In vitro response and gene expression of human Retinal Müller cells treated with different Anti-VEGF drugs

In the past decade, anti-vascular endothelial growth factor (VEGF) therapy has become more popular and the standard of care for several retinal diseases including exudative age-related macular degeneration (AMD) (Brown et al., 2006; Martin et al., 2012; Rosenfeld et al., 2006; Tufail et al., 2010), diabetic macular edema (DME) (Brown et al., 2013; Do et al., 2012; Do et al., 2013; TDRCR, 2015), and macular edema (ME) due to retinal vein occlusion (RVO) (Brown et al., 2010; Brown et al., 2013; Campochiaro et al., 2010; Wu et al., 2008). Ranibizumab (Lucentis®, Genentech, San Francisco, CA) is a humanized, recombinant, monoclonal antibody fragment. Intended for intraocular use, a 0.5mg/0.05ml dose is approved by the Food and Drug Administration for exudative AMD and ME due to RVO; a 0.3mg/0.05ml dose is approved by the FDA for DME, and diabetic retinopathy (DR). Bevacizumab (Avastin®, Genentech, San Francisco, CA) is a humanized monoclonal human immunoglobulin gamma 1 (IgG1) antibody that selectively binds to circulating VEGF and inhibit its binding to cell surface receptors. A 1.25mg/0.05ml is used off-label in the United States and is commonly used worldwide for exudative AMD, DME and RVO. Aflibercept (Eylea®, Regeneron Pharmaceuticals, Tarrytown, NY) is a recombinant protein receptor decoy composed of two VEGF receptors 1 and 2 fused with the Fc region of human IgG1. Compared to the former two anti-VEGF drugs, it has a higher binding affinity for VEGF and it also binds to VEGF-B and Placental growth factor (PGF). A concentration of 2.0mg/0.05ml is approved by the FDA for wet AMD, DME, ME due to RVO, and DR in patients with DME. Ziv-aflibercept (Zaltrap®, Sanofi Aventis, Bridgewater, NJ and Regeneron Pharmaceuticals, Tarrytown, NY), also known as VEGF Trap-oncologic, contains 25 mg/ml ziv-aflibercept, however contains higher sucrose concentrations than aflibercept (Eylea), which results in higher osmolality (Bayer, 2014; Group, 2012). Ziv-aflibercept is approved by the FDA for treatment of resistant or progressing metastatic colorectal cancer. However, Mansour et al. (Mansour et al., 2015; Mansour et al., 2017a; Mansour et al., 2017b); de Oliveira Dias et al. (de Oliveira Dias et al., 2015; de Oliveira Dias et al., 2017; de Oliveira Dias et al., 2016); and Chhablani et al., (Chhablani, 2015; Chhablani et al., 2017), among other authors, have published several reports finding good outcomes and no apparent safety issues in a series of patients treated with intravitreal ziv-aflibercept. Previous studies have shown that anti-VEGF drugs in clinically significant doses do not affect cell viability in vitro in a human retinal pigment epithelium (RPE) cell line (ARPE-19) (Luthra et al., 2006; Malik et al., 2014b). Nonetheless, subtle cytotoxic changes in RPE cells, such as decrease in mitochondrial membrane potential, could be observed even at clinical doses for some anti-VEGF drugs (Malik et al., 2014b). Numerous publications have described in vitro differential responses to the different anti-VEGF drugs in various retinal cells (Deissler et al., 2012; Klettner et al., 2010; Schnichels et al., 2013). Muller cells are predominant glial cells in the retina and provide structural and metabolic support to retinal neurons. Several studies have described numerous functions of Muller cells, including regulation of cellular homeostasis and pH, modulation of neurotransmitter recycling, contribution to the blood-retinal barrier by surrounding retinal capillaries with glial processes (Limb et al., 2002; Reichenbach and Bringmann, 2013), acting as light collectors by directing light to photoreceptors (Franze et al., 2007; Reichenbach and Bringmann, 2013), and secretion and regulation of VEGF and pigment epithelium derived factor (PEDF) (Garcia and Vecino, 2003; Limb et al., 2002; Reichenbach and Bringmann, 2013). Muller cells have also been associated with retinal neuroprotection, wound healing and regeneration (Garcia and Vecino, 2003). The purpose of this study was to evaluate the in vitro response and differences in gene expression of human retinal Muller cells treated with different concentrations of ranibizumab, bevacizumab, aflibercept or ziv-aflibercept. Assays for cell viability, metabolic activity, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) and apoptosis were used to evaluate responses to the anti-VEGF drugs. In addition, differences in the expression levels for angiogenesis-related, pro-apoptotic, inflammation and oxidative stress genes were assessed.