MMP9 associated with choroidal neovascularization in AMD is expressed in human immune cells

Abstract

Purpose : Immune-mediated mechanisms play an important role in AMD pathogenesis and choroidal neovascularization (CNV), which may explain why many with exudative AMD are incomplete responders to anti-VEGF therapy. The MMP9 gene encodes matrix metallopeptidase 9, aka type IV collagenase, associated with degradation of the extracellular matrix that is implicated in angiogenesis and fibrosis. We recently confirmed that MMP9 increases risk only for exudative AMD. We sought to determine the cell-specific localization of MMP9 gene expression and the levels of MMP9 expressed by human cells.

Methods : Data from single cell RNA sequencing enriched for CD31+ expressing cells of the human posterior segment were analyzed for MMP9 gene expression. THP-1 monocyte cells were differentiated into M0 polarized macrophages with PMA and M2 macrophages with PMA/interleukin(IL)-4/IL-13. THP-1 cells were differentiated into immature dendritic cells (iDCs) with IL-4/GM-CSF, and into mature DCs (mDCs) with IL-4/GM-CSF/TNF-alpha/ionomycin. ELISA for MMP9 was performed on the conditioned media of M0, M2, iDCs and mDCs followed by normalization of the total cell numbers. Zymography was used to detect active and latent forms of MMP9. Immunostaining was performed with CD86 and CD206 to label mDCs and M2 macrophages, respectively.

Results : scRNA-seq of human donor eyes with AMD enriched with CD31+ cells reveal that MMP9 expression is highest in a choroidal DC-like cluster that includes DCs and macrophages with very low to no expression in other cell types or regions of the posterior eye. DCs (mature>immature) expressed higher levels of MMP9 compared to M0 and M2 macrophages (see figure). The band intensity of MMP9 from zymography showed similarly high signal for DCs relative to macrophages. Verification of mature DCs and M2 macrophages was confirmed with immunolabeling.

Conclusions : MMP9 plays a role in CNV through immune cells found in the choroid. Protein expression of MMP9 from dendritic and macrophage cells confirm the plausible mechanism of its role in AMD. Elucidating the disease mechanisms associated with MMP9 is of importance to reveal new targets for therapeutic intervention, the next milestone in exudative AMD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

a) mDCs and M2 macrophages differentiated from the THP-1 cell line immunostained for CD86 and CD206, respectively. MMP9 levels from conditions media of iDCs and mDCs and M0 and M2 macrophages using b) ELISA and c) zymography.