Knockdown of lncRNA TUG1 suppresses corneal angiogenesis through regulating miR-505-3p/VEGFA.

OBJECTIVES: Vascular endothelial growth factor A (VEGFA) is one of the major factors initiating and regulating angiogenesis. LncRNA taurine up-regulated gene 1 (TUG1) has been implicated in the pathological neovascularization. The aim of this study is to explore the function of TUG1 in regulating VEGFA-mediated angiogenesis in endothelial cells. METHODS: A total of 12 corneal neovascularization (CRNV) samples were collected form patient undergoing corneal transplantation at Tongji Hospital, Wuhan, China. qRT-PCR and Western blotting were performed to examine gene expression and protein levels. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro angiogenesis model. CCK-8 proliferation assay was used to determine cell proliferation capacity and wound healing was performed to analyze cell migration ability. Dual luciferase reporter assay was used for functional interaction validation between miR-505-3p and its targets. The in vitro angiogenic potential was evaluated by tube formation assay. RESULTS: TUG1 and VEGFA were upregulated in CRNV tissues and VEGFA-treated HUVECs. TUG1 knockdown inhibited proliferation, migration and tube formation capacity of HUVECs. TUG1 regulated the angiogenesis of HUVECs by modulating VEGFA expression through targeting miR-505-3p. CONCLUSIONS: Our results suggest that lncRNA TUG1 promotes the angiogenesis of HUVECs through modulating miR-505-3p/VEGFA axis.

Ocular Graft-versus-Host Disease in a Chemotherapy-Based Minor-Mismatch Mouse Model Features Corneal (Lymph-) Angiogenesis.

Ocular graft-versus-host disease (oGVHD) is a fast progressing, autoimmunological disease following hematopoietic stem cell transplantation, leading to severe inflammation of the eye and destruction of the lacrimal functional unit with consecutive sight-threatening consequences. The therapeutic "window of opportunity" is narrow, and current treatment options are limited and often insufficient. To achieve new insights into the pathogenesis and to develop new therapeutic approaches, clinically relevant models of oGVHD are desirable. In this study, the ocular phenotype was described in a murine, chemotherapy-based, minor-mismatch GVHD model mimicking early-onset chronic oGVHD, with corneal epitheliopathy, inflammation of the lacrimal glands, and blepharitis. Additionally, corneal lymphangiogenesis was observed as part of oGVHD pathogenesis for the first time, thus opening up the investigation of lymphangiogenesis as a potential therapeutic and diagnostic tool.

Platelet and Erythrocyte Extravasation across Inflamed Corneal Venules Depend on CD18, Neutrophils, and Mast Cell Degranulation.

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.

Macrophage-Mediated Tissue Vascularization: Similarities and Differences Between Cornea and Skin.

Macrophages are critical mediators of tissue vascularization both in health and disease. In multiple tissues, macrophages have been identified as important regulators of both blood and lymphatic vessel growth, specifically following tissue injury and in pathological inflammatory responses. In development, macrophages have also been implicated in limiting vascular growth. Hence, macrophages provide an important therapeutic target to modulate tissue vascularization in the clinic. However, the molecular mechanisms how macrophages mediate tissue vascularization are still not entirely resolved. Furthermore, mechanisms might also vary among different tissues. Here we review the role of macrophages in tissue vascularization with a focus on their role in blood and lymphatic vessel formation in the barrier tissues cornea and skin. Comparing mechanisms of macrophage-mediated hem- and lymphangiogenesis in the angiogenically privileged cornea and the physiologically vascularized skin provides an opportunity to highlight similarities but also tissue-specific differences, and to understand how macrophage-mediated hem- and lymphangiogenesis can be exploited for the treatment of disease, including corneal wound healing after injury, graft rejection after corneal transplantation or pathological vascularization of the skin.

Effects on the Ocular Surface from Reading on Different Smartphone Screens: A Prospective Randomized Controlled Study.

The purpose of this study was to investigate the influence of smartphone reading on the ocular surface and to compare the various effects of different screens and light conditions on the ocular surface. One hundred nineteen volunteers were randomly divided into: light + organic light-emitting diode (OLED), light + electronic ink (eINK), dark + OLED, and dark + eINK. Ocular surface examinations, including noninvasive break-up time (NIBUT), noninvasive keratograph tear meniscus height (NIKTMH), ocular redness, fluorescein break-up time (FBUT), corneal fluorescein staining, meibomian gland assessment, Schirmer I Test, and blinking frequency, were performed before and after a reading task. Symptoms were evaluated using the Ocular Surface Disease Index (OSDI) and Computer Vision Syndrome Questionnaire (CVS-Q). NIBUT and FBUT were decreased statistically significantly after participants read on an OLED screen for 2 hours compared with the baseline in light and dark environments, whereas no statistically significant decrease was observed on an eINK screen. NIKTMH was statistically significantly decreased after reading on an OLED screen in light and dark settings, and the eINK screen had a lesser effect on NIKTMH. An obvious increase in the ocular redness, OSDI and CVS-Q scores was observed after reading on an OLED screen, whereas the eINK screen had a lesser effect on these indicators. Blink rate increased gradually in OLED subgroups during the reading task, whereas no statistically significant difference was observed in the eINK subgroups. Our research suggested that reading on an OLED screen can cause ocular surface disorder and obvious subjective discomfort, whereas reading on an eINK screen can minimize ocular surface disorder in both dark and light environments.

An Immunohistochemical Study of the Increase in Antioxidant Capacity of Corneal Epithelial Cells by Molecular Hydrogen, Leading to the Suppression of Alkali-Induced Oxidative Stress.

Corneal alkali burns are potentially blinding injuries. Alkali induces oxidative stress in corneas followed by excessive corneal inflammation, neovascularization, and untransparent scar formation. Molecular hydrogen (H2), a potent reactive oxygen species (ROS) scavenger, suppresses oxidative stress and enables corneal healing when applied on the corneal surface. The purpose of this study was to examine whether the H2 pretreatment of healthy corneas evokes a protective effect against corneal alkali-induced oxidative stress. Rabbit eyes were pretreated with a H2 solution or buffer solution, by drops onto the ocular surface, and the corneas were then burned with 0.25 M NaOH. The results obtained with immunohistochemistry and pachymetry showed that in the corneas of H2-pretreated eyes, slight oxidative stress appeared followed by an increased expression of antioxidant enzymes. When these corneas were postburned with alkali, the alkali-induced oxidative stress was suppressed. This was in contrast to postburned buffer-pretreated corneas, where the oxidative stress was strong. These corneas healed with scar formation and neovascularization, whereas corneas of H2-pretreated eyes healed with restoration of transparency in the majority of cases. Corneal neovascularization was strongly suppressed. Our results suggest that the corneal alkali-induced oxidative stress was reduced via the increased antioxidant capacity of corneal cells against reactive oxygen species (ROS). It is further suggested that the ability of H2 to induce the increase in antioxidant cell capacity is important for eye protection against various diseases or external influences associated with ROS production.

Simultaneous fluorescence imaging of distinct nerve and blood vessel patterns in dual Thy1-YFP and Flt1-DsRed transgenic mice.

Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.

Suppression of pathological ocular neovascularization by a small molecule, SU1498.

Selective inhibition of vascular endothelial growth factor receptor (VEGFR), particularly VEGFR-2, is an efficient method for the treatment of ocular neovascularization. SU1498 is a specific inhibitor of VEGFR-2. In this study, we investigated the role of SU1498 in ocular neovascularization. Administration of SU1498 did not show any cytotoxicity and tissue toxicity at the tested concentrations. Administration of SU1498 reduced the size and thickness of choroidal neovascularization and decreased the mean length and mean number of corneal neovascular vessels induced by alkali burn. Pretreatment of SU1498 significantly reduced the proliferation, migration, and tube formation ability of HUVECs. SU1498 played the anti-angiogenic role through the regulation of p38-MAPK signaling. Taken together, inhibition of VEGFR-2 by SU1498 provides a novel therapeutic approach for ocular neovascularization.

Comparison of the effect of topical bevacizumab and sorafenib in experimental corneal neovascularization.

PURPOSE: The purpose of this study was to compare the neovascularization inhibiting the effect of topical bevacizumab and sorafenib and to determine the effective dose of sorafenib. MATERIAL AND METHODS: Forty-two healthy Wistar albino rats were randomly divided into six groups. The right corneas of all rats except group 1 were cauterised with silver nitrate. Group 2 received DMSO, group 3 received topical bevacizumab (5 mg/dL, 3 times a day) and group 4, 5 and 6 received topical sorafenib (2.5 mg/dl, 5 mg/dL, 7.5 mg/dL, 2 times a day respectively), between days 1 and 7. Corneal photographs were taken on day 8 and the corneal neovascular area percentage was calculated. Following decapitation, the corneas were removed to determine the levels of VEGF ELISA and corneal immune staining. The Mann-Whitney U-test was used for statistical analysis. RESULTS: The neovascular corneal area percentage was statistically significantly lower in the treatment groups than group 2 (p < 0.05). The intensity of VEGF immune staining was also lower in groups 3, 5 and 6 from the group 2. Group 3, 5 and 6 were no significant differences compared to group 1. The VEGF ELISA levels were statistically significantly lower in group 3, 5 and 6 compared to group 2 (p < 0.05). There was no statistically difference between VEGF ELISA levels of group 2 and 4 (p > 0.05). CONCLUSIONS: Sorafenib was as effective as bevacizumab in the regression of corneal neovascularization. The effect of sorafenib seems to be dose-dependent. The low doses and twice a day administration are important advantages of sorafenib.

Pharmacological Inhibition of Caspase-8 Suppresses Inflammation-Induced Angiogenesis in the Cornea.

Inflammation-induced angiogenesis is closely related to many diseases and has been regarded as a therapeutic target. Caspase-8 has attracted increasing attention for its immune properties and therapeutic potential in inflammatory disorders. The aim of our study is to investigate the clinical application of pharmacological inhibition of caspase-8 and the underlying molecular mechanisms in inflammation-induced angiogenesis in the cornea. A model of alkali burn (AB)-induced corneal neovascularization (CNV) in C57BL/6 wild-type (WT) mice and toll-like receptor 4 knockout (Tlr4-/-) mice was used. We found that AB increased caspase-8 activity and the pharmacological inhibition of caspase-8 exerted substantial inhibitory effects on CNV, with consistent decreases in caspase-8 activity, inflammatory cell infiltration, macrophage recruitment and activation, VEGF-A, TNF-α, IL-1ß, MIP-1, and MCP-1 expression in the cornea. In vitro, caspase-8 mediated TLR4-dependent chemokines and VEGF-A production by macrophages. The TLR4 knockout significantly alleviated CNV, suppressed caspase-8 activity and downregulated expression of inflammatory cytokines and chemokines after AB. Taken together, these findings provide the first demonstration that the pharmacological inhibition of caspase-8 suppresses inflammation-induced angiogenesis and support the use of a pharmacological caspase-8 inhibitor as a novel clinical treatment for CNV and other angiogenic disorders.

Short-Term Results Analysis in the Allogenic Transplantation of Limbal Stem Cells Expanded on Amniotic Membrane in Patients with Bilateral Limbal Stem Cell Deficiency.

Purpose: The objective of this study was to describe the short-term results of allogenic transplantation of limbal stem cells expanded on amniotic membrane for the ocular surface reconstruction. Methods: Prospective nonrandomized, nonmasked study in a single ophthalmological center. Ten patients with bilateral total limbal stem cell deficiency (LSCD) were included. Expression and presence of ABCB5 and Δp63α in amniotic membrane-cultured limbal epithelial stem cells were analyzed, in relationship with clinical changes after allogenic transplantation. An objective evaluation was performed to determine corneal transparency and superficial vascularization. Results: In a median follow-up time of 11.6 months, 7 patients (70%) were considered as failure compared with the preoperative status. ABCB5 and Δp63α are expressed in similar amount in the limbal epithelial cells expanded in vitro and transplanted in patients with bilateral LSCD. Conclusions: Transplantation of allogenic epithelial limbal cells expanded in amniotic membrane could be considered in patients with LSCD due to burns or congenital etiologies such as aniridia, but its benefit is limited for patients with immunologic diseases.

Human antigen R protein modulates vascular endothelial growth factor expression in human corneal epithelial cells under hypoxia.

PURPOSE: Corneal avascularity is critical for corneal transparency; therefore, a tailored process has been presumed to minimize corneal neovascularization (NV). In most cell types, the transcription of vascular endothelial growth factor (VEGF) is up-regulated, and the stability of VEGF mRNA is sustained by human antigen R (HuR) during hypoxia; however, whether such response applies to corneal epithelial cells is unclear. METHODS: Human corneal epithelial cells (HCECs) and MCF-7 cells that serves as the control were incubated under 0.5% oxygen, and the levels of VEGF and HuR were examined time-dependently. The alteration of HuR was also examined in vivo using the closed-eye contact lens-induced corneal neovascularization rabbit model and immunohistochemistry. Additionally, the expression of HuR was modulated by transfection of plasmids encoding HuR or siRNA targeting HuR to validate the role of HuR in VEGF expression. RESULTS: We found that, unlike in control cells, the level of VEGF was not up-regulated, and the HuR expression was declined in HCECs following hypoxia. The HuR immunostaining intensities were decreased in corneal epithelial cells of rabbits wearing contact lenses. In addition, HuR overexpression restored the ability of HCECs to up-regulate VEGF under hypoxia; however, knockdown of HuR suppressed hypoxia-induced VEGF in control cells but did not further decrease VEGF in HCECs. These findings suggest that HCECs may modulate HuR to suppress hypoxia-mediated up-regulation of VEGF. CONCLUSION: Our study revealed a distinct regulation of VEGF via HuR in HCECs following hypoxia, which likely contributes to minimizing corneal NV and/or maintenance of corneal avascularity.

Optical Coherence Tomography Angiography Imaging to monitor Anti-VEGF treatment of Corneal Vascularization in a Rabbit Model.

Optical coherence tomography angiography (OCTA) is a well-established non-invasive retinal vascular imaging technique. It has been recently adapted to image the anterior segment and has shown good potential to image corneal vascularisation. The purpose of the study is to evaluate the usefulness of OCTA to monitor regression of corneal vessels following anti-VEGF (vascular endothelial growth factor) treatment using a previously established corneal vascularisation rabbit model. The regression of vessels following the treatment with aflibercept and ranibizumab anti-VEGFs using both topical instillation and sub-conjunctival injection was quantified using OCTA and compared with ICGA (indocyanine green angiography). Overall vessel density measurements using OCTA showed good correlation (r = 0.988, p < 0.001) with ICGA, with no significant difference between the two treatment groups (p = 0.795). It was also shown that OCTA provided good repeatability outcomes of the quantitative measurements. Using Bland-Altman plots, vessel growth density values between anti-VEGF treatments were compared to control saline group. It was observed that aflibercept provided longer lasting effect than ranibizumab. We also observed that in both drugs, the topical route of administration topical provided longer regression outcomes compared to one-time sub-conjunctival injection. Thereby, with this pilot study, it was demonstrated that OCTA is a reliable imaging technique to follow-up and monitor corneal vascularisation and its treatment quantitatively.

Sex differences in corneal neovascularization in response to superficial corneal cautery in the rat.

Sex-based differences in susceptibility have been reported for a number of neovascular ocular diseases. We quantified corneal neovascularization, induced by superficial silver nitrate cautery, in male and female inbred albino Sprague-Dawley, inbred albino Fischer 344, outbred pigmented Hooded Wistar and inbred pigmented Dark Agouti rats of a range of ages. Corneal neovascular area was quantified on haematoxylin-stained corneal flatmounts by image analysis. Pro-and anti-angiogenic gene expression was measured early in the neovascular response by quantitative real-time polymerase chain reaction. Androgen and estrogen receptor expression was assessed by immunohistochemistry. Male rats from all strains, with or without ocular pigmentation, exhibited significantly greater corneal neovascular area than females: Sprague-Dawley males 43±12% (n = 8), females 25±5% (n = 12), p = 0.001; Fischer 344 males 38±10% (n = 12) females 27±8% (n = 8) p = 0.043; Hooded Wistar males 32±6% (n = 8) females 22±5% (n = 12) p = 0.002; Dark Agouti males 37±11% (n = 9) females 26±7% (n = 9) p = 0.015. Corneal vascular endothelial cells expressed neither androgen nor estrogen receptor. The expression in cornea post-cautery of Cox-2, Vegf-a and Vegf-r2 was significantly higher in males compared with females and Vegf-r1 was significantly lower in the cornea of males compared to females, p<0.001 for each comparison. These data suggest that male corneas are primed for angiogenesis through a signalling nexus involving Cox-2, Vegf-a, and Vegf receptors 1 and 2. Our findings re-enforce that pre-clinical animal models of human diseases should account for sex-based differences in their design and highlight the need for well characterized and reproducible pre-clinical studies that include both male and female animals.

Changes in the Density of Corneal Endothelial Cells in Elderly Diabetic Patients After Combined Phacovitrectomy and Ex-PRESS Glaucoma Implants.

BACKGROUND & OBJECTIVE: Corneal endothelial cells (ECD) are characterized by limited regenerative potential, which is additionally impaired in patients with diabetes. This retrospective study included 27 patients aged 58.1±13.6, 16 female and 11 males, who underwent 23-gauge vitrectomy in combination with cataract surgery (phacovitrectomy) and further Ex-PRESS shunt implantation throughout 2013-2017 at St. Barbara Hospital in Sosnowiec, Poland. METHODS: In our study, we distinguished 4 periods: initial period; post phacovitrectomy and removal of oil tamponade; and 3 and 12 months post implantation of the Ex-PRESS shunt. Statistical analysis was performed at the level of statistical significance of p<0.05. It included an analysis of variance (ANOVA) and Tukey's post-hoc test in order to determine the differences in the density of ECD cells/mm2 between the periods of observation. The paired-samples t-Student test was also performed to determine whether the differences in visual acuity values before and after PPV and before and after Ex-PRESS shunt were statistically significant. RESULTS: The initial count of ECD cells was 2381.1±249, which decreased to 1872.8±350.7 cell/mm2 and finally to 1677.9±327 at the endpoint. Differences in the density of ECD cells/mm2 were observed to be statistically significant between the periods: after PPV vs. initial number of ECD (p = 0.000138); before 3 months after Ex-PRESS shunt vs. initial number of ECD (p = 0.000138); 12 months after Ex- PRESS shunt vs. initial number of ECD (p = 0000138). Analyzing the changes in visual acuity, we observed a deterioration both before and 3 months after Ex-PRESS shunt (p = 0.007944) and before and after PPV (p = 0.060334). In turn, correlation analysis indicated that there is a statistically significant, moderate, positive relationship. The relationship between visual acuity after Ex-PRESS shunt and ECD cells/mm2 density turned out to be statistically significant (r = +0.521381; p < 0.05). CONCLUSION: Regardless of the period of observation and the choice of ophthalmic treatment of diabetic complications, we observed a decrease in the number of ECD cells and a deterioration in visual acuity. It is, therefore, reasonable to provide the patient with complete information about the proposed procedures and to consider the risk-benefit balance.

Corneal neovascularization: updates on pathophysiology, investigations & management.

Objective. Corneal neovascularization is a sight-threatening condition affecting more than 1.4 million people per year. Left untreated, it can lead to tissue scarring, oedema, lipid deposition, and persistent inflammation that may significantly affect visual prognosis and quality of life. The aim was to review the recent evidence relating to the pathophysiology, investigations and management of corneal neovascularization. Methods. Literature review of prospective and retrospective studies, clinical trials and animal models relating to the pathophysiology, investigation and management of corneal neovascularization. Results. Corneal neovascularization is characterized by the invasion of new blood vessels into the cornea caused by an imbalance between angiogenic and antiangiogenic factors that preserve corneal transparency as a result of various ocular insults and hypoxic injuries. Risk factors that have been implicated in the pathogenesis of the disease include contact lens wear, ocular surface disease, trauma, previous surgery and herpes. The results highlighted the current and future management modalities of corneal neovascularization, which includes corneal transplantation, laser - phototherapy, injections and topical treatment. Conclusion. The future of corneal neovascularization is promising and this paper discusses the upcoming revolution in local gene therapy. Abbreviations. HSK = herpes stromal keratitis, VEGF = vascular endothelial growth factor, VEGFR-1 = VEGF Receptor-1, FGF = Fibroblast growth factor, PDGF = Platelet-derived growth factor, IL-6 = interleukin-6, IL-7 = interleukin-7, IL-8 = interleukin-8, IRS-1 = insulin receptor substrate-1.

Cucurbita argyrosperma Seed Extracts Attenuate Angiogenesis in a Corneal Chemical Burn Model.

Severe corneal inflammation produces opacity or even perforation, scarring, and angiogenesis, resulting in blindness. In this study, we used the cornea to examine the effect of new anti-angiogenic chemopreventive agents. We researched the anti-angiogenic effect of two extracts, methanol (Met) and hexane (Hex), from the seed of Cucurbita argyrosperma, on inflamed corneas. The corneas of Wistar rats were alkali-injured and treated intragastrically for seven successive days. We evaluated: opacity score, corneal neovascularization (CNV) area, re-epithelialization percentage, and histological changes. Also, we assessed the inflammatory (cyclooxigenase-2, nuclear factor-kappaB, and interleukin-1ß) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2, Il-1ß, and Vegf-a mRNA were also determined. After treatment, we observed a reduction in corneal edema, with lower opacity scores and cell infiltration compared to untreated rats. Treatment also accelerated wound healing and decreased the CNV area. The staining of inflammatory and angiogenic factors was significantly decreased and related to a down-expression of Cox-2, Il-1ß, and Vegf. These results suggest that intake of C. argyrosperma seed has the potential to attenuate the angiogenesis secondary to inflammation in corneal chemical damage.

Blood Vessels and Lymphatic Vessels in the Cornea and Iris After Penetrating Keratoplasty.

PURPOSE: To detect early growth of blood and lymphatic vessels in the mouse cornea and iris after penetrating keratoplasty. METHODS: Penetrating keratoplasty was performed with C57BL/6 mice as donors and BALB/c mice as recipients. Graft transparency and neovascularization were examined by slit-lamp microscopy. Whole mounts of the cornea and iris were processed for detection of the outgrowth of blood and lymph vessels. RESULTS: On day 3 after surgery, all corneal grafts were slightly edematous, and blood vessels in the corneoscleral limbus dilated. LYVE-1 lymphatic vessels and CD31 blood vessels were distributed in the peripheral cornea. In the iris, the density of blood vessels increased, and LYVE-1 cells nearly vanished. On day 7, the grafts became opaque, and blood vessels grew into the recipient bed. A great quantity of lymph vessels invaded the cornea. LYVE-1 arborescent cells were found around the lymphatic vessels. In the iris, blood vessels became bulky and stiff, and arborescent LYVE-1 cells increased in number. On day 14, corneal neovascular regression and graft clarity were found. Lymphatic vessels regressed more slowly than blood vessels in the cornea. In the iris, blood vessels remained coarse. Increasing arborescent LYVE-1 cells were also noted in the ciliary body. CONCLUSIONS: Our findings suggest that the iris-ciliary body could amplify immune signals and in part promote initiation of immune rejection after keratoplasty by providing a pathway for macrophages, which might participate in corneal lymphangiogenesis.

Local VEGF-A blockade modulates the microenvironment of the corneal graft bed.

The microenvironment plays an important role in several immunological processes. Vascular endothelial growth factor-A (VEGF-A) not only regulates angiogenesis, but is known as a modulator of the immune microenvironment. Modulating the site of transplantation might be beneficial for subsequent transplant survival. In this study, we therefore analyzed the effect that a local blockade of VEGF-A in the inflamed cornea as the graft receiving tissue has on the immune system. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation, which is an optimal model for local drug application. Mice were treated with VEGFR1/R2 trap prior to transplantation. We analyzed corneal gene expression, as well as protein levels in the cornea and serum on the day of transplantation, 2 and 8 weeks later. Local VEGF depletion prior to transplantation increases the expression of pro-inflammatory as well as immune regulatory cytokines only in the corneal microenvironment, but not in the serum. Furthermore, local VEGFR1/R2 trap treatment significantly inhibits the infiltration of CD11c+ dendritic cells into the cornea. Subsequent increased corneal transplantation success was accompanied by a local upregulation of Foxp3 gene expression. This study demonstrates that locally restricted VEGF depletion increases transplantation success by modulating the receiving corneal microenvironment and inducing tolerogenic mechanisms.

Protective Effect of TLR4 Ablation against Corneal Neovascularization following Chemical Burn in a Mouse Model.

PURPOSE: To determine whether Toll-like receptor 4 knockout protects mice from corneal neovascularization following chemical injury compared to wild-type (WT) mice. METHODS: A chemical burn (75% silver nitrate, 25% potassium nitrate) was created under anesthesia in the central right cornea of 32 WT and 31 Toll-like receptor 4 knockout mice. Corneal neovascularization was evaluated at 3, 4, 6, 8, 10, and 35 days after injury using digital photography, fluorescein angiography, gelatin perfusion with fluorescence vascular imaging, immunofluorescence staining, and molecular analysis. RESULTS: There was no significant between-group difference in relative corneal burn area at 10 days after injury (39.0 ± 2.4% vs. 38.8 ± 9.8%, respectively). Neovascularization was detected in all corneas in vivo and perfusion was detected by fluorescence vascular imaging, reaching maximum area on day 10. The relative area of neovascularization was significantly smaller in the knockout than the WT mice on days 6 (33.3 ± 4.2% vs. 46.8 ± 7.4%, respectively, p = 0.005) and 8 (36.6 ± 1.1% vs. 52.2 ± 6.4%, respectively, p = 0.027), although neovascularization was intensive in both groups. In line with the immunostaining findings of angiogenesis and inflammatory infiltration of damaged corneas, molecular analysis (performed on day 3) revealed elevated expression levels of angiogenesis-related genes (vascular endothelial growth factor, VEGFR2, VEGFR1) and inflammation-related genes (CD45 and TGFß1) in the WT mice. The knockout mice had higher TNF-α expression than the WT mice. CONCLUSION: In a mouse corneal chemical burn model, lack of Toll-like receptor 4 expression did not completely inhibit angiogenesis, but did have a relative effect to reduce neovascularization as compared to the WT.