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Extracellular Matrix and Outflow Resistance

细胞外基质和流出阻力

基本信息

批准号：
10650887
负责人：
Kate E Keller
金额：
$ 42.66万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10650887
关键词：
Actin-Binding Protein Actins Affect Anterior Antibodies Aqueous Humor Area Automobile Driving Binding Biogenesis Biological Biological Assay Blindness CD44 gene Cell Shape Cell membrane Cell physiology Cell surface Cells Cellular Structures Communication Complex Confocal Microscopy Cytoskeleton Data Extracellular Matrix Extracellular Matrix Degradation Eye Filopodia Funding Gelatinase A Genes Glaucoma Human In Situ Integral Membrane Protein Integrins Length Matrix Metalloproteinases Measures Mechanics Membrane Modeling Molecular Molecular Conformation Monitor Myosin ATPase Nanotubes Pathology Pathway interactions Patients Perfusion Persons Pharmaceutical Preparations Phenotype Phosphorylation Physiologic Intraocular Pressure Play Proteins RNA Interference Receptor Activation Regulation Research Resistance Rest Rho-associated kinase Risk Factors Role Site Source Stress Fibers Stretching Structure Testing Time Tissues Trabecular meshwork structure Vesicle aqueous cell cortex cellular imaging extracellular human tissue inhibitor insight kinase inhibitor knock-down live cell imaging mechanical force membrane model novel novel strategies novel therapeutics pharmacologic pressure receptor response rho rho GTP-Binding Proteins therapeutically effective ultra high resolution

项目摘要

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.

项目总结