Abstract
Purpose :
Extracellular matrix (ECM) plays a critical role in the regulation of trabecular meshwork (TM) aqueous humor (AH) outflow drainage resistance. Clusterin is known to regulate cell-matrix interactions. We studied the regulation of clusterin and its role in the modulation of ECM and intraocular pressure (IOP) homeostasis.
Methods :
Using qPCR, immunofluorescence, and immunoblotting assays, we studied the effects of - A) elevated pressure (2x) on clusterin expression in porcine ocular perfusion system ex vivo, B) clusterin loss on IOP and changes in collagen 1A and elastin distribution by Van Gieson elastin stain in the AH outflow pathway in clusterin knockout (Clu-/-) mice in vivo, and C) adenovirus-induced clusterin expression (AdCLU) on actin, ECM, matrix metalloproteases (MMPs), and clusterin receptors in primary human TM cells in vitro. Student’s t-test was used for statistical analyses and results were significant if p<0.05 with a sample size of n=3-8 in each experiment.
Results :
Pressure stress (2x) significantly decreased mRNA of clusterin (n=3, p< 0.04) and clusterin receptors- transforming growth factor beta receptor TGFBR1 & TGFBR2, and Very low-density lipoprotein receptor (VLDLR), whereas Low-density lipoprotein receptor 8 (LRP8) levels increased. Clu-/- mice demonstrated an age-dependent elevation in IOP with an increase at postnatal days 50 and significantly at 89 days (n=8, p<0.05) compared to the C57BL/6 wild type. Histological examination in Clu-/- mice showed a marked increase in Collagen 1A in the TM and a strong elastin band observed in the juxtacanalicular tissue and basement membrane region. AdCLU expression in HTM cells induced cell shape changes, decreased actin stress fibers, significantly decreased ECM mRNAs and protein including Elastin (n=3, p<0.001) and Collagen 1A (n=4, p<0.001) and fibronectin, whereas MMP2 was significantly increased (n=4, p<0.0009).
Conclusions :
We show that clusterin expression is negatively regulated under pathological pressure stress. This decrease of clusterin can in turn cause increased ECM deposition and sustained IOP elevation. However, increasing clusterin levels, lowers the ECM potentially by increasing the MMP expression and activity. Further studies on receptor-mediated signaling of clusterin will explain the mechanistic basis of clusterin on the regulation of ECM and IOP homeostasis and identify novel IOP lowering targets.
This is a 2020 ARVO Annual Meeting abstract.