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Mohammad Riazi Esfahani, Zahra Faghiri, Mohamed Tarek Moustafa, Mohamed Mohamed, Abdul Sami Memon, Baruch D Kuppermann, Cristina M Kenney; Effects of Ranibizumab on gene expressions in ARPE-19 cells during cobalt chloride-mediated hypoxia. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1058.
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© ARVO (1962-2015); The Authors (2016-present)
Hypoxia stimulates synthesis and release of vascular endothelial growth factor (VEGF) that is a key component in the pathogenesis of many retinal disorders. Cobalt chloride (CoCl2) is an iron analogue which can mimic hypoxic conditions when added to the medium. One of the main treatments for those disorders is the monoclonal antibody fragment Ranibizumab (Lucentis), which targets VEGF. However, some controversy exists regarding the effects of Ranibizumab on gene expressions in RPE cells. In this study we evaluated expression levels for pro-angiogenesis, pro-apoptosis and pro-inflammatory genes in RPE cells in response to hypoxia alone and in combination with Ranibizumab.
Hypoxia was induced in culture of ARPE-19 cells and after 24 hr cell viability was determined using MTT assay. For RNA isolation and qRT-PCR, ARPE-19 cultures were pretreated 24 hr with two concentrations of Ranibizumab (1x and 4x the clinical dose) and then stimulated with 25µM CoCl2 to induce hypoxia. qRT-PCR was used to evaluate the gene expression of VEGF, Bax, CASPASE-3 and IL-33.
25 µM Cocl2 had no significant effect on the viability of ARPE-19 cells. After treatment with 25 μM CoCl2 there was an increase of CASPASE-3 (3.1 fold, p=0.024) and IL-33 (7.5 fold, p=0.0025) compared to the untreated cultures, but it was reversed for both genes by pretreatment with 1x (CASPASE-3 0.47-fold, p=0.048 & IL-33 0.36-fold, p=0.0041) and 4x Ranibizumab (CASPASE-3 0.49-fold, p=0.046 & IL-33 0.33-fold, p=0.0031) compared to CoCl2 alone.Pretreatment with 1x Ranibizumab did not significantly change the CoCl2-induced increase in VEGF (3.79-fold, p<0.0001) and BAX (1.78-fold, p=0.0014) gene expressions. Ranibizumab at 4x concentration Significantly increased the VEGF (1.529-fold, p=0.0002) and BAX (1.508-fold, p=0.0007) compared to CoCl2 treatment alone.
Ranibizumab showed some protective effects on apoptosis (Caspase-3) and inflammation (IL-33) at both 1x and 4x, but had either no (1x) or increased (4x) effect on VEGF and BAX gene expressions during CoCL2 -induced hypoxia. Understanding variable effects of Ranibizumab in hypoxia may improve our current understanding of the mechanisms involved in pathogenesis of cell loss after long term use of this treatment in AMD patients.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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