Corneal Avascularity Research Articles

Angiogenesis: A special reference to corneal neovascularization.

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Corneal Neovascularization as a Target for Nucleotide-Based Therapies

The cornea is a transparent tissue with avascular characteristics (corneal avascularity). It can be compromised by imbalances of angiogenic factors due to chemical injuries, infections, or autoimmune diseases such as Stevens- Johnson’s syndrome, which can lead to corneal blindness. Clinically, the etiologies of corneal neovascularization (NV), such as inflammation, immune rejection, limbal stem cell deficiency, or hypoxia, are usually long-lasting; therefore, conventional treatment modalities, including antiangiogenic medications, laser, or surgeries have only a suboptimal effect, and the prognosis is even worse with multiple recurrences. In contrast, novel treatment modalities, such as gene therapy (enhanced intracellular expression of antiangiogenic factors) and nucleotide-based antiangiogenic therapy (antisense oligonucleotides, silence-RNA, and micro-RNA) have been accomplished in animal models or clinical trials in recent years. Because of its specific and long-lasting effect, as well as the improvement and verification of the safety of expression vectors and carriers, the clinical value of nucleotide-based therapy has been increasingly appreciated. This review summarizes and updates relevant research, and provides a better understanding regarding the mechanism and treatment of corneal NV.

Corneal Development: Different Cells from a Common Progenitor.

Development of the vertebrate cornea is a multistep process that involves cellular interactions between various ectodermal-derived tissues. Bilateral interactions between the neural ectoderm-derived optic vesicles and the cranial ectoderm give rise to the presumptive corneal epithelium and other epithelia of the ocular surface. Interactions between the neural tube and the adjacent ectoderm give rise to the neural crest cells, a highly migratory and multipotent cell population. Neural crest cells migrate between the lens and presumptive corneal epithelium to form the corneal endothelium and the stromal keratocytes. The sensory nerves that abundantly innervate the corneal stroma and epithelium originate from the neural crest- and ectodermal placode-derived trigeminal ganglion. Concomitant with corneal innervation is the formation of the limbal vascular plexus and the establishment of corneal avascularity. This review summarizes historical and current research to provide an overview of the genesis of the cellular layers of the cornea, corneal innervation, and avascularity.

Sema3A maintains corneal avascularity during development by inhibiting Vegf induced angioblast migration

Corneal avascularity is important for optical clarity and normal vision. However, the molecular mechanisms that prevent angioblast migration and vascularization of the developing cornea are not clear. Previously we showed that periocular angioblasts and forming ocular blood vessels avoid the presumptive cornea despite dynamic ingression of neural crest cells. In the current study, we investigate the role of Semaphorin3A (Sema3A), a cell guidance chemorepellent, on angioblast migration and corneal avascularity during development. We show that Sema3A, Vegf, and Nrp1 are expressed in the anterior eye during cornea development. Sema3A mRNA transcripts are expressed at significantly higher levels than Vegf in the lens that is positioned adjacent to the presumptive cornea. Blockade of Sema3A signaling via lens removal or injection of a synthetic Sema3A inhibitor causes ectopic migration of angioblasts into the cornea and results in its subsequent vascularization. In addition, using bead implantation, we demonstrate that exogenous Sema3A protein inhibits Vegf-induced vascularization of the cornea. In agreement with these findings, loss of Sema/Nrp1 signaling in Nrp1Sema− mutant mice results in ectopic angioblasts and vascularization of the embryonic mouse corneas. Altogether, our results reveal Sema3A signaling as an important cue during the establishment of corneal avascularity in both chick and mouse embryos. Our study introduces cornea development as a new model for studying the mechanisms involved in vascular patterning during embryogenesis and it also provides new insights into therapeutic potential for Sema3A in neovascular diseases.

Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.

Expression of pro‐ and anti‐angiogenic factors during the formation of the periocular vasculature and development of the avian cornea

During embryonic development, endothelial precursor cells (angioblasts) migrate relatively long distances to form the primary vascular plexus. The migratory behavior of angioblasts and localization of the primitive blood vessels is tightly regulated by pro-angiogenic and anti-angiogenic factors encountered in the embryonic environment. Despite the importance of corneal avascularity to proper vision, it is not known when avascularity is established in the developing cornea and how pro- and anti-angiogenic factors regulate this process. Using Tg(tie1:H2B:eYFP) transgenic quail embryos to visualize fluorescently labeled angioblasts, we show that the presumptive cornea remains avascular despite the invasion of cells from the periocular region where migratory angioblasts reside and form the primary vasculature. Semiquantitative reverse transcriptase polymerase chain reaction analysis and spatiotemporal examination of gene expression revealed that pro- and anti-angiogenic factors were expressed in patterns indicating their potential roles in angioblast guidance. Our findings show for the first time that chick corneal avascularity is established and maintained during development as the periocular vasculature forms. We also identify potential candidate pro- and anti-angiogenic factors that may play crucial roles during vascular patterning in the anterior eye.

Immunological Disruption of Antiangiogenic Signals by Recruited Allospecific T Cells Leads to Corneal Allograft Rejection

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival.

Reply to Chakrabarti et al.: Corneal angiogenesis in patients with null FOXC1 variants

In the 15 years since transcription factor mutation, and subsequently dosage alterations, were shown to account for a proportion of Axenfeld–Rieger syndrome cases, novel phenotypes continue to be defined. As illustrated by the spectrum of FOXC1 -attributable CNS anomalies (1, 2), these are frequently present in a subset of cases and only identified by meticulous phenotyping. Our original article in PNAS (3) demonstrated that a proportion of patients with a mutation or altered FOXC1 gene dosage exhibited corneal angiogenesis, confirming our extensive data from multiple murine mutants regarding Foxc1’s role in maintaining corneal avascularity. … [↵][1]1To whom correspondence should be addressed. E-mail: t-kume{at}northwestern.edu. [1]: #xref-corresp-1-1

Studies on Axenfeld–Rieger syndrome patients and mice reveal Foxc1's role in corneal neovascularization

Normal vision critically depends on the transparency of the cornea overlying the lens. This tissue is made up of precisely oriented layers of fibroblasts perfectly interwoven with the collagen and other ECM components that they produce. Importantly, in the healthy eye, there are no blood vessels or lymphatic vessels in the stroma, making it what is known as an “angiogenically privileged” site. However, in a number of human ocular disorders, and in response to injuries like chemical burns, blood vessels do grow into the cornea from around the junction between the iris and the sclera (the limbus). This pathological angiogenesis can severely disrupt clarity of vision and necessitate corneal transplants. Consequently, there is intense interest in understanding the processes that normally restrict blood vessel growth into the cornea. In PNAS, Seo et al. (1) provide compelling evidence from human and mouse studies that implicate one member of the large Forkhead box (Fox) transcription factor family [Forkhead box transcription factor c1 (Foxc1)] in the regulation of corneal avascularity. Specifically, they suggest that Foxc1 regulates the normal delicate balance between factors in the cornea that can promote angiogenesis (e.g., VEGF and VEGF receptor) and those that inhibit growth of blood vessels and lymphatics (e.g., soluble VEGF receptor).

Novel aspects of corneal angiogenic and lymphangiogenic privilege

In this article, we provide the results of experimental studies demonstrating that corneal avascularity is an active process involving the production of anti-angiogenic factors, which counterbalance the pro-angiogenic/lymphangiogenic factors that are upregulated during wound healing. We also summarize pertinent published reports regarding corneal neovascularization (NV), corneal lymphangiogenesis and corneal angiogenic/lymphangiogenic privilege. We outline the clinical causes of corneal NV, and discuss the angiogenic proteins (VEGF and bFGF) and angiogenesis regulatory proteins. We also describe the role of matrix metalloproteinases MMP-2, -7, and MT1-MMP, anti-angiogenic factors, and lymphangiogenic regulatory proteins during corneal wound healing. Established and potential new therapies for the treatment of corneal neovascularization are also discussed.

Matrix Revolution: Molecular Mechanism for Inflammatory Corneal Neovascularization and Restoration of Corneal Avascularity by Epithelial stem Cell Transplantation

Corneal neovascularization (CNV) associated with severe limbal stem cell (LSC) deficiency remains a challenging ocular surface disease in that corneal inflammation may persist and progress, and the condition will not improve without lsc transplantation. A prominent feature after successful LSC transplantation is the suppression of corneal inflammation and cnv, which is generally attributed to the endogenous anti-angiogenic/anti-inflammatory factors secreted by corneal epithelial cells. In addition, corneal epithelial basement membrane (EBM) plays a unique role in the regulation of angiogenesis; several potent anti-angiogenic factors are derived from the matrix component of EBM, such as endostatin (from collagen xviii) and restin (from collagen xv). Also, angio-inhibitory thrombospondin and tissue inhibitor of metalloproteinase-3 are deposited in EBM. Moreover, the heparan sulphate proteoglycan in EBM can bind and sequester vegf and FGF-2 from activation. Recently, cultivated corneal epithelial transplantation (CCET) and cultivated oral mucosal epithelial transplantation (comet) have emerged as promising techniques for the treatment of LSC deficiency. When human limbo-corneal epithelial (HLE) cells are cultivated on cryopreserved amniotic membrane, production of endostatin, restin, and IL-1ra is enhanced. This highlights the significance of delicate epithelial-matrix interactions in the generation of anti-angiogenic/anti-inflammatory factors by HLE cells, and this may, in part, explain the rapid restoration of corneal avascularity following CCET. In addition, whether epithelial stem cells can persist after transplantation is the key for CCET and COMET. Emerging evidence of long-term survival of cultivated epithelial cells after transplantation suggest that epithelial stem cells can be isolated and cultivated in vitro, and can re-establish the epithelial phenotype in vivo. Taken together, the merits of enhanced anti-angiogenic activity and the preservation of corneal epithelial stem cells encourage further application of this tissue engineering technique for ocular surface reconstruction.

Mediators of Neovascularization and the Hypoxic Cornea

The maintenance of corneal avascularity is essential to vision. The mechanisms by which the cornea becomes vascularized in response to inflammation or hypoxic stress are beginning to be elucidated. A detailed understanding of the molecular responses of the cornea to hypoxia is critical for prevention and development of novel treatments for neovascularization in a range of disease states. Here, we have examined the current literature on the major mediators of angiogenesis, which have previously been reported during hypoxia in the cornea in order to better understand the mechanisms by which corneal angiogenesis occurs in circumstances where the available oxygen is reduced. The normal cornea produces angiogenic factors that are regulated by the production of anti-angiogenic molecules. The various cell types of the cornea respond differentially to inflammatory and hypoxic stimuli. An understanding of the factors that may predispose patients to development of corneal blood vessels may provide an opportunity to develop novel prophylactic strategies. The difficulties with extrapolating data from other cell types and animal models to the cornea are also examined.

Emerging Roles for the CD36 Scavenger Receptor as a Potential Therapeutic Target for Corneal Neovascularization

Neovascularization (NV) of the normally avascular cornea arises from various causes including inflammation, infection, trauma, and contact lens wear. Corneal NV, whatever the cause, impairs vision and threatens the survival of corneal allografts, thus representing a serious clinical problem for which treatment is limited. Recent interest has focused on vascular endothelial growth factor (VEGF), a key angiogenic factor whose role in corneal NV is amply documented. While experimental studies underscore the efficacy of anti-VEGF targeted agents, there exists no clear consensus on the ideal treatment for this multifaceted pathology. This review discusses the therapeutic potential of CD36, a well established anti-angiogenic receptor. We present evidence that CD36 contributes significantly to the maintenance of corneal avascularity wherein its deficiency leads to age-related corneal NV. Data further reveal that activation of CD36 substantially attenuates and induces regression of inflammatory corneal NV via concerted inhibition of VEGFA, c-Jun N terminal kinase-1, and c-Jun. In parallel studies, we demonstrate that hypoxia, a fundamental stimulus of NV, markedly elevates CD36 corneal expression in a hypoxia-inducible factor-1 and reactive oxygen species dependent manner. Collectively, our findings unveil interesting avenues for future research on the involvement of CD36 in neovascular eye disease and suggest CD36 agonists as potential therapeutic agents for the management of corneal NV, possibly in combination with anti-VEGF therapies.

Genetic Ablation of CD36 Induces Age-Related Corneal Neovascularization

Corneal avascularity is tightly regulated by a balance between angiogenic and antiangiogenic factors (angiogenic privilege). In the current study, we tested the hypothesis that the CD36+/+ antiangiogenic receptor contributes toward the maintenance of corneal avascularity. Corneas of CD36 wild-type (CD36) and knockout (CD36) mice aged 4, 16, 52, and 78 weeks were histologically evaluated for corneal haze and neovascularization (NV). Quantitative real-time polymerase chain reaction was performed on corneal tissue from CD36+/+ and CD36-/- mice aged 4 and 52 weeks to examine the effect of CD36 deficiency on expression of relevant angiogenic factors. Corneal haze and NV were absent in CD36+/+ mice at all ages. Conversely, corneal haze and NV were evident at 52 and 78 weeks in CD36-/- mice, and the latter demonstrated a significant increase in vessel density at 52 and 78 weeks. Interestingly, compared with CD36+/+ mice, in the corneas of 52-week-old CD36-/- mice, thrombospondin-1 messenger RNA was repressed, and vascular endothelial growth factor A, c-Jun N-terminal kinase-1, and c-Jun levels were robustly upregulated. CD36-/- mice develop corneal NV that increases in severity with age, thus accentuating the role of CD36 in preserving corneal avascularity.

Ocular surface reconstruction: recent innovations, surgical candidate selection and postoperative management

Intricate homeostasis maintains the rich complexity and functionality of the ocular surface. Severe ocular surface diseases cause significant morbidities, including blindness. Ocular surface reconstruction strategies restore this homeostasis. Patients with limbal deficiency may benefit from limbal stem cell transplantation to alleviate persistent epithelial defects, maintain conjunctivalization regression and corneal avascularity, and restore vision. Amniotic membrane and other substrates function as tissue surrogates and substrates for expansion of stem cells. Maximum graft survivability necessitates aggressive and comprehensive preoperative management of inflammation, infection, microtrauma, dry eye conditions and HLA compatibility. Restoration of lacrimal gland function is important, as is implementation of aggressive immunosuppression. Future advancements in stem cell biology, tissue engineering, bioartificial microsystems or regenerative medicine will supplement the therapeutic armamentarium. The next decade will be an exciting time for corneal surgeons.

Cathepsin V, but not cathepsins L, B and K, may release angiostatin-like fragments from plasminogen

Abstract Cathepsin V is a lysosomal cysteine peptidase highly expressed in corneal epithelium; however, its function in the eye is still unknown. Here, we describe the capability of cathepsin V to hydrolyze plasminogen, which is also expressed in human cornea at levels high enough to produce physiologically relevant amounts of angiostatin-related molecules. The co-localization of these two proteins suggests an important role for the enzyme in the maintenance of corneal avascularity, essential for optimal visual performance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of plasminogen digestion by cathepsin V revealed the generation of three major products of 60, 50 and 40 kDa, which were electrotransferred to polyvinylidene difluoride membranes and excised for characterization. NH(2)-terminal amino acid sequencing of these fragments revealed the sequences EKKVYL, TEQLAP and LLPNVE, respectively. These data are compatible with cleavage sites at plasminogen F94-E95, S358-T359 and V468-L469 peptide bonds generating fragments of the five-kringle domains. In contrast, we did not detect any plasminogen degradation by cathepsins B, K and L. Using a Matrigel assay, we confirmed the angiogenesis inhibition activity on endothelial cells caused by plasminogen processing by cathepsin V. Our results suggest a novel physiological role for cathepsin V related to the control of neovascularization in cornea.

Regulation of corneal angiogenesis in limbal stem cell deficiency

Corneal angiogenesis is associated with a variety of corneal diseases, and is sometimes vision threatening. In recent years, with the discovery of major pro- and anti-angiogenic factors in the cornea, details of the angiogenic process are gradually unveiled. Of note, corneal inflammation and neovascularization associated with severe limbal stem cell (LSC) deficiency is a clinically challenging issue in that the condition persists long after the initial insult, and will not improve without transplantation of LSCs. However, to date the molecular mechanism by which LSC transplantation restores corneal avascularity is not fully understood. In addition to discussing major pro-angiogenic factors involved in corneal neovascularization, this review article also focuses on possible molecular mechanisms underlying persistent inflammation and neovascularization following severe LSC deficiency, and anti-angiogenic factors expressed by human limbo-corneal epithelial cells (HLCECs). Most of the recently discovered corneal anti-angiogenic factors belong to extracellular matrix proteins that acquire angio-inhibitory activity only after proper proteolytic processing. Our recent findings showed that the secretion of endostatin (derived from basement membrane collagen XVIII) and restin (from collagen XV) by HLCECs were enhanced when HLCECs were cultivated on amniotic membrane (AM). This adds to the advantage of transplanting ex vivo expanded HLCECs cultivated on AM in that the anti-angiogenic activity of the epithelial cells is augmented in a physiological way. Furthermore, proteomic profiling of HLCECs and human conjunctival epithelial cells (HCECs) identified a 14-3-3 protein (stratifin) preferentially expressed by HLCECs. In addition to functioning as a cell cycle controller, keratinocyte-derived stratifin induces MMPs which are involved in the generation of restin (by MMP-1) and endostatin (by MMP-3). These findings highlight the significance of delicate epithelial-matrix interactions in the maintenance of corneal avascularity.

Corneal avascularity is due to soluble VEGF receptor-1

Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision

Transparency of the cornea, the window of the eye, is a prerequisite for vision. Angiogenesis into the normally avascular cornea is incompatible with good vision and, therefore, the cornea is one of the few tissues in the human body where avascularity is actively maintained. Here, we provide evidence for a critical mechanism contributing to corneal avascularity. VEGF receptor 3, normally present on lymphatic and proliferating blood vascular endothelium, is strongly constitutively expressed by corneal epithelium and is mechanistically responsible for suppressing inflammatory corneal angiogenesis.

Unique Distribution of Thrombospondin-1 in Human Ocular Surface Epithelium

The study was conducted to elucidate the detailed expression pattern of angiogenesis-related factors in human ocular surface epithelium. The focus was factors with significantly higher gene expression in corneal epithelium (CE) than in conjunctival epithelium (CJE). The relative gene expression of 36 angiogenesis-related factors was compared in human CE and CJE, by using the introduced amplified fragment-length polymorphism (iAFLP) METHOD: Also examined were the expression patterns in the CE, limbal epithelium (LE), and CJE of factors with significantly higher expression in the CE, by using real-time PCR, in situ hybridization, immunohistochemistry, and immunoelectron microscopy. Only thrombospondin (TSP)-1 exhibited significantly higher expression in the CE. In situ hybridization and real-time PCR showed TSP-1 transcripts in the basal cells of the CE and LE. Compared with the CJE, they were significantly upregulated at those sites. Immunohistochemistry revealed that TSP-1 was strongly expressed in the basal region of the CE. Its expression was faint in LE and absent in CJE. Immunoelectron microscopy revealed that the CE and LE demonstrated TSP-1 labeling just below the epithelium, in the basal region of basal cells, and occasionally in the basal cell membrane. There was little or no labeling in the CJE. In the human ocular surface epithelium, basal cells of the CE and LE, but not of the CJE, synthesize TSP-1. High levels of TSP-1 are present only just below the CE. Its unique distribution may be related to corneal avascularity and integrity.