Abstract
Purpose :
Extracellular matrix (ECM) homeostasis is critical in the trabecular meshwork (TM) aqueous humor outflow pathway. TGFβ2, a profibrogenic factor, increases ECM production in TM and intraocular pressure (IOP). Cathepsin K (CTSK) is a potent collagenase known to degrade helical and non-helical regions of collagen 1. CTSK also regulates transforming growth factor beta-1 (TGFβ1)-mediated tissue fibrosis. The regulation and role of CTSK in TM is not well understood. We studied the role of CTSK on ECM production and remodeling and TGFβ2 regulation in TM.
Methods :
Quantitative polymerase chain reaction (qPCR), immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) assays were used to determine the - A) expression and distribution of CTSK in human trabecular meshwork (HTM) outflow pathway, B) regulation of CTSK expression with pressure (2x) in porcine ocular perfusion system, and C) effect of constitutive CTSK expression using adenovirus CTSK (AdCTSK) on actin cytoskeleton, ECM (collagen 1A and fibronectin), and TGFβ2 expression in HTM cells. 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 :
CTSK is expressed in human AH outflow pathway tissues, predominantly in TM region, and secreted into AH. HTM cultures displayed punctate staining for CTSK. Pressure stress increases CTSK mRNA. AdCTSK expression in HTM cells displayed – coalescence of actin with loss of stress fibers, significant decrease in collagen 1A mRNA (n=5, p<0.02) and protein (n=3, p<0.05), and loss of intracellular ECM content as well as extracellular fibrillar ECM arrangement. Most strikingly, AdCTSK expression decreased both TGFβ2 mRNA (n=9, p<0.001) and protein expression significantly in HTM cells (n=3, p<0.5).
Conclusions :
CTSK expression is tightly regulated by pressure indicating it as a potential response system to restore homeostasis in TM. Our results highlight the duality function of CTSK as a potential regulator of ECM and TGFβ2 via its protease activity as well as signaling towards transcriptional regulation. The effects of CTSK on ECM remodeling and TGFβ2 bioavailability makes it a promising target to lower IOP.
This is a 2020 ARVO Annual Meeting abstract.