Y）αvβ3整联蛋白的细胞将用于改变 HTM细胞和C57 BL/6 J小鼠体内的αvβ3整联蛋白。转导了NFAT-荧光素酶报告基因小鼠， Ad 5-αvβ3整联蛋白或Ad 5-生物活性TGFβ2转基因将用于检测αvβ3整联蛋白的作用， TGFβ2对体内NFATc 1活性的影响。Ca 2+螯合剂（BAPTA-AM）将用于抑制Ca 2+信号传导。 将如aim#1中所述测量TGFβ2和ECM表达的变化。目标3将测试 与IOP升高相关的机械力使Ca 2+升高持续的假设 增加TM中NFATc 1和αvβ3整合素活性。离体猴和人前段 将承受高压Ca 2+离子载体离子霉素将被用于激活NFATc 1， 用钙离子螯合剂（BAPTA-AM）阻断，用钙离子指示剂Fura 2-AM测定 程度.如目的#1所述，测量TGFβ2、αvβ3整联蛋白和ECM表达的变化