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.
|Effective start/end date||6/1/21 → 5/31/23|
- Kennedy Institute - National Eye Clinic (1K99EY032547-01)