NFAT and fibrosis in the trabecular meshwork

NFAT 和小梁网纤维化

基本信息

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
项目类别：
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

项目摘要

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) 眼压 (IOP) 升高的途径。 POAG 患者中 TGFβ2 表达的增加尚不清楚。转录因子、NFATc1 和 αvβ3 整合素信号通路形成正反馈环我们的研究表明，这会导致 POAG 中 TGFβ2 水平升高。 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（使用组合疗法） Aim#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 小鼠中表达组成型活性的腺病毒 (Ad5) 载体中的蛋白质。 (CA)-NFATc1 将用于激活 HTM 细胞和 C57BL/6J 小鼠中的 NFATc1 和 Lenti-NFATc1 shRNA。用 Ad5-cre 载体转导的 NFATc1flox/flox 小鼠将用于在体外沉默 NFATc1 表达，体内的 NFATc1 激活剂分别是地塞米松或 Ca2+ 离子载体离子霉素。用于确认 NFATc1 活性及其在 HTM 细胞和细胞中调节 TGFβ2 和 ECM 表达中的作用使用免疫荧光显微镜测量小鼠 TGFβ2 和 ECM 表达的变化，蛋白质印迹、PCR 和 AHO 将使用眼压计和前房插管进行测量， Aim#2 将检验 αvβ3 整合素活性分别驱动 TGFβ2 和 ECM 的假设。通过生成由 NFATc1 载体协调的 Ca2+ 依赖性反馈环路来表达。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 NFAT-荧光素酶报告小鼠转导。 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 NFATc1 活性的体内作用 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。假设机械力与眼压升高相关，使 Ca2+ 持续升高增加离体猴子和人类前段的 NFATc1 和 αvβ3 整合素活性。将受到高压作用，Ca2+离子载体离子霉素将用于激活 NFATc1。 Ca2+螯合剂（BAPTA-AM）将用于阻断Ca2+指示剂，Fura2-AM将用于测量Ca2+。 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。