Abstract
Purpose :
Vitronectin is a matricellular adhesive glycoprotein that interacts with several proteins in the extracellular milieu of trabecular meshwork (TM). Excessive extracellular matrix (ECM) deposition in the TM outflow pathway results in increased aqueous humor (AH) outflow resistance and intraocular pressure (IOP) elevation. Based on a recent (unpublished) work, we found reduction in vitronectin levels upon induction of the antifibrotic clusterin. Therefore, we evaluated the role of the poorly studied vitronectin on ECM homeostasis in human TM (HTM) cells.
Methods :
Co-immunoprecipitation (co-IP), immunoblotting (IB) and immunofluorescence (IF) were used to analyze the effects of - a) Transforming growth factor β2 (TGFβ2) mediated increase in vitronectin, plasminogen activator inhibitor (PAI-1) and integrin αV (INTαV), b) vitronectin induction on Integrins and ECM proteins-collagen 1 (COL1) and fibronectin (FN) in HTM cells. TMT based global proteomics to identify proteins that are differentially expressed upon vitronectin induction in HTM cells. Student’s t-test was used for statistical analyses with significance of p<0.05 with a sample size of 3-8.
Results :
IB analysis showed a significant increase (p=0.01) in vitronectin expression in aqueous humor of glaucoma patients compared to normal subjects. TGFβ2 induced vitronectin (p=0.05) and its interactors PAI-1 (p=0.03) and INTαV (p=0.04) expression. Co-IP and IF showed a strong interaction between vitronectin and INTαV under TGFβ2 and vitronectin induction. TMT based global proteomics showed increase in ECM associated, ECM biosynthesis and ECM stabilizing proteins under vitronectin induction. IB showed vitronectin induction significantly increased intracellular COL1 (p=0.02), profibrotic PAI-1 (p=0.05) and secretory FN (p=0.05). In addition, phosphorylation of ezrin/radixin/moesin (p-ERM) which links actin-cytoskeleton with membrane protein increased significantly (p=0.04). IF showed increased expression of COL1 and FN in vitronectin induced TM cells.
Conclusions :
For the first time, we show the importance of cell adhesion protein vitronectin and its interactions with integrin in TM, which affects ECM homeostasis and thus increase outflow resistance. Further evaluating the effect of vitronectin on IOP changes and understanding the importance in integrin activation and downstream signaling can identify its role in onset of ocular hypertension.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.