We characterized the presence of hemangiogenesis (HA) and lymphangiogenesis (LA) in human corneal specimens exhibiting 13 underlying pathologies.
Human corneal specimens were obtained from consenting subjects (n = 2 or n = 3 for each pathology; total sample size, n = 35). The pathological specimens were stained with hematoxylin and eosin (H&E) to determine the presence or absence of corneal neovascularization (NV) and superficial or deep stromal distribution of NV. Immunohistochemical staining was then performed to differentiate HA (positive for CD31) from LA (positive for lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1]).
The double-negative (CD31−/LYVE-1−) immunostaining, indicating the absence of NV, was exhibited by 21 specimens (60%). CD31+/LYVE-1−, indicating the presence of HA and absence of LA, was exhibited by 12 specimens (34%). The double-positive (CD31+/LYVE-1+) phenotype, indicating both HA and LA, was exhibited by 2 specimens (6%). Notably, the CD31−/LYVE-1+ phenotype, indicating the presence of LA and absence of HA, was not detected among the specimens. Deep stromal NV was exhibited in a 4:3 ratio to superficial stromal NV. The double-negative immunostaining was more prevalent in noninflammatory pathologies, particularly in comparison with combined neovascular phenotypes (ie, CD31+ or LYVE-1+). Among the neovascular phenotypes, HA was 7 times more common than LA. Specimens exhibiting LA presented only with the double-positive phenotype.
HA is the predominant component of NV in corneal pathologies. LA accompanies HA; however, isolated LA (from lymphatics in the conjunctiva) does not occur in these corneal pathologies. Our results suggest the potential therapeutic utility of targeting antineovascular therapies specifically for corneal HA and/or LA pathology.
corneal neovascularization; hemangiogenesis; lymphangiogenesis; LYVE-1; CD31
Corneal neovascularization is a serious condition that can lead to a profound decline in vision. The abnormal vessels block light, cause corneal scarring, compromise visual acuity, and may lead to inflammation and edema. Corneal neovascularization occurs when the balance between angiogenic and antiangiogenic factors is tipped toward angiogenic molecules. Vascular endothelial growth factor (VEGF), one of the most important mediators of angiogenesis, is upregulated during neovascularization. In fact, anti-VEGF agents have efficacy in the treatment of neovascular age-related macular degeneration, diabetic retinopathy, macular edema, neovascular glaucoma, and other neovascular diseases. These same agents have great potential for the treatment of corneal neovascularization. We review some of the most promising anti-VEGF therapies, including bevacizumab, VEGF trap, siRNA, and tyrosine kinase inhibitors.
anti-VEGF therapy; corneal neovascularization; bevacizumab; ranibizumab; VEGF Trap; silencing RNA
Decorin has been shown to have anti-angiogenic properties. In this study, we evaluate the involvement of membrane type 1-matrix metalloproteinase (MT1-MMP), a pro-angiogenic enzyme, in decorin cleavage in the cornea.
MT1-MMP expression was confirmed immunohistochemically in keratocytes and immortalized corneal fibroblast cell lines. Corneal micropockets of bFGF were used to assess the expression of decorin and MT1-MMP. Western blotting was used to evaluate decorin degradation by MT1-MMP. Aortic ring tube formation assays were used to assay the inhibitory effect of decorin and stimulatory effect of MT1-MMP on vascular endothelial cells in vitro.
We show that MT1-MMP expression is upregulated following bFGF pellet implantation in the cornea in vivo, and that MT1-MMP cleaves decorin in a time- and concentration-dependent manner in vitro. Furthermore, the addition of MT1-MMP reduces the inhibitory effects of decorin on aortic ring tube formation in vitro. Cleavage of decorin by MT1-MMP-deficient corneal cell lysates is diminished relative to that by wild-type corneal cell lysates, and an MT1-MMP knock-in restores decorin processing in vitro.
The pro-angiogenic role of MT1-MMP in the cornea may be mediated, in part, by facilitated cleavage of corneal decorin.
corneal neovascularization; angiogenesis; metalloproteinase; MMP-14; MT1-MMP; decorin; extracellular matrix
To characterize the involvement of Semaphorin 7A (Sema7a) in corneal neovascularization (NV).
We generated anti-Sema7A antibodies to detect protein expression. Corneal fibroblast cells were cultured, stimulated with bFGF, immunostained with anti-Sema7A antibodies, and visualized by confocal microscopy. bFGF pellets were implanted in mouse corneal micropockets for 3–10 days, and corneal sections were immunostained with anti-Sema7A antibodies. Mouse corneas were injected with naked Sema7A DNA and vector control for 3, 7, and 10 days. Mouse corneas were imaged by slit lamp microscopy, and areas of corneal NV were calculated using the ImageJ program. Mouse corneal sections were also immunostained with anti-macrophage marker (F4/80) and anti-vascular endothelial growth factor (VEGF)-A antibodies.
Our data showed enhanced Sema7A expression levels in bFGF-stimulated cultured corneal fibroblasts. bFGF corneal implantation also demonstrated enhanced Sema7A expression. Corneas injected with a Sema7A expression vector showed evidence of significant corneal NV compared to controls on day 10 (1.8 mm2 vs. 0.11 mm2; p<0.02). Additionally, immunolocalization of Sema7A DNA-injected corneas (at day 7) revealed macrophage recruitment and enhanced VEGF-A levels.
We demonstrated that Sema7A was expressed in vascularized corneas and showed pro-angiogenic properties in our corneal model. Understanding the mechanism of Sema7A in angiogenesis may provide a therapeutic target for the treatment of corneal angiogenesis-related disorders.
Semaphorin 7A; Sema7A; corneal neovascularization; angiogenesis
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 proangiogenic/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.
The introduction of the excimer laser for keratorefractive surgery in the 1990s permanently reshaped the treatment landscape for correcting refractive errors, such as myopia, hyperopia, and astigmatism. Until that point, these treatments had relied on less predictable techniques, such as radial keratotomy and automated lamellar keratectomy. In recent years, other new technologies, along with increased understanding of the basic science of refractive errors, higher-order aberrations, biomechanics, and the biology of corneal wound healing, have allowed for a reduction in the surgical complications of keratorefractive surgery. Novel technologies, such as eye tracking, anterior segment imaging, the femtosecond laser, and asphericity-optimized and wavefront-guided custom laser in situ keratomileusis, have assisted refractive surgeons in achieving greater predictability of their laser vision correction procedures. Understanding the cascade of events involved in the corneal wound healing process and examination of how corneal wound healing influences corneal biomechanics and optics are crucial to improve the efficacy and safety of laser vision correction.
angiogenesis; corneal wound healing; laser vision correction; metalloproteinases; refractive outcomes
To determine whether matrix metalloproteinase-7 (MMP-7) that is stably overexpressed by mouse corneal fibroblast cell lines exhibits proteolytic activity against the NC1 fragment of collagen XVIII.
Corneal fibroblasts isolated from MMP-7 knockout (7ko) mice were subjected to SV40 T-antigen immortalization and stably transfected with a bicistronic retroviral vector encoding green fluorescence protein and active MMP-7. The resulting MMP-7 knock-in fibroblasts (7ko-MMP-7 cells) were isolated and enriched by fluorescence activated cell sorting (FACS). Culture media samples from 7ko and 7ko-MMP-7 cells were then incubated with the recombinant NC1 fragment of collagen XVIII, and NC1 degradation was monitored by immunoblotting.
Immunoblot analysis revealed that MMP-7 was present in lysates and culture media from 7ko-MMP-7 fibroblasts, but not media from immortalized 7ko fibroblasts. Importantly, lower amounts of the NC1 fragment were present in in vitro enzymatic reaction mixtures containing concentrated 7ko-MMP-7 media than in those containing concentrated 7ko media.
Immortalized fibroblasts stably transfected with MMP-7 secrete active MMP-7 with proteolytic activity towards the NC1 fragment of collagen XVIII.
Cornea; Gene transfer; Matrilysin; Matrix metalloproteinases; MMP-7
The cornea is physiologically avascular. Following a corneal injury, wound healing often proceeds without neovascularization (NV); however, corneal NV may be induced during wound healing in certain inflammatory, infectious, degenerative, and traumatic states. Such states disrupt the physiologic balance between pro-angiogenic and anti-angiogenic mediators, favoring angiogenesis. Contributors to such states are matrix metalloproteinases (MMPs), which are key factors in both extracellular matrix remodeling and angiogenesis. Similarly, vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF) exert pro-angiogenic effects. Here, we elaborate on the facilitative role of MMPs—specifically Membrane Type 1 MMP (MT1-MMP, MMP14)—in corneal NV. Additionally, we provide new insight into the signaling relating to MT1-MMP, Ras, and ERK in the bFGF-induced VEGF-A expression pathways within the corneal fibroblasts.
Basic FGF; corneal angiogenesis; ERK; MT1-MMP; Ras; VEGF-A
To investigate potential risk factors associated with incident nuclear, cortical, and posterior subcapsular (PSC) cataracts and cataract surgery in participants in the Age-Related Eye Disease Study (AREDS).
Clinic-based prospective cohort study.
Persons (N=4425), aged 60 to 80 years of age enrolled in a controlled clinical trial of antioxidant vitamins and minerals, AREDS, for age-related macular degeneration (AMD) and cataract.
Lens photographs were graded centrally for nuclear, cortical, and PSC opacities using the AREDS System for Classifying Cataracts. Type-specific incident cataracts were defined as an increase in cataract grade from none or mild at baseline to a grade of moderate at follow-up, with also a grade of at least moderate at the final visit, or cataract surgery. Cox regression analyses were used to assess baseline risk factors associated with type specific opacities and cataract surgery.
Main Outcome Measures
Moderate cataract was defined as a grade of ≥4.0 for nuclear opacity, ≥10% involvement within the full visible lens for cortical opacity, and ≥5% involvement of the central 5 mm circle of the lens for PSC opacity. These were graded on baseline and annual lens photographs.
A clinic-based cohort of 4425 persons aged 55–80 years at baseline was followed for an average of 9.8 ± 2.4 years. The following associations were found: increasing age with increased risk of all types of cataract and cataract surgery; males with increased risk of PSC and decreased risk of cortical cataracts; non-whites with increased risk of cortical cataract; hyperopia with decreased risk of PSC, nuclear cataract, and cataract surgery; Centrum use with decreased risk of nuclear cataract; diabetes with increased risk of cortical, PSC cataract, and cataract surgery; higher educational level with decreased risk of cortical cataract; and smoking with increased risk of cortical cataract and cataract surgery. Estrogen replacement therapy in female participants increased the risk of cataract surgery.
Our findings are largely consistent with the results of previous studies, providing further evidence for possible modifiable risk factors for age-related cataract.
Decorin is a small chondroitin sulfate proteoglycan that inhibits vascular endothelial cell migration and tube formation. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown to be an important angiogenic enzyme in the cornea. We evaluated the specific role of MT1-MMP in decorin cleavage in the cornea.
Western blotting was used to evaluate decorin degradation by MT1-MMP. Aortic ring tube formation assays were used to assay the inhibitory effect of decorin and the stimulatory effect of MT1-MMP on vascular endothelial cells in vitro. Corneal micropocket assays employing basic fibroblast growth factor (bFGF) were used to assess changes in the levels of decorin and MT1-MMP.
MT1-MMP cleaves decorin in a time- and concentration-dependent manner in vitro. MT1-MMP levels were upregulated following in vivo bFGF pellet implantation in the cornea, and decorin cleavage products were detected in bFGF-implanted corneas but not in normal corneas. MT1-MMP reduced the inhibitory effects of decorin on aortic ring tube formation in vitro.
MT1-MMP may play an essential role in angiogenesis through proteolytic processing of decorin in the cornea.
angiogenesis; cornea; decorin; MT1-MMP
To determine the effects of corneal epithelial membrane-type 1 matrix metalloproteinase (MT1-MMP) on vascular endothelial migration and proliferation.
We generated immortalized wild-type, MT1-MMP knockout and MT1-MMP knockin corneal epithelial cells. Calf pulmonary arterial endothelial (CPAE) cell proliferation and Boyden chamber migration were assayed.
Conditioned media from MT1-MMP epithelial knockout cells significantly increased CPAE proliferation BrdU incorporation, and CPAE migration as compared with wild-type epithelial cells. Conditioned media from knockin cells reversed the increase in CPAE proliferation, BrdU incorporation and CPAE migration. Knockin cells transfected with mutant MT1-MMP (E240A) did not abrogate the reversal effect.
Corneal epithelial MT1-MMP is anti-angiogenic. This anti-angiogenic activity does not require the catalytic domain.
To compare the refractive and visual outcomes of Sub-Bowman Keratomileusis (SBK) and thick-flap LASIK for moderate-to-high myopia, and evaluate the effect of corneal flap thickness on outcomes.
Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
Two studies were performed. In the first study, we retrospectively analyzed the refractive and visual outcomes of 33 eyes that underwent SBK (flap thickness 82–120 µm) and 62 eyes that underwent thick-flap LASIK (flap thickness ≥ 160). Inclusion criteria were: spherical equivalent (SE) -4 to -10 diopters (D), astigmatism ≤ 3 D, and follow-up of ≥ 3 months. In the second study, we evaluated the influence of flap thickness. We performed a case-control matched study (n=21 pairs) in which we controlled for residual stromal bed (RSB) thickness.
The mean flap thickness in SBK was 110.2 ± 9.2 versus 179.2 ± 19.5 in thick-flap LASIK. There were no significant differences in the visual outcomes. In the second study with equivalent RSB thickness, case control matched comparisons between SBK (108.6 ± 8.0 µm) and thick flap LASIK (165.7 ± 12.6 µm) showed no differences in preoperative and postoperative refractive and visual outcomes. Comparison of the intended versus achieved correction revealed no significant differences between the two groups.
Our retrospective analyses showed that the safety, efficacy, and predictability of SBK are similar to conventional thick-flap LASIK for corneas with equivalent RSB thickness.
Sub-Bowman Keratomileusis; Laser in situ keratomileusis; LASIK; flap thickness; thin flap; thick flap; LASIK outcomes; myopia
We describe a simple proteomic approach to investigate the differential protein expression patterns and identify the physiologically relevant angiogenic and anti-angiogenic factors involved in the hyaloid vascular system regression. Differentially-expressed proteins were identified using two-dimensional gel electrophoresis followed by nano-flow chromatography coupled to tandem mass spectrometry. These proteins are expected to provide insight as to their function in the early maintenance and eventual regression of the hyaloid vascular system.
hyaloid vascular system; vasa hyaloidea propria; tunica vasculosa lentis; proteomic analysis; kininogen
To determine the effect of keratocyte-derived MT1-MMP on calf pulmonary artery endothelial cell (CPAE) proliferation and migration.
Keratocyte lines were generated from MT1-MMP knockout (KO) and wild type (WT) mice. WT keratocytes were transfected with WT or mutant MT1-MMP DNAs (ΔTC or E240A). The effect of keratocyte-conditioned media on CPAE proliferation and migration was assayed.
KO keratocyte conditioned media resulted in the greatest increase of CPAE cell proliferation (190.5±6.0%; P<0.01). WT keratocyte conditioned media showed higher CPAE proliferation (155.4±3.6%) than WT/MT1-MMP-transfected keratocytes (119.7±2.2%; P<0.001). Migration assays confirmed these findings.
Keratocyte-derived MT1-MMP has anti-angiogenic effects in CPAE cells.
keratocyte; angiogenesis; gene/expression; metalloproteinases; MT1-MMP; keratocyte-derived MT1-MMP
The significance of collagen XVIII in the regulation of corneal reinnervation remains largely unknown. We used whole mount immunoconfocal microscopy to localize collagen XVIII to the nerve basement membrane of wild-type (WT) mouse corneas. Transmission electron microscopy showed corneal nerve disorganization in collagen XVIII knockout mice (col18a1−/−). Antibody 2H3-specific neurofilament colocalized with collagens XVIII and IV and laminin-2 in WT mouse corneas, but did not colocalize with collagen IV and laminin-2 in col18a1−/− mouse corneas. Following keratectomy, col18a1−/− mice displayed decreased corneal neurite extension compared to WT mice. Our data indicate that collagen XVIII may play an important role in corneal reinnervation after wounding.
extracellular matrix; collagen XVIII
Neostatin-7, with an anti-angiogenic potential, is generated from the proteolytic action of matrix metalloproteinase-7 on collagen XVIII. We previously reported that neostatin-7 inhibited angiogenesis in vitro and in vivo. Here we demonstrate that neostatin-7/collagen XVIII may possess anti-lymphangiogenic activities by: 1) corneal micropellet implantation of neostatin-7 reduced bFGF-induced corneal lymphangiogenesis; 2) neostatin-7 bound to VEGF receptor-3 in vitro; and 3) enhanced corneal lymphangiogenesis and VEGF-C expression in collagen XVIII knockout mice in a corneal wounding model. Understanding the mechanism of neostatin-7/collagen XVIII on corneal lymphangiogenesis may provide therapeutic interventions to treat lymphangiogenesis-related disorders, such as lymphedema, transplantation rejection and cancers.
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
To report two cases of femtosecond laser-assisted small incision deep lamellar endothelial keratoplasty (DLEK) for patients with corneal endothelial decompensation by Fuchs dystrophy and glaucoma
Femtosecond laser (IntraLase®; IntraLase Corp., Irvine, CA) with 15 kHz of repetition rate, was used for a 9.5 mm diameter by 400 µm thickness donor corneal lamellar dissection.
In Case 1, the graft was clear and compact without interface haze, Orbscan showed smooth and regular corneal surface, specular microscopy was unremarkable without sign of corneal endothelial damage, and Optical coherence tomography showed uniform graft well attached to recipient stroma with minimal interface reflection at 2 months postoperation. In Case 2, the graft was clear and compact with minimal interface haze at 1 month postoperation. Femtosecond laser-assisted small incision DLEK was safe and technically feasible in our cases; however, further evaluation is required to determine long-term effects.
Deep Lamellar Endothelial Keratoplasty; Femtosecond Laser
To obtain objective light-scattering measurements to test a hypothesis that identical PTK treatments cause distinct low- and high-level light-scattering responses in rabbit corneas.
An excimer laser was used to produce identical 6-mm diameter phototherapeutic keratectomy treatments (PTK) in 32 pigmented rabbits. Eyes were treated by performing a 40-μm epithelial ablation, followed by a 100-μm stromal PTK. Objective scattering measurements were made before treatment, weekly up to 5 weeks, and then biweekly to 9 weeks. Confocal microscopy was performed on several corneas at 4 and 7 weeks.
Mean scattering levels split into distinct low- and high-scattering groups 2 weeks after treatment and remained distinct until week 7 (P < 0.003). Scattering in the low group reached a broad peak that lasted from weeks 2 to 4 at approximately 3 times the pretreatment level. Scattering in the high group peaked at 3 weeks at approximately 12 times the pretreatment level. Scattering levels diminished after reaching their peaks. Confocal images showed a band of highly reflective material in the anterior stroma that extended much deeper in corneas from the high group. The reflective band in the highly scattering corneas obscured the posterior stroma from view for up to 5 weeks.
Quantitative scattering data obtained with the scatterometer suggest that identical PTK treatments indeed result in distinct low- and high-level light-scattering responses in rabbits.
To determine the molecular basis of corneal avascularity during wound healing and determine the role of angiogenic and antiangiogenic factors in corneal vasculogenesis.
The expression of proangiogenic factors (vascular endothelial growth factor [VEGF]; basic fibroblast growth factor [bFGF]; matrix metalloproteinase-2 [MMP-2]; and membrane-type 1-MMP [MT1-MMP]) and antiangiogenic factors (pigment epithelium–derived factor [PEDF]; angiostatin; restin; and endostatin) was analyzed in avascular corneas and in models of corneal neovascularization (bFGF pellet implantation, intrastromal injection of MT1-MMP cDNA, and surgically induced partial limbal deficiency).
Immunohistochemistry demonstrated the presence of antiangiogenic factors (PEDF, angiostatin, restin, and endostatin) and proangiogenic molecules (VEGF, bFGF, MMP-2, and MT1-MMP) in the cornea after wounding. Proangiogenic MMPs were upregulated in stromal fibroblasts in the vicinity of invading vessels following bFGF pellet implantation. Corneal neovascularization (NV) was also induced by intrastromal injection of MT1-MMP naked cDNA in conjunction with de-epithelialization. Partial limbal deficiency (HLD-) resulted in corneal NV in MMP-7 and MMP-3 knockout mice but not in wild type controls.
Corneal angiogenic privilege is an active process involving the production of antiangiogenic factors to counterbalance the proangiogenic factors (which are upregulated after wound healing even in the absence of new vessels). Our finding that the potent antiangiogenic factors, angiostatin and endostatin, are colocalized with several MMPs during wound healing suggests that MMPs may be involved in the elaboration of these antiangiogenic molecules by proteolytic processing of substrates within the cornea.