Abstract
Purpose :
Secreted Protein Acidic and Rich in Cysteine (SPARC) is an important regulator of the homeostasis of extracellular matrix (ECM) turnover in the trabecular meshwork (TM) of the eye.1 This is of particular interest due to the role that the TM plays in draining aqueous humor from the eye and regulating intraocular pressure (IOP), which is critical in the pathogenesis of glaucoma.2 Our purpose is to elucidate the molecular pathways that SPARC utilizes to affect the expression of certain ECM proteins. Plasminogen Activator Inhibitor-1 (PAI-1) is a protein involved in the turnover of ECM and regulated by SPARC. We hypothesize that suppressing SPARC will lead to the decrease of IOP in the expression of PAI-1.
Methods :
Using primary TM cell culture (N=6) for in vitro experiments, we suppressed SPARC via SPARC suppressing lentivirus (lenti-shSPARC). TM endothelial cells were transduced by lenti-shSPARC at an MOI of 10. PAI-1 and SPARC proteins from cell lysates and conditioned media were analyzed by immunoblot. In addition, mRNA level of PAI-1 and SPARC from cell lysate were analyzed by quantitative polymerase chain reaction (qPCR).
Results :
We observed that SPARC was suppressed (0.28 ± 0.12, p= 0.006, N=6) with shSPARC compared to shcontrol. Suppressed SPARC induced the elevation of PAI-1 at the protein (79.79 ± 66.36%, p = 0.036, N=6) and RNA (2.89 ± 1.14, p = 0.010, N=6) level compared to shcontrol.
Conclusions :
SPARC suppression elevated the expression of PAI-1. PAI-1 may play a role for the IOP control in SPARC signaling pathway of human TM.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.