The intrinsic stiffness of human trabecular meshwork cells increases with senescence

Abstract

Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases.

Small-molecule inhibition of Wnt signaling abrogates dexamethasone-induced phenotype of primary human trabecular meshwork cells

“…The morphology of the cells we used in our study was similar to that reported by Rhee et al [24], Morgan et al [19], and Lin et al [22], where they were small in size, slightly elongated, exhibited a moderate to low number of processes, and their cell bodies were both rounder and wider than those of scleral fibroblasts. The predominant marker of TM cells is increased myocilin expression following treatment with Dex [10, 25, 26].…”

“…The presence of active Wnt signaling has also been reported on a cellular level in vitro within TM cells [16, 17]. This active Wnt signaling was found to be involved in TM cell mediated ECM expression [18] and TM cell stiffening [19]. Overall, Wnt signaling appears to be a key player in TM regulation, with strong evidence suggesting that abnormal Wnt signaling may promote IOP [15].…”

A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing

“…We also found that inhibition of the Wnt signaling pathway by DKK1 increased IOP [43]. One potential mechanism for this increase is through the stiffening of the trabecular meshwork [45,46]. Also, inhibition of the canonical Wnt signaling may promote ECM deposition [47].…”

Ocular Hypotension, Actin Stress Fiber Disruption and Phagocytosis Increase by RKI-1447, a Rho-Kinase Inhibitor

“…In general, factors that induce stress fiber formation increase TM stiffness and decrease outflow facility. For example, senescence and aging, a risk factor for primary open-angle glaucoma, has increases the stress fiber formation and TM stiffness 40 . In a different study, dexamethasone, an inducer of cross-linked actin network 41,42 , increased the stiffness of primary TM cells and extracellular matrix twofold and fourfold, respectively, after three days 38 , and caused an IOP elevation in an ex vivo perfusion model of human eyes 42 .…”