PEER REVIEWED

Proteomics in Practice: Potential Consequences of Long-term Anti-VEGF Therapy

K. V. Chalam, MD, PhD • Vikram S. Brar, MD

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 retinopathy² and AMD,³ in which it has been linked to increased expression of vascular endothelial growth factor. 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^26-30 and the retina.^31-32 Thus, total VEGF blockade with anti-VEGF agents may have unintended negative effects.³³ We summarize the role of oxidative stress in selected, irreversibly blinding ocular diseases, highlight the role of VEGF in neuroprotection and describe the potential consequences of anti-VEGF therapy on retinal ganglion cells.³¹

OXIDATIVE 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 and glutathione levels, while decreasing thiobarbituric acid reactive substances (a measure of lipid peroxidation).⁴¹ Several other antioxidant compounds are being evaluated in the treatment of glaucoma.⁴²

OXIDATIVE STRESS IN DIABETIC RETINOPATHY

In the United States, 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, in which hyperglycemia induces superoxide production by the mitochondria as the sentinel event.⁴⁴ In the polyol pathway, hyperglycemia results in the conversion of glucose to sorbitol by 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

Reactive oxygen species also lead to reduced activity of glyceraldehyde-3 phosphate dehydrogenase, 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.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 for the PKC inhibitor ruboxistaurin on vision loss in non-proliferative diabetic retinopathy.⁴⁹

OXIDATIVE STRESS IN AMD

Age-related macular degeneration is a major cause of blindness in the world.⁵⁰ There is increasing evidence that pathogenic oxidative mechanisms contribute to the progression of AMD.^3,51-54 Due to the retina's high metabolic activity, its concentration of polyunsaturated fatty acids and its 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 RPE results in the generation of the superoxide anion, hydroxyl radicals and hydrogen peroxide.⁵⁵ The subsequent oxidative stress results in the 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

VEGF

Vascular endothelial growth factor 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, such as in the retina.²⁷ Developmentally, astrocytes in the retinal ganglion cell layer, cells of the inner nuclear layer, Müller cells and RPE cells all express VEGF.^60-61 Physiologically, in the mature retina, VEGF is expressed in the absence of active neovascularization and maintains the homeostasis of adult retinal neurons.³³

Specifically, VEGF-A has been implicated in retinal vascular disease, in which increased VEGF expression was due to retinal ischemia.⁶² Increased VEGF expression occurs in many vascular conditions of the retina, including AMD 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 promotes survival in aortic endothelial cells exposed to cytotoxic agents, including hydrogen peroxide.⁶³ In the retina, such induction of VEGF results in the development of neovascular AMD⁵⁶ and conversely protects against oxidative cell death in an autocrine fashion in RPE cells.³²

VEGF-MEDIATED NEUROPROTECTION

VEGF has also been shown to be neuroprotective in many models of central nervous system injury. Early reports described VEGF protection following ischemic insults in the rat brain.²² VEGF also protected spinal cord neurons in vitro from glutamate-induced excitotoxicity.²³ With regard to the eye, VEGF-A reduced apoptosis in retinal neurons following ischemic injury²⁴ and delayed degeneration in retinal ganglion cells following axotomy.²⁵

Oxidative stress related to mutations in superoxide dismutase (SOD) contributes to the pathogenesis of amytrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder, in which VEGF has also been implicated.⁶⁴ A more severe form of the disease was encountered in a VEGF-deficient mouse model of ALS, compared to the wild type.²⁶ In SOD-1 mutant mice, enhanced VEGF expression decreased oxidative stress and improved neuronal cell survival.²⁷ This result led to attempts to reduce motor neuron degeneration with an intraventricular injection of VEGF in a rat model of ALS.²⁸ Further, both intraperitoneal²⁹ and intramuscular³⁰ injections of VEGF increased survival in mouse models. VEGF has demonstrated promise in the management of oxidative stress–related neurodegenerative disease, although its exact mechanism and role in the future is being evaluated.⁶⁴

MECHANISMS 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 oxidative stress induced by 3-nitropropionic acid (3-NP), an inhibitor of succinic acid dehydrogenase. This result was accomplished through induction of SOD, and 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 a rise in NF-κB.⁶⁸ In a cell culture model, VEGF was shown to protect motor neurons from hydrogen peroxide–mediated oxidative stress through the antiapoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway.⁶⁹ This mechanism was confirmed in human RPE cells in culture, in which 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 antioxidant agents (SOD and HO-1^70-71) and pathways associated with cell survival (NF-κB⁷² and PI3-K/Akt⁷³).

ANTI-VEGF THERAPY

Numerous anti-VEGF agents have been introduced for clinical use.^74-78 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 AMD,^20-21 as well as in other vascular diseases of the posterior segment.^13-19

With regard to bevacizumab, numerous reports have failed to demonstrate any toxicity of this agent.^79-83 However, in lieu of VEGF-mediated neuroprotection, anti-VEGF therapy may have negative effects on cells in the retina. Animal studies have shown that systemic neutralization of VEGF with soluble VEGF receptors resulted in reduced thickness of the inner and outer nuclear layer in the adult mouse retina.³³ Thus, repeated treatment with anti-VEGF agents may negate the physiologic function and neutralize VEGF mediated neuroprotection.

VEGF PROTECTION AGAINST OXIDATIVE STRESS IN RETINAL GANGLION CELLS

We evaluated the protective role of VEGF against oxidative stress in differentiated retinal ganglion cells⁸⁴ and the effect of anti-VEGF therapy on the process. Oxidative stress was induced using hydrogen peroxide to simulate conditions faced by retinal ganglion cells in various ocular diseases. Co-treatment with VEGF₁₆₅ protected cells from hydrogen peroxide–mediated cell toxicity, and this effect was neutralized by bevacizumab.

The glutathione reductase inhibitor buthionine sulfoxime (BSO) also eliminated VEGF-mediated cytoprotection. This result is supported by the fact that VEGF induces SOD,⁶⁶ one product of which, hydrogen peroxide, is converted to water and oxygen by glutathione peroxidase through oxidation of glutathione. BSO treatment depletes the store of reduced glutathione and reduces the ability of the cell to respond to oxidative stress. This protective effect is especially relevant in the setting of combination therapy with anti-VEGF agents and of brachytherapy in the management of CNV membranes, in which radiation-mediated induction of ROS induces choroidal endothelial cell death.^85-86

SUMMARY

Oxidative stress–mediated toxicity is common in several sight-threatening ocular conditions, in which VEGF plays both a pathologic and protective role. Anti-VEGF therapy can negate this role and enhance oxidative stress and, thus, should be administered with caution, as long-term intravitreal usage of bevacizumab may have collateral effects on retinal cells, although these results have been borne out in animal models only. RP

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