Abstract
Purpose :
Overexpression of matrix metalloproteinases-2 (MMP-2), an extracellular matrix (ECM) proteolytic enzyme, has shown to be associated with aging and retinal degenerative diseases such as age-related macular degeneration (AMD). Cytosolic MMPs is now known to interact with intracellular proteins, potentially disrupting essential functions. Aberrations in the phagocytosis of photoreceptor outer segments (POS) by the retinal pigmented epithelium (RPE) is common in age-related retinal degeneration and can be considered its precursor. However, the exact mechanisms of how aging affects RPE-POS phagocytosis remains unclear. Here, we investigated the effects of MMP-2 overexpression on critical proteins associated with RPE-POS phagocytosis.
Methods :
Human RPE cells (ARPE-19) were cultured in triplicates and transfected with an MMP-2 overexpression plasmid using lipofectamine. POS isolated from bovine eyes using sucrose gradient ultracentrifugation were introduced onto the ARPE-19 cells and incubated for 4 hours in a cell incubator at 37°C to initiate phagocytosis. After termination of phagocytosis, proteins in the ARPE-19 cells were extracted and the expression of phagocytosis-related proteins were assessed using western blot.
Results :
POS-fed RPE cells overexpressing MMP-2 expressed significantly less proteins associated with phagocytosis including digestive enzyme, cathepsin D (p = 0.025), and protein receptor, MerTK (p = 0.001). Expression of rhodopsin, associated with POS exposure and/or internalization, was significantly less (p = 0.002) in RPE overexpressing MMP-2, indicating a potential loss of internalization mechanism. Lysosome-associated protein, LAMP2, was significantly expressed in RPE cells transfected with MMP-2 with (p = 0.038) and without POS exposure (p = 0.037), indicating increased intracellular protein degradation.
Conclusions :
MMP-2 overexpression in RPE cells was able to significantly affect proteins critical to POS phagocytosis. Overexpressing MMP-2 affected molecules involved in the activation and digestion steps of phagocytosis. In addition, cytosolic MMP-2 could promote lysosomal activation and potentially intracellular degradation. Further studies are needed to determine the exact molecular action of MMP-2 in RPE cells and how they could impact its essential functions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.