Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

inflammatory or angiogenic conditions, thus maintaining corneal transparency and preventing loss of vision. A study with corneas from stillborn patients showed that this privilege is already present during intrauterine development at least from gestational age of 17 weeks. Contrary to the conjunctiva, in which blood and lymphatic vessels are detectable, no vessels are found in the cornea. This leads to the presumption that due to an early expression of anti-angiogenic and anti-lymphangiogenic factors, the cornea is primarily devoid of blood and lymphatic vessels and not as a result of regression of already existing vessels. The occurrence of neovascularization by angiogenic activity is initiated by a disbalance of angiogenic and antiangiogenic factors caused by upregulating angiogenic molecules as well as by downregulating angiogenesisinhibiting molecules. Interestingly, homozygote TSP-1 or TSP-2-knockout mice and even TSP-1/2-knockout mice showed no spontaneous corneal angiogenesis. The deficiency of an important anti-angiogenic factor like TSP-1 or TSP-2 does not result in a breakdown of the angiogenic privilege. This leads to the conclusion that at least during embryonic development the angiogenic privilege seems to be redundantly regulated by several anti-angiogenic factors. However, secondary to severe inflammation and several other diseases the angiogenic privilege can be overcome and an initial parallel outgrowth of blood and lymphatic vessels occurs.

Several factors comprising angiogenic and anti-angio- genic molecules, the cornea itself, and adjacent structures like the limbus are known to be involved in affecting the angiogenic privilege (see Figure 1). Deprivation of the angiogenic privilege can lead to corneal neovascularization and, in consequence, loss of vision.

Angiogenic and Anti-Angiogenic Molecules

Involved in Corneal Neovascularization

Numerous angiogenic and anti-angiogenic molecules have been identified in the cornea over the last years. Angiogenic molecules include vascular endothelial growth factors (VEGFs), basic fibroblast growth factors (bFGFs), and matrix metalloproteinases (MMPs). Angiostatin, endostatin, thrombospondins, and pigment epithelium-derived factor (PEDF) are some of the anti-angiogenic molecules detected in the cornea.

The regulation of angiogenesis is due to the interaction of pro-angiogenic molecules and angiogenesis inhibitors, where tilting the balance toward pro-angiogenic factors can lead to neovascularization. Lymphangiogenesis seems to proceed in a similar way as angiogenesis, and can be activated in the adult during inflammation, immune responses, or malignant processes.

Stimuli like hypoxia, for example, in the context of wound healing, can also trigger the induction of hemangiogenesis via hypoxia-inducible transcription factor (HIF), a key transcriptional regulator for VEGF-A. In contrast, lymphangiogenic VEGF-C cannot be upregulated by hypoxia but only by proinflammatory cytokines.

Vascular endothelial growth factors

The VEGF growth factor family currently consists of five members, VEGF/VEGF-A, PIGF, VEGF-B, VEGF-C, and VEGF-D. The growth factors are recognized by different VEGF receptors, namely VEGFR-1, VEGFR-2, and VEGFR-3. VEGF-A originally isolated from a human histiocytic lymphoma cell line U937 is secreted

in five different isoforms, generated by alternative splicing: VEGF115, VEGF121, VEGF165, VEGF189, and VEGF206.

Vascular endothelial growth factor

VEGF-A shows numerous activities such as inducing endothelial cell proliferation and migration, proteolytic activity, and stimulating microvascular leakage – all of them promoting angiogenesis. VEGF-A mediates its function through receptors VEGFR-1 and VEGFR-2. Additionally, VEGF-A was reported to promote angiogenesis via an indirect pathway by upregulating NRP1, a neuronal receptor that has recently been shown to act as an isoform-specific receptor for VEGF165.

VEGF-A can be released during hypoxia, in inflammatory situations, and during glucose deficiency. It was shown that the expression of VEGF-A is upregulated in inflamed and vascularized human corneas. In conclusion, VEGF-A seems to play an important role in inducing wound and inflammation-related corneal neovascularization. This was confirmed by the fact that corneal neovascularization could be suppressed after implantation of VEGF-A neutralizing antibodies in the corneal stroma of rats and rabbits. Whereas early data proposed that VEGF stimulates selectively hemangiogenesis but not lymhangiogenesis, recent data also suggest an (indirect) lymphangiogenic role: endogenous VEGF can promote lymphangiogenesisvia the recruitment of bone marrow-derived macrophages, releasing lymphangiogenic growth factors such as VEGF-C and -D. This broadens the impact of VEGF-A, not only for pathological hemangiogenesis, but also for lymphangiogenesis – at least in context of inflammation-induced neovascularization. A specific role for VEGF-A in the regulation of lymphangiogenesis was also described for primary tumors. The tumors were shown to overexpress VEGF-A, thereby inducing sentinel lymph node lymphangiogenesis. In a mouse model of delayed-type hypersensitivity (DTH), lymphangiogenesis was promoted by VEGF-A that was produced in the inflamed tissue.

In addition, genetic variety, that is, single-nucleotide polymorphisms in the gene coding for VEGF-A, is associated with eye diseases like neovascular age-related macular degeneration and diabetic retinopathy.

VEGF-C and VEGF-D

VEGF-C and VEGF-D are the main growth factors for lymphangiogenesis and both mediate their function by binding to receptors VEGFR-2 and VEGFR-3, present on endothelial cells. VEGF-C stimulates migration of cultured endothelial cells in vitro and increases – in its fully processed form – vascular permeability, migration, and proliferation of endothelial cells. Recently, the decisive role for VEGF-C during lymphangiogenic development was demonstrated in a study where homozygote as well as heterozygote VEGF-C-lacking mice were shown

to have severe defects in the formation of lymphatic vessels. A study undertaken to analyze VEGF-C and its role in corneal neovascularization suggests VEGF-C to induce corneal lymphangiogenesis by binding to its cognate receptor VEGFR3 on lymphatic vessels in the conjunctiva. Inflammatory cells invading the cornea were identified as the main source of VEGF-C that was strongly upregulated 3 days following the injury. Besides its role as classic lymphangiogenic growth factor, VEGF- C was reported to induce angiogenesis in vivo. VEGF-D also can act as a potent angiogenic factor, controlled by the nuclear oncogene c-fos and thus playing an important role in tumor invasion and tumor cell growth.

Basic fibroblast growth factor

Another important pro-angiogenic molecule – the basic fibroblast growth factor (bFGF/ FGF-2) – belongs to the FGF family. bFGF and acidic FGF (aFGF) expression has been demonstrated immunohistochemically in the outer retina of rat and mouse. bFGF was analyzed in several corneal neovascularization models and was recently demonstrated to induce angiogenesis as well as lymphangiogenesis in vivo in a mouse model corneal micropocket assay. Lymphangiogenesis was mediated by bFGF in an indirect way via VEGFR3 and was suppressed after inhibition of VEGFR3 signaling with anti-VEGFR3 antibodies.

Both factors, bFGF and aFGF, were detectable in retinal pigment epithelial cells from choroidal neovascular membranes from human subjects with age-related macular degeneration (AMD), whereas there was only little immunoreactivity for the growth factors in retinal pigmented epithelial (RPE) cells from healthy eyes. This suggests an important role for aFGF and bFGF in the development of choroidal neovascularization. bFGF might play an indirect role in initiation of neovascularization and interacts with the VEGF signal-transduction pathways. This is supported by the fact that bFGF was found to be colocalized with VEGF in cells of epiretinal and choroidal neovascular membranes, suggesting that more than one growth factor may contribute to pathological angiogenesis. In favor for that theory is that mice with a disruption of the bFGF-coding gene can still develop choroidal neovascularization.

Recently, bFGF was thought to take a role in progression and survival of retinoblastoma, a tumor producing significant amounts of bFGF. The differential production and response to isoforms of bFGF reveal bFGF as a growth factor influencing pathogenesis and chemoresistence of retinoblastoma.

Inhibitory PAS (Per/Arnt/Sim) domain protein

As mentioned earlier, the upregulation of angiogenic molecules like VEGF-A and angiopoietin-4 (Ang-4)

during hypoxia is mediated by hypoxia-inducible transcription factor-1 (HIF-1) and can induce an angiogenic response. Interestingly, hypoxic conditions in the cornea appearing, for example, during overnight closure of the eye, do not induce corneal neovascularization, indicating the presence of factors suppressing hypoxia-induced angiogenesis. However, prolonged contact lens use has been associated with corneal angiogenesis and hypoxia has been implicated in this process. IPAS, a basic helix–loop–helix (bHLH)/PAS protein, expressed in mouse corneal epithelium, was suggested as a negative regulator of HIF-mediated control of gene expression: only low levels of IPAS mRNA were detectable in primary cultures of mouse corneal cells under normoxic conditions, whereas under hypoxic conditions IPAS mRNA expression was upregulated. Following transfection of primary corneal cells with an IPAS antisense vector, VEGF mRNA expression – under normoxic and hypoxic conditions – was upregulated. Furthermore, in vivo experiments with mouse corneas containing pellets with IPAS antisense oligonucleotides showed a significantly induced neovascularization compared to the eyes treated with the IPAS sense oligonucleotide.

Cornea and Corneal Epithelium

Consistent with the assumption of a redundantly organized angiogenic privilege, numerous endogenous anti-angiogenic factors in the cornea are described to be implicated in the regulation of angiogenesis.

Years ago, the corneal epithelium itself was found to have angiogenic activity. In 1978, Eliason reported data from an in vivo system suggesting corneal epithelium as a source for an unknown vasostimulating substance. In vitro experiments from Eliason and Elliott also showed a stimulating effect of corneal epithelial homogenate and epithelial-conditioned medium on the proliferation of cultured rabbit vascular endothelial cells. Recent research attributes corneal epithelium an anti-angiogenic function: an intact corneal epithelium can suppress inflammation and corneal neovascularization in the graft following orthotopic transplantation in mice. Secondly, mice with de-epithelialized corneas have significantly increased recruitment of CD45þ inflammatory cells and an increased neovascular response compared to mice with an intact epithelium. One potent mechanism for an antiangiogenic function of corneal epithelium is the ectopic constitutive expression of VEGFR-3 on normal human corneal epithelial cells. VEGFR-3 can act as a decoy receptor to bind VEGF-C, thus functioning as a sink for the angiogenic molecules and inhibiting inflammation induced corneal hemangiogenesis and lymphangiogenesis. A similar task fulfils the soluble form of VEGFR-1 – expressed in the cornea – where it can neutralize VEGF-A.

Limbal Barrier Function

The border between sclera/conjunctiva and the transparent cornea, the limbus, is of great importance for angiogenesis: the loops and arcades of conjunctival capillaries as well as the lymphatic capillaries end in the limbal region. Corneal neovascularization, however, arises in the limbal area from preexisting pericorneal vessels (hemangiogenesis as well as lymphangiogenesis).

Stem cells, required for corneal epithelial cell proliferation and differentiation, are located in the basal epithelium at the corneoscleral limbus. They were described to act as a barrier between conjunctival and corneal epithelium and as important for corneal wound healing. In normal situations, the limbal stem cells prevent conjunctival epithelial cells from migrating to the ocular surface thereby inhibiting corneal neovascularization. This limbalbarrier concept may contribute to the maintenance of the angiogenic privilege. The theory is supported by the observation of conjunctivalization of the corneal surface with subsequent vascularization in situations of loss or malfunction of the stem cells.

Angiogenic Privilege and Immune Privilege

Ingrowth of blood and lymphatic vessels into the cornea is incompatible with good vision. Visual acuity is impaired not only by vascularization itself, but also by secondary changes such as lipid keratopathy, corneal edema, or bleeding into the cornea, thereby reducing corneal clarity and transparency. As mentioned earlier, actively maintaining the avascularity even under inflammatory or other angiogenic conditions is ensured by the angiogenic privilege. It contributes, at least partly, to the occurrence of the prolonged graft survival of corneal allografts, called the immune privilege of the eye. The phenomenon of the immune-privileged site was first proposed by Medawar, in 1948, and has built a foundation for numerous research. Nowadays, ocular immune privilege is commonly seen as the fact that vulnerable organs or tissues are protected from pathogens without an immunogenic inflammation that would permanently damage those tissues and/or would lead to a loss of specialized functions.

An immune response after transplantation in so-called low-risk eyes can only be noticed in around 10%, although under normal circumstances there is no HLA matching and only a topical, but no systemic, immunosuppression. In contrast, immune reactions in high-risk eyes with preceding corneal inflammation or neovascularization occur in over 50%. The pathologically vascularized recipient bed prior to corneal transplantation (i.e., penetrating keratoplasty), therefore, lowers the outcome of corneal transplantation and is an important risk factor for subsequent immune reactions.

Immune responses are primarily mediated by corneal lymphatic vessels which form the afferent arc of the immune response. Via the lymphatic vessels invading antigen-presenting cells (APCs; dendritic cells from the graft or host) and antigenic material from the graft can be transported via conjunctival lymph vessels to the regional lymph nodes. The draining cervical lymph nodes were shown to be critically involved in promoting alloimmunity and allograft rejection. Following surgical removal of the cervical lymph nodes and following orthotopic corneal transplantation of fully mismatched high-risk allografts, over 90% of the hosts accepted the allograft.

The importance of the lymphatic vessels, being the afferent arc of the immune response, offers new therapeutic opportunities for improving graft survival. Interfering with this pathway might restore the immune privileged status of the eye and ensures prolonged graft survival in lowand high-risk eyes. Early studies have shown that induction of donor-specific anterior chamber-associated immune deviation (ACAID) – manifestation of the ocular immune-privilege induced prolonged graft survival in high-risk eyes of C57BL/6 mice. Recently, it was shown that Integrin a5-blockade could significantly block the outgrowth of lymphatic vessels in the cornea. The angiostatic drug bevacizumab, a recombinant humanized monoclonal antibody against VEGF-A, inhibits corneal hemangiogenesis and lymphangiogenesis in vitro and in vivo. Furthermore, inhibition of corneal hemangiogenesis and lymphangiogenesis by a molecular VEGF-A trap leads to improved long-term graft survival. In addition to the inhibition of inflammatory lymphangiogenesis, alternative strategies like induction of regression of established lymphatic vessels in prevascularized corneas and influencing the recruitment of APCs could be possible methods for corneal anti-lymphangiogenic treatment. Recently, it was shown that even hemangiogenesis and lymphangiogenesis occurring following transplantation increase the risk for graft rejection after high-risk corneal transplantation.

See also: Avascularity of the Cornea; Corneal Angiogenesis.

Further Reading

Azar, D. T. (2006). Corneal angiogenic privilege: Angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis).

Transactions of the American Ophthalmological Society 104: 264–302.

Cebulla, C., Jockovich, M. E., Pina, Y., et al. (2008). Basic fibroblast growth factor impact on retinoblastoma progression and survival.

Investigative Ophthalmology and Visual Science 49(12): 5215–5221. Chang, J. H., Gabison, E. E., Kato, T., and Azar, D. T. (2001). Corneal neovascularization. Current Opinion in Ophthalmology 12: 242–249.

Churchill, A. J., Carter, J. G., Ramsden, C., et al. (2008). VEGF polymorphisms are associated with severity of diabetic retinopathy.

Investigative Ophthalmology and Visual Science 49: 3611–3616. Cursiefen, C. (2007). Immune privilege and angiogenic privilege of the

cornea. Chemical Immunology and Allergy 92: 50–57. Cursiefen, C., Chen, L., Dana, M. R., and Streilein, J. W. (2003a).

Corneal lymphangiogenesis: Evidence, mechanisms, and implications for corneal transplant immunology. Cornea 22: 273–281.

Cursiefen, C., Seitz, B., Dana, M. R., and Streilein, J. W. (2003b). Angiogenesis and lymphangiogenesis in the cornea. Pathogenesis, clinical implications and treatment options. Ophthalmologe 100: 292–299.

Folkman, J. and Shing, Y. (1992). Angiogenesis. Journal of Biological Chemistry 267: 10931–10934.

Hori, J. and Niederkorn, J. Y. (2007). Immunogenicity and immune privilege of corneal allografts. Chemical Immunology and Allergy 92: 290–299.

Makino, Y., Cao, R., Svensson, K., et al. (2001). Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 414: 550–554.

Niederkorn, J. Y. (1999). The immune privilege of corneal allografts.

Transplantation 67: 1503–1508.

Niederkorn, J. Y. (2007). Immune mechanisms of corneal allograft rejection. Current Eye Research 32: 1005–1016.

Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992). Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 359: 843–845.

Streilein, J. W. (2003a). Ocular immune privilege: The eye takes a dim but practical view of immunity and inflammation. Journal of Leukocyte Biology 74: 179–185.

Streilein, J. W. (2003b). Ocular immune privilege: Therapeutic opportunities from an experiment of nature. Nature Reviews Immunology 3: 879–889.

Glossary

Angiogenesis – The formation of new vessels from preexisting vascular structures.

Allograft – The tissue taken from one person for transplantation into another.

Autograft – The tissue transplanted from one part of the body to another part of the same individual. Chemotaxis – The movement of neutrophils toward bacteria or an area of tissue damage.

Conjunctival metaplasia – An abnormal epithelial differentiation represented by a spectrum of skin-like changes of conjunctival epithelium.

Corneal extracellular matrix – The tissue that provides structural support to the cells in the cornea. Corneal pannus – The fibrovascular connective tissue that proliferates in the anterior layers of the peripheral cornea in inflammatory corneal disease. Growth factor – A naturally occurring protein capable of stimulating cellular growth, proliferation, and differentiation.

Hypercapnia – High levels of carbon dioxide. Hypoxia – Oxygen deficiency.

Limbal stem cell deficiency – The loss of stem cells in the limbus (ring around the base of the cornea which supports health of the corneal epithelium).

Matrix metalloproteinases (MMPs) – The zincdependent endopeptidases capable of degrading all kinds of extracellular matrix proteins; they can also process bioactive molecules.

Penetrating keratoplasty (PK) – The procedure in which a full-thickness button of cornea is removed from the recipient and replaced with a similar-sized or larger button of tissue from a donor. Vasculogenesis – The formation of new blood vessels from bone-marrow-derived angioblasts that occurs mainly during embryogenesis.

Introduction

Under homeostatic conditions, the cornea is avascular, which is critical for corneal light transmission and proper optical performance. Corneal avascularity is maintained by tightly controlled biological anti-angiogenic events that counterbalance the effects of pro-angiogenic factors in the

cornea. Under pathological conditions, the balance may be shifted toward angiogenesis, leading to the formation of new blood vessels and lymphatic channels. New blood vessel formation or corneal neovascularization (NV) is a sight-threatening condition usually associated with inflammatory or infectious disorders. It is a major contributor to the loss of corneal transparency. The presence of corneal NV, in turn, elevates the risk of graft rejection and decreases the success of penetrating keratoplasty (PK).

Angiogenesis

Angiogenesis is the main process of blood vessel formation in nonembryonic tissue. It involves the formation of new vessels from preexisting vascular structures and is the primary mechanism underlying corneal NV. New blood vessels also form during vasculogenesis, which is the formation of new blood vessels from bone-marrow-derived angioblasts and occurs mainly during embryogenesis.

Angiogenesis is a complex process that starts with vasodilation of existing vessels and an increase in vascular permeability. This leads to extravasation of plasma proteins (such as fibrin), growth factors, and inflammatory mediators. The accumulation of plasma proteins in the surrounding tissue provides a supporting structure for subsequent endothelial cell (EC) migration. The combined presence of growth factors and inflammatory mediators stimulates the degradation of the extracellular matrix (ECM), making room for EC migration as well as releasing angiogenic factors anchored in the matrix. The newly released angiogenic factors then continue to activate ECs, which migrate from preexisting vessels and form sprouting tubes.

The avascularity of the cornea dictates that nutrients for this tissue be obtained from adjacent tissues. The three major sources of nutrients are tear film, aqueous humor, and the pericorneal capillary plexus at the limbus. This plexus nourishes the peripheral cornea and is derived from ciliary arteries, which are branches of the ophthalmic artery. These vessels do not normally enter the cornea. However, new blood vessels may sprout from capillaries and venules of the pericorneal plexus under pathological conditions, leading to corneal NV.

Etiology and Epidemiology of Corneal NV

Although the exact incidence of corneal NV is not known, it was estimated that this condition affects 1.4 million

patients in the United States annually and that 20% of corneal buttons obtained during PK show evidence of NV. The causes of corneal NV include immune, inflammatory, infectious, degenerative, and traumatic disorders. Corneal infections are the most common worldwide causes of corneal NV leading to vision loss. A classic example is trachoma, an infectious disease characterized by the formation of a superior pannus, which can extend to the central cornea and is often associated with corneal scarring, opacification, and loss of visual acuity. The incidence of trachoma in the US is low; however, this condition remains a major cause of blindness in other parts of the world (Figure 1).

Corneal NV is also commonly associated with other severe bacterial and viral infections. The herpes virus family (primarily herpes simplex and herpes zoster viruses) is the primary cause of keratitis-induced NV in transplant buttons. In herpes-simplex-induced stromal keratitis, NV is essential for the pathogenesis of keratitis, and inhibition of angiogenesis can reduce the formation of corneal lesions.

Although infections account for many US cases of corneal NV, the most common cause of corneal NV in the US is the use of contact lenses. In this case, hypoxia and hypercapnia are thought to be associated with the induction of NV (Table 1).

The incidence of corneal NV after PK can be as high as 40% at 6–9 months after surgery. The prognosis of transplanting grafts into heavily vascularized corneas is poor. Graft failure has been reported to contribute to >30% of the histopathological diagnoses obtained from vascularized corneal buttons. Risk factors for corneal NV after PK in patients without active inflammation, previous corneal NV, or persistent epithelial defects include suture knots buried in the host stroma, active blepharitis, and a large recipient bed.

Figure 1 Salzman’s nodular degeneration with associated superficial corneal neovascularization.

Immune disorders also contribute to corneal NV (Figure 2). These disorders can result in significant vision loss and include ocular pemphigoid, rosacea, atopic keratoconjunctivitis, and Stevens–Johnson syndrome. The incidence of corneal NV among patients with these disorders can be considerable. Long-term follow-up of patients with atopic keratoconjunctivitis, for example, revealed that the rate of corneal NV was as high as 60% during the disease course.

Limbal stem cell deficiency, which may occur following trauma and chemical burns, is another cause of corneal NV. It may be also associated with aniridia and autoimmune disorders. Limbal stem cell deficiency produces not only corneal NV, but also corneal inflammation and conjunctivalization of the corneal epithelium. The restoration of corneal avascularity after successful limbal stem cell transplantation underscores the importance of the antiangiogenic and anti-inflammatory activity of normal corneal epithelial cells.

Clinical Manifestations

Corneal NV can be classified as pannus or stromal NV. In the former, fibrovascular tissue is visible between the epithelium and Bowman layer. Inflammatory pannus is associated with prominent leukocyte infiltration and disruption of Bowman’s layer. In contrast, degenerative pannus is characterized by fewer inflammatory cells, an intact Bowman’s layer, and regression of the vascular component that leaves a layer of fibrous tissue. Stromal NV, located posterior to Bowman’s layer, is more commonly seen in the anterior two-third of the stroma. In herpetic and syphilitic interstitial keratitis, deep stromal vessels (and ghost vessels in the late quiescent stages) are seen just anterior to Descemet’s membrane (Figure 3).

Visual acuity is reduced by corneal NV. Reduced visual acuity may be secondary to multiple factors. For example, opacity caused by circulating blood cells may interfere with visual acuity. Acuity may also be reduced by irregular architecture of vascular walls, a feature that induces higher-order aberrations. Other effects contributing to diminished visual acuity may include alteration in the spacing of stroma collagen fibers, fluid leakage, edema and lipid deposition in the surrounding tissue, and corneal surface irregularities (Figure 4).

Mechanisms Underlying the Maintenance

of Corneal Avascularity

Several mechanisms have been proposed to contribute to corneal avascularity. These include:

Figure 2 Corneal neovascularization and infectious keratitis in a patient with underlying severe dry eye syndrome secondary to rheumatoid arthritis.

Figure 4 Lipid deposition secondary to corneal neovascularization. Note relative small vessel crossing the host-graft junction responsible for significant lipid deposit.

Figure 3 Superficial and deep stromal neovascularization in a patient with neurotrophic cornea.

1.Corneal dehydration resulting in tightly packed collagen lamellae. The relationship between corneal NV and corneal edema was first reported in 1949 by Cogan, who postulated that the distention and bursting of the vessels preceding formation of the capillary sprouts were due to a decrease in external pressure that reduced

vessel wall support. However, subsequent studies showed that corneal edema alone is not sufficient to trigger corneal NV.

2.The angiostatic nature of corneal epithelial cells. Blood vessels are known to be capable of growing into corneas in the absence of epithelium. Early research suggested that corneal epithelial cells are a source of angiogenic factors. More recent studies suggest that the corneal epithelium has an anti-angiogenic effect. The presence of soluble vascular endothelial growth factor (VEGF) receptors 1 and 3, and other naturally occurring antiangiogenic factors, in the corneal epithelium contributes to its angiostatic nature.

3.The immune privilege of the cornea. The mechanisms underlying corneal immune privilege include low expression of major histocompatibility complex (MHC) antigens on corneal cells, expression of Fas ligand by the cornea, a relative paucity of mature antigen-presenting cells, and the presence of immunomodulatory molecules in the anterior chamber. Importantly, this state can be reversed by inflammation, and such reversal may contribute to vascularization. For example, polymorphonuclear leukocytes have the potential to initiate corneal NV through

the release of chemical mediators. Accordingly, there is a clear association between corneal inflammation and NV. Nevertheless, corneal NV may occur in the absence of inflammation.

4.Low corneal temperature, extensive innervation, and movement of the aqueous humor across the cornea. The roles of these factors in the maintenance of corneal avascularity are currently unclear.

5.The barrier function of limbal cells. The limbus is thought to prevent corneal NV by acting as a barrier to conjunctival growth over the cornea. This barrier function may be attributable to the ability of limbal stem cells to replenish the corneal epithelium, thus preventing invasion of conjunctival epithelium and avoiding NV. This hypothesis has been used to account for corneal NV following experimental limbal damage and stem cell dysfunction. It is also one of the explanations for corneal pannus observed in aniridia. This hypothesized barrier function of limbal cells supports the use of limbal stem cell transplantation as a definitive treatment for ocular-surface disorders. However, a physical barrier may not completely explain corneal avascularity.

6.Low levels of angiogenic factors and active production of potent anti-angiogenic factors in the cornea during homeostasis.

7.Active production of potent anti-angiogenic factors. Although the five previously mentioned factors likely contribute to maintenance of corneal avascularity, available evidence supports this as the main mechanism responsible for maintaining corneal avascularity.

Table 2 Factors involved in the regulation of angiogenesis

Corneal Angiogenic Privilege: The Balance between Angiogenesis and Anti-Angiogenesis

Multiple local and systemic signals are responsible for regulating growth and regression of new blood vessels. These signals include cyclic adenosine monophosphate (cAMP), steroid hormones, protein kinase C (PKC) agonists, polypeptide growth factors, oxygen, free radicals, glucose, cobalt, and iron. In the cornea, the tight equilibrium between these proand anti-angiogenic signals may be disrupted under pathological conditions. Ultimately, this may tip the balance toward an upregulation of proangiogenic factors or a downregulation of anti-angiogenic factors, in either case leading to corneal NV (Table 2).

When the cornea is injured, wound healing often occurs in the absence of NV. This is the case for most adequately treated corneal infections. Healing after corneal surgery is also usually avascular. Corneal wound healing involves four phases. In the first phase, keratocytes near the area of epithelial debridement undergo apoptosis. In the second phase, adjacent keratocytes proliferate to repopulate the wound within 24–48 h after wounding. These keratocytes

transform into fibroblasts and migrate into the wound area. This process may take up to a week and is not accompanied by corneal NV. In the third phase, fibroblasts may transform into myofibroblasts. This occurs in laser-inflicted wounds lacking Bowman’s layer and in incisional wounds. Myofibroblasts may take up to a month to become apparent. Corneal NV is also absent in this phase of wound healing. The fourth and final phase involves stromal remodeling and is dependent on the original wound. When wound healing is accompanied by ECM turnover, angiogenesis in granulation tissue is usually observed.

Some of the molecules that regulate angiogenesis are discussed below.

Angiogenic Molecules

Vascular endothelial growth factor

VEGF is a dimeric 46-kDa glycoprotein. This growth factor stimulates angiogenesis by increasing EC proliferation, migration, proteolytic activity, and capillary tube formation. It also significantly increases vascular permeability. The VEGF family includes VEGF-A, -B, -C, -D, placenta growth factor (PlGF), and the viral VEGF

homolog VEGF-E. VEGF-B promotes nonangiogenic tumor progression, while VEGF-C and -D participate in angiogenesis and lymphangiogenesis. VEGF-A also participates in angiogenesis and increases vascular permeability.

Five isoforms of VEGF-A (VEGF115, 121, 165, 189, and 206) can be generated by alternative splicing of the same gene. The longer isoforms (VEGF189 and 206) are matrixbound, whereas the shorter isoforms (VEGF121 and 165) are freely diffusible. These VEGF isoforms produce different actions when secreted. For example, all isoforms increase in vascular permeability, but only VEGF121 and VEGF165 have mitogenic activity. VEGF121 has greater angiogenic activity than VEGF165 or VEGF189. On the other hand, VEGF165 is more potent than VEGF121 in induction of inflammation, intercellular adhesion mole- cule-1 (ICAM-1) expression in ECs, and chemotaxis of monocytes. This suggests that alternate splicing of VEGF messenger RNA (mRNA) can be regulated to achieve a range of physiologic actions.

The VEGF family members act through binding to high-affinity receptor tyrosine kinases. Two high-affinity receptor tyrosine kinases have been identified for VEGF- A: VEGFR-1 (fms-like tyrosine kinase-1 or Flt-1) and VEGFR-2 (kinase insert domain-containing receptor or KDR). VEGFR-3 (fms-like tyrosine kinase-4 or Flt-4) serves as a high-affinity receptor for VEGF-C and -D. Both VEGFR-1 and -2 are expressed primarily in vascular ECs, while VEGFR-3 is predominantly expressed in lymphatic ECs. VEGF-B binds to VEGFR-1 and has mild mitogenic activity. In contrast, binding of VEGF-D and -C to VEGFR-3 regulates the growth and differentiation of blood vessels and lymphatic endothelium.

VEGF is produced by macrophages, T cells, astrocytes, pericytes, fibroblasts, retinal pigment epithelial cells, and smooth muscle cells. In addition, VEGF is expressed in all three cellular layers of the cornea. It is highly expressed in vascular ECs of limbal vessels and in new stromal vessels. Under inflammatory conditions, VEGF expression is increased in epithelial and vascular ECs, particularly near macrophage infiltrates and fibroblasts in corneal scars. Following corneal cautery, VEGF165 and 189 mRNA is increased at 48 h and returns to baseline by day 7. Immunohistochemistry has revealed that VEGF is initially expressed in neutrophils and later expressed in macrophages, demonstrating that VEGF production by leukocytes is associated with corneal NV. In addition, VEGF concentration is significantly increased in vascularized corneas as compared to normal corneas. In limbal-deficiency-induced corneal NV, VEGF mRNA and protein are induced after injury and are both temporally and spatially correlated with inflammation and NV. VEGF is not only induced during NV, but is also required for corneal angiogenesis. The indispensable role of VEGF in angiogenesis is shown by the finding that

stromal implantation of anti-VEGF antibodies inhibits NV in a rat model. Conversely, implantation of a Hydron pellet containing VEGF into the stroma induces severe corneal NV without significant inflammation.

The effects of VEGF in the cornea are not limited to NV, as this growth factor has also been shown to regulate goblet cell migration. Studies analyzing the correlation between cornea NV and conjunctivalization showed that VEGFR-1 is present in the conjunctiva-like epithelium covering the cornea as well as in goblet cells, invading leukocytes, and the corneal vasculature. Inhibition of VEGF activity inhibited not only corneal NV, but also goblet cell density, suggesting that VEGF may promote goblet cell migration.

Evidence suggests that VEGF also participates in corneal lymphangiogenesis. Corneal lymphangiogenesis may contribute to graft sensitization and rejection, following high-risk keratoplasty of vascularized corneas. VEGF-C binds to VEFGR-3 and induces lymphatic growth in the cornea. Interestingly, inhibition of lymphatic growth is observed after administration of a VEGF trap that neutralizes VEGF-A, but not VEGF-C or -D. This could be explained by the chemotactic effect on macrophages that release VEGF-C in inflamed corneas observed with VEGF-A. Thus, VEGF-A amplifies signals essential for lymphatic growth. In general, corneal lymphangiogenesis seems to correlate well with the degree of corneal hemangiogenesis.

Recent studies have shown that VEGF, although present in the cornea, does not promote angiogenesis under normal conditions. VEGF-A found in corneal tissue is mostly bound to an alternative spliced secreted isoform of VEGFR-1 (sflt-1), which acts as a trap for secreted VEGF-A and in this way contributes to maintenance of corneal avascularity. In addition, VEGFR-3 is expressed in endothelial as well as epithelial cells in the cornea. When VEGF-C and -D bind to endothelial VEGFR-3, they stimulate proangiogenic signaling. In contrast, VEGFR-3 expressed by corneal epithelium acts as a decoy receptor sequestering VEGF but yet rendering it available when an angiogenic response is needed to enhance the immune defense. This VEGFR-3 sink system is a potent mechanism that inhibits inflammatory-induced angiogenesis.

Basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is another potent angiogenic factor. It is a member of the fibroblast growth factor (FGF) family, which includes 23 heparin-binding peptides widely expressed during cell differentiation, angiogenesis, mitogenesis, and wound healing. bFGF functions are mediated by the receptors FGFR-1, -2, -3, and-4. FGF recptor-1 (FGFR-1) is expressed in normal corneal epithelium, while bFGF is upregulated following injury. It is also upregulated following co-culture of corneal epithelial cells with vascular EC and keratocytes. The affinity of bFGF for its receptor differs according to the extent of

maturation of new vessels. This may be due to varying expression of heparan sulfate proteoglycans and highlights the role of ECM proteins in the regulation of corneal angiogenesis.

Matrix metalloproteinases

The matrix metalloproteinases (MMPs) constitute a multigene family of zinc-binding proteolytic enzymes that participate in ECM remodeling. Many of the growth factors that modulate angiogenesis also influence MMP expression. These growth factors include VEGF, FGF-2, and tumor necrosis factor-alpha (TNF-a). Vascular ECs respond by secreting proteolytic enzymes that degrade the ECM to facilitate migration and differentiation of ECs. The MMPs that have identified in the cornea are collagenases I and II (MMP-1 and -13), stromelysin (MMP-3), matrilysin (MMP-7), membrane-type MMP (MT-MMP-14), and gelatinases A and B (MMP-2 and -9). Both MMP-2 and MMP-9 are proteolytically activated primarily by MT1MMP during capillary formation. Several reports suggest that these MMPs participate in vascular invasion by directly degrading the matrix or releasing matrix-bound cytokines and growth factors. Accordingly, inhibition of MMP-9 activity in the cornea decreases angiogenesis. However, given their ability to degrade ECM, MMPs exhibit a dual action in angiogenesis. For example, MMP-2 activation may release anti-angiogenic fragments, allow the production of potent angiostatic factors, or facilitate angiogenesis.

Lipid mediators

One of the initial events that occurs after corneal injury is the release of arachidonic acid. In the corneal epithelium, arachidonic acid is then metabolized by cyclooxygenase (COX) to generate eicosanoids (such as 12and 15-HETE), lipoxin A4 (LXA4), and prostaglandins. 12(S)-HETE is a powerful angiogenic factor, and COX inhibitors have been shown to reduce corneal angiogenesis in animal models. Plateletactivating factor is another potent lipid mediator released from the cell membrane after corneal injury. It contributes to corneal NV by increasing expression of VEGF, MMP-9, and urokinase plasminogen activator (uPA), all of which subsequently stimulate vascular EC migration.

Anti-Angiogenic Molecules

Angiostatin

Angiostatin results from the cleavage of plasminogen. Several MMPs can cleave plasminogen to generate angiostatinlike molecules. The inhibitory effect of angiostatin on vascular ECs may be due to inhibition of adenosine triphosphate (ATP) synthesis in these cells, an effect that decreases EC migration and proliferation. Angiostatin binds to integrin alpha-v beta-3 (avb3) and affects angiogenesis as well as

developmental NV. It also induces vascular EC apoptosis mainly in areas of NV.

All three layers of the cornea are able to synthesize plasminogen and angiostatin. Tears collected after overnight eye closure contain a significant amount of angiostatinrelated molecules known to have anti-angiogenic properties. This has also been shown in tears of contact lens-bearing patients, suggesting that these molecules play a role in preventing NV under hypoxic conditions. Corneal NV occurs following injection of anti-angiostatin antibodies into corneas having undergone post-excimer laser keratectomy. This supports the idea that plasminogen and angiostatin are important for the maintenance of corneal avascularity.

Endostatin and neostatins

Endostatin is a 20-kDa proteolytic fragment of collagen XVIII that exhibits anti-angiogenic activity. It was originally discovered as an angiogenic inhibitor purified from conditioned media of murine hemangioendothelioma cells. Endostatin inhibits bFGF-induced corneal NV as well as VEGF-induced vascular EC migration and proliferation. Collagen XVIII is localized mainly in the corneal vascular and epithelial basement membrane. Smaller fragments of collagen XVIII, known as Neostatins -7 and -14, are generated by the enzymatic activity of MMPs -7 and -14, respectively. They have potent antiangiogenic and anti-lymphangiogenic properties. Local production of endostatin and Neostatins -7 and -14 may occur during wound healing. Endostatin is Food and Drug Administration (FDA)-approved for the treatment of cancer-related NV.

Pigment-epithelial-derived factor

Pigment-epithelial-derived factor (PEDF) is a potent anti-angiogenic and neurotrophic factor that is found in multiple eye tissues including the cornea. In contrast to VEGF, which is induced under low oxygen conditions, PEDF expression is suppressed during hypoxia. PEDF induces EC apoptosis. It also has antipermeability and anti-inflammatory activity that counterbalances VEGF actions. Studies have shown that PEDF-blocking antibodies induce corneal NV when implanted into the stroma and that recombinant PEDF inhibits bFGF-induced corneal NV. These findings are consistent with an essential role for PEDF in maintaining the avascularity of ocular tissues. Given its effectiveness at countering VEGF activity, PEDF may be a good pharmacological inhibitor of angiogenesis.

Arresten, canstatin, and tumstatin

Arresten is a 26-kDa protein derived from the noncollagenous (NCl) domain of the type IV collagen a1 chain. This molecule has been shown to inhibit bFGFstimulated proliferation, migration, and tube formation of