**5. Paraclinical evaluation of corneal vascularization**

cally. The level of vascularization is chiefly related to the level of pathology rather than to the etiology. Superficial corneal pathology results in superficial vascularization, and deep pathol‐ ogy results in deep vessels. Often when the disease process extends through the thickness of

red with a slow circulation, and some parts of the complex are less visible or have undergone attrition.

**Figure 8.** Partially regressed vessels with lipid keratopathy (asterisk) at the donor‐recipient interface in a patient who underwent deep anterior lamellar keratoplasty (DALK). Vessels arising from the limbus sharply dip into a deep suture track and continue to the deep lamellar plane created by the DALK procedure, before fanning out. The vessels are dull

A detailed clinical evaluation of corneal neovascularization, including extension (the num‐ ber of quadrants involved) and depth, is crucial for treatment planning. In addition to the extent and level of corneal vascularization, the state of vessel activity is also important [30]. Clinically, corneal vascularization can be classified as active young, active old, mature, par‐ tially regressed, and regressed. This often corresponds with the stage of activity or chronicity of the disease. Active young vessels are freshly formed vessels that are full of blood, appear bright red in color, have minimal surrounding fibrous tissue sheathing, and are actively progressing in the cornea with a well‐defined arborizing network of fine (capillary) vessels (**Figures 4A** and **7**). The corneal stroma surrounding the vessels shows signs of leakage and edema. Active old vessels appear less bright and maintain a brisk circulation (**Figure 5**). This represents the stage when the vessels have reached and surrounded or covered the offending lesion in the cornea. Their progression ceases but consolidation continues. Mature vessels are relatively large vessels, with minimal arborization and regressed or absent capillary net‐ works, seen to persist in scar tissue or in the corneal stroma after the corneal pathology has healed. These vessels contain blood and maintain a circulation (**Figure 9**). Partially regressed vessels are seen when the corneal pathology has abated in response to therapy or the arrival

the cornea, superficial and deep vessels are seen in the same cornea.

64 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Accurate evaluation and documentation of corneal neovascularization are essential to moni‐ tor the effect of any treatment modality employed. Case note entries can be used to assess the extent of corneal vascularization, and the depth of penetration and the centripetal progression of vessels, which allows a semiquantitative measurement of corneal neovascularization. It is neither time efficient nor practical, however, to manually trace the corneal vessels in each fol‐ low‐up examination. Furthermore, the reproducibility is questionable, and the opportunity for variability and human error is very high.

The need to measure corneal neovascularization motivated researchers to explore measurement tools. An ideal measurement tool should allow rapid, reproducible, accurate, and objective mea‐ surement of corneal neovascularization. Digitized photographs with good contrast can be ana‐ lyzed, based on the grayscale values, to evaluate the progression of vascularization [32]. Corneal vessels can be quantified on the basis of contrast enhancement, density threshold identification for the blood vessels, and pixel measurement [33]. A more novel automatic approach on the basis of gray filter sampling and threshold analyses of digital photographs using an image analysis software has also been investigated [34, 35]. Despite the recent progress in the graphic editing software, automated methods have some limitations. First, the optimization and validation of any automated quantitative tool are questionable [36–38]. Second, it does not allow sufficient appreciation of details on vessel extent, localization, leakage, origin, and differentiation of the afferent and efferent systems. This information is of importance for guidance of clinical judg‐ ment and treatment [39].

Corneal angiography, using fluorescein and indocyanine green, provides excellent details of the neovascular complexes, thus enabling an enhanced clinical assessment and decision‐mak‐ ing even in patients with complex corneal neovascularization [39]. The required technological equipment for corneal angiography is readily available in most ophthalmologic centers, as angiography is widely used to diagnose vascular disorders of the retina of various origin. It is a relatively inexpensive and safe diagnostic intervention, and serious adverse events like anaphylaxis to the intravenous dye are extremely rare [40, 41].

Fluorescein angiography gives an indication of the vessel maturity and leakage activity, whereas indocyanine green angiography allows better depiction of capillaries and deeper corneal neo‐ vascularization, particularly in the presence of vessel obscuration because of corneal haze and scarring [39]. It is possible to calculate the area of corneal neovascularization, the time to first detection of fluorescein dye leakage, corneal neovascular vessel diameter, and vascular tortuos‐ ity and activity. These parameters reliably quantify changes in corneal neovascularization over time [39]. Therefore, it allows monitoring of the natural course and treatment success [42].

## **6. Treatments**

The treatment for corneal neovascularization aims at the occlusion of afferent corneal blood vessels to reduce exudative lipid keratopathy, and stromal edema and inflammation or as a preoperative conditioning intervention before keratoplasty to increase chances of graft sur‐ vival [17, 43]. Current treatments for corneal neovascularization consist of topical nonste‐ roid anti‐inflammatory and corticosteroid medications [44], photodynamic therapy [45], laser photocoagulation [46, 47], fine needle diathermy [48], and limbal, conjunctival, and amniotic membrane transplantation (AMT) [49]. More recently, manipulation of VEGF activity and manipulation of proangiogenic mediators like interleukin have been under investigation [50, 51]. Unfortunately, all of these approaches have a limited clinical efficacy, especially when the vessels are large because large vessels are difficult to occlude and easily recanalized. In addi‐ tion, a multitude of undesirable side effects can occur after the treatment of corneal neovas‐ cularization. The following section reviews the available treatment approaches for corneal neovascularization and their limitations.

#### **6.1. Corticosteroid therapy**

Inflammation is a potent driver for corneal neovascularization. When inflammation set‐ tles, spontaneous regression of corneal neovascularization can occur and lead to gradual resolution of lipid keratopathy if present. Topical and periocular steroids have been popu‐ lar and can effectively reduce inflammation and consequently corneal neovascularization in various disease conditions. However, the risks of superinfection, glaucoma, and cata‐ ract associated with the long‐term use of corticosteroids have been a limiting factor [44]. Additionally, steroids have only limited antiangiogenic effects [52]. Cyclosporine A and nonsteroidal anti‐inflammatory agents were reported to be largely ineffective in controlling or limiting corneal angiogenesis [53].

## **6.2. Laser photocoagulation**

for the blood vessels, and pixel measurement [33]. A more novel automatic approach on the basis of gray filter sampling and threshold analyses of digital photographs using an image analysis software has also been investigated [34, 35]. Despite the recent progress in the graphic editing software, automated methods have some limitations. First, the optimization and validation of any automated quantitative tool are questionable [36–38]. Second, it does not allow sufficient appreciation of details on vessel extent, localization, leakage, origin, and differentiation of the afferent and efferent systems. This information is of importance for guidance of clinical judg‐

Corneal angiography, using fluorescein and indocyanine green, provides excellent details of the neovascular complexes, thus enabling an enhanced clinical assessment and decision‐mak‐ ing even in patients with complex corneal neovascularization [39]. The required technological equipment for corneal angiography is readily available in most ophthalmologic centers, as angiography is widely used to diagnose vascular disorders of the retina of various origin. It is a relatively inexpensive and safe diagnostic intervention, and serious adverse events like

Fluorescein angiography gives an indication of the vessel maturity and leakage activity, whereas indocyanine green angiography allows better depiction of capillaries and deeper corneal neo‐ vascularization, particularly in the presence of vessel obscuration because of corneal haze and scarring [39]. It is possible to calculate the area of corneal neovascularization, the time to first detection of fluorescein dye leakage, corneal neovascular vessel diameter, and vascular tortuos‐ ity and activity. These parameters reliably quantify changes in corneal neovascularization over time [39]. Therefore, it allows monitoring of the natural course and treatment success [42].

The treatment for corneal neovascularization aims at the occlusion of afferent corneal blood vessels to reduce exudative lipid keratopathy, and stromal edema and inflammation or as a preoperative conditioning intervention before keratoplasty to increase chances of graft sur‐ vival [17, 43]. Current treatments for corneal neovascularization consist of topical nonste‐ roid anti‐inflammatory and corticosteroid medications [44], photodynamic therapy [45], laser photocoagulation [46, 47], fine needle diathermy [48], and limbal, conjunctival, and amniotic membrane transplantation (AMT) [49]. More recently, manipulation of VEGF activity and manipulation of proangiogenic mediators like interleukin have been under investigation [50, 51]. Unfortunately, all of these approaches have a limited clinical efficacy, especially when the vessels are large because large vessels are difficult to occlude and easily recanalized. In addi‐ tion, a multitude of undesirable side effects can occur after the treatment of corneal neovas‐ cularization. The following section reviews the available treatment approaches for corneal

Inflammation is a potent driver for corneal neovascularization. When inflammation set‐ tles, spontaneous regression of corneal neovascularization can occur and lead to gradual

anaphylaxis to the intravenous dye are extremely rare [40, 41].

66 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

ment and treatment [39].

**6. Treatments**

neovascularization and their limitations.

**6.1. Corticosteroid therapy**

Photocoagulation of vessels has been shown to be an effective method to obliterate corneal vas‐ cularization [46, 47]. The argon laser [46] and the 577 nm yellow dye lasers [47] have been used effectively for treating vascularization in lipid keratopathy and graft rejection. Laser obliteration of corneal efferent vessels is comparatively easy as they are wider and have a relatively slower blood flow. Conversely, the afferent vessels are narrower and deeper, have a rapid blood flow, and are more difficult to obliterate. Consequently, reopening of the afferent vessels takes place in a high proportion of patients. In such cases, the procedure can be repeated more than once. Laser photocoagulation may not be effective in cases with extensive corneal neovascularization [46]. Other drawbacks include damage to iris and accidental suture lysis, which has a signifi‐ cant implication for grafts with running sutures. Furthermore, the expense of this equipment and the lack of availability in most centers make the treatment inaccessible to most surgeons.

#### **6.3. Fine needle diathermy**

Fine needle diathermy (FND) is an inexpensive and useful procedure that can serve as an adjunct or alternative to laser photocoagulation for the management of established corneal vessels. FND is simple and inexpensive and can be performed under topical anesthesia by any ophthalmologist. It can be applied at any depth to obliterate both afferent and efferent ves‐ sels with equal efficacy. However, it may have to be repeated to obtain the desired result [48]. Corneal microperforation is a potentially serious adverse event that can occur during passage of the needle. This is particularly so when the vascularized cornea is thin [48]. Other adverse events, such as striae, whitening, and intracorneal hemorrhages, are reversible [48]. Transient opacification of the cornea is observed in the stroma immediately surrounding the needle in all patients and persists for 24–48 h, with complete resolution. Intracorneal hemorrhage occurring intraoperatively or immediately postoperatively is the commonest adverse event. Though dramatic in appearance, intracorneal hemorrhages all resolve over a week or two. Sometimes, crystalline deposits can develop in the site of hemorrhage [48].

#### **6.4. Corneal anti‐angiogenesis target therapies**

The advent of anti‐VEGF agents has introduced a new dimension to the management of cor‐ neal vessels [54]. Active young vessels which usually indicate an underlying ongoing pathol‐ ogy continuing to induce further vascularization are probably best treated with anti‐VEGF drops or subconjunctival injections. There is a growing list of therapeutic agents that target corneal angiogenesis (**Table 2**). Currently, only limited experience using anti‐VEGFs on the cornea and only in an off‐label setting is available [54].


HIF‐1a: hypoxia‐inducible factor 1a, CYP: cytochrome P450 mono‐oxygenase, PDGF: platelet‐derived growth factor, SSAO: semicarbazide‐sensitive amine oxidase, PEDF: pigment epithelium‐derived factor, VAP‐1: vascular adhesive protein‐1, sVEGFR: soluble form of vascular endothelial growth factor receptor, VEGF: vascular endothelial growth factor, VEGFR: vascular endothelial growth factor receptor.

**Table 2.** Corneal antiangiogenesis target therapies.

## *6.4.1. Anti‐VEGF antibody*

**Targets Mechanisms Therapeutics**

68 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

receptors

Soluble or modified VEGF

Anti–VEGF‐A antibodies Bevacizumab

VEGF‐A aptamer Pegaptanib

PEDF direct effect PEDF

PDGF receptor inhibitor AG 1296

Angiostatin Angiostatin direct effect Angiostatin pump

kinase inhibitor

mono‐oxygenase

Decorin Decorin direct effect Decorin gene therapy

Vasohibin‐1 Vasohibin‐1 directly effect Vasohibin‐1 gene therapy

HIF‐1a: hypoxia‐inducible factor 1a, CYP: cytochrome P450 mono‐oxygenase, PDGF: platelet‐derived growth factor, SSAO: semicarbazide‐sensitive amine oxidase, PEDF: pigment epithelium‐derived factor, VAP‐1: vascular adhesive protein‐1, sVEGFR: soluble form of vascular endothelial growth factor receptor, VEGF: vascular endothelial growth

factors

Vascular adhesion protein VAP‐1/SSAO inhibitor U‐V002

Cannabinoid receptor CB1 CB1 antagonist Rimonabant

Platelet‐derived growth factor Multitargeted receptor tyrosine

12‐Hydroxyeicosatrienoic acid siRNA for cytochrome P450

Hypoxia‐inducible factors shRNA for hypoxia‐inducible

factor, VEGFR: vascular endothelial growth factor receptor.

**Table 2.** Corneal antiangiogenesis target therapies.

Ranibizumab

VEGFR‐2‐Fc

VEGFR‐1 morpholino

Recombinant dimeric

(Flt23k, Flt24k)

PEDF gene therapy PEDF‐derived peptide

Sunitinib

RNAi‐A)

LJP1207

sVEGFR‐3 overexpression gene therapy

sVEGFR‐1 overexpression gene therapy

VEGFR intraceptor gene therapy

Aflibercept/VEGF‐Trap(R1R2)

CYP4B1 siRNA gene therapy

HIF‐1a shRNA gene therapy (HIF‐1a

Vascular endothelial growth

Pigment epithelium‐derived

factor

factor

Inhibition of VEGF activity by a specific neutralizing anti‐VEGF antibody is one possible strategy for treating corneal angiogenesis. VEGF inhibitors such as pegaptanib sodium (Macugen™, OSI/Eyetech), off‐label bevacizumab (Avastin™, Genentech), and ranibizumab (Lucentis™, Genentech) are currently used for the treatment of different retinal pathologies including wet‐type age‐related macular degeneration [55]. Both animal models and clinical trials have demonstrated that these agents are effective in reducing corneal neovasculariza‐ tion. Both ranibizumab and bevacizumab use the same mechanisms and nonspecifically inhibit the VEGF‐A isoforms [56]. Nevertheless, differently from ranibizumab and bevaci‐ zumab, pegaptanib specifically binds to VEGF‐A165 and does not inhibit all of the VEGF iso‐ forms. Subconjunctival ranibizumab, pegaptanib sodium, and bevacizumab are effective with no epitheliopathy in reducing corneal angiogenesis. Repeated subconjunctival injections with higher doses and concentrations and combination therapy with other antiangiogenic agents may be valid options to improve the effectiveness of treatments [57].

Treating corneal new vessel with the anti‐VEGF antibody has some limitations. In contrast to superficial and active vascularization, in which clear regression is observed, anti‐VEGF agents have a lower effect on deep vascularization. The effect of the anti‐VEGF antibodies depends on the time of the treatment after the onset of neovascularization. In contrast to newly formed vessels, stable vessels are less affected by VEGF blockade [58]. The vessels mature in chronic neovascularization, and pericytes are recruited to the area around the region of corneal neo‐ vascularization [59]. Such coverage may reduce the influence of anti‐VEGF agents on the regression of newly formed immature vessels. Anti‐VEGF therapy is only a symptomatic treatment of corneal neovascularization that does not cure the underlying pathology, mak‐ ing it necessary to repeat the treatment to maintain its positive effect over a span of time [27].

**Bevacizumab**, which is FDA approved for intravenous administration in the treatment of various cancers, is a full‐length, humanized murine monoclonal antibody with a molecular weight of 149 kD. Bevacizumab recognizes all isoforms of VEGF and is in widespread use, off‐ label, as an intravitreal injection to treat different retinal diseases [60]. Additionally, studies have demonstrated that topical, subconjunctival, and intraocular application of bevacizumab can partially reduce corneal angiogenesis and inflammatory response, resulting in an increase in corneal transparency [61, 62]. Bevacizumab can inhibit macrophage migration to the cor‐ neal stroma in early but not late treatment. Macrophages are known to trigger neovascu‐ larization in ischemic or inflamed corneas [63]. There is a concern about the interference of the topical form but not subconjunctival form of bevacizumab with nerve regeneration and delayed wound healing [54, 64, 65].

**Ranibizumab**, which has VEGF‐binding characteristics similar to bevacizumab, is a recombi‐ nant humanized monoclonal antibody fragment that binds and inhibits all VEGF‐A isoforms. Bevacizumab and ranibizumab are related to each other, but ranibizumab is the Fab fragment from the same antibody used to create bevacizumab. Therefore, ranibizumab has a molecular weight of 48 kD, making it approximately one‐third the size of bevacizumab and theoretically allowing a better corneal penetration. In addition, it has been affinity matured to optimize the VEGF‐A binding potential. These characteristics may enable ranibizumab to reduce cor‐

neal angiogenesis more effectively than bevacizumab [66]. Subconjunctival ranibizumab sig‐ nificantly reduces VEGF levels not only in the bulbar conjunctiva and cornea but also in the iris and aqueous humor [67]. Clinically, stable corneal neovascularization can be effectively treated by topical ranibizumab 1% as evidenced by a significant reduction in vessel caliber and neovascular area with no significant change in invasion area. These findings suggest that the main outcome of ranibizumab treatment for stable corneal neovascularization is to induce the narrowing of vessels more than a reduction in their length.

#### *6.4.2. Pigment epithelium‐derived factor*

PEDF is a glycoprotein with neurotrophic, antitumorigenic, and antiangiogenic functions. PEDF can inhibit FGF, VEGF, and interlukin‐8 (IL‐8/CXCL8)‐mediated angiogenesis by inducing the cells' apoptosis and reducing endothelial cell migration simultaneously [68, 69]. It is also found to play an important role in the antiangiogenic effect of AMT [70]. Topical PEDF or PEDF‐derived (P5‐2 and P5‐3) peptides can downregulate VEGF expression and inhibit corneal neovascularization in a chemical‐induced corneal model [71].

#### *6.4.3. Tyrosine kinase inhibitors*

Anti‐VEGF antibodies block the effect of VEGF before it attaches to the endothelial recep‐ tors. Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 (TIE2) that is predominantly or exclusively expressed in endothelial cells is an important regulator of angiogenesis. Tyrosine kinase inhibitors inhibit the activity of VEGF by block‐ ing tyrosine kinase in the intracellular part of the VEGF cell membrane receptor. This may offer a different opportunity for the management of the angiogenesis process in corneal dis‐ eases. Regorafenib is a multikinase inhibitor that targets various kinases, including PDGF β, VEGFR1, VEGFR2, and VEGFR3, mutant oncogenic kinases, TIE2, and the FGF receptor, which are involved in neovascularization. The inhibitory effects of topical regorafenib are comparable to those of topical bevacizumab and dexamethasone [72]. Sunitinib is a multitar‐ geted receptor tyrosine kinase inhibitor that blocks both VEGF and PDGF. Topically adminis‐ tered sunitinib can reduce corneal neovascularization more effectively than bevacizumab [73].

Trastuzumab is a monoclonal antibody that interferes with the HER2/ neu receptor. Lapatinib is a dual tyrosine kinase inhibitor, which interrupts the epidermal growth factor receptor (EGFR) and HER2/ neu pathways. Lapatinib used in the form of lapatinib ditosylate is an orally active drug for solid tumors such as breast cancer. In recent studies, both substances were compared for the treatment of experimental corneal angiogenesis. The results suggested that systemically administered lapatinib is more effective than systemically administered trastuzumab in preventing corneal angiogenesis [74].

## **7. Conclusion**

Corneal neovascularization is a common clinical feature in different corneal diseases includ‐ ing ocular traumatic or chemical injury, autoimmune diseases, chronic contact lens wear, infectious keratitis, and keratoplasties. Although corneal neovascularization can serve a beneficial role in arresting stromal melts, wound healing, and the clearing of infections, its disadvantages are numerous and it frequently results in edema, tissue scarring, persistent inflammation, and lipid deposition that may significantly reduce vision. Furthermore, it plays a major role in corneal graft rejection by breaching corneal immune privilege. VEGF, which plays a crucial role in angiogenesis and the pathologic neovascularization associated with a variety of eye diseases, is the most important target for antiangiogenic therapies. Experience indicates that anti‐VEGFs are effective in occluding actively growing corneal neovascularization but not established vessels. Surgical procedures, including laser photo‐ coagulation or fine needle diathermy, are useful particularly to obliterate large, established corneal vessels.
