Abstract
Purpose :
Clusterin is a secretory chaperone protein known to regulate cell-matrix interactions. Altered cytoskeletal rearrangement and excessive extracellular matrix (ECM) deposition in the trabecular meshwork (TM) outflow pathway results in increased AH outflow resistance and elevated IOP. This study evaluated the role and mechanism of clusterin in regulating IOP.
Methods :
Immunoblotting (IB) was used to analyze the effect of– a) adenovirus-mediated clusterin expression (AdCLU) on proteins related to actin organization, ECM and fibrosis; b) effect of recombinant clusterin (rhCLU) on TGFβ2-mediated ECM changes in TM. Immunofluorescence (IF) and live-cell imaging using SiR-actin was used to analyze the effect of clusterin on ECM and actin fibers, respectively. The effect of clusterin on TGFβ2-mediated IOP elevation was studied using human anterior segment perfusion cultures by perfusing with TGFβ2 followed by rhCLU in the presence of TGFβ2. Students t-test was used for statistical analyses with significance of p<0.05.
Results :
The IB analysis showed AdCLU significantly reduced- a) actin-cytoskeleton associated proteins- PRK2(p=0.0006), LIMK1(p=0.04); b) Integrins- INTαV(p=0.04), INTβ5(p=0.01); c) tight junction protein Zo-1(p=0.007) d) ECM- COL1A(p=0.04), FN(p=0.03), ELN(p=0.005), and secretory FN(p=0.002); e) pro-fibrotic proteins- TGFβ2(p=0.03), TSP-1(p=0.005), and secretory PAI-1(p=0.007) and TSP-1(p=0.05). rhCLU treatment significantly reduced pro-fibrotic effect of TGFβ2, evidenced by significant reduction in COL1A(p=0.002) and TSP1(p=0.01), and secretory TSP1(p=0.01) and PAI-1(p=0.02). IF showed reduced COL1A and FN distribution in AdCLU. Live cell imaging showed reduction in F-actin distribution with rhCLU, which was initially increased by TGFβ2 treatment. Perfusion of TGFβ2 in the human anterior segment increased IOP significantly (n=5, p=0.006) by the third day which was significantly reduced on day eight (p=0.008) due to rhCLU perfusion.
Conclusions :
For the first time, we show importance of clusterin in lowering actin-based tension and attenuating profibrotic ECM accumulation in TM, possibly facilitating the increase in AH outflow and, thereby regulating IOP homeostasis. Further understanding of the functional role of clusterin and its binding partners in mediating cell-matrix interactions in TM will pave way for novel therapeutic approaches for lowering elevated IOP.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.