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理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激酶信号通路调节小梁网细胞的细胞骨架张力和细胞-细胞外基质相互作用，在细胞外基质的合成和转换中起关键作用，并有助于房水流出阻力的稳态。因此，预测流出通路内Rho/Rho激酶信号传导的异常激活有助于青光眼眼的房水流出阻力增加。这一假说的有力证据来自我们最近的研究，激动剂和RhoA诱导的TM细胞收缩张力和房水流出设施灌注模型的变化。收缩激动剂（溶血磷脂和凝血酶）、TGF β、细胞外基质（ECM）蛋白和眼内压升高均刺激TM细胞中的肌球蛋白II磷酸化和肌动球蛋白组装，这是细胞骨架张力升高的公认特征。这些作用进一步与TM细胞中Rho GT3活化增加相关，并且与灌注的猪和人眼中房水流出设施的显著降低相关。此外，组成型活性RhoA的表达显着降低了器官培养的猪眼前段的水流出设施和增加的基因编码的各种ECM蛋白和细胞因子在人TM细胞的表达。总之，这些观察结果表明Rho GT3信号传导、细胞骨架张力、ECM合成和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 GT3诱导的细胞骨架张力和细胞ECM相互作用在调节房水流出阻力中的作用，我们在本申请中提出研究：1. Rho GT3诱导的人TM细胞骨架张力与ECM合成和细胞因子表达之间的关系; 2.在活体啮齿动物模型中持续激活Rho GT3对眼内压和流出途径中ECM合成的体内作用，以及3. Rho/Rho激酶信号传导组分在眼内压升高的眼睛的流出通路中以及在高血压开角型青光眼的人类供体眼睛中的表达和活化状态。这些研究的完成应解开的作用，TM细胞骨架张力和细胞ECM的相互作用，在体内平衡的房水流出阻力，确定调节ECM合成的Rho/Rho激酶信号通路，并澄清这些事件的意义，眼内压升高，在青光眼的眼睛。与公共卫生的相关性：更好地了解调节房水流出阻力稳态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。在这项研究中，我们探索了一种新的范式，即细胞骨架信号和房水流出阻力的稳态之间存在着很强的功能联系。探索这种联系将为我们理解正常和高眼压性青光眼眼中房水流出阻力的生理和病理调节提供重要见解，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.