Wnt inhibition induces persistent increases in intrinsic stiffness of human trabecular meshwork cells

Abstract

Wnt antagonism has been linked to glaucoma and intraocular pressure regulation, as has increased stiffness of human trabecular meshwork (HTM) tissue. We have shown culturing HTM cells on substrates that mimic the elevated stiffness of glaucomatous tissue leads to elevated expression of the Wnt antagonist secreted frizzled related protein 1 (SFRP1), suggesting a linkage between SFRP1 and HTM mechanobiology. In this study, we document biomechanical consequences of Wnt antagonism on HTM cells. Cells were treated with the Wnt antagonists (SFRP1, KY02111, and LGK-974) for 8 days and allowed to recover for 4 days. After recovery, intrinsic cell stiffness and activation of the Wnt pathway via β-catenin staining and blotting were assayed. Basal cell stiffness values were 3.71±0.37, 4.33±3.07, and 3.07± kPa (median±S.D.) for cells derived from 3 donors. Cell stiffness increased after 0.25 μg/mL (4.32±5.12, 8.86±8.51, 4.84±3.15 kPa) and 0.5 μg/mL (16.75±5.59, 13.18±7.99, and 8.54±5.77 kPa) SFRP1 treatment. Stiffening was observed after 10μM KY02111 (10.72±5.63 and 6.57±5.53 kPa) as well as LGK-974 (9.60±7.41 and 11.40±9.24 kPa) treatment compared with controls (3.79±1.01 and 5.16±2.14 kPa). Additionally, Wnt inhibition resulted in decreased β-catenin staining and increased phosphorylation at threonine 41 after recovery. In conclusion, this work demonstrates a causal relationship between Wnt inhibition and cell stiffening. Additionally, these findings suggest transient Wnt inhibition resulted in durable modulation of the mechanical phenotype of HTM cells. When placed in context with previous results, these findings provide a causal link between Wnt antagonism and cell stiffness and suggest a feedback loop contributing to glaucoma progression.

Aqueous outflow - A continuum from trabecular meshwork to episcleral veins

“…Such investigations are necessary to determine the relationship between flow rates and TM stiffness since low flow regions have increased TM stiffness compared to high flow regions (Ethier, 2016). Investigations of stiffness between normal and glaucomatous TM (Morgan et al, 2015; Russell and Johnson, 2012; Vranka et al, 2015a) combined with the stiffness of the high and low flow regions will give new insight into AH resistance in the TM; in particular, identifying the spatiotemporal molecular changes associated with the increased resistance of the TM tissues (Morgan et al, 2015). Current developments in high throughput approaches (Savas et al, 2012; Toyama et al, 2013) will be likely to provide important, fundamental insights.…”

Steroid-induced ocular hypertension/glaucoma: Focus on pharmacogenomics and implications for precision medicine

“…Expression of the canonical Wnt pathway antagonist SFRP1 was increased in trabecular meshwork cell lines from POAG patients and transfer of an SFRP1 gene to perfused human anterior segments increased IOP. Also importantly, Wnt pathway inhibition was shown to induce a persistent increase in intrinsic stiffness of human trabecular meshwork cells (Morgan et al, 2015). Treatment of trabecular meshwork cells with glucocorticoids stimulates expression of the non-canonical Wnt ligand WNT5A and treatment of trabecular meshwork cells with WNT5A induces CLAN formation (Yuan et al, 2013).…”

Segmental outflow of aqueous humor in mouse and human

“…TM stiffness (elastic modulus) measurements have demonstrated normal TM to possesses lower stiffness than glaucomatous TM (Last et al, 2011). Recent investigations suggest low flow regions to have increased TM stiffness compared to high flow regions (Russell and Johnson, 2012; Morgan et al, 2015; Vranka et al, 2015a). The molecular changes underlie the increased resistance of TM (Morgan et al, 2015).…”