MMP2-mediated sterile inflammation contributes to drusen accumulation in iRPE models of age-related macular degeneration and related macular dystrophies.

Abstract

Purpose : Accumulation of sub-retinal pigment epithelium (RPE) drusen deposits and extracellular matrix (ECM) are primary hallmarks of age-related macular degeneration (AMD) and related macular dystrophies (MDs) that are recapitulated in induced pluripotent stem cell derived-RPE (iRPE) models of AMD/MDs. However, the mechanism underlying drusen biogenesis and accumulation in AMD/MD iRPE models is not established. The purpose of this study was to evaluate the role of RPE-secreted matrix metalloproteinase-2 (MMP2), a key regulator of ECM turnover and sterile inflammation, in drusen accumulation in AMD/MD iRPE models.

Methods : Control and AMD/MD iRPE cells were analyzed longitudinally for i) MMP2 activity by gelatin zymography and ii) pro-inflammatory damage-associated molecular pattern (DAMP) proteolytic substrates (HMGB1, S100A6) of MMP2 and their modulators (RAGE and sPLA2-IIA) by Western blotting. Aged AMD/MD iRPE cultures were supplemented with active MMP2 or treated with i) an antagonist specific for DAMP-mediated RAGE activation (RAP, 5µM), or ii) an inhibitor of sPLA2-IIA (sPLA-I, 5µM) for up to 14 days and analyzed for drusen by immunocytochemistry (ICC) by measuring the count and area of co-localized sub-RPE deposits (Nile Red-APOE). Additionally, control iRPE cells were treated with MMP2 inhibitor I (MMP2-I1, 5µM) for up to 14 days and monitored for MMP2 activity and drusen by ICC as described above.

Results : Compared to control iRPE, longitudinal analyses showed that AMD/MD iRPE cells displayed reduced MMP2 activity, increased levels of both MMP2-regulated DAMP (HMGB1) and downstream signaling modulators (RAGE and sPLA2-IIA) prior to drusen accumulation. Linking MMP2-mediated sterile inflammation to drusen, MMP2-I1 treated control iRPE showed i) reduced MMP2 activity, ii) increased levels of RAGE, HMGB1 and sPLA2-IIA and iii) elevated count/area of sPLA2-IIA/APOE co-localized sub-RPE deposits when compared to untreated iRPE cells. Therapeutically, basal supplementation with active MMP2, as well as RAP and sPLA2-I treatment reduced count and area of drusen in AMD/MD iRPE cultures.

Conclusions : Altogether, our data suggests that MMP2-mediated sterile inflammation contributes to drusen accumulation in AMD/MD iRPE cultures. Therapeutically, drusen can be targeted by MMP2 supplementation, RAP and sPLA2-I in AMD/MD iRPE cultures.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.