Trabecular Meshwork Cytoskeletal Signaling-Regulation of Aqueous Humor Outflow

描述(由申请人提供)：确定控制房水流出阻力的潜在调节机制对于了解青光眼和开发有效的治疗方法具有重要意义。我们推测，Rho/Rho激酶信号通路调节的小梁细胞骨架张力和细胞外基质相互作用在细胞外基质的合成和周转中起关键作用，并有助于房水流出阻力的动态平衡。因此，流出通路中Rho/Rho激酶信号的异常激活被预测为青光眼房水流出阻力增加的原因之一。这一假说的有力证据来自我们最近对激动剂和RhoA在灌流模型中引起的TM细胞收缩张力和房水流出设施的变化的研究。收缩激动剂(溶血磷脂和凝血酶)、转化生长因子β、细胞外基质(ECM)蛋白和眼压升高都能刺激TM细胞的肌球蛋白II磷酸化和肌动球蛋白组装，这是公认的细胞骨架张力增加的特征。这些效应还与TM细胞中Rho GTPase活性的增加以及猪和人眼灌流后房水流出能力的显著降低有关。此外，结构性活性RhoA的表达显著降低了器官培养的猪眼前段房水流出的便利性，并增加了人TM细胞中各种ECM蛋白和细胞因子的基因表达。综上所述，这些观察表明Rho GTPase信号、细胞骨架张力、ECM合成和房水流出阻力之间存在潜在的相互作用。鉴于有令人信服的证据支持Rho GTPase诱导的细胞骨架张力和细胞外基质相互作用在房水流出阻力调节中的作用，我们建议在本应用中研究：1.Rho GTPase诱导的细胞骨架张力和ECM合成与人TM细胞中细胞因子表达的关系；2.在活体啮齿动物模型中，Rho GTP酶持续激活对眼压和流出通路中ECM合成的影响；3.在高眼压和高压性开角型青光眼供体眼的流出通路中Rho/Rho激酶信号成分的表达和激活状态。这些研究的完成将揭开TM细胞骨架张力和细胞ECM相互作用在房水流出阻力动态平衡中的作用，明确Rho/Rho激酶信号通路对ECM合成的调节，并阐明这些事件在青光眼眼内压升高中的意义。与公众健康相关：更好地理解调节房水流出阻力的动态平衡的分子机制，可以为设计治疗青光眼的靶向疗法提供新的途径。了解原发性开角型青光眼的病因和开发有效的治疗方法需要确定控制房水流出的调节机制。在拟议的研究中，我们探索了一种新的范式，即细胞骨架信号与房水流出阻力的动态平衡之间存在着强大的功能联系。探索这种联系将为我们理解正常和高眼压性青光眼眼房水流出阻力的生理和病理调节提供重要的见解，并对青光眼新疗法的翻译应用具有重要意义。

DESCRIPTION (provided by applicant): Identifying potential regulatory mechanisms controlling aqueous humor outflow resistance has important implications for understanding glaucoma and developing effective therapies. We hypothesize that Rho/Rho kinase signaling pathway regulated cytoskeletal tension and cell extracellular matrix interactions of trabecular meshwork (TM) cells play a critical role in the synthesis and turnover of extracellular matrix, and contribute to the homeostasis of aqueous humor outflow resistance. Abnormal activation of Rho/Rho kinase signaling within the outflow pathway is therefore predicted to contribute to the increased aqueous outflow resistance in glaucomatous eyes. Compelling evidence for this hypothesis derives from our recent studies of agonist and RhoA induced changes in TM cell contractile tone and aqueous humor outflow facility in perfused models. Contractile agonists (lysophospholipids and thrombin), TGF beta, extracellular matrix (ECM) proteins and increased intraocular pressure, all stimulated myosin II phosphorylation and actomyosin assembly in TM cells, which are recognized characteristics of increased cellular cytoskeletal tension. These effects were further associated with increased Rho GTPase activation in TM cells, and with significant decreases in aqueous outflow facility in perfused porcine and human eyes. Moreover, expression of constitutively active RhoA significantly decreased aqueous outflow facility in organ cultured porcine eye anterior segments and increased expression of genes encoding various ECM proteins and cytokines in human TM cells. Taken together, these observations indicate the existence of a potential interaction between Rho GTPase signaling, cytoskeletal tension, ECM synthesis and aqueous outflow resistance. Given the convincing evidence in support of a role for Rho GTPase induced cytoskeletal tension and cell ECM interactions in regulation of aqueous humor outflow resistance we propose in this application to investigate: 1. the association between Rho GTPase induced cytoskeletal tension and ECM synthesis and the expression of cytokines in human TM cells; 2. in vivo effects of sustained activation of Rho GTPase on intraocular pressure and ECM synthesis in the outflow pathway in a live rodent model, and 3. the expression and activation status of the Rho/Rho kinase signaling components in the outflow pathway of eyes subjected to elevated intraocular pressure, and in hypertensive open angle glaucomatous human donor eyes. Completion of these studies should unravel the role of TM cytoskeletal tension and cell ECM interactions in the homeostasis of aqueous humor outflow resistance, define the regulation of ECM synthesis by the Rho/Rho kinase signaling pathway, and clarify the significance of these events in increased intraocular pressure in glaucomatous eyes. Relevance to public health: Better understanding of the molecular mechanisms regulating homeostasis of aqueous humor outflow resistance could provide novel avenues for designing targeted therapies for treatment of glaucoma. Understanding the etiology of primary open angle glaucoma and developing effective therapies requires identification of regulatory mechanisms which control aqueous humor outflow. In the proposed study we explore the novel paradigm that there is a strong functional connection between the cytoskeletal signaling and the homeostasis of aqueous humor outflow resistance. Exploring this connection will provide important insights into our understanding of physiological and pathological regulation of aqueous outflow resistance in normal and ocular hypertensive glaucoma eyes and have an important bearing on translational application to novel therapies for glaucoma.