Abstract
Purpose :
Elevated intraocular pressure (IOP) is a primary risk factor for glaucoma. IOP homeostasis relies on the regulation of aqueous outflow through trabecular meshwork (TM). ECM accumulation in TM elevates IOP. Clusterin, a secretory stress response chaperone protein, has been reported to regulate cell-matrix interactions. Studies have shown a putative linkage between clusterin and primary open angle glaucoma pathogenesis. We aim to study the- 1) effect of stressors known to increase IOP on clusterin expression in TM, 2) understand the role of clusterin in ECM remodeling, and 3) effect of loss of clusterin on IOP.
Methods :
Using qPCR and immunoblotting, we studied the effects of - A) CTGF, Endothelin, Dexamethasone, TGFβ2, elevated pressure on clusterin expression, B) adenovirus-induced clusterin expression (AdCLU) on ECM, pro-fibrotic proteins, and ECM regulatory matrix metalloproteinases (MMPs) activity using zymography, C) recombinant clusterin (rhCLU) on TGFβ2-induced fibrosis and ECM accumulation, in vitro. TM cells were transfected with expression clones Clusterin-RFP-GFP (CLU-RG) or Signal peptide-RFP-GFP (SP-RG), followed by live cell imaging post TGFβ2 treatment. IOP was measured in clusterin knockout (Clu-/-) mice by tonometry. Student’s t-test was used for statistical analyses and results were significant if p<0.05 with a sample size of n=3-7 per experiment.
Results :
AdCLU significantly decreased ECM mRNAs and proteins including, Collagen 1A (n=3, p=0.04) and fibronectin (n=3, p=0.01), Elastin (n=3, p=0.05), whereas MMP2 expression was increased. CTGF (n=3, p=0.006), Endothelin (n=3, p=0.05), TGFβ2 (n=3, p=0.04) and Pressure stress (n=4, p=0.04) significantly reduced clusterin protein expression. rhCLU attenuated significantly the effects of TGFβ2 on ECM proteins- COL1A (n=3, p=0.01), Fibronectin (n=3, p=0.05). TM cells transfected with CLU-RG showed decrease in clusterin availability under TGFβ2 treatment. Clu-/- mice demonstrated an age-dependent elevation in IOP with significant increase at postnatal day 90 compared to wild type (n=7, p=0.03).
Conclusions :
Our novel finding demonstrates the role of clusterin on ECM remodeling and loss of clusterin function elevates IOP signifying the importance of clusterin in IOP homeostasis. Further studies on the signaling events of clusterin-mediated ECM remodeling and IOP changes can help us identify novel IOP lowering therapies.
This is a 2021 ARVO Annual Meeting abstract.