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

青光眼患者对房水流出的大部分抵抗力增加发生在 Schlemm管(SC)内壁内皮细胞附近。巨大空泡(GV)和毛孔 与SC内皮细胞相关的是房水进入管内的唯一开放空间。 因此，它们被认为在调节流出阻力方面发挥着重要作用。GVS表单响应基本的 到顶端压力梯度，使SC细胞发生实质性变形。这种变形的程度是 在干细胞力学的作用下。我们最近发现青光眼患者流出阻力升高 人眼与其干细胞原位僵硬的增加有关。这些观察结果使SC 细胞硬度是GV形成和流出动态平衡的关键因素。然而，人们对这种机制知之甚少(S) 调节干细胞和GV的生物力学特性。我们之前已经证明，干细胞会变成 在体外培养在更硬的底物上时会更硬。我们最近还发现，青光眼干细胞和它们的 潜在的细胞外基质比健康的原位基质更坚硬。这些发现表明， 干细胞的机械性能依赖于底物。在这个项目中，我的目标是研究 波形蛋白中间丝(VIF)细胞骨架对SC细胞生物力学特性的调节及其机制 关联的GV。我对VIF感兴趣的原因有两个：首先，他们被证明是主要的贡献者 对于一般的细胞力学来说，它们是大变形时细胞力学的主要决定因素，并且 它们具有底物刚性依赖的组装状态；其次，VIF在SC细胞中高表达，以及 研究表明，它们在体内与GVS结合，并在体外影响GVS的生命周期。要检查这一点 我将首先击倒培养的人SC细胞中的波形蛋白，并使用原子力显微镜和牵引器 强制显微镜以确定VIF在干细胞僵硬和收缩中的作用。这些研究的发现 将作为使用超分辨率成像、生物化学和 微加工以确定VIF在SC细胞中的底物依赖性表达和组装状态 会影响它们的硬度和伸缩性。接下来，我将通过体外研究VIF在GV形成中的作用 野生型和波形蛋白基因敲除小鼠的眼睛灌流，随后表征和比较GV 沿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流出设备来产生流出阻力。最后，我将击倒Vimentin 在正常血压的小鼠内壁中，确定靶向vif作为调节流出设施的可行性。我 然后将这种方法扩展到高眼压小鼠身上，以衡量这种方法作为一种新方法的有效性 青光眼的治疗。通过检测VIF对SC内壁生物力学的贡献，我寻求 过渡到独立的职业，以研究外流增加的机械基础 在青光眼的耐药性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.