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Zeljka Smit-McBride, Yan Yi Chen, Minh-Tam C. Bui, Hannah R. Schmitz, Zhishan Liu, Julia M. Camilleri, Lawrence S Morse; Effect of DR microRNA biomarkers on angiogenesis assay activity in vitro. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2658.
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© ARVO (1962-2015); The Authors (2016-present)
To test whether circulatory microRNAs highly expressed in surgical specimens of ocular fluids from patients with proliferative diabetic retinopathy have biological activity in stimulation of angiogenesis.
Mimics of the 5 microRNAs and scrambled control (Ambion) were transfected using RNAiMAX reagent into the ARPE19 cells at 0pmol, 10pmol, 25pmol concentrations. The ARPE19 conditioned cell media were harvested at 12h, 24h, and 48h. Tube formation assay was performed by incubating HUVEC cells with ARPE-19 conditioned media, plated on Geltrex™ LDEV-Free basement membrane matrix, in 96-well plates. The cells were labeled with CellTrace calcein red-orange AM after 12h incubation. Tube formation images were acquired using a 2X objective on the ImageXpress Micro high-content screening system (Molecular Devices) at IDDRC Biological Analysis Core. Images were then analyzed using MetaXpress software MX5 built-in analysis for angiogenesis.
We used the tube formation assay by HUVECS to test whether ARPE19 conditioned media from cells transfected with miRNA biomarkers (miR-155, let-7b, miR-486, miR-92a, and miR-320b) can regulate angiogenesis. The total of 14 statistical parameters were calculated by MetaExpress software: total tube length, area, nodes, etc. At the 24h time point, the strongest inhibition of total tube length was with 10nm let-7b (Fold Change, FC=-1.9), 25nm miR-486 (FC=-1.8), and miR-92A (FC=-2.6), compared to non-conditioned ARPE-19 media. At a 48h time point, strongest stimulation of total tube length was observed with 10nm miR155 (FC=1.8), followed by 25nM miR-320b and 25nM let7b (FC=1.4).
We have shown that our microRNA biomarkers have biological activity on the regulation of angiogenesis in vitro. Regulation by microRNAs is complex, as they can demonstrate stimulatory or inhibitory effects, dependent on dose and length of exposure. Our results show that HUVECs are responsive to changes in factors in ARPE-19 conditioned media transfected with miRNAs. Such miRNA-induced changes in retinal epithelial cells to secrete angiogenic factor may negatively impact the integrity of the microvasculature in the diabetic retina leading to angiogenesis and vascular leakage.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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