Alessandro Abbouda, MD; Bijan Khaksari, BA; and Pedram Hamrah, MD, FRCS

Corneal Neovascularization

Corneal avascularity is fundamental in maintaining a clear cornea and to guarantee optimal vision. The cornea is one of the few avascular tissues in the body. However, several corneal diseases, such as inflammatory conditions, infectious keratitis, pterygium, ocular surface neoplasia, limbal stem cell deficiency, chemical burns, and corneal graft rejection can induce corneal neovascularization.¹ Corneal neovascularization is defined as the development of new vessels originating from the limbus that invade the cornea. It can promote lipid exudation, resulting in lipid keratopathy, and corneal scarring, due to alterations of the stromal collagen architecture and spacing between collagen fibrils.²

The lack of avascularity in the normal cornea has been termed angiogenic privilege.² Several factors have been associated with maintaining corneal angiogenic privilege:

• Corneal dehydration. The relation between corneal edema and neovascularization was mentioned initially in 1949 by Cogan.³ He noted that in corneal burns, new vessels were found at the site where corneal edema was greatest.
  
• Preservation of the corneal epithelium. Corneal epithelial cells exhibit anti-angiogenic effects, thus preventing the growth of new vessels.^4,5
  
• Extensive corneal innervation, diffusion of aqueous humor across the cornea, and lower corneal temperature.²
  
• Limited levels of pro-angiogenic factors, including low levels of pro-angiogenic matrix metalloproteases (MMPs),^6,7 during hemostasis and wound healing.
  
• Barrier by limbal stem cells. The parallel orientation of band fibrils and the constant renewal of epithelial cells blocks the development of vessels from the limbus during homeostasis.²
  
• Active expression of anti-angiogenic factors and active suppression of pro-angiogenic factors in the cornea (Table 17-1).⁸

Among pro-angiogenic factors, vascular endothelial growth factor (VEGF) is the most important in the pathogenesis of corneal neovascularization and is the target used for many treatments. Although several different isoforms exist,¹⁰ VEGF-A₁₆₅ is considered to be the dominant pro-angiogenic isoform related to pathologic angiogenesis.¹¹ VEGF-A is produced by pericytes, smooth muscle cells, and macrophages during inflammation and hypoxia.^12–14 The binding of VEGF-A to its receptor VEGFR-2 promotes pathologic angiogenesis, as well as vascular dilation and enhanced permeability.^15–17

During corneal insult, pro-angiogenic growth factors cross the tissue, bind to respective receptors on endothelial cells located near the lesion site, and result in proliferation and migration of endothelial cells. Subsequently induced intracellular signaling pathways then generate the production of specific proteolytic enzymes that are capable of degrading the basement membrane collagen of capillaries and venules. Endothelial cells then proliferate and form capillary tubes, forming the backbone of future new vessels. Once proteolytic enzymes degrade stromal collagen, enhancing permeability, the new capillary tubes are able to penetrate the tissue. These capillaries dilate and then branch out, forming vascular loops and connections with neighboring vessels. Finally, specialized smooth muscle cells and pericytes stabilize capillaries by providing structural support. It is only at the end of this last step that the vascular blood flow will commence in the capillary tubes.^15,17

Bevacizumab (Avastin) is a full-length, humanized murine monoclonal antibody and has a molecular weight of 149 kD. It recognizes all VEGF-A isoforms.18 A healthy corneal epithelial barrier precludes its penetration in normal eyes; however, during corneal neovascularization, the integrity of the epithelial tight junction is lost, allowing the antibody to penetrate into the corneal stroma.¹⁹ Several studies have reported the successful use of bevacizumab for corneal neovascularization via different approaches, including topical eye drops and subconjunctival injections.^9,19–28 Both methods of administration have been shown to induce regression of new vessels^27,28 and decrease inflammation, resulting in return of normal corneal function.²⁷ Reported side effects include corneal epitheliopathy, stromal thinning, and descemetocele with prolonged use.^24,25,28

Ranibizumab

Ranibizumab (Lucentis) is a humanized recombinant monoclonal antibody fragment that binds to and inhibits all VEGF-A isoforms. The molecular weight of ranibizumab is 48 kD, making it approximately one-third the size of bevacizumab, thus in theory allowing for better corneal penetration. Further, ranibizumab has been affinity-matured and optimized for improved VEGF-A binding potential. These characteristics may allow ranibizumab to treat corneal neovascularization more effectively than bevacizumab.²⁹

Pegaptanib Sodium

Pegaptanib sodium (Macugen) is an RNA aptamer that is directed against VEGF₁₆₅. This VEGF isoform is primarily responsible for pathological corneal neovascularization and increased vascular permeability. Currently, no clinical studies using pegaptanib sodium have been performed in patients with corneal neovascularization.

Aflibercept

Aflibercept (Eylea) is a humanized recombinant fusion protein with a molecular weight of 115 kDa that is constructed from the extracellular domains of the human VEGFR-1 and VEGFR-2.³⁰ It is derived through fusion of the fragment crystallizable regions of human immunoglobulin G1 to the second binding domain of VEGFR-1 and third binding domain of VEGFR-2.³¹ Aflibercept acts like a VEGF-trap by binding to circulating VEGF molecules. In contrast to other anti-VEGF agents that solely target VEGF-A, aflibercept also blocks VEGF-B, placental growth factor-1, and placental growth factor-2. The affinity of aflibercept for VEGF-A₁₆₅ is approximately 120 times greater than bevacizumab and approximately 100 times greater than ranibizumab.³² There are a few murine preclinical studies that have demonstrated its efficacy in treating or inhibiting corneal neovascularization.^33,34

The total incidence of corneal neovascular disease in the United States has been estimated to be 1.4 million patients per year, with extended contact lens wearers, corneal transplantation, and infectious keratitis (Figure 17-1) being the most common diseases associated with corneal neovascularization.³⁵

Corneal Transplantation and Graft Rejection

Corneal transplantation is the most common organ transplantation performed in the United States and has a higher success rate compared to other forms of solid organ transplantation.^36–38 For uncomplicated low-risk patients, the survival rate is 90% for the first year.³⁹ However, in high-risk patients, such as in the setting of corneal neovascularization, the survival rate can drop to as low as 20%⁴⁰ with significantly increased rates of immune rejection.⁴¹ The number of quadrants with neovascularization in the host has been associated with a higher risk of rejection.⁴² In addition, promotion of new vessels into the donor cornea due to sutures and suturing technique, which can act as an inflammatory stimulus, has been associated with increased rate of graft rejection. This is particularly more common if the knots are placed on the limbal or host side, instead of on the donor side.⁴⁰

Recently, the use of anti-angiogenic therapies, in particular anti-VEGF agents, have led to increased success rates in the setting of high-risk corneal transplantation. Anti-angiogenic therapy has been used as an adjunct with topical or subconjunctival applications. Dekaris et al.²⁰ proposed different treatment protocols according to the number of corneal quadrants involved. In a large prospective clinical trial of 50 patients with 3 years of follow-up, all patients received subconjunctival bevacizumab (0.5 ml of 25 mg/ml). In cases with more than 2 quadrants of corneal neovascularization, patients also received topical bevacizumab (25 mg/ml, 4 times daily, not exceeding 12 weeks). The authors observed decreased neovascularization in 84% of patients and clear corneal grafts in 70% of patients, thus demonstrating successful management in the majority of high-risk cases.20

An alternative approach was described by Elbaz et al,⁴³ who proposed the use of corneal fine-needle diathermy (FND) combined with intrastromal bevacizumab injections in children. The authors inserted FND intrastromally 1 mm from the limbus to the level of the corneal vessels. Unipolar diathermy was used to blanch the vessels without observation of collagen shrinkage. Once the vessels were cauterized, bevacizumab (25 mg/ml) was injected into the stroma in proximity of the vessels. Of 9 treated eyes, 8 eyes obtained complete resolution. The rationale for this approach is to combine the efficacy of bevacizumab for new immature vessels, which is effective prior to vessels acquiring perycites,⁴⁴ with the use of FND for more mature vessels that are poorly responsive to topical bevacizumab. However, the use of FND in general has also been shown to induce pro-angiogenic factors.⁴⁵

Another approach was proposed in a prospective case-control series of 27 patients with corneal neovascularization requiring corneal transplantation.²¹ The authors pretreated the case group with 3 cycles of subconjunctival and/or intrastromal injections of bevacizumab (5 mg/0.2 ml). In the presence of a large stromal vessel, the authors injected half the dose in the conjunctiva and the other half in the stroma. After an average of six months, all patients underwent corneal transplantation; the case group also received a subconjunctival injection of bevacizumab at the end of the keratoplasty. During a mean follow-up of 26 months, none of the bevacizumab-treated patients demonstrated corneal graft rejection, while 6 out of 13 eyes in the control group demonstrated graft rejection at a mean of 3.8 months, suggesting that treatment of high-risk eyes with corneal neovascularization can improve the survival rate and decrease corneal rejection rate. In addition to the above studies, a multicenter clinical trial is currently underway to assess the safety and efficacy of topical bevacizumab 1% in high-risk corneal transplantation (clinicaltrials.gov identifier NCT01996826).

Both bevacizumab 1% and ranibizumab 1% have been shown to be effective in the treatment of corneal neovascularization as demonstrated by a decrease in the corneal neovascular area as well as vascular caliber.^46–48 However, the lack of change in the invasion area suggests treatment does not reduce vascular length, but rather the caliber alone. Thus, anti-VEGF therapy is more effective in the treatment of new immature vessels, rather than in stable neovascularization with the presence of mature vessels.⁴⁶ Preclinical experimental studies comparing ranibizumab to bevacizumab in a rabbit model of corneal neovascularization have shown that subconjunctival injection of either therapy results in the inhibition of new vessel growth in an equivalent fashion.⁴⁸ Similar to data in patients, the effect of the anti-VEGF therapy was dependent on the timing of initial therapy after induction of angiogenesis.^26,48

In addition to corneal neovascularization in the setting of full-thickness penetrating keratoplasty, corneal neovascularization can develop in the stromal interface after deep anterior lamellar keratoplasty procedures. Bevacizumab treatment has also been successfully applied for the treatment of corneal neovascularization in these cases.^9,22,23 It has been injected perilimbally22 or intrastromally into the interface adjacent to the vessels at doses of 1.25 mg²³ or 2.5 mg.⁹

In summary, anti-angiogenic treatments for corneal neovascularization before and after corneal transplantation has been shown to be safe and effective in inducing regression of vessels and promoting graft survival.

Herpes Simplex Keratitis

The recurrent nature of herpes simplex keratitis makes it the most common infectious cause of corneal blindness in the Western world. The incidence and prevalence of herpes simplex keratitis has been estimated to be around 20.7 and 149 cases/100,000 patients a year, respectively.⁴⁹ Corneal neovascularization is a main feature of herpetic stromal keratitis caused by the herpes simplex virus (HSV)-1. Several mechanisms have been associated with angiogenesis and the subsequent corneal scarring and lipid keratopathy in HSV keratitis: upregulation of VEGF-A from HSV-infected epithelial cells and infiltrating macrophages⁵⁰ and additional matrix-degrading proteases, such as MMP-9.⁵¹ As such, the inhibition of angiogenesis is advantageous, as it prevents or reduces corneal opacities due to scarring or lipid deposition. An initial case report described the positive outcome of subconjunctival bevacizumab (1.25 mg in 0.05 ml) in an elderly woman with herpes stromal keratitis.⁵² The injection resulted in rapid regression of the corneal vessels. Furthermore, no relapse was seen during the 3-month follow-up. Another case report of a young woman with corneal pannus due to HSV keratitis treated with bevacizumab demonstrated no initial change in corneal opacity and vascularization after treatment.⁵³ However, subsequent treatment with both intrastromal and subconjunctival ranibizumab injections resulted in a significant regression of the vascular area. Several other larger studies have demonstrated the efficacy of anti-VEGF therapy for the treatment of corneal neovascularization in patients with HSV keratitis.^28,43,46 No study to date, however, has directly compared different VEGF inhibitors for corneal neovascularization. In order to justify the increased cost of ranibizumab and other drugs as compared to bevacizumab, it may be necessary to demonstrate their superiority in randomized trials.

Contact Lenses Wear

Corneal neovascularization is relatively common among contact lens wearers. The prevalence is estimated to be between 10% to 30% of cases, with a variable degree of vascularization.⁵⁴ Corneal neovascularization in contact lens wearers has been described in 4 levels⁵⁵:

1. Limbal hyperemia with dilation of existing limbal capillaries
   
2. Penetration of vessels into the superficial cornea with a superficial pannus
   
3. Deep stromal vessels
   
4. Intracorneal hemorrhage

Furthermore, limbal stem cell deficiency due to chronic contact lens wear can result in variable levels of corneal neovascularization. Currently, no studies have been conducted assessing the efficacy of anti-VEGF therapies in these patients. However, a case series of 5 patients with corneal neovascularization reported the effective use of the prosthetic replacement of the ocular surface (PROSE) scleral lens as a delivery device for bevacizumab.56

Image Analysis of Corneal Neovascularization

The evaluation of corneal neovascularization by direct slit-lamp biomicroscopy does not provide a reliable and scientific methodology that can be applied to clinical trials. While vessels are visible, it is not easy to discern the variation in size, length, and diameter for all vessels. Previously, investigators used software for a more quantitative approach to assess changes in corneal vessels.^28,46 More recently, the application of corneal angiography has been an innovative and effective approach to visualize vessels beyond slit-lamp examination.^57,58 Romano et al⁵⁷ reported the application of corneal angiography using both fluorescein angiography and indocyanine green angiography using the Heidelberg system with a 15-degree, 20-degree, or 30-degree lens, and a 32 D or 53 D focus. This application was able to distinguish between presumed active and inactive vessels. Moreover, this technique provided important information about the area of corneal neovascularization, the corneal vessel diameter, as well as vascular tortuosity.⁵⁸ Corneal angiography has also been used to demonstrate vascular changes due to fine-needle diathermy.⁵⁹

Furthermore, en-face anterior segment optical coherence tomography (AS-OCT) angiography has allowed visualization of corneal vessels without the use of invasive dye.⁶⁰ The quality and resolution of images may not reach the resolution of standard corneal angiography; however, AS-OCT angiography has the ability to visualize blood flow within each layer and may be the gold standard in the years to come. Images obtained by any methodology can be analyzed using the new ImageJ plugin, VesselJ,⁶¹ which was developed to quantify vascular changes. With the increased utility of imaging devices for quantification and assessment of corneal neovascularization and its changes following therapy, centralized anterior segment reading will be required for objective and standardized analysis of vascular alterations.

Pterygium

A pterygium is a common ocular surface condition that presents as a wing-shaped fibrovascular lesion over the nasal or temporal cornea, typically extending from the bulbar conjunctiva. It can be associated with inflammation, superficial corneal neovascularization (Figure 17-2), alterations of limbal epithelial cells, squamous metaplasia, and proliferation of fibroblasts. Patients may have chronic discomfort, conjunctival hyperemia, corneal astigmatism, and, in advanced cases, significant vision loss. As such, the main purpose of pterygium excision is to remove the abnormal tissue from both the cornea and conjunctiva to rehabilitate vision and prevent recurrence.⁶²

Several surgical approaches, such as conjunctival autograft and amniotic membrane transplantation in conjunction with excision, have been advocated to reduce recurrence in these patients. The incidence of recurrence with the conjunctival autograft technique is reported to be 5.3%, whereas patients treated with the bare sclera technique have recurrence rates as high as 89%. Notably, the recurrence rate after placement of a conjunctival autograft with glue has been reported to be 9.8%,⁶² and the recurrence rate with amniotic transplantation is 20% higher than that for conjunctival autograft.⁶³

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