Extracellular Matrix and Outflow Resistance

项目摘要 高眼压是青光眼的主要危险因素，青光眼影响着6600万人。 全世界。降低眼压仍然是阻止青光眼进展的唯一有效的治疗策略 视力丧失。小梁网络(TM)是眼压调节的主要部位，但很少有药物能特异性地流出 以TM为目标，这是病理的地方。最新一类青光眼药物，Rho激酶抑制剂，抑制Rho- 拆解肌动蛋白应力纤维的岩石路径。然而，最近的研究未能在人类体内检测到应激纤维 体外TM组织。因此，Rho激酶抑制剂如何降低眼压的确切生物学机制仍不清楚。 相反，皮质肌动蛋白网络在原位占主导地位。由于Rho GTP酶调节皮质肌动蛋白，所以Rho激酶 抑制剂可能针对皮质肌动蛋白。这是TM中一条尚未探索的研究途径。大脑皮质的收缩能力 肌动蛋白位于细胞膜的正下方，施加张力并产生机械力来驱动 细胞形状的改变，如内陷和细胞突起。在本应用程序中，我们将重点介绍 皮质肌动蛋白、质膜和细胞外基质的关系，因为它与两个TM细胞突起有关： 丝状/隧道状纳米管(TNTs)和足体和类蹄盖状结构(PILs)。我们会调查的 膜组织的‘尖桩-栅栏’模式是否影响丝状足纲/TNTs。在此模型中， 膜被锚定的跨膜‘Pickket’蛋白隔开，例如CD44 细胞外和亚膜内的“栅栏”(皮质肌动蛋白)。‘尖桩栅栏’分子‘畜栏’其他 跨膜蛋白，如整合素，进入膜室。在目标1中，我们将测试 肌动蛋白结合蛋白和/或CD44‘Pickket’蛋白的操纵将导致大脑皮层局部解体。 肌动蛋白‘栅栏’，以启动TNT的形成。影响将由超分辨率共聚焦显微镜，TNT来确定 囊泡转移试验、活体TM细胞成像和CD44磷酸化试验。特定肌动蛋白的作用- 将研究结合蛋白抑制物在灌流培养中对外流的调节作用，以评估它们的潜力。 来调节眼压。目的#2研究正常人和正常人的质膜张力和整合素的激活 青光眼TM细胞。细胞膜张力的差异将使用Flipper-TR型荧光测定仪进行测量 探针和整合素的激活状态将使用构象特异性抗体来确定。这将提供 关于膜张力如何影响跨膜受体激活的新信息。最后，在目标3中， 我们将研究肌球蛋白-X，这是一种肌动蛋白结合蛋白，是PILS的一个组成部分。我们将调查 Myo10在TM细胞中协调整合素和基质金属蛋白酶降解复合体活性中的作用 在静息状态和机械拉伸状态下，以及以1x的速度灌流的人前段TM组织 和2倍的压力。了解影响细胞突起的分子机制(丝状伪足/TNTs和 PILS)将为皮质肌动蛋白细胞骨架在眼压调节中的作用提供新的见解，将修订我们的 目前对Rho激酶抑制剂如何降低眼压并可能导致新的青光眼治疗方法的了解。

Project Summary Elevated intraocular pressure (IOP) is a primary risk factor for glaucoma, which affects over 66 million people worldwide. Lowering IOP remains the only effective therapeutic strategy to stop the progression of glaucomatous vision loss. The trabecular meshwork (TM) is the primary site of IOP regulation, but few outflow drugs specifically target the TM, the site of pathology. The newest class of glaucoma drugs, Rho kinase inhibitors, inhibit the Rho- ROCK pathway to disassemble actin stress fibers. However, recent studies failed to detect stress fibers in human ex vivo TM tissue. Thus, the exact biological mechanisms of how Rho kinase inhibitors lower IOP remain unclear. Instead, cortical actin networks predominate in situ. Since Rho GTPases regulate cortical actin, Rho kinase inhibitors may target cortical actin. This is an unexplored research avenue in the TM. Contractility of cortical actin, which sits just beneath the cell membrane, exerts tension and produces a mechanical force that drives changes in cell shape such as invaginations and cellular protrusions. In this application, we will focus on the relationship between cortical actin, the plasma membrane and ECM as it pertains to two TM cellular protrusions: filopodia/tunneling nanotubes (TNTs) and podosome and invadopodia-like structures (PILS). We will investigate whether the `picket-fence' model of membrane organization influences filopodia/TNTs. In this model, the membrane is compartmentalized by transmembrane `picket' proteins, e.g. CD44, which are anchored extracellularly and to sub-membrane intracellular “fences” (cortical actin). `Picket-fence' molecules `corral' other transmembrane proteins, such as integrins, into membrane compartments. In Aim #1, we will test whether manipulation of actin-binding proteins and/or CD44 `picket' proteins will lead to local disassembly of the cortical actin `fence' to initiate TNT formation. Effects will be determined by super-resolution confocal microscopy, TNT vesicle transfer assays, live TM cell imaging and CD44 phosphorylation assays. The effects of specific actin- binding protein inhibitors on outflow regulation in perfusion culture will be investigated to evaluate their potential to modulate IOP. Aim #2 will investigate plasma membrane tension and integrin activation in normal and glaucomatous TM cells. Differences in cell membrane tension will be measured using a Flipper-TR fluorogenic probe and integrin activation state will be determined using conformation-specific antibodies. This will provide novel information on how membrane tension influences transmembrane receptor activation. Finally, in Aim #3, we will investigate Myosin-X, an actin-binding protein that is a component of PILS. We will investigate the role of Myo10 in coordinating integrins and matrix metalloproteinase activity at these degradative complexes in TM cells at rest and when subjected to mechanical stretch, and in TM tissue of human anterior segments perfused at 1x and 2x pressure. Understanding the molecular machinery influencing cellular protrusions (filopodia/TNTs and PILS) will provide novel insights into the role of the cortical actin cytoskeleton in IOP regulation, will revise our current understanding of how Rho kinase inhibitors lower IOP and may lead to new glaucoma therapies.