Abstract
Purpose :
To evaluate the effect of aflibercept and ranibizumab on the expression of genes related to angiogenesis and oxidative stress in cybrid cells.
Methods :
Cybrid cells (CYB) were created by fusing Rho0 cells, ARPE-19 cells depleted of mitochondrial DNA (mtDNA), with platelets isolated from wet AMD patients n=7. These CYB have the same nuclear genome but different mtDNA. CYB were cultured for 48 hours, then treated with aflibercept or ranibizumab at 1X or 4X concentrations of the clinical intravitreal dose (equivalent to 0.05 ml of drug in 4 ml of vitreous volume). After 24 hours, the gene expression levels for VEGFA, SOD2 and HIF1α were measured by qRT-PCR. Results were normalized to untreated cells. All the experiments were repeated three times and unpaired t test was used to analyze data.
Results :
CYB 14-145 showed increased expression levels of all angiogenic genes with ranibizumab (ΔΔCT Folds: HIF1α 1X 1.04, 4X 2.03 and VEGFA 1X 1.43, 4X 1.45; p<0.05), in contrast to aflibercept that showed decreased expression levels in all drug concentrations (fold: HIF1α 1X 0.96, 4X 0.80 and VEGFA 1X 0.78, 4X 0.56, p<0.05) compared to untreated. Oxidative stress gene expression showed a decreased level with ranibizumab and an increased level with aflibercept (Folds: SOD2 1X 0.99, 4X 0.53; SOD2 1X 2.15, 4X 2.37; p<0.05).
Three CYB showed statistically significant reduction in HIF1α, VEGFA and increased expression of SOD2 in all drug concentrations: CYB 14-136 (aflibercept/ranibizumab Fold): [HIF1α 1X 0.86/1.07, 4X 0.85/0.87; VEGFA 1X 0.87/0.93, 4X 0.75/0.75; SOD2 1X 1.93/1.47, 4X 2.17/1.76; p<0.05], CYB 15-151 [HIF1α 1X 0.93/0.97, 4X 0.88/0.97; VEGFA 1X 0.86/0.83, 4X 0.73/0.80; SOD2 1X 1.21/1.40, 4X 1.38/1.89; p<0.05] and CYB 15-157 [HIF1α 1X 0.80/0.98, 4X 0.89/0.79; VEGFA 1X 0.71/0.96, 4X 0.77/0.82; SOD2 1X 1.90/1.02, 4X 1.94/1.70; p<0.05].
CYB 14-14-146, 14-147 and 15-159 had unresponsive gene expression compared to untreated cells.
Conclusions :
Since all cybrid cells had identical nuclei but unique mitochondria, our data suggests that the difference in gene expression levels after anti-VEGF treatment is related to mitochondrial interaction. Further investigation is needed to better understand the interaction between anti-VEGF drugs and the role mitochondria play in patients with wet AMD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.