The Effects of Mechanical Stretch on Integrins and Filopodial-Associated Proteins in Normal and Glaucomatous Trabecular Meshwork Cells

Abstract

The trabecular meshwork (TM) is the tissue responsible for regulating aqueous humor fluid egress from the anterior eye. If drainage is impaired, intraocular pressure (IOP) becomes elevated, which is a primary risk factor for primary open angle glaucoma. TM cells sense elevated IOP via changes in their biomechanical environment. Filopodia cellular protrusions and integrin transmembrane proteins may play roles in detecting IOP elevation, yet this has not been studied in detail in the TM. Here, we investigate integrins and filopodial proteins, such as myosin-X (Myo10), in response to mechanical stretch, an in vitro technique that produces mechanical alterations mimicking elevated IOP. Pull-down assays showed Myo10 binding to α5 but not the β1 subunit, αvβ3, and αvβ5 integrins. Several of these integrins colocalized in nascent adhesions in the filopodial tip and shaft. Using conformation-specific antibodies, we found that β1 integrin, but not α5 or αvβ3 integrins, were activated following 1-h mechanical stretch. Cadherin -11 (CDH11), a cell adhesion molecule, did not bind to Myo10, but was associated with filopodia. Interestingly, CDH11 was downregulated on the TM cell surface following 1-h mechanical stretch. In glaucoma cells, CDH11 protein levels were increased. Finally, mechanical stretch caused a small, yet significant increase in Myo10 protein levels in glaucoma cells, but did not affect cellular communication of fluorescent vesicles via filopodia-like tunneling nanotubes. Together, these data suggest that TM cell adhesion proteins, β1 integrin and CDH11, have relatively rapid responses to mechanical stretch, which suggests a central role in sensing changes in IOP elevation in situ.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation

“…This study strengthens the correlation between lipid changes and tissue stiffness and reinforces the significance and role of actin‐cell adhesive interactions in bringing about changes in IOP 106 . It has been characterized that the focal adhesion vinculin but not paxillin directly interacts with actin 107,108 .…”

“…This study strengthens the correlation between lipid changes and tissue stiffness and reinforces the significance and role of actin-cell adhesive interactions in bringing about changes in IOP. 106 It has been characterized that the focal adhesion vinculin but not paxillin directly interacts with actin. 107,108 Additional mechanistic evidence in our study confirms that SREBPs inactivation regulates vinculin distribution but not expression confirming the requirement of SREBPs activity in modulating the recruitment of vinculin to the focal adhesion site.…”

Digital spatial profiling of segmental outflow regions in trabecular meshwork reveals a role for ADAM15

“…All other genes identified in this study and the biological processes that they are associated with are listed in S2 Table . Interestingly, none of the genes for ECM proteins (i.e SPARC, VCAN, THBS2, FN1, DCN) previously associated with affecting outflow facility [20,21,23,25,67,68] were found to be statistically different between high and low outflow regions of the TM in this study (Fig 6). Nor did we see any statistical differences in the various integrins expressed in the low outflow [29,[69][70][71]. However, boxplot analysis of the distribution of the DCN and ITGA8 integrin subunits did show an enrichment of these genes in high outflow regions compared to low outflow regions.…”

“…One of the striking findings of this study was the observation that none of the ECM proteins [20,21,23,25,67,68] and integrins [29,[69][70][71] previously identified to affect IOP or outflow showed any significant differences in gene expression between low and high outflow regions. As shown in the boxplots for these genes and S3 Table, there was a lot of variation in the expression of genes within a high or low flow region that could not be attributed to the donor eye.…”

Regulatory role of cholesterol in modulating actin dynamics and cell adhesive interactions in the trabecular meshwork

“…Integrins are known to be involved in TM biomechanics and glaucoma pathogenesis (20, 21). Integrins act as mechanotransducers in TM cells to respond to the extracellular environment (22), control matrix assembly by tethering fibronectin, and its activation also regulates actin cytoskeleton remodeling (20). Moreover, deletion of integrin β3 results in a significant reduction in IOP in animal models, and its activation increases the IOP (82).…”