Abstract
Purpose:
Wnt antagonism has been linked to glaucoma and we have shown that the stiffness of the trabecular meshwork in the juxtacanalicular region increases with glaucoma. We wished to determine the effects of cell passage on the Wnt antagonist, secreted frizzled related protein 1 (sFRP1), and to determine if its expression would alter HTM cell stiffness.
Methods:
Primary human trabecular meshwork (HTM) cells from corneoscleral rims unsuitable for transplant were cultured, and the expression of sFRP1 was measured in early passage and later passage HTM cells. HTM cells were also treated with various concentrations of sFRP1 for eight days followed by a recovery for four days. The concentrations of sFRP1 used ranged from 0 to 1 µg/ml. The elastic moduli of the cells were measured using the atomic force microscope (AFM) and compared to control cells. HTM cell stiffness was measured as the cells approached senescence in latter passage. In addition, the canonical Wnt pathway was inhibited by LGK-974, an O-acyltransferase inhibitor, known to antagonize Wnt and another inhibitor of the pathway KY02111 at 10 µM each. Cell stiffness was measured in cells incubated with these inhibitors after the four day recovery point.
Results:
The expression of sFRP1 increased between 2 and 5 fold as passage number increased depending on the donor. As HTM cells approached senescence, the cell modulus increased 2 to 3 fold. The sFRP1 at 0.5 µg/ml increased the stiffness of the HTM cells between 3 and 6 fold depending on the donor. sFRP1 at a concentration of 1 µg/ml had minimal effects on cell stiffness consistent with previous reports in other cells. Both LGK-974 and KY02111 increased HTM cell stiffness 3-4 fold compared to control cells.
Conclusions:
sFRP1 expression increased as HTM cells were passaged. This antagonist of the Wnt pathway increased HTM cell stiffness. Two other inhibitors of the Wnt signaling pathway also increased HTM cell stiffness. These findings provide a causal link between the Wnt pathway and cell stiffness and suggest a feedback loop contributing to glaucoma progression.