Abstract
Purpose :
A sustained increase in intraocular pressure (IOP) is a major risk factor for primary-open angle glaucoma (POAG). Extracellular matrix (ECM) accumulation in the trabecular meshwork (TM) outflow pathway results in IOP elevation. Among the major ECM components in the TM outflow pathway, increased accumulation of collagen 1A is found in POAG. Cathepsin K (CTSK) is a lysosomal protease and a potent collagenase known to degrade helical and non-helical regions of collagen 1A. We studied the role of CTSK activity on ECM modulation in the TM outflow pathway and on IOP.
Methods :
Porcine TM (PTM) cells were isolated, cultured and were treated with 10uM Balicatib, a potent cell-permeable CTSK activity inhibitor, for 1, 4, 8, 16, 24hrs to study time-dependent inhibition of CTSK activation and assessed for its effect on ECM modulation using immunoblotting. Porcine anterior segment organ culture perfusion was used to assess the effect of Balicatib on - a) IOP, b) changes in tissue distribution and expression of ECM like collagen, fibronectin, and elastin using immunofluorescence and immunoblotting. Student’s t-test was used for statistical analyses and results were significant if p<0.05 with a sample size of n=3-9 in each experiment.
Results :
Balicatib treated PTM cells decreased CTSK activity by 80%. Time-dependent balicatib treatment in porcine TM cells showed no changes in active CTSK but a significant increase in pro-CTSK (p=0.001) as well as in ECM proteins like COL1A (p=0.004), FN (p=0.009). Perfusion of balicatib in the porcine anterior segment perfusion cultures increased IOP nearly two-fold (n=5, p<0.05). TM isolated from perfused globes showed increased elastin protein expression by nearly seven-fold (n=2, p=0.023). Immunofluorescence data showed an increase in collagen 1A and fibronectin distribution in the TM outflow pathway
Conclusions :
Firstly, this proof of concept study shows that directly perturbing the activity or function of an ECM degrading enzyme-like CTSK increases intraocular pressure by altering the ECM degradation. Secondly, we demonstrate that CTSK is a critical regulator of IOP homeostasis. This study also reinforces the fact that ECM in the outflow pathway is very important for maintaining IOP. Finally, activation of CTSK is an attractive therapeutic strategy to lower IOP
This is a 2021 ARVO Annual Meeting abstract.