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

青光眼患者房水流出阻力增加的主要原因是： 施累姆氏管（SC）的内壁内皮附近。巨大的空泡和气孔 与SC内皮细胞相关的是用于房水进入管的唯一开放空间。 因此，它们被认为在调节流出阻力中起重要作用。GVs的形成是对基础 顶压梯度使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，脑水肿患者的流出阻力升高， 人眼睛与其原位SC细胞的硬度增加有关。这些观察使SC 细胞硬度是GV形成和流出体内平衡的关键因素。然而，人们对这一机制知之甚少。 调节SC细胞和GVs的生物力学特性。我们以前的研究表明，SC细胞成为 当在较硬的体外基质上培养时较硬。我们最近还表明，肿瘤SC细胞及其 底层细胞外基质比原位健康基质更坚硬。这些发现表明 SC电池的机械性能依赖于基底。在这个项目中，我的目标是研究的作用， 波形蛋白中间丝（VIF）细胞骨架在调节SC细胞生物力学特性中的作用及其 相关的GV。我对VIF感兴趣的原因有两个：首先，它们是主要的贡献者 对于一般的细胞力学，它们是大变形下细胞力学的主要决定因素， 它们具有基底刚度依赖性组装状态;其次，VIF在SC细胞中高度表达，并且 已经显示它们与GV原位结合，且还影响它们体外生命周期。审查这个 我将首先在培养的人类SC细胞中敲低波形蛋白，并使用原子力显微镜和牵引 力显微镜以确定VIF在SC细胞刚度和收缩性中的作用。这些研究的结果 将被用作采用超分辨率成像，生物化学， 微加工以确定VIF在SC细胞中的基底依赖性表达和组装状态 影响它们的硬度和收缩力。接下来，我将研究VIFs在GV形成中的作用，通过体外实验， 灌注来自野生型和波形蛋白敲除小鼠的眼睛，然后表征和比较GV 尺寸和密度沿着其SC. I还将确定VIF的存在或不存在对 通过测量这些眼睛中的流出便利性来产生流出阻力。最后，我会击倒维曼汀 以确定靶向VIF调节流出功能的可行性。我 然后将这种方法扩展到高眼压小鼠，以评估这种方法作为一种新的方法的有效性。 青光眼的治疗通过检测VIFs对SC内壁生物力学的贡献， 过渡到一个独立的职业生涯，以调查增加外流的机械基础 抗青光眼和开发新的治疗方法的疾病。

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.