Clusterin Modulates Extracellular Matrix Homeostasis in the Trabecular Meshwork

Abstract

Purpose : To understand the functional role of clusterin, an N-glycosylated secretory chaperone protein, in the trabecular meshwok (TM) outflow pathway.

Methods : Glaucomatous human TM (GTM) and normal TM cells lines were obtained from Dr. Abe Clark. Primary human TM (HTM) cells were transfected with constitutively hypoglycosylated clusterin mutants (CLU^MUT) or secretory clusterin (sClu) and subjected to phagocytosis assay by imaging pHRodo labeled E.coli uptake after 48h and apoptosis assay by flow cytometry using ApoDetect Annexin V-FITC kit after 72h. Immunofluorescence staining on paraffin sections of outflow pathway tissues from C57BL/6J wild types (WT) and Clusterin null (Clu^-/-) mice was performed and quantified in the TM-juxtacanalicular (JCT) region across groups and the corrected mean fluorescence intensity (CMF) calculated using ImageJ. N=4-8 was for all experiments. Students t-tests were used for statistical analyses and p<0.05 were significant.

Results : GTM cells revealed higher levels of intracellular unglycosylated clusterin and decreased sClu compared to NTM. Treatment of HTM cells with ocular hypertension-inducing transforming growth factor-β2 (TGFβ2) decreased intracellular and sClu levels. To understand the functional role of clusterin, siRNA-mediated knockdown of clusterin in vitro in HTM cultures demonstrated a significant increase in pro-fibrogenic α-smooth muscle actin (αSMA) and collagen-1A. Immunofluorescence analyses performed on the outflow pathway of 8 weeks old (wo) Clu^-/- mice revealed increased immunostaining of extracellular matrix - collagen 1A, collagen IV, fibronectin and total collagen stained with Sirius red in the TM-JCT region with a significant increase in collagen 1A and collagen IV compared to the WT in vivo. Contrarily, induced expression of clusterin by transfection of sClu in HTM cells in vitro decreased TGFβ2-mediated induction of collagen. Induced expression of CLU^MUT stimulated apoptosis and decreased phagocytosis [~50%] compared to sClu-transfected cells and to no-transfection control. Conversely, sClu transfected HTM cells increased phagocytosis by 67% compared to no-transfection control.

Conclusions : Clusterin regulates fibrogenic activation in TM and clusterin glycosylation modulates the phagocytic properties of TM and TM cellularity via apoptosis. Therefore a dysregulation in clusterin production, glycosylation, and its secretion in TM can elevate intraocular pressure.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.