Extracellular Matrix and Outflow Resistance

项目摘要 眼内压升高是青光眼的主要危险因素，影响超过6600万人 国际吧降低眼压仍然是阻止青光眼进展的唯一有效的治疗策略。 视力丧失小梁网是眼压调节的主要部位，但很少有外流药物特异性调节眼压。 目标是TM，病理部位。最新的一类青光眼药物，Rho激酶抑制剂，抑制Rho- ROCK途径分解肌动蛋白应力纤维。然而，最近的研究未能检测到人类的应力纤维， 离体TM组织。因此，Rho激酶抑制剂如何降低IOP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，Rho激酶 抑制剂可以靶向皮质肌动蛋白。这是TM中一个未开发的研究途径。皮质收缩力 位于细胞膜下方的肌动蛋白施加张力并产生机械力， 细胞形状的变化，如内陷和细胞突起。在此应用程序中，我们将重点关注 皮质肌动蛋白，质膜和ECM之间的关系，因为它涉及两个TM细胞突起： 丝状伪足/隧穿纳米管（TNT）和足体和侵入伪足样结构（PILS）。我们将调查 膜组织的“栅栏”模型是否影响丝状伪足/TNT。该模型 膜被跨膜“尖桩”蛋白（例如CD 44）分隔，这些蛋白锚定在 细胞外和亚膜细胞内“栅栏”（皮质肌动蛋白）。“栅栏”分子“包围”其他分子 跨膜蛋白，如整合素，进入膜区室。在目标1中，我们将测试是否 肌动蛋白结合蛋白和/或CD 44“picket”蛋白的操作将导致皮质的局部分解 起“栅栏”作用以启动TNT的形成。将通过超分辨率共聚焦显微镜，TNT 囊泡转移测定、活TM细胞成像和CD 44磷酸化测定。特定肌动蛋白的作用- 将研究结合蛋白抑制剂对灌注培养中流出调节的影响，以评估其潜力 以调节IOP。目的#2将研究正常和高血压患者的质膜张力和整合素活化， 昏迷的TM细胞。将使用Flipper-TR荧光分析仪测量细胞膜张力的差异。 探针和整联蛋白活化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，一种肌动蛋白结合蛋白，是PILS的一个组成部分。我们将研究 Myo 10在TM细胞中协调整合素和基质金属蛋白酶在这些降解复合物中的活性 在静止时和机械拉伸时，以及在1x灌注的人眼前节TM组织中 2倍压力了解影响细胞突起的分子机制（丝状伪足/TNT和 PILS）将为皮质肌动蛋白细胞骨架在IOP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IOP的理解可能会导致新的青光眼治疗。

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.