Chapter 29

VEGF Inhibitor Induced Oxidative Stress

in Retinal Ganglion Cells

Vikram S. Brar and K.V. Chalam

Abstract Oxidative stress mediated toxicity is common to several sight-threatening ocular conditions, in which vascular endothelial growth factor (VEGF) plays both a pathologic and protective role. Anti-VEGF therapy can negate the protective role of endogenous VEGF and enhance oxidative stress and thus should be administered with caution as long-term intravitreal usage of bevacizumab may have collateral negative effects on retinal cells.

29.1Introduction

Reactive oxygen species (ROS) are generated in normal metabolic processes and the imbalance between their production and detoxification generates oxidative stress. In the setting of increased ROS, cell membranes, nucleic acids, and proteins are vulnerable to chemical modification, and promote cell death via apoptosis. Oxidative stress has been implicated in the pathogenesis of many ocular diseases, including glaucoma [1], diabetic retinopathy [2], and age related macular degeneration (ARMD) [3], where it has been linked to increased expression of vascular endothelial growth factor (VEGF). VEGF has been implicated in a variety of retinal vascular conditions [4–10] and treatment with anti-VEGF agents has emerged as the standard of care in the management of these diseases [11–21]. However, VEGF has also been described as a neuroprotectant [22–25], particularly against oxidative stress in the central nervous system (CNS) [26–30] and the retina [31, 32]. Thus, total VEGF blockade with anti-VEGF agents may have unintended negative effects [33]. The focus of this chapter is to summarize the role of oxidative stress in select irreversibly

V.S. Brar (*) • K.V. Chalam

Department of Ophthalmology, University of Florida College of Medicine, 580 West 8th Street, Plaza II 3rd floor, Jacksonville, FL 32209, USA e-mail: s2vsingh@vcu.edu

in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-606-7_29, © Springer Science+Business Media, LLC 2012

blinding ocular diseases, highlight the role of VEGF in neuroprotection, and describe the potential consequences of anti-VEGF therapy on retinal ganglion cells [31].

29.1.1Oxidative Stress in Glaucoma

Oxidative stress has also been implicated in glaucoma [1], another significant cause of irreversible blindness [34]. Elevated oxidative markers have been demonstrated in the aqueous humor of glaucomatous eyes [35, 36] and treatment with topical dorzolamide has been shown to decrease them [36]. Retinal ganglion cells are vulnerable to oxidative stress via their high metabolic demand and exposure to light [37, 38]. Subsequent damage to cell membranes and nucleic acids, both mitochondrial and nuclear, result in cell death, classically through apoptosis [39, 40]. Melatonin, a known antioxidant, exhibits neuroprotection independent of elevated intraocular pressure, by positively influencing oxidative markers and increasing superoxide dismutase (SOD) and glutathione levels while decreasing thiobarbituric acid reactive substances (TBARS—measure of lipid peroxidation) [41]. Several other antioxidant compounds are being evaluated in the treatment of glaucoma [42].

29.1.2Oxidative Stress in Diabetic Retinopathy

In the USA, diabetic retinopathy is the main cause of permanent vision loss in the working population [43]. Oxidative stress plays a central role in the development of diabetic retinopathy, where hyperglycemia induced superoxide production by the mitochondria is the sentinel event [44]. In the polyol pathway, hyperglycemia results in the conversion of glucose to sorbitol, by the aldose reductase [45, 46]. This pathway consumes NADPH, a required cofactor in the regeneration of glutathione. Therefore, deprivation of NADPH diminishes defense against oxidative stress [47]. ROS also lead to reduced activity of glyceraldehyde-3 phosphate dehydrogenase (GADPH), which regulates several pathways implicated in oxidative stress, one of which is the protein kinase C (PKC) pathway [44].

PKC activation results in the production of proinflammatory advanced glycation end products (AGE) [48]. Further, PKC has also been shown to have effects on vascular permeability [2]. This and other pathological effects in the retina culminated in a multicenter prospective randomized clinical trial, which demonstrated a positive role of the PKC inhibitor ruboxistaurin on vision loss in nonproliferative diabetic retinopathy [49].

29.1.3Oxidative Stress in Age Related Macular Degeneration

ARMD is a major cause of blindness in the world [50]. There is increasing evidence that pathogenic oxidative mechanisms contribute to the progression of ARMD

[3, 51–54]. Due to the retina’s high metabolic activity, oxygen rich environment, concentration of polyunsaturated fatty acids, and continuous exposure to light, it is highly susceptible to oxidative stress and the subsequent generation of ROS [3, 54]. More specifically, phagocytosis of outer segments by the retinal pigment epithelium (RPE) results in the generation of the superoxide anion, hydroxyl radical, and hydrogen peroxide [55]. The subsequent oxidative stress can eventually result in induction of apoptosis in RPE cells [32, 53]. Further, oxidative stress has been shown to induce VEGF-A and VEGF-C secretion in RPE cells, leading to the development of choroidal neovascularization [56]. However, VEGF-A also plays a protective role against oxidative stress induced apoptosis [32].

29.1.4Vascular Endothelial Growth Factor

VEGF acts primarily as an angiogenic as well as a vasopermeable agent [57, 58]. The VEGF family includes VEGF-A-E and placental growth factor, with VEGF-C primarily involved in lymphangiogenesis [59]. In addition to its effects on endothelial cells, VEGF plays an essential role in the development and maturation of neural tissue, including the retina [27]. Developmentally, astrocytes in the retinal ganglion cell layer, cells of the inner nuclear layer, Müller cells, and retinal pigment epithelial cells all express VEGF [60, 61]. Physiologically in mature retina, VEGF is expressed in the absence of active neovascularization and helps maintain the homeostasis of adult retinal neurons [33].

Specifically, VEGF-A has been implicated in retinal vascular disease, where increased VEGF expression was due to retinal ischemia [62]. Increased VEGF expression occurs in many vascular conditions of the retina including ARMD and diabetic retinopathy [4–10].

In different cell types, VEGF expression is initiated under the influence of oxidative stress, which may have both pathologic and protective effects on surrounding cells. Moreover, increased VEGF expression promoted survival in aortic endothelial cells exposed to cytotoxic agents including hydrogen peroxide [63]. In contrast, in the retina, such induction of VEGF results in the development of neovascular ARMD [56] and conversely protects against oxidative cell death, in an autocrine fashion in retinal pigment epithelial cells [32].

29.1.5VEGF Mediated Neuroprotection

VEGF has also been shown to be neuroprotective in many models of CNS injury. Early reports describe VEGF protection following ischemic insults in rat brain [22]. VEGF also protected spinal cord neurons in vitro from glutamate induced excitotoxicity [23]. In the eye, VEGF-A reduced apoptosis in retinal neurons following ischemic injury [24] and delayed degeneration in retinal ganglion cells following axotomy [25].

Oxidative stress related to mutations in SOD contributes to the pathogenesis of amytrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder, where VEGF has also been implicated [64]. A more severe form of CNS pathology was encountered in a VEGF deficient mouse model of ALS compared to wild-type [26]. In SOD-1 deficient VEFG-overexpressing double-transgenic mutant mice, enhanced VEGF expression decreased oxidative stress and improved neuronal cell survival [27]. This lead to attempts to reduce motor neuron degeneration with intraventricular injection of VEGF in a rat model of ALS [28]. Further, both intraperitoneal [29] and intramuscular [30] injections of VEGF increased survival in mice models. VEGF has therefore demonstrated promise in the management of oxidative stressrelated neurodegenerative disease, though its exact mechanism and role in this context is still being evaluated [64].

29.1.6Mechanisms of VEGF Protection Against Oxidative Stress

Several different pathways have been described to explain the cytoprotective effect mediated by VEGF against oxidative stress. VEGF has been shown to induce mitochondrial SOD, a major enzyme in the defense against oxidative stress [65]. Subsequently, enhanced VEGF expression was shown to protect neuronal cells from 3-nitropropionic acid (3-NP: inhibitor of succinic acid dehydrogenase) induced oxidative stress. This was accomplished through induction of SOD; treatment with anti-VEGF antibodies abrogated this effect [66]. Similarly, VEGF was shown to protect against ROS through induction of heme oxygenase-1 (HO-1), an oxidative enzyme, in an animal model of hyperoxic acute lung injury [67]. In another study, involving endothelial cell response to cytotoxic levels of hydrogen peroxide, VEGF expression was associated with increased cell survival, which was coupled with an induction of NF-kB [68]. In a cell culture model, VEGF was shown to protect motor neurons from hydrogen peroxide mediated oxidative stress and death through the anti-apoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway [69]. This mechanism was confirmed in human RPE cells in culture where treatment with VEGF-A protected RPE cells from hydrogen peroxide induced apoptosis [32]. In summary, VEGF appears to protect against oxidative stress through induction of anti-oxidant agents (SOD, HO-1 [70, 71]) and pathways associated with cell survival (NF-kB [72], PI3-K/Akt [73]).

29.1.7Anti-VEGF Therapy

Numerous anti-VEGF agents have been introduced for clinical use [11, 12, 20, 21, 74]. However due to lower cost, efficacy, and expanding clinical applications, bevacizumab has emerged as the most commonly employed agent. Intravitreal injections of bevacizumab, a humanized monoclonal antibody, are widely used in the treatment of neovascular ARMD [20, 21] as well as in other vascular diseases of the