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 水平升高被认为是导致该病的主要原因 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（POAG）眼内压（IOP）升高。然而，途径 POAG 患者中 TGFβ2 表达的增加尚不清楚。我们假设协调激活 转录因子、NFATc1 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 整合素信号的激活会触发人小梁中 TGFβ2 表达的增加 网状 (HTM) 细胞，αvβ3 整合素表达受 NFATc1 转录活性控制。 了解 NFATc1 和 αvβ3 整合素活性如何共同控制 TGFβ2 水平非常重要，因为它 可以展示控制 POAG 的新方法（使用联合疗法）。目标#1 将测试 假设 NFATc1 的激活参与调节 TGFβ2 和 ECM 的表达 HTM 细胞和 C57BL/6J 小鼠中的蛋白质。表达组成型活性的腺病毒 (Ad5) 载体 (CA)-NFATc1 将用于激活 HTM 细胞和 C57BL/6J 小鼠中的 NFATc1。 Lenti-NFATc1 shRNA 和 用 Ad5-cre 载体转导的 NFATc1flox/flox 小鼠将用于在体外沉默 NFATc1 表达， 分别在体内。地塞米松或 Ca2+ 离子载体离子霉素（已知的 NFATc1 激活剂）将被 用于确认 NFATc1 活性及其在 HTM 细胞和细胞中调节 TGFβ2 和 ECM 表达中的作用 老鼠。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。 IOP 和 AHO 将使用眼压计和前房插管进行测量， 分别。目标#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+ 依赖性反馈环路来表达。 Ad5载体 表达 CA-αvβ3 整合素或失活 (D119Y) αvβ3 整合素将用于改变 HTM 细胞和 C57BL/6J 小鼠体内的 αvβ3 整合素。转染 NFAT 荧光素酶报告基因的小鼠 Ad5-αvβ3 整合素或 Ad5 生物活性 TGFβ2 转基因将用于检测 αvβ3 整合素的作用和 TGFβ2 对体内 NFATc1 活性的影响。 Ca2+-螯合剂 (BAPTA-AM) 将用于抑制 Ca2+ 信号传导。 TGFβ2 和 ECM 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 假设机械力与眼压升高相关，使 Ca2+ 持续升高 增加 TM 中 NFATc1 和 αvβ3 整合素的活性。离体猴子和人类前段 将受到高压。 Ca2+ 离子载体离子霉素将用于激活 NFATc1，同时 Ca2+螯合剂（BAPTA-AM）将用于阻断它。 Ca2+ 指示剂 Fura2-AM 将用于测量 Ca2+ 水平。 TGFβ2、αvβ3 整合素和 ECM 表达的变化将按照目标#1 中的描述进行测量