Session Title: Free Paper Session 22: AMD V
Session Date/Time: Sunday 10/09/2017 | 08:00-09:30
Paper Time: 09:18
Venue: Room 115
First Author: : R.Costa BRAZIL
Co Author(s): : M. Moustafa J. Caceres-del Carpio M. Mohamed K. Thaker M. Kenney B. Kuppermann
Cybrids were created with donor carriers of age-related macular degeneration (AMD) that had been treated with an anti-angiogenic drug (Ranibizumab). Cybrids were treated with different ranibizumab concentrations and tested to compare reactive oxygen species production and the VEGF production expression after anti-VEGF treatment.
AMD cybrid cell lines and Ranibizumab
Cybrids were created by fusing mitochondria-depleted RPE cells from an immortalized cell line (ARPE-19) with donor platelets. ARPE-19 cells were treated with ethidium bromide to deplete them of their mitochondria (Rho0) and were then fused with AMD donor’s platelets which are rich in mitochondria to create individual cybrid cell lines consisting of nuclear DNA from the immortalized RPE cells and mitochondrial DNA from the donor patients (n=06). The cybrids containing mitochondria from AMD subjects (AMD) were treated with Ranibizumab at 4 different concentrations and then the levels of reactive oxygen species (ROS) were measured. Two AMD cybrids with different behaviors were identified and treated with Ranibizumab at 1X and 4X the human clinical dose concentration for 24 hours and then the gene expression levels for VEGF-A, SOD2 and HIF1a were measured by qRT-PCR. VEGF production expression of the cybrids from the two AMD-donors under anti-VEGF treatment with Ranibizumab were correlated with the molecular and clinical findings. All the experiments were repeated at least three times.
Cybrids from Patient-01 showed down-regulation of gene expression of VEGF A and HIF1 A at both 1X and 4X Ranibizumab concentrations. The Patient-01 cybrid cultures had an increase in the ROS levels at 1X (P=0.0317), no changes at 2X (P=0.8350) and a decrease at 4X (P=0.0015) and 10X (P=0.0011) of Ranibizumab. The Patient-02 cybrids demonstrated an up-regulation of gene expression of VEGF A and HIF1 A at Ranibizumab 1X and 4X concentrations. There was a decrease in ROS levels at 1X (P=0.1606), at 2X (P=0.0388), 4X (P=0.0010) and 10X (P=<0.0001).
Although the cybrids have the same nuclei from the immortalized RPE cell lines, they have mitochondria from the individual patients. Our results show a differential response between AMD cybrid cell lines. The analysis of VEGF production feedback after anti-VEGF treatment can be useful to help optimize the anti-VEGF treatment regimen. Further investigation is needed to better understand the role that the mitochondria and the drugs play in these observed changes.