Angiogenesis in Glaucoma Filtration Surgery and Neovascular Glaucoma-A Review

Abstract

Angiogenesis may pose a clinical challenge in glaucoma, for example during the wound healing phase after glaucoma filtration surgery and in a severe form of secondary glaucoma called neovascular glaucoma (NVG). Up regulation of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, occurs in eyes that have undergone glaucoma filtration surgery, as well as those with NVG. This has led to studies investigating the ability of anti-VEGF therapy to improve outcomes, and we examine their findings with respect to the safety and efficacy of anti-VEGF agents, mainly bevacizumab and ranibizumab, in eyes that have undergone glaucoma filtration surgery or have NVG. Combining conventional therapies—such as anti-metabolites after filtration surgery and panretinal photocoagulation in NVG—and anti-VEGF drugs may achieve a synergetic effect, although further studies are required to evaluate the long-term efficacy of combination treatments.

I. Introduction

A. Angiogenesis

Angiogenesis is the process of new blood vessel growth from existing blood vessels. This essential process occurs naturally in growth, reproduction, and wound healing to supply nutrients and oxygen to tissues.^1,5 Pathologically, aberrant angiogenesis is associated with rheumatoid arthritis,¹⁰⁸ tumor growth and metastasis,^7,34 and eye disorders such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusions, and age-related macular degeneration.^25,37,121 The vascular endothelial growth factor (VEGF) family of cytokines promotes angiogenesis in both normal development and disease.^{1,7,25,34,37,102,108,121}

The endogenous members of the VEGF family are placenta growth factor (PlGF) and VEGF-A, -B, -C, and –D. VEGF-A serves as the principal ligand, and soluble forms of VEGF-A include VEGF-121, VEGF-145, and VEGF-165.¹¹⁶ The various members of the VEGF family and their isoforms bind to various VEGF receptors (VEGFR)-1, -2, or -3.^47,93 The signaling pathways that are activated after association of VEGFRs with their ligands and the cellular responses are summarized in Table I. The important roles of the VEGFs in angiogenesis have been demonstrated in cancers, where inhibition of the VEGF pathway inhibits the angiogenic process in various tumors.^52,73,129 Additionally, hypoxia promotes VEGF transcription, indicating that the metabolic requirements of tissues can regulate angiogenesis in order to maintain the delivery of vital nutrients to hypoxic tissues through the proliferation of new capillaries.^90,122 Because angiogenesis plays a major role in a variety of pathological conditions, angiogenic inhibitors have been the focus of numerous clinical studies,^{10,18,31,32,57,125,127} with a recent focus on anti-VEGF therapies including antibodies such as bevacizumab and ranibizumab, VEGF trap/aflibercept, and small interfering RNA directed against VEGF or VEGF receptors.^2,19,88

Table I.

VEGF Family and VEGF Receptors

Receptors	Known Signaling Pathways	Ligands15	Responses
VEGFR-1 (Flt1)	ERK/MAPK PI3K/PKB/AKT111	VEGF-A	Weak angiogenesis81 Soluble VEGFR-1 prevents VEGF-A binding to VEGFR-2 (negatively regulates angiogenesis)123
		VEGF-B	Sustains newly formed blood vessels128
		PlGF	Arteriogenesis89
VEGFR-2 (KDR)	Ras/RAFK/ERK/MAPK63,103	VEGF-A	10-fold stronger angiogenic response than VEGFR-112 Endothelial cell proliferation, survival, migration and permeability13
VEGFR-3 (Flt4)	PI3K/AKT23	VEGF-C	Lymphangiogenesis33,56,71
		VEGF-D

B. Glaucoma

Glaucoma, one of the leading causes of irreversible blindness worldwide, is normally associated with aging.²⁶ The number of people with glaucoma is predicted to increase from 64.3 million in 2013 to 111.8 million in 2040, disproportionately affecting Asian and African populations.¹¹² Glaucoma is not a single entity, but rather a term that describes a group of ocular disorders of diverse etiologies that are clinically defined as intraocular pressure (IOP)-associated optic neuropathy.¹⁶ All forms are potentially progressive and may lead to blindness,¹⁶ but the most prevalent form is primary open-angle glaucoma (POAG). POAG is characterized by changes to the optic nerve head with corresponding defects in the visual field, but retention of a normalanterior chamber.⁴⁵ Other types of glaucoma include angle-closure glaucoma, normal tension glaucoma, and secondary glaucoma such as neovascular glaucoma (NVG), exfoliative glaucoma, and uveitic glaucoma.

Normal IOP in human is between 10 and 20 mmHg. The IOP is mainly determined by the production of the aqueous humor and its drainage mainly through the trabecular meshwork at the chamber angle (so called conventional outflow pathway). ^9,16 Some aqueous humor, however, also leaves the eye via the ciliary body, through the uveoscleral or non-conventional outflow pathway.⁴⁰ The pressure gradients and resistance to the aqueous outflow are likely altered in the various types of glaucoma. POAG is frequently associated with elevated intraocular pressure (IOP).

As many as half of glaucoma cases are diagnosed in later stages of disease, because most forms of chronic glaucoma are asymptomatic.²⁸ Current therapy is targeted at the reduction of IOP to slow the progression of glaucoma.⁹ Because of their efficacy and tolerability, the conventional first-line drugs are β-blockers and prostaglandin analogs, which reduce IOP by decreasing aqueous formation and increasing uveoscleral aqueous outflow, respectively. Other antihypertensive glaucoma medications include carbonic anhydrase inhibitors, cholinergic agonists, and α2-adrenoceptor agonists. In patients who do not respond to any of the antihypertensive medications, laser trabeculoplasty or glaucoma filtration surgery may be performed to control IOP.²¹ Trabeculectomy, the most common type of glaucoma filtration surgery, is considered the mainstay of incisional antiglaucomatous surgeries.⁵⁸ The surgical goal is to bypass the TM by allowing aqueous humor to exit through a subconjunctival bleb, thereby relieving IOP.⁹¹

With the advent of anti-VEGF therapies, many clinical studies have focused on targeting VEGF in ocular disorders, including glaucoma. Anti-VEGF therapy is expected to be an effective addition to the glaucoma treatment regimen because angiogenesis occurs in the wound healing phase after glaucoma filtration surgery to maintain the intentionally created bleb and is fundamental to the underlying pathophysiology of NVG. Inhibition of angiogenesis through anti-VEGF therapy has therefore the potential to improve the success of glaucoma filtration surgery, as well as the outcome in NVG. We describe the angiogenic events that occur following glaucoma filtration surgery and in NVG and summarize pertinent findings from recently published studies evaluating the use of anti-VEGF therapy.

II. Angiogenesis in the Medical Management of Glaucoma

A. Angiogenic response to glaucoma filtration surgery

Although glaucoma is often controlled with antihypertensive medications, surgical intervention becomes necessary in certain situations such as poor patient compliance, progression of disease despite maximum medical therapy, or both.⁹⁸ Unlike most other types of surgery, a completely healed wound after filtration surgery constitutes failure. The surgery aims to create a filtering bleb that functions to drain the intraocular fluid through the sclera, which enhances the aqueous outflow and thereby reduces the IOP. Postoperative conjunctival scarring at the site of the filtering bleb, however, promotes adhesion to episcleral tissue, which leads to resealing of the bleb and thus inhibition of the aqueous flow and poor control of IOP.^39,67 The failure to maintain the bleb occurs via increased angiogenesis and fibroblast migration in the conjunctiva, leading to fibroblast proliferation with collagen deposition.¹⁰⁰

Seet et al⁹⁹ developed a systematic spatio-temporal analysis of the phases in the wound healing response to glaucoma filtration surgery using a mouse model. They observed that this post-surgical tissue response can be separated into two distinctive phases. The early “acute inflammatory” phase is characterized by significantly increased transcriptional expression of VEGF, chemokine (C-X-C motif) ligand (CXCL), and matrix metalloproteinase (MMP), as well as increased infiltration of inflammatory cells. The late “fibrotic” phase is marked by increased expression of transforming growth factor (TGF)-β2 and extracellular matrix genes with a concurrent reduction in the inflammatory cell infiltration. The increase in VEGF expression during the early phase, on both transcription and protein levels, indicates that angiogenesis is an early response in the process of bleb wound healing.

Clinically, increased bleb vascularity is associated with a poorer prognosis for trabeculectomy.^14,75 This prompted the hypothesis that decreasing vascularity via inhibition of angiogenesis could improve the outcome. Currently, anti-fibrotic medications are used as adjuncts to inhibit bleb healing; however, despite their efficacy, these are associated with several sight-threatening complications.⁹⁸ Interestingly, VEGF expression was shown to be increased in the Tenon tissue of patients who experienced failed glaucoma filtration surgeries compared to patients in whom the surgery was successful and patients without glaucoma.⁷² Such results confirm that a significant correlation exists between VEGF expression and the outcome of glaucoma surgery and suggest the potential usefulness of anti-VEGF therapy in promoting the success of glaucoma filtration surgery.

B. Angiogenesis in NVG

NVG is an aggressive form of secondary glaucoma commonly associated with proliferative diabetic retinopathy (PDR), ischemic central retinal vein occlusion (CRVO), and ocular ischemic syndrome.⁴⁴ NVG often results in poor visual outcomes. The term “neovascular glaucoma” was first coined by Weiss et al in 1963 to describe glaucoma associated with the presence of new iris and angle vessels.⁸⁵ NVG occurs when new fibrovascular tissue proliferates onto the iris and chamber angle structures including the trabecular meshwork, usually in response to ischemia of various etiologies.¹⁰⁵ In approximately 97% of NVG cases, neovascularization of the iris and angle occurs in response to retinal ischemia, whereas only a small fraction of the cases is caused by inflammation without ischemia.¹⁰⁵ Retinal ischemia induces production of pro-angiogenic factors that diffuse into the anterior segment and promote neovascularization of the iris, the angle, or both.³ The fibrovascular membrane that is created inhibits the aqueous flow and leads to an increase in IOP. Contraction of this abnormal tissue induces the development of peripheral anterior synechiae and progressive angle closure, which further increases IOP to harmful levels that cannot be controlled via conventional antiglaucoma therapy.³

III. Therapy in Glaucoma Filtration Surgery

A. Current therapy

In order to maintain the bleb created in filtration surgery, medications are used as adjuncts to inhibit wound healing of the bleb. 5-Fluorouracil (5-FU) and mitomycin C (MMC) are anti-metabolites commonly used to limit wound healing via the induction of widespread fibroblast cell death.¹⁰⁶ MMC is preferentially used as the intraoperative antifibrotics, more than twice as often as 5-FU,⁶⁰ and has been validated by Cochrane meta-analysis to reduce significantly the IOP and the risk of surgical failure in eyes that have undergone no previous surgery and in eyes at high risk of failure.¹²⁰ Despite their efficacy in reducing post-operative scarring,⁶⁷ however, anti-metabolites are associated with complications such as hypotony with maculopathy, cystic avascular bleb, bleb leak, bleb infection, and endophthalmitis.^39,53,95

Because TGF-β2 has been reported to stimulate proliferation and migration of human Tenon fibroblasts,²² CAT-152, a human, monoclonal antibody against TGF-β2, was recently evaluated for its efficacy in a large, multicenter, randomized clinical trial.⁴³ Although the effects of CAT-152 in animal models have been encouraging,⁷⁸ the clinical study revealed no significant efficacy for the prevention of bleb failure over a 12-month follow-up period.⁴³ The limitations regarding the safety and efficacy of anti-fibrotic pharmaceutical agents as adjuncts have thus motivated researchers to continue searching for better alternatives.

Several other types of adjunctive agents such as Rho-associated protein kinase inhibitor,⁴⁹ β irradiation,⁹² and triamcinolone acetonide⁴⁸ have been investigated as potential alternatives, but are not yet routinely used in clinical settings for trabeculectomies. Anti-VEGF therapy, on the other hand, has demonstrated clinical effectiveness. Anti-VEGF adjuncts have been tested in preliminary in vitro and animal studies, as well as small clinical trials, that have shown promising results in reducing postoperative scar formation.

B. Anti-VEGF approach

a. Role of VEGF in bleb scarring

VEGF is most commonly known as a stimulator of endothelial growth and vascular permeability, but it is also an important mediator in wound healing and scar formation. To achieve these functions, VEGF stimulates the angiogenic cascade to provide conduits for oxygen, nutrients, and other mediators involved in wound healing,⁶ which is required for the formation of granulation tissue.⁶⁸ There is enhanced healing upon stimulation of angiogenesis,^{38,82,118,124} as well as delayed healing when angiogenesis is inhibited.^8,76,94

VEGF not only regulates fibrosis via angiogenesis, but also acts as a mediator in a signaling pathway that promotes fibroblast migration, proliferation, and collagen production.^6,119 VEGF has been shown to induce proliferation of Tenon fibroblasts in vitro during the post-trabeculectomy wound healing process.⁶⁹

VEGF directly stimulates both vascular endothelial cells and fibroblasts and may be the link between angiogenesis and scar formation.¹¹⁹ Among different VEGF isotopes, VEGF-A is the only one showing significantly decreased expression in later stages of wound healing.⁹⁹ This suggests that VEGF-A may be involved in the transition from the early to late phases of wound healing. Among the different isoforms of VEGF-A, VEGF-121, VEGF-165, and VEGF-189 are expressed in rabbit Tenon fibroblasts.⁶⁹ In vitro the addition of VEGF-121 and VEGF-165 stimulates endothelial cell proliferation, whereas the addition of VEGF-121 and VEGF-189 increases fibroblast growth.¹¹³ Although VEGF-121 stimulates proliferation of both endothelial cells and fibroblasts, its effect is more prominent in endothelial cells. This suggests that VEGF-121 and VEGF-165 predominantly affect blood vessel growth, whereas VEGF-189 may be more important in fibrosis. Since VEGF signaling is involved in both angiogenesis and fibrosis, two critical processes in scar formation, inhibition of all isoforms of VEGF may delay bleb healing after glaucoma filtration surgery.

b. Anti-VEGF therapy

A number of studies, including small clinical trials, have investigated anti-VEGF antibodies such as bevacizumab and ranibizumab as potential adjunctive agents in glaucoma filtration surgery. Both antibodies bind to all of the isoforms of VEGF-A; however, ranibizumab is a mature antibody designed to have a significantly stronger binding affinity than bevacizumab.⁸⁸

1. Bevacizumab

Li et al⁶⁹ reported that administration of bevacizumab significantly inhibited VEGF-induced Tenon fibroblast proliferation in human (P=0.04) and rabbit (P=0.02) in a dose-dependent manner. Similarly, O’Neill et al⁸⁴ demonstrated, in an in vitro model of wound healing with human Tenon fibroblasts, that bevacizumab disrupted fibroblast proliferation, inhibited collagen gel contractility, and induced fibroblast death at concentrations greater than 7.5 mg/mL in serum-free conditions. Li et al⁶⁹ also showed that a single application of bevacizumab into the subconjunctival space and anterior chamber at the time of trabeculectomy resulted in a larger bleb area in a rabbit model (n=34; P<0.05). The IOP, however, was similar in the treated and control eyes 29 days after surgery. Memarzadeh et al⁷⁹ reported similar results in a larger animal study in which 42 randomized rabbits received seven subconjunctival injections of bevacizumab, 5-FU, or balanced salt solution during the first 14 days after trabeculectomy. There was no significant difference in the mean IOP, but bevacizumab did more than double the bleb survival time (P<0.05) compared to the other two treatments. Ozgonul et al⁸⁶ also demonstrated the efficacy of anti-VEGF therapy through a study that compared the efficacy between subconjunctival and intravitreal applications of bevacizumab in rabbit models. They reported that subconjunctival injection of bevacizumab resulted in a greater area and height of the bleb and lower mean IOP compared to intravitreal bevacizumab, 5-FU, and control groups. Inflammation (P=0.030), neovascularization (P=0.004), and fibrosis were also lower in the subconjunctival bevacizumab group. Although achieving significant differences in IOP is difficult in animal models, the bleb morphologic features and bleb survival time support the efficacy of bevacizumab in improving outcomes of glaucoma filtration surgery.

The safety and efficacy of bevacizumab in trabeculectomy have in addition been tested in a number of clinical trials. Vandewalle et al¹¹⁴ studied the effect of a single intracameral bevacizumab injection in a 12-month prospective, randomized trial with 138 patients. The IOP at 1 year postoperatively was significantly lower than baseline in the placebo (P<0.01) and the treated (P<0.01) group. The bevacizumab-treated group, however, had a higher absolute success rate (P=0.02) and required less IOP-lowering interventions (P=0.003). Grewal et al⁴¹ evaluated the effect of subconjunctival bevacizumab through a nonrandomized, open-label, prospective, interventional case series of 12 patients. After a follow-up of 182 days, IOP control was observed in 92% of the eyes, with an average IOP reduction of 52%. An interesting observation in their study was that the bleb vascularity began to increase 3 months after administration of bevacizumab. While this might have decreased the development of cystic avascular blebs that frequently develop following MMC-augmented surgery, it also raised a concern for possible future bleb failure.

To gauge its value, anti-VEGF therapy was compared to the commonly used anti-metabolites, 5-FU and MMC. Sengupta et al¹⁰¹ analyzed the conventional MMC and both subconjunctival and topical administrations of bevacizumab in 38 patients undergoing single-site phacotrabeculectomy. All three groups had a significant reduction in mean IOP that was sustained at 6 months. However, 90% of patients treated with subconjunctival bevacizumab showed complete success, which was defined as IOP <18 mm Hg or at least a 20% reduction from the baseline IOP at the end of the follow-up period, as opposed to 60% in the other two groups (P=0.04). They also observed a gradual increase in the vascularity over a 6-month period in blebs of patients who received bevacizumab. Nilfoushan et al⁸³ compared the outcome of trabeculectomy with subconjunctival bevacizumab (n=18) versus MMC (n=18) in a prospective, randomized study. Although IOP was significantly reduced in both groups compared to baseline, the reduction was more prominent in the MMC-treated group at 12 months, with 34% and 56% IOP reductions in the bevacizumab- and MMC-treated groups, respectively. Similarly, Jurkowska et al⁵⁴ compared the effects of bevacizumab and 5-FU in a nonrandomized, prospective, interventional case study in which 21 patients received intraoperative 5-FU and 32 patients received subconjunctival bevacizumab immediately before and after surgery and again 1 and 7 days after surgery. A significant reduction in IOP occurred in both group, but a greater percentage of 5-FU–treated patients (86.7%) experienced a 30% reduction in initial IOP compared to bevacizumab-treated patients (78.1%) at the end of 12-month follow-up (P=0.38). There were no notable differences between the two groups in terms of visual field indices and postoperative complications; however, more patients in the bevacizumab-treated group required additional medical interventions to successfully control IOP.

2. Ranibizumab

Ranibizumab significantly induces human Tenon fibroblast death compared to serum-free control condition in vitro (P<0.05)⁷⁷ and also significantly reduced collagen type 1 α1 (COL1α1) mRNA, but not fibronectin (FN) mRNA, expression. COL1α1 and FN protein levels, however, were upregulated in the cells treated with ranibizumab compared to the untreated control (P<0.01 and P<0.05, respectively), and such opposing results for protein and mRNA expression following ranibizumab administration hinders any attempts to draw conclusions from the study regarding VEGF’s effects on collagen expression. Nevertheless, these results confirm the direct effect of anti-VEGF antibodies on Tenon fibroblast proliferation and possibly collagen production. The effects of bevacizumab and ranibizumab in filtration surgery are summarized in Table II.

Table II.

Application of Anti-VEGF Agents in Glaucoma Filtration Surgery

Anti-VEGF Agents	Mechanism of Action	Method of Administration	Outcome			References
			In vitro	Animal Model	Clinical Trial
Bevacizumab (Bvb)	Binds to all isoforms of VEGF-A, inhibiting ligand-receptor (VEGFR-1 and VEGFR-2) interaction97	Intracameral	Inhibition of rabbit and human Tenon fibroblast proliferation and collagen gel contractility in a dose-dependent manner	--	No significant effect in terms of IOP control but higher absolute success rate compared to placebo No significant additive effect when combined with 5-FU	Li et al69 O’Neill et al84 Memarzadeh et al79 Ozgonul et al86 How et al50 Vandewalle et al114 Grewal et al41 Sengupta et al101 Nilforushan et al83 Jurkowska-Dudzinska et al54 Suh et al107 Chua et al20
		Subconjunctival		Larger, more diffused, and less vascular bleb with longer bleb survival time but no significant effect on IOP Combination with 5-FU has better antifibrotic effect than monotherapy	No significant difference in terms of safety and efficacy compared to 5-FU or MMC No significant additive effect when combined with 5-FU Gradual increase in bleb vascularity starting 3 months after treatment
Ranibizumab (Rbb)	Affinity - matured to stronger affinity than Bvb, Rbb binds to all isoforms of VEGF-A inhibiting ligand-receptor binding (VEGFR-1 and VEGFR-2)88,97	Intravitreal	Inhibition of human Tenon fibroblast proliferation and reduction of COL1α1 transcription	--	Larger, more diffuse blebs with lower vascularity when combined with MMC	Noh et al77 Kahook et al55

3. Combination therapy

As a number of studies have shown that anti-VEGF therapy alone may not be sufficient to prevent scar formation, researchers have investigated whether combined therapy can offer additive or synergistic effects. How et al⁵⁰ analyzed the effect of combining bevacizumab and 5-FU and found that this combination results in a superior antifibrotic effect compared to monotherapy with 5-FU or bevacizumab in rabbit models, as evidenced by a decrease in FN and mature COL1 expression and deposition (P<0.05). Additionally, 100% bleb survival at 28 days was observed in the combined treatment group, whereas bleb survival in the monotherapy groups (50% bevacizumab [P<0.05] and 25% 5-FU [P<0.001]) was significantly lower. A significant reduction in conjunctival vascularity also was observed in the combined therapy group as well as the bevacizumab-only group.

Clinical studies, however, did not show any additive advantages of bevacizumab and 5-FU combination therapies. Suh et al¹⁰⁷ recently performed a clinical study comparing outcomes in 12 patients who received combination therapy with intracameral and subconjunctival bevacizumab and subconjunctival 5-FU to those in 24 patients who received only subconjunctival 5-FU at the time of trabeculectomy. They found no significant difference between the two groups with regards to visual acuity, postoperative IOP, and anti-glaucoma medication use after surgery (P>0.05), indicating that the addition of bevacizumab did not have a significant additive benefit compared to 5-FU monotherapy. Similarly, Chua et al²⁰ analyzed a combination therapy of subconjunctival bevacizumab and 5-FU (n=22) versus monotherapy of 5-FU (n=21) and observed no significant differences in terms of visual acuity, IOP, or postoperative interventions between the two groups. By 18 months, central bleb avascularity was more frequent in the combined group than the 5-FU group (47.4% and 21.1%, respectively; P=0.17), but this effect was not statistically significant.

By contrast, Kahook et al⁵⁵ reported positive results from their pilot study in which 10 patients were randomized to either trabeculectomy with only MMC or trabeculectomy with a combination of intravitreal ranibizumab and MMC. There were statistically significant differences in peripheral bleb area (P=0.02), peripheral bleb vascularity (P=0.02), and non-bleb-related peripheral conjunctiva vascularity (P=0.0003), with the combined group exhibiting more diffuse blebs with a lower degree of vascularity (Table II). The positive results, however, should be regarded with caution because it was insufficiently powered for a long-term follow-up and the conclusions drawn based mainly on bleb morphology, not mean IOP.

4. Other anti-VEGF agents

In addition to bevacizumab and ranibizumab, other relatively uncommon anti-VEGF agents also have been explored. Van Bergen et al¹¹³ evaluated the effect of specific VEGF inhibition in vitro using pegaptinib, an antibody specifically targeting the VEGF-165 isoform. Although proliferation of human umbilical vascular endothelial cells was effectively inhibited by pegaptinib in a dose-dependent manner, growth of human Tenon fibroblasts was only significantly reduced by the highest dose of this selective inhibitor. They also tested the effect of pegaptinib in a rabbit trabeculectomy model. A single administration of pegaptanib at the time of surgery significantly reduced angiogenesis during the initial phase (P≤0.03) compared to the control, but there were no significant differences in inflammatory responses and collagen deposition.

Additionally, a preliminary study was performed using trehalose, an anti-angiogenic agent that indirectly targets VEGF. After discovering that trehalose inhibits conjunctival neovascularization and fibroblast proliferation in their previous study¹¹⁰, Takeuchi et al¹⁰⁹ investigated the effect of trehalose on VEGF-stimulated angiogenesis and myofibroblast proliferation in vitro. They observed a significant dose-dependent inhibition of neovascularization (P<0.01), as well as a partial inhibition of myofibroblast proliferation via stimulation of mesenchymal-epithelial transition. Although trehalose is a rather unconventional and indirect anti-VEGF agent, the promising outcomes from the in vitro study suggest the possibility of its use as an adjunctive antiscarring agent in the future.

IV. Anti-VEGF Therapy in NVG

A. Current therapy

Treatment of NVG involves reducing IOP as well as treating the underlying disease process responsible for NVG. Panretinal photocoagulation (PRP) is the current gold standard treatment for NVG. PRP reduces the global retinal oxygen demand by destroying the nonessential ischemic retinal tissue to remove the stimulus for production of vasoproliferative factors.⁴ A decrease in VEGF levels was reported in patients who received PRP, along with a resultant decrease in neovascularization.¹⁷ Although PRP has shown effectiveness in treating NVG, this treatment leads to death of healthy cells and permanently diminishes visual fields.³⁶ PRP also does not result in rapid regression of iris and angle neovascularization, and patients continue to experience high IOP and discomfort for a period of time after the procedure. Moreover, PRP is difficult to perform in patients with NVG with media opacities such as corneal edema and cataract. Hence, treatments aimed at reducing the pro-angiogenic factors directly, such as the use of anti-VEGF antibodies, could help reduce and possibly reverse the neovascularization. Although anti-VEGF therapy does not directly address the underlying ischemia in NVG, it can control the pathologic process without destroying healthy retinal cells.

While PRP and/or anti-VEGF interventions may be sufficient to control the IOP in open-angle NVG, subsequent glaucoma therapy (with or without surgery) is usually indicated when most of the angle is closed due to synechiae.¹⁰⁴ Aqueous drainage devices,⁸⁰ cyclocryocoagulation,⁶⁵ transscleral diode laser,³⁵ Nd-Yag cyclophotocoagulation,²⁷ and vitrectomy with PRP and trabeculectomy⁶² are the possible methods of treatment. Although the aqueous drainage device has traditionally been preferred over trabeculectomy, advent of anti-VEGF therapy has resulted in increasing use of the latter. The use of anti-VEGF agents has been shown to decrease IOP, possible complications, and hyphema postoperatively, thereby improving the surgical success rates.^61,96

B. Anti-VEGF approach

a. Role of VEGF in NVG

VEGF is synthesized by multiple types of retinal cells in response to retinal ischemia. Studies have found that VEGF levels are significantly elevated in the aqueous humor of patients with rubeosis and NVG.^51,59,130 In addition, the aqueous and vitreous levels of VEGF are higher in patients with NVG associated with diabetes than in diabetic patients with only proliferative retinopathy.⁶⁶ These results confirm the importance of VEGF signaling in neovascularization in NVG. Therefore, the use of VEGF inhibitors to reduce VEGF levels has been explored as a treatment option for NVG, and multiple case series have indicated the treatment efficacy of anti-VEGF agents, predominantly intravitreal injections of bevacizumab and ranibizumab, in reducing neovascularization in the iris and angle in NVG. These are summarized in Table III and described in more detail below.

Table III.

Application of Anti-VEGF Agents in Neovascular Glaucoma

Anti-VEGF Agents	Mechanism of Action	Administration	Clinical Trial Results	References
Bevacizumab (Bvb)	Refer to Table II	Intravitreal	Reduced iris neovascularization and IOP Combination therapy with PRP and retinal ablative procedure showed a longer lasting open angle, improved visual acuity, and decreased IOP Reduced pain within 1 week after first administration	Yazdani et al126 Brouzas et al11 Ehelers et al30 Vasudev et al115 Kotecha et al64 Costagliola et al24 Lim et al70 Duch et al29 Waisbourd et al117
		Intracameral	Decreased concentration of VEGF in aqueous humor No significant change in IOP Allowed or eliminated need for angle filtration surgery
		Topical	Reduction of mean IOP
Ranibizumab (Rbb)		Intravitreal	Reduced and maintained IOP in patients with rubeosis and NVG better than in patients with rubeosis only	Luke et al74

b. Anti-VEGF therapy

1. Bevacizumab

Many clinical trials have evaluated the use of anti-VEGF antibodies to treat NVG. In one of the first randomized clinical trials evaluating the efficacy of intravitreal bevacizumab, NVG patients treated with three intravitreal bevacizumab injections at 4-week intervals exhibited a significant decrease in IOP compared to their baseline IOP as well as significant regression in iris neovascularization at 1 month (P=0.007 and P=0.01, respectively), 3 months (P=0.058 and P=0.004), and 6 months (P=0.047 and P=0.004) post-treatment time points.¹²⁶ An observational case series tracked 50 adults with NVG who could not be treated with PRP. Six months after these patients were treated with intravitreal bevacizumab, they reported a reduction in pain from their baseline pain score starting 1 week after administration (P<0.001).⁶⁴ Additionally, a short-term clinical study reported that patients with NVG displayed a considerable reduction in VEGF concentration in the aqueous humor (P=0.04) without a significant change in IOP 2 weeks after receiving an intracameral injection of bevacizumab.⁷⁰ In a similar pilot study evaluating the effect of intracameral bevacizumab before any surgical treatment for NVG, some patients were found to no longer require any surgical intervention, whereas other patients became candidates for filtration surgery.²⁹ Upon testing the use of bevacizumab as a topical treatment, another pilot study found that after topical application of bevacizumab 4 times a day consistently for 2 weeks the mean IOP of eight patients was reduced by 17.5%.¹¹⁷

2. Ranibizumab

The efficacy of ranibizumab was investigated in a clinical series by Luke et al⁷⁴ who showed that patients with both rubeosis and NVG experienced considerable iris neovascularization regression (P<0.001) and a rapid IOP reduction from their baseline (P=0.005) 2 weeks after ranibizumab injection. Moreover, the improvement in IOP was maintained for the follow up period of 12 months (P<0.05). They concluded that ranibizumab is effective as an adjuvant treatment for NVG and rubeosis because it preserves the anterior chamber angle.

3. Combination therapy

Brouzas et al¹¹ published a clinical series evaluating the safety and efficacy of monthly intravitreal bevacizumab with standard NVG treatment. The case series demonstrated that NVG treatment along with monthly injections of bevacizumab was effective in decreasing iris and anterior chamber angle neovascularization. After these combined treatments, however, acceptable IOP levels were not maintained, requiring additional medical interventions. Ehelers et al³⁰ compared combination therapy consisting of PRP and same-day intravitreal bevacizumab injection to PRP alone. They showed that total neovascular reduction occurred more often (P<0.001) and more rapidly (P<0.0001) in the combined group. In addition, IOP decreased more rapidly in the combined treatment group (P=0.03). Similarly, Vasudev et al¹¹⁵ compared the effects of intravitreal bevacizumab with PRP treatment to PRP treatment alone on the angle of the anterior chamber. From the 1-year follow-up, patients treated with bevacizumab and PRP were seen to have a longer lasting open angle than those treated with PRP only (P<0.05).

In addition to PRP, the value of adding bevacizumab to current NVG treatments was also investigated in retinal ablative procedures²⁴. After 12 months, NVG patients who received intravitreal bevacizumab injection after a retinal ablative procedure had reduced vasculature in the iris, better visual acuity (P=0.0001), and decreased IOP from their baseline (P=0.0001; Table III).

V. Future Directions

A. Glaucoma filtration surgery

Additional studies with larger sample sizes and a more systematic approach are necessary to gain a better understanding of the effect of anti-VEGF agents in patients undergoing glaucoma filtration surgery. More evidence is needed to support the use of VEGF inhibitors for preventing postoperative angiogenic and fibroblastic activities, including determination of the most effective doses and the optimal timing and route of administration (intravitreal, subconjunctival, or intracameral). The gradual increase in bleb vascularity by 3 months after administration of VEGF inhibitors, as observed in multiple clinical studies,^41,101 must be addressed to avoid bleb failure associated with increased bleb vascularity. Multiple administrations of anti-VEGF agent may be necessary to suppress angiogenesis and achieve long-term preservation of the bleb.

Several comparison studies have shown that the safety and efficacy of anti-VEGF therapy are not significantly different from those of current antiscarring medications. To confirm these results, a larger randomized clinical trial is planned.⁵⁴ Although only a few clinical studies have examined the efficacy of bevacizumab combined with an anti-metabolite in comparison to that of an anti-metabolite alone, most of these studies have reported no significant additive effect. A clinical study with a larger cohort and longer follow-up period is required to confirm and better understand these findings.

Moreover, combination therapy using anti-angiogenic and anti-fibrotic agents should be further investigated as these processes occur at different times in wound healing. TGF-β2 exerts anti-inflammatory as well as pro-fibrotic effects in subconjunctival scarring. The difficulty of mediating these contradictory effects may explain, in part, the lack of success to date of anti-TGF-β2 therapy as an adjunctive agent following glaucoma filtration surgery.⁴² The use of anti-VEGF agents in combination with anti-TGF-β2 agents not only allows abrogation of both early and late stages of scar formation, but may also enhance the anti-fibrotic effect of anti-TGF-β2 therapy by potentially reducing inflammation. Defining the early and late phases of bleb healing in humans will also be advantageous for improving the specificity of molecularly targeted therapies and outlining the therapeutic windows for optimal use.

B. Neovascular glaucoma

The main concerns regarding clinical trials using anti-VEGF antibodies in NVG are the length of evaluation and sample size. Although bevacizumab and ranibizumab were found to be effective for reducing iris and anterior chamber neovascularization, larger studies evaluating patients over longer time periods will further determine their long-term effect. This is important because reappearance of neovascularization may recur.^11,30,126 More studies are needed to evaluate the effect of these antibodies on IOP, especially given that the finding of Brouzas et al¹¹ conflicts with those of other studies,^30,117,126 with Brouzas et al reporting that IOP could not be maintained without the use of additional drugs. This suggests that combination therapy with another drug may more effectively treat NVG.

Although there has been a focus on using bevacizumab in combination with PRP, another therapeutic strategy employing bevacizumab involves administering the drug beforehand to allow PRP in patients who initially are unable to undergo PRP.²⁹ Further studies on this treatment approach are warranted. Likewise, it would be worthwhile to investigate the effects of intracameral bevacizumab injections on IOP and neovascularization, because the decrease in VEGF concentration with this treatment is a promising finding.⁷⁰

In addition to antibodies such as bevacizumab and ranibizumab, aflibercept could potentially be another anti-VEGF agent for treating NVG. The NCT01711879 a 52-week randomized controlled clinical trial investigating the efficacy of intravitreal aflibercept injections in participants with NVG compared to standard PRP, is underway. Aflibercept, which is a novel humanized recombinant fusion protein designed to bind all isoforms of VEGF-A as well as PIGF,² is known to be effective in treating neovascular age-related macular degeneration⁴⁶ and is reported to have broader anti-angiogenic effect and higher binding affinity for VEGF-A isoforms than other anti-VEGF therapies.⁸⁷ With evidence demonstrating excellent responses to anti-VEGF therapy in NVG, such an addition to the available anti-VEGF agents may improve treatment outcomes.

VI. Method of Literature Search

In order to prepare this review, we conducted a Medline and PubMed search of the medical literature for the period between 1963 and 2015 using the following key words in various combinations: angiogenesis, glaucoma, VEGF, neovascularization, anti-VEGF therapy, bevacizumab, pegaptanib, ranibizumab, glaucoma filtration surgery, neovascular glaucoma, topical, subconjunctival, intravitreal, and intracameral. In addition, reference lists from the selected articles were used to identify additional articles not included in the electronic database. Articles were appraised critically and pertinent information was included in this review and cited accordingly.

VII Disclosure

The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. Publication for this article was supported by National Institutes of Health grants, EY021886, EY023691, VA I01 BX002386 (JHC), UL1TR000050 (UIC CCTS), and EY01792 (UIC core grant); an unrestricted grant from Research to Prevent Blindness, New York, NY; and an award from the Illinois Society for the Prevention of Blindness (HY).