•Novel antiangiogenic and antilymphangiogenic therapies can improve graft survival both by regression of corneal vessels prior to grafting as well as after low-risk as well as high-risk keratoplasty by reducing the incidence of immune rejections (novel therapeutic concept).

•Topical and subconjunctival anti-VEGFs are a potent way to treat immature actively outgrowing corneal blood vessels off-label. Safety concerns are development of a neurotrophic keratopathy and inhibition of epithelial and stromal wound healing.

•Targeting Insulin Receptor Substrate −1 is another promising new approach to target both corneal hem-and lymphangiogenesis with an ongoing phase III clinical trial.

•Steroids not only are anti-inflammatory, but also antihemand antilymphangiogenic. In the transplant context, Prednisolone as the most potent inhibitor of lymphangiogenesis should be used.

•Corneal lymphangiogenesis is not only the prime mediator of corneal graft rejection, but is also involved in the induction of immune-mediated dry eye disease and the induction of ocular surface tumor metastasis.

Introduction

Corneal avascularity is of paramount importance to maintain cornel transparency, the latter being essential for good visual acuity. Therefore, in all higher animals depending on good vision, the cornea normally is devoid of blood and lymphatic vessels (“corneal angiogenic privilege”) [1–3]. Nevertheless, several diseases and surgical manipulations can lead to corneal (hem)angiogenesis (i.e., ingrowths of visible blood vessels from the limbal vascular arcade into the cornea) and lymphangiogenesis (i.e., ingrowths of invisible lymphatic vessels from the limbal vascular arcade into the cornea [1, 2]). Corneal hemand lymphangiogenesis can cause a significant reduction in visual acuity and blindness as well as render these corneas high-risk in case of a subsequent penetrating or lamellar keratoplasty [1, 2]. In fact, corneal angiogenesis is associated with the most common cause of corneal blindness worldwide (trachoma) as well as the most common form of infectious blindness in western countries (herpetic keratitis [1, 2]). Whereas the animal cornea has been used as in vivo model to study the mechanisms of angiogenesis for decades, the molecular pathways being responsible for maintaining normal avascularity of the human cornea (“angiogenic privilege”) are only starting to evolve in recent years [4]. Nonetheless, great progress has been made in recent years to unravel corneal angiogenic and lymphangiogenic privilege [5–8]. Corneal lymphangiogenesis has recently been shown to be of essential importance

in the induction of immune responses after corneal transplantation so that novel antihemand antilymphangiogenic therapies start to emerge as new tools to improve graft survival both in the low-risk as well as the high-risk setting of corneal transplantation [5, 9].

“Angiogenic Privilege of the Cornea” or “How Does the Normal Corneal Maintain Its Avascularity?”

Although the cornea – due to its anatomically exposed position – constantly is in contact with numerous minor inflammatory and thereby angiogenic stimuli, the normal cornea remains avascular [1, 3, 4]. Even after more severe trauma – such as refractive surgery – the cornea in contrast to other tissues – does not respond with angiogenesis. This so-called “angiogenic privilege” [1, 3, 4] is not only essential for good visual acuity but also is responsible for the excellent survival of corneal grafts placed into avascular low-risk recipient beds since in these eyes the graft is physically separated both from the afferent (lymphatic) as well as the efferent (blood vascular) arm of a so-called immune reflex arc leading to immune rejection after keratoplasty [1, 2, 5, 9, 10]. Recent research has shown that the cornea uses several and redundant mechanisms to maintain its avascularity against numerous subthreshold angiogenic stimuli. In fact, several of these mechanisms are very elaborate and examples of how evolutionary important corneal avascularity was. First, the cornea contains numerous endogenous inhibitors of angiogenesis and lymphangiogenesis (such as PEDF, thrombospondins 1 and 2, antiangiogenic matrix cleavage products such as angiostatin and endostatin, IL1RA, etc.) [1, 11]. It seems that these antiangiogenic factors are strategically located at the inner and outer linings of the cornea (Descemet’s membrane and epithelial basement membrane) to counteract angiogenic stimuli both from inside (e.g., high concentrations of angiogenic growth factors in the aqueous humor during proliferative diabetic retinopathy) or from outside (e.g., against angiogenic growth factors from the tear film [1, 11]). Animal experiments using mice deficient of one or more antiangiogenic factors (such as thrombospondins 1 and 2) have shown that the corneal angiogenic privilege is redundantly organized so that absence of one or two factors does not cause spontaneous ingrowths of limbal blood vessels [3, 4]. This is in contrast to other intraocular tissues such as the iris, where absence of these factors causes increased vascularity [4]. This demonstrates that evolutionary, the cornea has acquired a robust and redundant antiangiogenic system, normally maintaining avascularity unless it is overrun by overwhelmingly strong (usually inflammatory/infectious) stimuli for angiogenesis which threaten the integrity of the whole eye or even the whole body [3, 4]. In addition, secondly, the cornea has several receptor decoy mechanisms in place which use “false” receptors to bind and neutralize angiogenic growth factors which would normally cause (lymph) angiogenesis. Examples are the ectopic expression of a VEGF receptor (VEGFR3; Fig. 5.1) on the corneal epithelium, the soluble form of VEGF receptor 1 and 2 as well as Interleukin 1 receptors. A third strategy of the

cornea is to block hypoxia-induced forms of angiogenesis by molecularly interfering with hypoxia (HIF)-induced upregulation of angiogenic growth factors in the cornea such as VEGF.

Summary for the Clinician

•Cornea and cartilage are the only avascular tissues of the human body.

•Corneal avascularity is achieved by numerous elaborate and redundant mechanisms in place and actively maintained, e.g. after refractive surgery (“corneal angiogenic privilege”).

•Loss of angiogenic privilege, i.e. corneal angiogenesis is associated with and potentially causative for the most common causes of corneal blindness worldwide (trachoma) and the most common form of infectious corneal blindness in industrialized countries (herpetic keratitis)

Fig. 5.1 Corneal angiogenic privilege is maintained by multiple and redundant mechanisms. One interesting strategy is the expression of decoy receptors in the cornea, amongst them ectopically expressed VEGF receptor 3 (VEGFR3) in the corneal epithelium. In this ectopic location, the receptor binds and neutralizes angiogenic growth factors such as VEGF-C and -D and prevents their ligation to the normal receptors in adjacent conjunctival vessels, thereby maintaining corneal avascularity (From Ref. [12])

Corneal Hemangiogenesis and Lymphangiogenesis: General

Mechanisms and Clinical Features

General Mechanisms

According to FOLKMAN, a balance between angiogenic and antiangiogenic factors in each tissue and situation determines whether angiogenesis occurs or not. If the balance is tipped toward angiogenic growth factors, vessel outgrowth starts (“angiogenic switch”), whereas if inhibitors prevail, angiogenesis is prohibited. Several angiogenic growth factors (primarily growth factors from the VEGF family [VEGF-A, VEGF-C, VEGF-D], FGF, IL1, etc.) as well as inhibitors of angiogenesis have been identified in recent years [13]. Pathologic angiogenesis (to clearly separate this process from lymphangiogenesis, we will subsequently refer to it as “hemangiogenesis”) and lymphangiogenesis into the cornea mainly occur in settings of an inflammatory “insult” to the cornea, corneal hypoxia, or limbal barrier defects, all overriding the angiogenic privilege of the cornea, which is actively maintained [3, 4, 12, 14]. Clinical conditions most commonly associated with corneal neovascularization include keratitis (herpetic and bacterial in nature), contact lens wear as well as inherited or acquired limbal deficiency states (primarily chemical burns [3, 4, 12, 14]). In addition, “secondary” corneal angiogenesis can occur after surgical manipulations at the cornea, which primarily involve placement of corneal sutures (e.g., after corneal wound repair, after corneal transplantation, after block excision, etc. [15]). Growth factors of the VEGF family have been identified as key players in both inflammation-driven hemas well as lymphangiogenesis into the normally avascular cornea [1, 9, 16]. Release of angiogenic growth factors generally is induced primarily by two factors: (a) inflammation and inflammatory cytokines (at the cornea: e.g., keratitis) and (b) hypoxia (at the cornea: e.g., contact lens-induced). The sources for (lymph)angiogenic growth factors within the cornea are multifold, but inflammatory cells and, here especially, macrophages seem to be key players. Early removal of macrophages during an inflammatory insult to the cornea can nearly completely block hemand lymphangiogenesis in the cornea.

Clinical Consequences of Corneal Hemand Lymphangiogenesis

Corneal angiogenesis can lead to reduced visual acuity not only by the physical presence of blood vessels itself, but also due to leakage of products from immature corneal blood vessels. This includes corneal edema due to water leakage, corneal lipid keratopathy due to lipid leakage, and intrastromal or subepithelial hemorrhage (e.g., in contact lens patients). In addition, neovascularization can occur into the interface after deep anterior lamellar keratoplasty and cause significant vision loss. Furthermore, as outlined below, corneal angiogenesis impairs the prognosis of corneal grafts placed into vascularized high-risk corneas. In fact, the Collaborative Corneal Transplantation Study [17] (and numerous other clinical as well as