Impact of Matrix Stiffness and Active Flow on Actin Stress Fiber Formation in Normal and Glaucomatous Trabecular Meshwork Cells: 2D and 3D Culture Models

Abstract

Purpose : Studies have shown a significant increase in matrix stiffness within the trabecular meshwork (TM) tissue in primary open angle glaucoma (POAG). We hypothesized that formation of actin stress fibers is directly influenced by substrate stiffness and flow in both normal (NTM) and glaucomatous (GTM) cells.

Methods : NTM and GTM cells were cultured on 2D and 3D polyacrylamide (PAM) gels with embedded FluoSpheres. 3D gels were fabricated using a mold that was 3D printed from serial block-face scanning electron microscopy images (Fig. 1). The 2D experimental set was maintained in a static environment. The 3D models were subjected to static and active fluid-structure interaction (FSI) environment. Gels at elastic moduli of 1.5 and 21.7 kPa were placed on glass coverslips. Cells were imaged for 20-hour on a confocal microscope equipped with an incubator. Post imaging, the samples were fixed in 8% paraformaldehyde, followed by phalloidin staining. Actin microfilament visualization was performed with a Zeiss Airyscan confocal microscope, employing a 40X 1.2 NA water immersion lens (Fig. 2).

Results : Actin stress fibers were observed spanning across NTM and GTM cells on both 1.5 and 21.7 kPa gels. Notably, GTM cells showed increased actin bundling at these stiffnesses compared to NTM cells. Stress fiber formation in NTM cells was more pronounced on 3D matrices than on 2D matrix. GTM cells, in contrast, displayed podosome-like structures on their ventral attachment surfaces and a reduced number of actin stress fibers. Exposing NTM cells to the FSI environment resulted formation of distinct stress fiber orientation and decreased lamellipodia formation. This pattern was echoed in GTM cells, where actin bundles were primarily localized at the cell periphery on both 1.5 and 21.7 kPa gel environments. In GTM cells cultured on 1.5 kPa gels, no lamellipodia were observed, whereas those on 21.7 kPa gels showed extensive peripheral actin organization.

Conclusions : Matrix stiffness critically influences actin organization in human TM cells, with NTM and GTM cells exhibiting distinct actin patterns in 2D versus 3D cultures. Fluid dynamics further alters these patterns, particularly in GTM cells. These findings, reflecting glaucoma's biomechanical characteristics, highlight the physiological relevance of 3D TM cultures in POAG research and therapeutic development.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.