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Peter Shaw, Victor Lin, Zhigang Lu, Adam May, Briana Che, Kyle Tran; HTRA1 synergizes with oxidized phospholipids in activation of VEGF and inflammtory factors in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2636. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
HTRA1 is a major genetic risk factor associated AMD but its molecular mechanism in disease pathology is still unclear. In this study, we aim to investigate the interplay of HTRA1 protein with oxidized phospholipids (oxPLs), which induces abnormal expression of VEGF, and other inflammatory factors in RPE cells. We also seek to inhibit HTRA1 activity with antibodies to interrupt the association to RPE. We then evaluate the expression of VEGF and inflammatory factors that involve in the pathogenic angiogenesis of AMD development.
APRE-19 cells are cultured in MEM medium with 10 % FCS and seeded on to 6 well plates for treatment. The cells were administered with fresh MEM containing following materials and concentrations: Nat-LDL (50ug/ml), oxLDL (50ug/ml), HTRA1-wt (100ng/ml), and HTRA1 (100ng/ml) + ox-LDL (50ng/ml), followed by 24 hours incubation period. The RNA was extracted with Direct-zolTM RNA MiniPrep kit and used to synthesize cDNA with a Reverse Transcription Kit. QPCR was performed using SYBR green Supermix with pre-tested target gene primers. All gene expression data were normalized to β-actin.
1. Comparing to native-LDL, the ox-LDL resulted in moderate increase in HTRA1 (3.4 ± 1.3 vs 4.4 ± 1.7) and mild increase in VEGF expression (1.08 ± 0.4 vs 1.3 ± 0.4) (data are relative fold change to control (BSA) ± SEM) in ARPE-19 cells.2. The HTRA1+oxLDL stimulate higher expression of HTRA1 (11.6 ± 2.3 vs 3.9 ± 1.1); IL-6 (35 ± 8.4 vs 5.0 ± 0.8); IL-8 (506 ± 132 vs 76 ± 8.3) and VEGF (4.2 ± 1.1 vs 1.2 ± 0.3), (Data are HTRA1+oxLDL vs HTRA1 alone and expressed as Relative Fold Change to control (BSA) ± SEM).3. Under the standard assy condition, when 500 ng/ml of purified anti-HTRA1 antibody was pre-incubated with 100 ng/ml of purified HTRA1-wt protein, the relative protease activity was 55% of those HTRA1 protein alone; when anti-HTRA1 increased to 1 ug/ml, the relative protease activity was further reduced to 12%. This indicates a dose-dependent inhibition.
Either HTRA1 protein or oxPLs can induce the expression of VEGF and other inflammatory cytokines. Incubated together, they display synergitic effect in elevated stimulation of HTRA1 itself as well as VEGF and other inflammatory factors. The antibody targeting HTRA1 can greatly nutralize the HTRA1 activity indicating that HTRA1 can be the potential target for treating wet AMD.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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