Comparative effects of bevacizumab, ranibizumab and pegaptanib at intravitreal dose range on endothelial cells
Introduction
Angiogenesis is a key aspect in many ocular pathologies that are leading causes of blindness in the world, such as neovascular age-related macular degeneration (AMD), diabetic retinopathy, retinopathy of prematurity (ROP), central retinal vein occlusion and other diseases associated with ischemia and neovascularization (Eichler et al., 2006).
Although angiogenesis is a highly complex and coordinated process requiring multiple receptors and ligands in endothelial cells, vascular endothelial growth factor (VEGF) appears to be a pivotal element required for the process in a variety of normal and pathological circumstances (Ferrara et al., 2004, Ferrara et al., 2006). Since VEGF appears to be the most important activating factor of angiogenesis and vascular permeability, new molecules that bind and inhibit VEGF were developed in recent years.
Pegaptanib sodium (Peg) (Macugen ®, Pfizer), an aptamer that specifically binds extracellular VEGF isoforms that are ≥165 amino acids in size, was the first VEGF-binding drug to be approved for intravitreal treatment of neovascular AMD (Gragoudas et al., 2004, Quiram et al., 2007).
In July 2006, Ranibizumab (Lucentis®, Genentech and Novartis)-, a high-affinity, recombinant, humanized, antigen-binding fragment Fab that neutralizes all active isoforms of VEGF, derived from the same anti-VEGF murine monoclonal antibody that originated Bevacizumab – was also approved by the Food and Drug Administration (FDA) for the treatment of wet AMD. Administered by intravitreal injection, Ranibizumab (Ran) prevents visual loss and improves mean visual acuity in treated patients (Rosenfeld et al., 2006, Brown et al., 2006).
Bevacizumab (Bev) (Avastin ®) was the first humanized anti-VEGF neutralizing antibody approved by the FDA for treatment of metastatic colon cancer (Ferrara et al., 2004). Additionally, Bev combined with chemotherapy has been used in clinical trials for several types of cancer (Shojaei and Ferrara, 2007). More recently, following the first intravitreal application of Avastin® in 2005 (Rosenfeld et al., 2005), the off-label administration of intravitreal Bev in the treatment of exsudative AMD is now widely used (Avery et al., 2006, Spaide et al., 2006), with positive visual results, at a lower price. There are no head to head studies that compare the efficacy of the three different molecules in the treatment of ocular neovascular pathologies (Steinbrook, 2006). On the other hand, the efficacy of the three drugs in other pathologies such as diabetic macular edema (DME), proliferative diabetic retinopathy, central retinal vein occlusion and ROP is being investigated. The FDA has not approved the use of any of the three drugs for the treatment of other angiogenic ocular diseases apart from wet AMD. However, their off-label use has started a new era in the treatment of neovascular blinding ophthalmologic diseases.
Although largely used in clinical practice, there are no studies comparing the three agents in order to evaluate their relative safety and efficacy in vivo and only few reports compare the molecules in vitro.
In the present work, we evaluate the precise effects of Ran, Peg and Bev in several steps of angiogenesis. Human umbilical vein endothelial cells (HUVEC) were incubated with the three agents at concentrations within the clinically established range, or identical amounts of the excipients; cell cytotoxicity, proliferation, apoptosis, migration and vessel assembly were assessed. Additionally, the expression of the phosphorylated (active) form of VEGFR2 in HUVEC cultures previously treated with Bev, Ran or Peg was assessed by Western blotting assays.
Section snippets
Cell culture experiments
Human umbilical vein endothelial cells (HUVECs) were obtained from ScienceCell Research Labs (San Diego, USA). Cells were used between passages 3 and 8. HUVECs were cultured in M199 medium (Sigma–Aldrich, Portugal) supplemented with 20% FBS (Invitrogen Life Technologies, Scotland, UK), 1% penicillin/streptomycin (Invitrogen Life Technologies, Scotland, UK), 0.1% heparin (Sigma–Aldrich, Portugal) and 0.1% endothelial cell growth supplement (ECGS) (Sigma–Aldrich, Portugal), and maintained at 37 °C
Effect of anti-VEGF agents on cell viability
We first attempted to evaluate whether the three anti-VEGF agents developed cytotoxic effects. HUVEC were cultured until 90% confluency and cytotoxicity was assessed by MTT assay. As observed in Fig. 1, cell viability was not affected by Bev, Ran or Peg at any concentration tested, as compared to excipient-treated cell cultures (controls). MTT assays were performed in highly confluent cell cultures, thus presenting an already slow replication rate. This ensures that the possible toxic effect is
Discussion
Although widely used as intravitreal therapies for many vasoproliferative ocular diseases worldwide, there are only few and limited comparative studies in the literature to evaluate the relative safety and efficacy of Peg, Ran and Bev in vitro. In the current study, we aimed at an overview of the effects observed after treating HUVEC with these three anti-VEGF agents on the multiple steps that comprise angiogenesis. We first evaluated whether these agents induced cytotoxicity in confluent HUVEC
Acknowledgements
The authors would like to thank Professor Isabel Azevedo for the helpful discussions. This study was partially funded by FCT (POCI/BM/55556/04, Feder and Programa Comunitário de Apoio), by University of Porto, undergraduation project (PG/07) and by “Sociedade Portuguesa de Oftalmologia”.
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