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Kay D Beharry, Charles L Cai, Taimur Ahmad, Gloria B Valencia, Douglas R Lazzaro, Jacob V Aranda; Response of Human Retinal Endothelial Cells (HRECs) to Small Interfering RNA (SiRNA) Silencing of Notch-1, Delta-Like Ligand 4 (Dll4), and Jagged-1 During Hyperoxia with Intermittent Hypoxia (IH).. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5375.
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To test the hypothesis that IH disturbs the Notch-Dll4 signaling pathway and is associated sVEGFR-1.
SiRNA was used to silence Notch, Dll4 and Jagged-1 in human retinal ECs (HRECs) exposed to normoxia (Nx, 5% CO2) or IH (50% O2 with brief 10% O2 (8 cycles/day). Biomarkers of angiogenesis (Notch-1, VEGF, sVEGFR-1, and IGF-I) were determined in the media at 24, 48, and 72 hours. Expression of genes involved in the Notch signaling pathway was examined in the cells using qPCR. Cell migration and tube-forming capacity were determined using casein AM labeled tube-formation plates.
HRECs were more responsive to SiRNA silencing in IH. VEGF levels were generally low in the media while sVEGFR-1 and IGF-I levels were high. Notch-1 silencing resulted in significant reductions in sVEGFR-1 levels and this effect was more robust in IH. In contrast, IGF-I levels increased with Notch-1, Dll4, & Jagged-1 silencing. Notch-1 silencing resulted in downregulation of >70% genes and was associated with increased migration but decreased tube forming capacity in IH.
IH increases the knockdown efficiency of SiRNAs possibly via structural remodeling of the cell wall. Notch-1 and sVEGFR-1 are likely cor-regulators of sprout guidance and tip cell quiescence. Conversely, IGF-I may promote tip cell phenotype and induce migration. These results provide new insights regarding the effects of IH on HREC responses to Notch-Dll4-Jagged knockdown.
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