MFAP4 supports retinal endothelial cell proliferation

Abstract

Purpose : Current therapeutic approaches for age-related macular degeneration (AMD) are not optimal and cause considerable difficulties. Thus, development of new treatment strategies for AMD is required to improve the disease management. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein and a ligand for a_vb₃ and a_vb₅ integrins. We have previously shown that anti-MFAP4 antibodies effectively block pathological angiogenesis and inflammation in a mouse model of laser-induced choroidal neovascularization. Now we aim to evaluate the ocular expression of MFAP4 and characterize its mechanistic role in vitro. We hypothesize that MFAP4 contributes to AMD pathology by influencing retinal pigment epithelial (RPE) as well as endothelial cell growth.

Methods : Human eye sections were stained with anti-MFAP4 antibody.
Anterior chamber (AC) humor (N=29, incl. 3 with neovascular AMD) and vitreous humour (N=54, incl. 42 with nAMD) were collected during elective cataract surgery and pars plana vitrectomy, respectively. MFAP4 levels were measured by AlphaLISA and analyzed by Wilcoxon rank-sum test.
Primary retinal endothelial cell (REC) and ARPE-19 cell cultures were used for in vitro experiments. Proliferation was assessed by bromodeoxyuridine incorporation 48 h after stimulating serum-starved cells with immobilized MFAP4 (10 mg/ml), a relevant growth factor (VEGF for RECs, PDGF for ARPE-19 cells; 100 ng/ml) or MFAP4/growth factor combination. Results were analyzed by one-way ANOVA.

Results : MFAP4 was expressed in the choroid and RPE layers.
Median MFAP4 was significantly lower in the AC compared to the vitreous (7.9 U/ml vs 17.9 U/ml, p<0.0001). There was no difference in median MFAP4 between nAMD cases and controls (AC: 8.1 U/ml vs 7.9 U/ml, p=0.33; vitreous: 17.9 U/ml vs 17.8 U/ml, p=0.79).
MFAP4 did not influence ARPE-19 cell proliferation regardless of PDGF stimulation (p=0.7). However, MFAP4 stimulation significantly upregulated proliferation of RECs (p=0.002) compared to non-stimulated cells.

Conclusions : We demonstrated MFAP4 expression within the human eye. Vitreal MFAP4 does not appear to be regulated with nAMD, supporting that MFAP4 is constitutively expressed and permissive for integrin activation during neovascularization. Our in vitro results are in accordance with the hypothesis that MFAP4 modulates REC proliferation. However, further investigation will be needed to understand the exact role of MFAP4 in AMD-related pathology.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.