The Role of Vimentin Cytoskeleton in the Mechanobiology of Schlemm's Canal Endothelium.

波形蛋白细胞骨架在施累姆氏管内皮力学生物学中的作用。

基本信息

批准号：
10874866
负责人：
Amir Vahabikashi
金额：
$ 1.53万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10874866
关键词：
Adrenal Cortex Hormones Affect Apical Aqueous Humor Atomic Force Microscopy Biochemistry Biomechanics Cells Characteristics Cytoskeletal Filaments Cytoskeleton Disease Effectiveness Endothelial Cells Endothelium Extracellular Matrix Eye Eye Enucleation Generations Glaucoma Homeostasis Human In Situ In Vitro Intermediate Filaments Knockout Mice Life Cycle Stages Measures Mechanics Mediating Methods Microfabrication Microscopy Mus Ocular Hypertension Pathway interactions Perfusion Physiologic Intraocular Pressure Play Property Regulation Resistance Role Structure of sinus venosus of sclera Technology Testing Therapeutic Trabecular meshwork structure Traction Force Microscopy Vacuole Vimentin Western Blotting career density ex vivo perfusion feasibility testing interest knock-down mechanical properties mouse model normotensive novel novel therapeutic intervention pressure response superresolution imaging therapeutic target

项目摘要

Project Summary The bulk of increased resistance to the outflow of aqueous humor in glaucoma occurs at the vicinity of the inner wall endothelium of the Schlemm’s Canal (SC). The giant vacuoles (GVs) and pores associated with the SC endothelial cells are the only open spaces for the aqueous humor to enter the canal. Thus, they are thought to play an important role in regulating outflow resistance. GVs form in response to a basal to apical pressure gradient that subjects SC cells to substantial deformation. The extent of this deformation is mediated by SC cell mechanics. We recently discovered that the elevated outflow resistance in glaucomatous human eyes is associated with the increased stiffness of their SC cells in situ. These observations render SC cell stiffness a key factor in GV formation and outflow homeostasis. Yet, little is known about the mechanism(s) that regulate the biomechanical properties of SC cells and GVs. We previously showed that SC cells become stiffer when cultured on stiffer substrates in vitro. We also recently showed that glaucomatous SC cells and their underlying extracellular matrix are stiffer than their healthy counterparts in situ. These findings suggest that the mechanical properties of SC cells are substrate dependent. In this project, I aim to examine the role of the vimentin intermediate filament (VIF) cytoskeleton in regulating the biomechanical properties of SC cells and their associated GVs. The reasons for my interest in VIFs are twofold: first, they are shown to be major contributors to cell mechanics in general, they are the dominant determinant of cell mechanics at large deformations, and they have a substrate stiffness dependent assembly state; secondly, VIFs are highly expressed in SC cells, and it has been shown that they associate with GVs in situ and also impact their life cycle in vitro. To examine this role, I will first knockdown vimentin in cultured human SC cells and use atomic force microscopy and traction force microscopy to establish the role of VIFs in SC cell stiffness and contractility. The findings from these studies will be used as a basis for additional studies employing super-resolution imaging, biochemistry, and microfabrication to determine how substrate dependent expression and assembly states of VIFs in SC cells affects their stiffness and contractility. I will next investigate the role of VIFs in GV formation through ex vivo perfusion of eyes from wildtype and vimentin knockout mice followed by characterizing and comparing the GV size and density along their SC. I will also determine the impact of the presence or absence of VIFs on the generation of outflow resistance by measuring the outflow facility in these eyes. Finally, I will knockdown vimentin in normotensive mouse inner wall to determine the feasibility of targeting VIFs for modulating outflow facility. I will then extend this method to ocular hypertensive mice to gauge the effectiveness of this approach as a novel treatment for glaucoma. Through examining the contribution of VIFs to the biomechanics of SC inner wall, I seek to transition into an independent career in order to investigate the mechanical basis of increased outflow resistance in glaucoma and to develop novel therapeutic approaches for the disease.

项目摘要在青光眼中对水幽默出口的大部分耐药性发生在 Schlemm的运河（SC）的内壁内皮附近。巨型吸尘器（GVS）和毛孔与SC内皮细胞相关的是水性幽默进入运河的唯一开放空间。那就是他们认为他们在控制流出阻力方面发挥了重要作用。响应基本的GVS形式至顶压力梯度，使SC细胞发生实质性变形。这种变形的程度是由SC细胞力学介导。我们最近发现青光眼的出口电阻升高人眼与原位SC细胞的刚度增加有关。这些观察结果使SC 细胞刚度GV形成和出口稳态的关键因素。然而，对机制知之甚少调节SC细胞和GV的生物力学特性。我们以前表明SC细胞成为在体外培养在更硬的底物上培养时。我们最近还表明，青光眼的SC细胞及其潜在的细胞外基质比其健康的原位更稳固。这些发现表明 SC细胞的机械性能取决于底物。在这个项目中，我旨在研究波形蛋白中间细丝（VIF）细胞骨架在调节SC细胞的生物力学特性及相关的GV。我对VIF感兴趣的原因是双重的：首先，它们被证明是主要贡献者通常，对于细胞力学，它们是大变形时细胞力学的主要决定因素，并且它们具有底物刚度依赖性组装状态；其次，VIF在SC细胞中高度表达，并且已经表明，它们与原位GV相关，并在体外影响其生命周期。检查一下角色，我将首先在培养的人类SC细胞中敲除波形蛋白，并使用原子力显微镜和牵引力力显微镜确定VIF在SC细胞刚度和收缩力中的作用。这些研究的发现将使用超分辨率成像，生物化学和微结构以确定SC细胞中VIF的底物依赖性表达和组装状态如何影响他们的僵硬和收缩力。接下来，我将通过离体研究VIF在GV形成中的作用野生型和波形蛋白敲除小鼠的眼睛灌注，然后表征和比较GV 沿其SC的大小和密度。我还将确定vif的存在或不存在对通过测量这些眼睛中的出口设施来产生出口阻力。最后，我将击倒波形蛋白在正常的小鼠内壁中，确定靶向VIF用于调节出口设施的可行性。我然后将这种方法扩展到眼部高血压小鼠，以评估这种方法的有效性青光眼治疗。通过检查VIF对SC内壁的生物力学的贡献，我寻求过渡到独立职业，以调查增加出口的机械基础青光眼的耐药性并开发了新型疾病治疗方法。