Effect of SPARC Suppression in Mice, Perfused Human Anterior Segments, and Trabecular Meshwork Cells.
MacDonald William W, Swaminathan Swarup S, Heo Jae Young, Castillejos Alexandra, Hsueh Jessica, Liu Brian J, Jo Diane, Du Annie, Lee Hyunpil, Kang Min Hyung
AI Summary
Suppressing SPARC protein lowered intraocular pressure by ~20% in mice and human eye tissues, correlating with decreased structural ECM proteins. This suggests SPARC is a promising therapeutic target for glaucoma.
Abstract
Purpose
Secreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors.
Methods
A lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively.
Results
Suppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography.
Conclusions
Suppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.
MeSH Terms
Shields Classification
Key Concepts6
Suppression of Secreted protein, acidic and rich in cysteine (SPARC) decreased intraocular pressure (IOP) in mouse eyes and perfused human anterior segments by approximately 20%.
Histologically, SPARC suppression correlated to a decrease in collagen I, IV, and VI in the mouse trabecular meshwork (TM) and human juxtacanalicular trabecular meshwork (JCT) regions.
In mouse eyes, SPARC suppression also decreased fibronectin, but this effect was not observed in human JCT regions.
In human trabecular meshwork (TM) cells, SPARC suppression decreased collagen I, collagen IV, fibronectin, matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of MMP-1.
SPARC suppression in human trabecular meshwork (TM) cells did not change the messenger RNA levels of collagen I, collagen IV, fibronectin, matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of MMP-1, suggesting that the observed changes are not due to transcriptional regulation.
Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting following SPARC suppression in human trabecular meshwork (TM) cells.
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