Crosstalk of TGF-beta and TLR4 pathways in the trabecular meshwork

小梁网中 TGF-β 和 TLR4 通路的串扰

• 批准号: 10256019
• 负责人: Colleen Mary McDowell
• 金额: $ 37.59万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10256019
• 关键词: Activins; Address; Age-Months; Anterior; Aqueous Humor; Binding; Binding Sites; Blocking Antibodies; Cells; Characteristics; Data; Dermal; Development; Disease; Extracellular Matrix; Eye; Eye Development; Fibroblasts; Fibronectins; Fibrosis; Gene Proteins; Glaucoma; Human; Immunohistochemistry; Impairment; In Vitro; Integrin Binding; Integrin alpha4beta1; Integrins; Knock-out; Label; Laminin; Lead; Lentivirus; LoxP-flanked allele; Measures; Membrane; Modeling; Molecular; Morphology; Mus; Neurons; Ocular Hypertension; Optic Nerve; Organ Culture Techniques; Pathogenicity; Pathologic; Pathway interactions; Patients; Peptides; Perfusion; Phenotype; Physiologic Intraocular Pressure; Quantitative Reverse Transcriptase PCR; Regulation; Reporting; Research; Resistance; Risk Factors; Role; Signal Pathway; Signal Transduction; TLR4 gene; Testing; Therapeutic; Trabecular meshwork structure; Transforming Growth Factor beta; Transmission Electron Microscopy; Virus; Visual; Western Blotting; age effect; cost; immunocytochemistry; in vivo; inhibitor/antagonist; integrin alpha9 beta1; knock-down; mutant; nerve damage; new therapeutic target; novel; overexpression; pressure; prevent; receptor; tonometry

Elevated intraocular pressure (IOP) is one of the major risk factors for the development and progression of glaucoma and is due to damage to the trabecular meshwork (TM). We have identified a novel molecular pathway of TGFβ2 and toll-like receptor 4 (TLR4) signaling crosstalk involved in the development of ocular hypertension and glaucomatous TM damage. We identified that the TLR4 signaling pathway is involved in the regulation of the extracellular matrix (ECM) in the TM and activation of TLR4 can enhance the effect of TGFβ2- induced glaucomatous phenotypes, while inhibition of TLR4 blocks the effect. We have identified both BMP and activin membrane bound inhibitor (BAMBI) and fibronectin extra domain A (FN-EDA) as important regulators of pathogenic TLR4 and TGFβ2 signaling in the TM. FN-EDA contains both integrin α4β1 and α9β1 binding sites which are known to regulate FN-EDA function. Recently integrin α4β1 was identified as a TLR4 co-receptor necessary for EDA-initiated activation of TLR4 in dermal fibroblasts. A20, an endogenous regulator of downstream TLR4 signaling, has been identified as an important regulatory molecule in other fibrotic diseases. Here we propose to determine the molecular mechanism of FN-EDA regulation of TLR4, determine the role of endogenous regulators BAMBI and A20 on pathological TGFβ2-TLR4 signaling, and identify novel therapeutic targets to prevent glaucomatous TM damage and elevated IOP. Our hypothesis is TGFβ2-TLR4 signaling crosstalk leads to pathological glaucomatous TM damage and elevated IOP, which can be controlled by both therapeutic and endogenous regulators. We will address this hypothesis with 4 specific aims: Specific Aim #1 will determine whether FN-EDA alters the TM morphology and function through TLR4 signaling. We will utilize B6.EDA+/+ mice which constitutively express only FN containing EDA and will analyze the IOP profiles, TM morphology, and ECM makeup. Specific Aim #2 will determine whether the EDA-binding integrins a4b1 and a9b1 are necessary for EDA activation of TLR4 signaling and ocular hypertension utilizing primary human TM cells as well as a4 and a9 integrin floxed mice. Specific Aim #3 will determine whether the endogenous negative regulators of TGFβ2-TLR4 signaling, BAMBI or A20, can block IOP elevation in mice. We will overexpress Bambi (using Ad5.Bambi virus) or A20 (using Ad5.A20 virus) in the TM and determine whether they can block Ad5.TGFβ2-induced ocular hypertension in C57BL/6J mice. Specific Aim #4 will determine whether inhibiting FN-EDA expression can block IOP elevation in mice and in a human anterior segment perfusion organ culture model (POC). Studies will use a fibronectin-binding peptide called FUD to block EDA+/+-induced and TGFβ2-induced ocular hypertension. These studies aim to explore a novel pathway involved in the development of glaucomatous TM damage. The data will be invaluable to the field of glaucoma research and could provide new targets to lower IOP and further explain the mechanisms involved in the development of glaucomatous TM damage.

眼内压（IOP）升高是发生和进展的主要危险因素之一， 青光眼是由于小梁网（TM）的损伤。我们发现了一种新的分子 TGFβ2和Toll样受体4（TLR 4）信号转导通路参与眼内肿瘤的发生 高血压和昏迷性TM损害。我们发现TLR 4信号通路参与了 调节TM中的细胞外基质（ECM）和TLR 4的活化可增强TGFβ2- 诱导的肿瘤表型，而TLR 4的抑制阻断了该作用。我们已经鉴定出两种BMP 激活素膜结合抑制因子（BAMBI）和纤连蛋白胞外结构域A（FN-EDA）是重要的 TM中致病性TLR 4和TGFβ2信号传导的调节剂。FN-EDA含有整合素α4β1和α9β1 结合位点，其已知调节FN-EDA功能。最近整合素α4β1被鉴定为TLR 4 在皮肤成纤维细胞中，EDA启动TLR 4活化所必需的辅助受体。A20，一种内源性调节因子 下游TLR 4信号传导，已被确定为其他纤维化的重要调控分子， 疾病在这里，我们建议确定FN-EDA调节TLR 4的分子机制，确定 内源性调节剂BAMBI和A20在病理性TGFβ2-TLR 4信号传导中作用，并鉴定新的 治疗靶点，以预防青光眼TM损伤和IOP升高。我们的假设是TGFβ2-TLR 4 信号串扰导致病理性青光眼TM损伤和IOP升高，这可能是 由治疗性和内源性调节剂控制。我们将用4个具体的假设来解决这个问题。 目的：特定目的#1将确定FN-EDA是否通过TLR 4改变TM形态和功能 发信号。我们将利用仅组成型表达含有EDA的FN的B6.EDA+/+小鼠，并将分析 IOP曲线、TM形态和ECM组成。具体目标#2将决定是否EDA结合 整合素a4 b1和a9 b1是EDA激活TLR 4信号传导和眼高血压所必需的， 原代人TM细胞以及α 4和α 9整联蛋白floxed小鼠。具体目标#3将决定 TGFβ2-TLR 4信号传导的内源性负调节因子BAMBI或A20可以阻断小鼠的IOP升高。 我们将在TM中过表达Bambi（使用Ad5.Bambi病毒）或A20（使用Ad5.A20病毒），并确定 能否阻断Ad 5、TGF β2诱导的C57 BL/6 J小鼠高眼压。具体目标#4 确定抑制FN-EDA表达是否可以阻断小鼠和人前房的IOP升高， 节段灌注器官培养模型（POC）。研究将使用一种称为FUD的纤维连接蛋白结合肽， 阻断EDA+/+和TGFβ2诱导的高眼压。这些研究旨在探索一种新的途径 参与了昏迷性TM损伤的发展。这些数据对青光眼领域将是无价的 研究，并可能提供新的目标，以降低眼压，并进一步解释参与的机制， 形成昏迷性TM损伤。