Corneal neovascularization (NV) and lymphangiogenesis are sight-threatening conditions that introduce vascular conditions into the normally avascular cornea ( Box 10.1 ). Corneal NV is induced by various stimuli and is mainly associated with inflammation, trauma, transplantation, and infection of the ocular surface ; lymphangiogenesis is usually concurrent with hemangiogenesis in the human cornea. Both corneal NV and lymphangiogenesis are promoted or inhibited by a balance of factors, including the dynamics between vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, sFlt, VEGFR3, endostatin, and thrombospondin-1 and -2 contained in the cornea ( Box 10.2 ). Recently, evidence has shown that soluble VEGF receptor (VEGFR-1) and ectopic VEGFR-3 are expressed in corneal epithelial cells, and act as decoy receptors for VEGF-A and VEGF-C/-D, respectively. These decoy receptors function to maintain corneal clarity and prevent corneal NV and lymphangiogenesis. In corneas that are diseased by inflammation, infection, degeneration, transplantation, or trauma, the normal balance of pro- and antiangiogenic factors is shifted toward proangiogenic status, leading to corneal NV and/or lymphangiogenesis. The pathogenesis of corneal NV and lymphangiogenesis may be influenced by growth factors, cytokines, matrix components, and matrix metalloproteinases (MMPs). New medical and surgical treatments that have been effective in corneal NV/lymphangiogenesis in animals and humans include immunosuppressant agents, angiostatic steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), argon laser photocoagulation, and both photodynamic and antiangiogenic therapies.
Corneal NV and lymphangiogenesis are sight-threatening conditions that introduce vascular conditions into the normally avascular cornea
Corneal NV and lymphangiogenesis are derived from:
Degenerative congenital disorders
Trauma and other causes
Both corneal neovascularization and lymphangiogenesis are promoted or inhibited by a balance of proangiogenic, antiangiogenic, prolymphangiogenic, and antilymphangiogenic factors:
Basic fibroblast growth factor, vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D
sFlt, VEGFR3, endostatin, thrombospondin-1, -2, and others
The main purpose of this chapter is to describe the pathophysiology of corneal NV and lymphangiogenesis. The current treatments and potential antiangiogenic and/or antilymphangiogenic therapies will also be addressed at the end of this chapter.
Corneal NV and lymphangiogenesis together represent major public health burdens in the USA, affecting an estimated 1.4 million patients in any given year. These conditions are caused by a wide range of inflammatory, infectious, degenerative, toxic, and traumatic disorders; major ocular complications include corneal scarring, edema, lipid deposition, and inflammation ( Box 10.3 ). Corneal NV and lymphangiogenesis not only significantly alter visual acuity, but also worsen the prognosis of subsequent penetrating keratoplasty.
Corneal neovascularization and lymphangiogenesis together represent major public health burdens and can cause many ocular complications, including:
Corneal NV originates from the perilimbal plexus of conjunctival venules and capillaries and may invade the cornea at any level. Two types of corneal NV can be clinically discerned: pannus and stromal NV. In pannus NV, the proliferation of blood vessels spreads between the epithelium and Bowman’s layer and is usually associated with ocular surface disorders such as infection, trauma, or metabolic dysfunction. In stromal NV, the vessels are usually in a straight line, following the anatomical divisions of the corneal lamellae and branching in a brushlike manner. This is most common in inflammatory status of the cornea, like stromal keratitis.
Clinically, patients with corneal NV may complain of a decrease in visual acuity. The diagnostic workup should include slit-lamp examination, which will identify the origin of the NV, as well as the depth of its invasion in the cornea. If corneal edema is presented at the same time, employment of pachymetry, specular microscopy, or confocal microscopy can aid in confirmation of the diagnosis.
Treatment of corneal NV has been widely investigated both medically and surgically. Current treatments for corneal NV in humans include steroids, NSAIDs, ciclosporin A, anti-VEGF-A antibody, argon laser, electrocoagulation, limbal transplantation, amniotic membrane transplantation, and conjunctival transplantation.
In corneal pannus, a fibrous tissue with a significant vascular component is seen between the epithelium and Bowman’s layer; this is called “subepithelial fibrovascular pannus.” There are two types of corneal pannus: inflammatory pannus and degenerative pannus. Inflammatory pannus is associated with prominent leukocytic infiltration and includes polymorphonuclear leukocytes in the active stages. However, by the time the pathologist detects the inflammatory pannus, it is commonly constituted by overwhelming numbers of lymphocytes and plasma cells. Frequently, the Bowman’s layer is disrupted, and the vessels wander haphazardly through the anterior stroma. In degenerative pannus, there are fewer inflammatory cells. The vascular component has never been prominent and is liable to regress, leaving a hyalinized, relatively acellular layer of fibrous tissue. This type of pannus is especially common in conditions that give rise to chronic epithelial edema, such as glaucoma.
Stromal NV differs from its superficial counterparts and is located beneath the Bowman’s layer. Although it can occur anywhere in the stroma, it is usually identified at the upper and middle third of this layer.
Inflammatory disorders: ocular pemphigoid, atopic keratoconjunctivitis, rosacea, graft rejection, Lyell’s syndrome, Stevens–Johnson syndrome, and graft-versus-host disease.
Infectious diseases: viral keratitis (i.e., herpes simplex keratitis, herpes zoster keratitis), bacterial keratitis (i.e., Pseudomonas infection, syphilis), fungal keratitis (i.e., Candida , Fusarium , Aspergillus ), and parasitic infection (i.e., onchocerciasis).
Degenerative: congenital disorders: pterygium, Terrien marginal degeneration, and aniridia.
Traumatic: iatrogenic disorders and miscellaneous: contact lens wear, chemical burns, iatrogenic injury, and stem cell deficiency.
|Ocular pemphigoid||Yes||NA||Figure 10.1|
|Atopic conjunctivitis||Yes||NA||Figure 10.2|
|Stevens–Johnson syndrome||Yes||NA||Figure 10.1|
|Degenerative congenital disorders|
|Terrien’s marginal degeneration||Yes||NA|
|Traumatic and others|
|Chemical burn||Yes||Yes||Figures 10.1 and 10.2|
|Stem cell deficiency||Yes||Yes||Figure 10.3|
Multiple steps involved in corneal NV and lymphangiogenesis
Corneal NV consists of the formation of new vascular structures in previously avascular areas. In an in vivo experimental corneal model, the growth of a capillary involves an ordered sequence of events: the release of angiogenic factors, vascular endothelial cell activation, lysis of the basement membrane of a parent venule, vascular endothelial cell proliferation, directional migration of capillary endothelial cells towards the angiogenic stimulus, lumen formation, development of branches, and anastomosis of the tip of one tube with another to form a loop. Similarly, lymphangiogenesis consists of the growth of new lymphatic vessels that are derived from pre-existing lymphatic endothelial cells. In adulthood, lymphangiogenesis is primarily associated with pathological processes, such as chronic inflammation, tissue injury, lymphedema, and tumor metastasis. Lymphatic vessels differ from blood vessels in that they do not have a continuous basement membrane. Moreover, the initial lymphatics display an irregular shape, intercellular openings, intracellular channels, and phagocytotic power, which constitute major paths for transport of matrix components in inflammatory diseases.
Localization of corneal vascular and lymphatic vessels
Under normal conditions, the cornea is transparent and without vascular and lymphatic vessels. Recent findings suggest that maintenance of the cornea devoid of corneal vascular and lymphatic vessels is an active process for preventing and modulating angiogenic and lymphangiogenic reactions. The active mechanism for maintaining the corneal avascularity has been termed “corneal angiogenic privilege”. The onset of corneal NV is characterized by blood supply that arises from the ciliary arteries branching off from the ophthalmic artery, which subsequently divide and end in the pericorneal plexus within the limbus. Corneal NV can be derived from stroma, which is mainly associated with stromal keratitis. Corneal NV can also develop from the superficial corneal periphery, which is mainly associated with ocular surface disorders, such as Stevens–Johnson syndrome, ocular pemphigoid, and thermal or chemical burns. Although NV may involve several corneal layers, a study has demonstrated that the main locations of vascularized corneal buttons are in the upper and middle third areas of the anterior stroma. Similarly, induced lymphatic vessels are localized to the corneal subepithelium and stroma layers in the wounded cornea.
Matrix involvement in corneal NV and lymphangiogenesis
The extracellular matrix, an active regulator of cellular proliferation, migration, adhesion, and invasion, can influence corneal NV and lymphangiogenesis. The MMPs comprise a large family of proteolytic enzymes that are responsible for matrix degradation. These MMPs may also modulate vascular and lymphatic endothelial cell sprouting and extension.
Cornea provides a tool for evaluating angiogenic/lymphangiogenic and antiangiogenic/lymphangiogenic factors
The “corneal angiogenic and immunogenic privileged” site has been used to assay the molecular basis of angiogenesis and lymphangiogenesis both in vivo and in vitro. The cornea is a good model for evaluating proangiogenic/lymphangiogenic and antiangiogenic/lymphangiogenic factors, due to the absence of blood and lymphatic vessels. Corneal avascularity requires low levels of angiogenic factors and high levels of antiangiogenic factors under basal conditions. Shifting of the balance towards higher levels of angiogenic and lymphangiogenic factors in the cornea is associated with pathological processes ( Box 10.4 ). Here we review certain corneal disorders associated with NV and lymphangiogenesis, the molecular basis of such complications, and current potential therapeutics.
Modulation of corneal angiogenic/lymphangiogenic factors regulates corneal neovascularization. For example:
Limbal stem cell deficiency (loss of limbal stem cells)
Defects in renewal and repair of ocular surface caused by:
Herpes simplex virus infection
Surgical modality to replenish or repopulate the ocular surface epithelium
Function of vascular and lymphatic vessels
During development, vascular vessels function to foster new tissue growth, but they become tightly regulated during adulthood. The major role of lymphatic vessels, on the other hand, is to maintain tissue fluid homeostasis by transporting lymph fluid from tissues to the circulatory system. Tissue fluid may readily be transported in the lymph to the nearest lymph node. During corneal inflammation, a rapid upregulation of proinflammatory cytokines takes place, attracting the migration of inflammatory cells into the cornea. Interactions between resident or infiltrated cells with extracellular matrices may induce cytokine productions in a paracrine fashion. These cytokines are beneficial to the cornea because they protect against the invasion of bacteria and other microorganisms. While inflammation is part of a physiological process for repairing damage, uncontrolled inflammation may actually cause damage. Therefore, understanding the lymphatic status in structure and function is an important step towards the control of unwanted corneal inflammation, edema, and transplant rejection.
Corneal NV and lymphangiogenesis-related disorders
Immunologic and infectious disorders of the cornea and conjunctiva, including ocular pemphigoid, graft rejection, viral and bacterial infection, pterygium, and aniridia, may involve the production of angiogenic and lymphangiogenic molecules responsible for corneal NV and lymphangiogenesis. Accordingly, a long-term follow-up of patients with inflammatory disorders, such as atopic keratoconjunctivitis, has revealed that the percentage of corneal NV may be as high as 60% during the course of their diseases. Corneal transplant rejection has been correlated with alloantigen-specific delayed-type hypersensitivity, infiltration of CD4+ T cells, and an increased amount of interferon-γ. Additionally, corneal lymphangiogenesis may play a role in explaining why certain patients (particularly younger ones) have increased corneal transplant rejection. Among different infectious agents, the herpes virus family (mostly herpes simplex virus (HSV) and herpes zoster) appears to be the primary cause of keratitis-induced NV in penetrating keratoplasty buttons. This complication occurs after interstitial, necrotizing, or recurrent keratitis and is not solely dependent on the host reaction. HSV-1 virus-infected cells can also produce interleukin-6 to stimulate noninfected resident corneal cells and other inflammatory cells to secrete VEGF, a potent angiogenic factor, in a paracrine manner. Following ocular HSV-1 infection, the NV of the avascular cornea is a critical event in the pathogenesis of herpetic stromal keratitis. There are approximately 300 000 cases of ocular HSV-1 infection diagnosed annually in the USA. The initial infection involves the corneal epithelium, and the neovascularized cornea lacks stromal NV. Repeated episodes of recurrent disease can lead to the involvement of the underlying stroma.
Corneal NV may occur in degenerative disorders, such as pterygium and Terrien’s marginal degeneration, as well as in congenital disorders such as aniridia. Recently, Ambati et al have shown that patients with aniridia have mutated PAX6 genes and deficiencies in corneal sFlt-1 expression. This study demonstrates that antiangiogenic factors are involved in the pathogenesis of corneal NV.
Corneal NV and lymphangiogenesis: molecular basis and factors involved
NV and lymphangiogenesis occur in a tissue when the balance between angiogenic and antiangiogenic factors is tilted towards angiogenic molecules. In animal models, corneal NV and lymphangiogenesis are induced not only by the upregulation of angiogenic and/or lymphangiogenic factors (such as VEGF, VEGF-C, or -D), but also by the downregulation of antiangiogenic or lymphangiogenic factors (sFlt-1, ectopic expressed VEGFR3).
While many factors have been characterized and published, only a few of these factors have reached clinical trials. Here we discuss the potential benefits of using anti-VEGF antibodies, MMP inhibitors, and proteolytic fragments of extracellular matrix (endostatin and angiostatin).
Vascular endothelial growth factors
The VEGF family is structurally related to four other members: placenta growth factor, VEGF-B, -C, and -D. VEGF-A and VEGF-C are highly specific mitogens for vascular and lymphatic endothelial cells, in vitro and in vivo, respectively. This VEGF family of proteins binds selectively with varying affinities to distinct VEGF receptors. The binding of VEGF to endothelial-specific receptor tyrosine kinases, VEGFR1 and VEGFR2 (expressed primarily on vascular endothelial cells), mediates angiogenic responses. In one case, VEGF-C bound to VEGFR3 (flt-4, which is predominantly expressed in lymphatic endothelial cells in adult tissues) induced corneal lymphangiogenesis.
During corneal NV and lymphangiogenesis, an upregulation of angiogenic and lymphangiogenic factors is usually present. For example, it has recently been shown that the vascular endothelial growth factor (VEGF, VEGF-C or VEGF-D) was upregulated in inflamed and vascularized human and animal corneal models. VEGFs are secreted growth factor peptides generated by alternative splicing in five isoforms (VEGF115, VEGF121, VEGF 165, VEGF 189, and VEGF 206). VEGF is produced by macrophages, T cells, astrocytes, and smooth-muscle cells in corneal hypoxia and inflammatory conditions. The requirement of VEGF and VEGF-C in corneal NV and lymphangiogenesis has been demonstrated by the inhibition of NV or lymphangiogenesis after stromal implantation of anti-VEGF neutralizing antibodies: the soluble recombinant molecules VEGFR1 or VEGFR3.
Corneal extracellular matrix turnover by MMPs is usually associated with wound healing during corneal NV and lymphangiogenesis. MMPs are a group of zinc-binding proteolytic enzymes that participate in extracellular matrix remodeling, NV, and lymphangiogenesis. They are produced as proenzymes and are activated by a variety of proteinases, including MMPs and serine proteases. Among the 25 MMPs already described, at least 11 have been identified in the cornea, including collagenases (MMP-1, -8, and -13), gelatinases A and B (MMP-2 and -9), stromelysins (MMP-3, -10, -11), matrilysin (MMP-7), and membrane-type (MT)-MMP (MMP-14). Their upregulation during corneal NV has been published, and their roles in the regulation of NV are gradually being demonstrated. Individual MMPs may have more distinct roles in corneal NV. For example, MMP-2 and MT1-MMP possess proangiogenic potential because experiments demonstrated that MMP-2 and MT1-MMP knockout mice displayed a diminished basic fibroblast growth factor (bFGF)-induced corneal NV. However, MMP-7 has been shown to have antiangiogenic functions, since: (1) MMP-7 knockout mice displayed enhanced keratectomy-induced corneal NV; and (2) MMP-7 may cleave corneal extracelluar matrix to generate antiangiogenic fragments. The roles of MMPs in corneal lymphangiogenesis are under current investigation. Understanding the functions of MMPs in corneal NV and lymphangiogenesis may lead to new venues for the development of therapeutic interventions, in conjunction with anti-VEGF therapy for disorders related to corneal NV and lymphangiogenesis.
Endostatin, a putative antiangiogenic factor, is a 20-kDa proteolytic fragment of collagen XVIII. Recombinant endostatin and its related fragments have been shown to inhibit bFGF-induced corneal NV in vivo and bFGF- and VEGF-induced vascular endothelial cell migration and proliferation in vitro. Specifically, endostatin implanted in the cornea demonstrated an inhibition of the bFGF-induced NV. Collagen XVIII is a nonfibrillar collagen localized mainly to the corneal vascular and epithelial basement membrane. Cleavage of collagen XVIII by proteases (including MMPs, cathepsin L, and elastase) generates endostatin-like fragments that may display antiangiogenic properties. Local production of endostatin may occur during corneal wound healing, as both the cleaving enzymes (MMPs) and the substrate (collagen XVIII) are present in the basement membrane area to prevent corneal NV. Endostatin has been approved by the US Food and Drug Administration (FDA) for the treatment of NV-related cancer; thus, it may be an additional drug that can be added to anti-VEGF therapy to treat corneal NV- and lymphangiogenesis-related disorders.
Corneal NV and lymphangiogenesis management
Current therapy of the vascularized corneas includes using antiangiogenic/lymphangiogenic factors for the blocking of chemokines and cytokines produced by the inflammatory, vascular, and lymphatic cells. Specifically, inhibitors for the vascular endothelial growth factors (VEGF-A, VEGF-C, VEGF-D), bFGF, ang1, insulin-like growth factor, platelet-derived growth factor-BB, CCL21, interleukin-6, and CD4+ T cells have been shown to be effective in preventing and regressing corneal NV and lymphangiogenesis, based on animal models and clinical trials. Additionally, clinical experience with angiogenesis signaling inhibitors has focused on VEGF blockers. Anti-VEGF therapies have been extensively applied to corneal NV and lymphangiogenesis-related disorders. Several antiangiogenic factors derived from collagens or the extracellular matrix (endostatin, angiostatin, arrestin, thrombospondins) have also been discovered, but their roles in corneal NV have not been fully characterized. Recently, ectopic expression of VEGFR3 in corneal epithelial cells has been shown to have a direct inhibitory effect on corneal lymphangiogenesis. Administration of anti-VEGFR3 neutralizing antibodies in the cornea diminishes the epithelium’s ability to dampen injury-induced corneal lymphangiogenesis. In addition, the application of chimeric VEGFR3 in the cornea can prevent cautery-induced corneal lymphangiogenesis. Thus, modulation of VEGFR3 function may be able to provide a therapeutic intervention for the treatment of lymphangiogenesis-related corneal disorders.
Neostatin-7, a 28-kDa fragment, is generated by MMP-7 cleavage of type XVIII collagen. Neostatin-7 is one of several naturally occurring endostatin-spanning fragments. Neostatin-7 possesses antilymphangiogenic activity and may provide therapeutic interventions to treat lymphangiogenesis-related disorders, such as lymphedema, transplantation rejection, and cancer ( Box 10.5 ).