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

高眼压是青光眼发生、发展的主要危险因素之一。 青光眼是由于小梁网络(TM)受损所致。我们发现了一种新的分子 转化生长因子β-2和Toll样受体4信号串扰信号通路参与眼部发育 高血压和青光眼的TM损害。我们发现TLR4信号通路参与了 调节TM中的细胞外基质和激活TLR4可增强转化生长因子β2的作用。 诱导青光眼的表型，而抑制TLR4的作用被阻断。我们已经确认了两个BMP 而激活素膜结合抑制物(BAMBI)和纤维连接蛋白额外结构域A(FN-EDA)是重要的 TM中致病TLR4和转化生长因子β-2信号的调节。FN-EDA含有整合素α4β1和α9β1 已知的调节FN-EDA功能的结合位点。最近，整合素α4β1被鉴定为TLR4 EDA激活真皮成纤维细胞TLR4所必需的辅受体。A20，一种内源性调节因子 TLR4信号的下游，已被确定为其他肝纤维化的重要调节分子 疾病。在这里我们建议确定FN-EDA调节TLR4的分子机制，确定 内源性调节因子BAMBI和A20在病理性转化生长因子β2-TLR4信号转导中的作用 预防青光眼TM损害和眼压升高的治疗目标。我们的假设是转化生长因子β2-TLR4 信号串扰导致病理性青光眼TM损害和眼压升高，这可能是 由治疗性和内源性调节因子共同控制。我们将用4个具体的例子来说明这一假设 目的：具体目标#1将决定FN-EDA是否通过TLR4改变TM的形态和功能 发信号。我们将利用B6.EDA+/+小鼠，它们结构性地只表达含有EDA的FN，并将分析 眼压、眼内膜形态和细胞外基质成分。具体目标#2将决定EDA绑定是否 整合素a4b1和a9b1是EDA激活TLR4信号和利用高眼压所必需的 原代人TM细胞以及A4和A9整合素诱导的小鼠。具体目标#3将决定是否 内源性转化生长因子β2-TLR4信号负性调节因子BAMBI或A20可阻断小鼠眼压升高。 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Ad5、TGFβ2诱导的C57BL/6J小鼠高眼压。具体目标#4将 确定抑制FN-EDA的表达是否可以阻止小鼠和人类前房的眼压升高 节段灌流器官培养模型(POC)。研究将使用一种名为FUD的纤维连接蛋白结合肽来 阻断EDA+/+和转化生长因子β2诱导的高眼压。这些研究旨在探索一种新的途径 参与了青光眼TM损害的发生。这些数据对青光眼领域将是无价的。 研究并可能为降低眼压提供新的靶点，并进一步解释 青光眼TM损害的发生。

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.