Upadacitinib inhibits corneal inflammation and neovascularization by suppressing M1 macrophage infiltration in the corneal alkali burn model.

Alkali burn-induced corneal inflammation and subsequent corneal neovascularization (CNV) are major causes of corneal opacity and vision loss. M1 macrophages play a central role in inflammation and CNV. Therefore, modulation of M1 macrophage polarization is a promising strategy for corneal alkali burns. Here, we illustrate the effect and underlying mechanisms of upadacitinib on corneal inflammation and CNV induced by alkali burns in mice. The corneas of BALB/c mice were administered with 1 M NaOH for 30 s and randomly assigned to the vehicle group and the upadacitinib-treated group. Corneal opacity and corneal epithelial defects were assessed clinically. Quantitative real-time PCR (qRT-PCR), immunohistochemistry, and western blot analysis were performed to detect M1 macrophage polarization and CD31+ corneal blood vessels. The results showed that upadacitinib notably decreased corneal opacity, and promoted corneal wound healing. On day 7 and 14 after alkali burns, upadacitinib significantly suppressed CNV. Corneal alkali injury caused M1 macrophage recruitment in the cornea. In contrast to the vehicle, upadacitinib suppressed M1 macrophage infiltration and decreased the mRNA expression levels of inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1ß, and vascular endothelial growth factor A (VEGF-A) in alkali-injured corneas. Moreover, upadacitinib dose-dependently inhibited M1 macrophage polarization by suppressing interferon (IFN)-γ-/lipopolysaccharide-stimulated STAT1 activation in vitro. Our findings reveal that upadacitinib can efficiently alleviate alkali-induced corneal inflammation and neovascularization by inhibiting M1 macrophage infiltration. These data demonstrate that upadacitinib is an effective drug for the treatment of corneal alkali burns.

Impaired Autophagy Causes Severe Corneal Neovascularization.

PURPOSE: To investigate the role of macrophage autophagy in the process of corneal neovascularization (CNV). METHODS: In vivo, mice CNV was induced by alkali injury and compared with rapamycin-treated alkaline burn mice. Western blot was used to determine the autophagic status of the macrophages. We quantified the levels of macrophage polarization markers (CD86, INOS, CD163, CD206) by RT-qPCR and measured inflammatory factors through ELISA (IL-6 and TNF-α) in the early phase after injury. In vitro, the human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium (MCM) induced by the THP-1 cell line to simulate the neovascular microenvironment. The vascularization capacity of HUVECs was examined using the CCK-8 assay kit, tube formation assay, and scratch wound-healing assay. RESULTS: In vivo, the mRNA expression of Beclin-1 and ATG5 was increased, together with the upregulation of M1 macrophage markers (CD86 and INOS) in corneas after early alkali injury. The area of CNV is effectively relieved in the rapamycin-treated mice. In vitro, upregulation of autophagy level by pretreatment with 3-methyladenine (3-MA) could increase the mRNA expression of the M1 markers. Macrophage-conditioned medium with impaired autophagy contains more IL-6 and TNF-α compared to the M1 macrophage-conditioned medium, promoting HUVEC proliferation, migration, and tube formation capacity. Enhancing the autophagy level with rapamycin (RAPA) could reverse this phenomenon. CONCLUSIONS: Impaired autophagy promoted macrophage polarization toward M1 type and increased the expression of IL-6 and TNF-α, which led to severe CNV. Using the autophagy activator (RAPA) could effectively alleviate CNV by promoting autophagy.

Effect of Nintedanib Nanothermoreversible Hydrogel on Neovascularization in an Ocular Alkali Burn Rat Model.

PURPOSE: To compare the therapeutic effects of different forms of nintedanib ophthalmic preparations on neovascularization corneal alkali burns in rats. METHODS: Forty rat models of left eye corneal alkali burns were constructed, and the five groups (N = 8) were treated with normal saline, dexamethasone ointment (dexamethasone), 0.2% nintedanib aqueous solution and nintedanib nano thermoreversible hydrogel (NNTH). A slit lamp microscope was used to observe the area of neovascularization. The levels of the inflammatory factors were detected by ELISA. HE staining was performed on the rat corneas. Vascular endothelial growth factor (VEGFA) was detected by immunohistochemistry, and the expression of corneal VEGFA and CD31 was detected by western blotting. An MTT assay was performed to detect the cytotoxicity of nintedanib on human corneal epithelial cells (HCECs) and human umbilical vein vascular endothelial cells (HUVECs). Cell migration was detected by a cell scratch assay, and the proportion of apoptotic cells was detected by Annexin/PI double staining. Immunofluorescence and western blotting were performed to detect the protein expression of VEGFA and CD31. RESULTS: NNTH had a stronger inhibitory effect on corneal neovascularization (CNV) in alkali-burned rats while reducing the level of inflammatory factors. NNTH had a longer drug duration of release than nanoformulations in vitro. Nintedanib at low concentrations (<8 µM) had no significant cytotoxicity to HCECs but significantly induced apoptosis and inhibited the expression of VEGFA and CD31 and the migration of HUVECs. CONCLUSIONS: Nanomorphic thermoreversible hydrogel is superior among the nintedanib ophthalmic preparations, showing better inhibition of CNV in alkali-burned eyeballs and it blocked the migration and proangiogenic ability of HUVECs.

AIP1 suppresses neovascularization by inhibiting the NOX4-induced NLRP3/NLRP6 imbalance in a murine corneal alkali burn model.

BACKGROUND: Apoptosis signal-regulating kinase 1-interacting protein 1 (AIP1) participates in inflammatory neovascularization induction. NADPH oxidase 4 (NOX4) produces reactive oxygen species (ROS), leading to an imbalance in nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) and NLR family pyrin domain containing 6 (NLRP6) expression. The mechanisms of AIP1, NOX4, ROS and inflammasomes in corneal neovascularization were studied herein. METHODS: C57BL/6 and AIP1-knockout mice were used in this study. The alkali burn procedure was performed on the right eye. Adenovirus encoding AIP1 plus green fluorescence protein (GFP) (Ad-AIP1-GFP) or GFP alone was injected into the right anterior chamber, GLX351322 was applied as a NOX4 inhibitor, and then corneal neovascularization was scored. The expression of related genes was measured by quantitative real-time polymerase chain reaction, western blotting and immunofluorescence staining. 2',7'-Dichlorofluorescin diacetate staining was used to determine the ROS levels. RESULTS: The expression of AIP1 was decreased, while that of cleaved interleukin-1ß (clv-IL-1ß) and vascular endothelial growth factor A (VEGFa) was increased after alkali burn injury. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. NLRP3/NLRP6 expression was imbalanced after alkali burns. GLX351322 reversed the imbalance in NLRP3/NLRP6 by reducing the ROS levels. This treatment also reduced the expression of clv-IL-1ß and VEGFa, suppressing neovascularization. CONCLUSIONS: AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burn injury. Based on the pathogenesis of corneal neovascularization, these findings are expected to provide new therapeutic strategies for patients. Corneal alkali burn injury is a common type of ocular injury that is difficult to treat in the clinic. The cornea is a clear and avascular tissue. Corneal neovascularization after alkali burn injury is a serious complication; it not only seriously affects the patient's vision but also is the main reason for failed corneal transplantation. Corneal neovascularization affects approximately 1.4 million patients a year. We show for the first time that AIP1 and NOX4 can regulate corneal inflammation and neovascularization after alkali burns. The expression of AIP1 was decreased, while that of clv-IL-1ß and VEGFa was increased after alkali burns. We tried to elucidate the specific molecular mechanisms by which AIP1 regulates corneal neovascularization. NOX4 activation was due to decreased AIP1 expression in murine corneas with alkali burns. NOX4 expression was increased, the imbalance in NLRP3/NLRP6 was exacerbated, and corneal neovascularization was increased significantly in AIP1-knockout mice compared with those in C57BL/6 mice after alkali burns. These effects were reversed by AIP1 overexpression. Additionally, NLRP3/NLRP6 expression was unbalanced, with NLRP3 activation and NLRP6 suppression in the corneal alkali burn murine model. Eye drops containing GLX351322, a NOX4 inhibitor, reversed the imbalance in NLRP3/NLRP6 by reducing ROS expression. This treatment also reduced the expression of clv-IL-1ß and VEGFa, reducing neovascularization. Therefore, we provide new gene therapeutic strategies for patients. With the development of neovascularization therapy, we believe that in addition to corneal transplantation, new drug or gene therapies can achieve better results. Video Abstract.

Forkhead Domain Inhibitor-6 Suppresses Corneal Neovascularization and Subsequent Fibrosis After Alkali Burn in Rats.

Purpose: The purpose of this study was to investigate the effects of Forkhead Domain Inhibitor-6 (FDI-6) on regulating inflammatory corneal angiogenesis and subsequent fibrosis induced by alkali burn. Methods: A corneal alkali burn model was established in Sprague Dawley rats using NaOH and the rat eyes were topically treated with FDI-6 (40 µM) or a control vehicle four times daily for 7 days. Corneal neovascularization, inflammation and epithelial defects were observed on days 1, 4, and 7 under a slit lamp microscope after corneal alkali burn. Analysis of angiogenesis-, inflammation-, and fibrosis-related indicators was conducted on day 7. Murine macrophages (RAW264.7 cells) and mouse retinal microvascular endothelial cells (MRMECs) were used to examine the effects of FDI-6 on inflammatory angiogenesis in vitro. Results: Topical delivery of FDI-6 significantly attenuated alkali burn-induced corneal inflammation, neovascularization, and fibrosis. FDI-6 suppressed the expression of angiogenic factors (vascular epidermal growth factor, CD31, matrix metalloproteinase-9, and endothelial NO synthase), fibrotic factors (α-smooth muscle actin and fibronectin), and pro-inflammatory factor interleukin-6 in alkali-injured corneas. FDI-6 downregulated the expression of monocyte chemotactic protein-1, pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-alpha), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3, and vascular endothelial growth factor in RAW264.7 cells and inhibited the proliferation, migration, and tube formation of MRMECs in vitro. Conclusions: FDI-6 can attenuate corneal neovascularization, inflammation, and fibrosis in alkali-injured corneas.

Pharmacological Inhibition of Glutaminase 1 Attenuates Alkali-Induced Corneal Neovascularization by Modulating Macrophages.

Corneal neovascularization (CoNV) in response to chemical burns is a leading cause of vision impairment. Although glutamine metabolism plays a crucial role in macrophage polarization, its regulatory effect on macrophages involved in chemical burn-induced corneal injury is not known. Here, we elucidated the connection between the reprogramming of glutamine metabolism in macrophages and the development of alkali burn-induced CoNV. Glutaminase 1 (GLS1) expression was upregulated in the mouse corneas damaged with alkali burns and was primarily located in F4/80-positive macrophages. Treatment with a selective oral GLS1 inhibitor, CB-839 (telaglenastat), significantly decreased the distribution of polarized M2 macrophages in the alkali-injured corneas and suppressed the development of CoNV. In vitro studies further demonstrated that glutamine deprivation or CB-839 treatment inhibited the proliferation, adhesion, and M2 polarization of bone marrow-derived macrophages (BMDMs) from C57BL/6J mice. CB-839 treatment markedly attenuated the secretion of proangiogenic factors, including vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) from interleukin-4- (IL-4-) regulated M2 macrophages. Our findings revealed that GLS1 inhibition or glutamine deprivation prevented alkali-induced CoNV by inhibiting the infiltration and M2 polarization of macrophages. This work suggests that pharmacological GLS1 inhibition is a feasible and effective treatment strategy for chemical burn-related CoNV in humans.

The Possible Protective Effect of Boric Acid in an Alkaline-Induced Corneal Neovascularization Rat Model.

It is known that boric acid (BA) exerts it antioxidant and anti-inflammatory effects by activating the activating transcription factor 4 (ATF4) and nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. This pathway has been reported to control antioxidant status in the eye. The aim of this study was to investigate the possible preventive effects of boric acid administration on oxidative damage and corneal neovascularization (CNV). Sixteen adult female Wistar albino rats were divided into two groups: (I) control (n = 8); the CNV model was applied to the right eye of the rats, and the left eyes were used as healthy controls. (II) CNV + BA (n = 8): After the CNV model was applied to the right eyes, a single subconjunctival dose (0.05 mL) of 0,018 g/mL BA was injected into the right and left eyes of the rats. Biochemical, histopathological, and immunohistochemical analyses were performed. Moderate VEGF positivity was observed in the vessels of the CNV group, a decrease in vessel proliferation, and weak VEGF positivity in the CNV + BA group. The TAS level in the CNV + BA group was significantly higher than that in the other groups. The TOS level was significantly higher in all groups than it is in the control group. The OSI value was increased in all groups when compared to the control group, but only the CNV and BA groups were statistically significant. BA not only reduced alkaline-induced corneal damage histologically but also showed a protective effect on oxidative stress biochemically.

Exploring the Mechanism of the miRNA-145/Paxillin Axis in Cell Metabolism During VEGF-A-Induced Corneal Angiogenesis.

Purpose: Paxillin (PXN) is a key component of focal adhesions and plays an important role in angiogenesis. The aim of the present study was to investigate the effect of PXN in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were transfected with PXN overexpression and PXN interference vectors. Biochemical detection was used to detect adenosine triphosphate and lactic acid production. The morphology of mitochondria was observed under an electron microscope, and flow cytometry was conducted to measure mitochondrial membrane potential. Transwell experiments were used to detect the migration and tube formation ability of each group of cells. The expression of hexokinase (HK)1, HK2, glucose transporter 1 (GLUT1), phosphorylated phosphatidylinositol 3-kinase (PI3K), phosphorylated AKT, and phosphorylated mechanistic target of rapamycin (mTOR) was evaluated by western blot. Results: PXN silencing reduced the levels of lactic acid and adenosine triphosphate, downregulated HK1, HK2, and GLUT1, suppressed PI3K/AKT/mTOR signaling activation, and inhibited VEGF-A-induced mitochondria injury in VEGF-A-induced HUVECs. We also determined that miR-145-5p decreased the VEGF-A-induced expression of PXN and inhibited the invasion and angiogenesis of HUVECs. Also, miR-145-5p inhibition blocked the protective effect of PXN interference on VEGF-A-induced HUVEC injury. Furthermore, PXN interference significantly decreased lactic acid and adenosine triphosphate levels, inhibited PI3K/AKT/mTOR activation, and decreased the levels of HK1, HK2, and GLUT1 in VEGF-A-treated mouse corneal. Conclusions: The results indicate that PXN silencing inhibited the VEGF-A-induced invasion and angiogenesis of HUVECs via regulation of cell metabolism and mitochondrial damage, suggesting that PXN may be a potential target for antiangiogenic therapies.

Effect of Topical Instillation of Pegaptanib Sodium Upon Inflammatory Corneal Neovascularization in Rabbits.

Purpose: To evaluate the effect of topical instillation of pegaptanib sodium upon inflammatory angiogenesis induced in the rabbit cornea by alkaline cauterization. Methods: Inflammatory angiogenesis was induced by alkaline (sodium hydroxide) cauterization in the corneas of 29 male New Zealand rabbits. The animals were divided into 4 groups: a control group treated with 0.5% carboxymethylcellulose sodium eye drops, a group treated with 1.0% prednisolone acetate eye drops, a group treated with 0.5% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium, and a group treated with 1.0% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium. After cauterization, eye drops were administered every 12 hours for 21 days. The animals were evaluated every 3 days after cauterization, and the newly formed vessels were quantified from photographs. The treatment effectiveness was analyzed with 3 parameters of antiangiogenic response: neovascularization area (NA), total vascular length (TVL), and number of blood vessels (BVN). Results: Average NA, TVL, and BVN values were significantly higher in both pegaptanib groups than in the prednisolone group. A nonstatistically significant reduction in parameters on days 18 and 21 was the minimum achieved in both pegaptanib groups. The efficacy of the treatments in relation to the control was significantly greater in the prednisolone group than in the 0.5% pegaptanib group or the 1.0% pegaptanib group (P < 0.001). Conclusion: Topical instillation of 0.5% and 1.0% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium had no inhibitory effect on corneal neovascularization in this rabbit model.

Inhibition of PDGF-BB reduces alkali-induced corneal neovascularization in mice.

The aim of the present study was to investigate the role of platelet­derived growth factor (PDGF)­BB/PDGF receptor (R)­ß signaling in an experimental murine corneal neovascularization (CrNV) model. Experimental CrNV was induced by alkali injury. The intra­corneal expression of PDGF­BB was examined using immunohistochemistry. The effect of PDGF­BB on CrNV was evaluated using immunofluorescence staining. The expression levels of PDGFR­ß in human retinal endothelial cells (HRECs) under normal conditions or following cobalt chloride treatment, which induced hypoxic conditions, was assessed using reverse transcription­quantitative PCR. The effect of exogenous treatment of PDGF­BB on the proliferation, migration and tube formation of HRECs under normoxic or hypoxic conditions was evaluated in vitro using Cell Counting Kit­8, wound healing and 3D Matrigel capillary tube formation assays, respectively. The results indicated that the intra­corneal expression levels of the proteins of PDGF­BB and PDGFR­ß were detectable on days 2 and 7 following alkali injury. The treatment with neutralizing anti­PDGF­BB antibody resulted in significant inhibition of CrNV. The intra­corneal expression levels of vascular endothelial growth factor A, matrix metallopeptidase (MMP)­2 and MMP­9 proteins were downregulated, while the expression levels of thrombospondin (TSP)­1, TSP­2, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­1 and ADAMTS­2 were upregulated significantly in mice treated with anti­PDGF­BB antibody. The expression levels of PDGFR­ß were upregulated in HRECs under hypoxic conditions compared with those noted under normoxic conditions. Recombinant human PDGF­BB promoted the proliferation, migration and tube formation of HRECs under hypoxic conditions. The data indicated that PDGF­BB/PDGFR­ß signaling was involved in CrNV and that it promoted endothelial cell proliferation, migration and tube formation. The pro­angiogenic effects of this pathway may be mediated via the induction of pro­angiogenic cytokine secretion and the suppression of anti­angiogenic cytokine secretion.

Development of mucoadhesive cationic polypeptide micelles for sustained cabozantinib release and inhibition of corneal neovascularization.

Corneal neovascularization (CNV) is one of the leading risk factors for vision loss. Anti-angiogenic drugs can theoretically be extended to the treatment of CNV. However, the application of these drugs is often hindered by traditional administration methods, e.g., eye drops, which is ascribed to the unique structure of the cornea and tear film. In this study, cationic polypeptide nanoparticles with mucoadhesive ability that carry lipophilic cabozantinib (a tyrosine kinase inhibitor), called Cabo-NPs, were developed for sustained cabozantinib release and inhibition of CNV. The polypeptides were synthesized via N-carboxyanhydride ring-opening polymerization and could self-assemble into micelles with cabozantinib in aqueous solution. The Cabo-NPs possessed good biocompatibility both in corneal epithelial cells and mouse corneas. More importantly, in vitro angiogenesis assays demonstrated the strong inhibitory effect of Cabo-NPs on cell migration and tube formation. Furthermore, the Cabo-NPs exerted superior anti-angiogenic effects with remarkable reductions in the neovascular area, which were as effective as the clinical dexamethasone but without apparent side effects. The therapeutic mechanism of the Cabo-NPs is closely related to the significant decrease in proangiogenic and proinflammatory factors, suppressing neovascularization and inflammation. Overall, cationic Cabo-NPs offer a new prospect for safe and effective CNV treatment via enhancing the bioavailability of lipophilic cabozantinib.

Activation of Toll-like receptor 3 promotes pathological corneal neovascularization by enhancement of SDF-1-mediated endothelial progenitor cell recruitment.

Toll-like receptors (TLRs) play an important role in inflammatory and immunological responses, which are intimately related to neovascularization. However, the precise mode of action of TLR3 in neovascularization still remains ambiguous. In this study, we sought to investigate the role of TLR3 in pathological corneal neovascularization (CNV) using a mouse model of alkali-induced CNV. CNV was attenuated in TLR3-deficient mice, and the absence of TLR3 led to decreased production of stromal cell-derived factor 1 (SDF-1), a well-characterized cytokine that regulates the recruitment of endothelial progenitor cells (EPCs) to the sites of neo-angiogenic niches in the injured tissues. Topical administration of polyinosinic-polycytidylic acid [poly (I:C)], a synthetic ligand for TLR3, to the injured cornea promoted CNV in wild type (WT) mice but not in TLR3-deficient mice. In addition, the effect of poly (I:C) on WT mice was abolished by addition of SDF-1 receptor antagonist AMD 3100. Furthermore, poly (I:C) treatment in vitro enhanced the migration of EPCs, whereas the enhanced migration was abolished by AMD 3100. These results indicate an essential role of TLR3 signalling in CNV that involves upregulating SDF-1 production and recruiting EPCs to the sites of injury for neovascularization. Thus, targeting the TLR3 signalling cascade may constitute a novel therapeutic approach for treating neovascularization-related diseases.

Prevention of Corneal Neovascularization by Adenovirus Encoding Human Vascular Endothelial Growth Factor Soluble Receptor (s-VEGFR1) in Lacrimal Gland.

Purpose: The aims of this study were (1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (s-VEGFR1) gene transfer to the lacrimal gland (LG); (2) to investigate whether expression of s-VEGFR1 prevents corneal neovascularization (CNV) induced by alkali burns; and (3) to evaluate the safety of the procedure. Methods: AdVEGFR1 vectors (25 µL, 1 × 1010 pfu/mL) were injected in the right LGs of rats and were compared with AdNull vector (25 µL, 1 × 1010 pfu/mL) or 25 µL of saline (Control) before cornea alkali burns with 1 M NaOH. After 7 days, CNV was documented at the slit lamp. Tear secretion was measured with phenol red threads. The animals were tested for s-VEGFR1 mRNA and protein in the LG by quantitative (q)PCR and immunohistochemistry staining, respectively. qPCR was used to compare the mRNA levels of IL-1ß, IL-6, and TNF-α in the LG and ipsilateral trigeminal ganglion (TG). Results: Ad-VEGFR1 transfected 83% (10/12) of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals in the Ad-VEGFR1 group, compared with the Ad-Null (3:10) and Control groups (1:10) (P = 0.0317). The tear secretion and cytokine mRNA levels in the LG and TG were similar in all three groups (P > 0.05). Conclusions: Adenoviral vector gene transfer was safe for LG structure and function. The LG as the target tissue showed local expression of human s-VEGFR1, and CNV was prevented in most of the eyes exposed to alkali burns.

Comparison of the Effects of Subconjunctival Injections of Bevacizumab and Interferon Alpha-2a on Corneal Angiogenesis in a Rat Model.

BACKGROUND AND OBJECTIVE: Corneal neovascularization (CNV) is a vision-threatening condition arising from various corneal diseases. The aim of this study is to compare the effectiveness of bevacizumab and interferon alpha-2a (IFNα-2a) treatment on corneal neovascularization. MATERIALS AND METHODS: Twenty-four Wistar albino rats were used in this study. After cauterization of the cornea with a silver nitrate applicator stick, the control group received 0.1 mL saline solution, the second group received 0.1 mL IFNα-2a (IFNα-2a, 6 million international units [MIU]/0.5 mL), and the third group received 2.5 mg bevacizumab by subconjunctival injection. An additional injection was administered to each group on the fourth day. After one week, the corneal neovascularization rate and the longest neovascular sprout length were determined. RESULTS: The neovascularization rate (saline 0.65 ± 0.05; IFNα-2a 0.62 ± 0.07; bevacizumab 0.42 ± 0.11) with bevacizumab was significantly lower, more than those with IFNα-2a and saline (p < 0.001 and p < 0.001). The longest neovascular sprout length (saline, 4.00 ± 0.6 mm; IFNα-2a, 3.63 ± 0.52 mm; bevacizumab, 2.81 ± 0.65 mm) with bevacizumab was significantly shorter than those with saline and IFNα-2a (p = 0.001 and p = 0.012). CONCLUSIONS: Subconjunctival IFNα-2a has limited efficacy in the treatment of corneal neovascularization.

Anti-angiogenic effect of a humanized antibody blocking the Wnt/ß-catenin signaling pathway.

PURPOSE: Our previous study demonstrated that Mab2F1, a murine monoclonal antibody blocking the Wnt/ß-catenin signaling pathway, has beneficial effects on experimental diabetic retinopathy and choroidal neovascularization (NV). The aforementioned antibody has been humanized. This study evaluated effects of the humanized antibody, H1L1, on NV. METHODS: H1L1 was evaluated in the alkali burn-induced corneal NV rat model. Rats with corneal NV were injected subconjunctivally with Mab2F1 or H1L1 using non-specific mouse or human IgG as controls. Corneal NV and opacity were evaluated using corneal NV area and inflammatory index. Expression of angiogenic and inflammatory factors and components of the Wnt/ß-catenin pathway in both the corneas of the animal model and human corneal epithelial (HCE) cells exposed to Wnt3a conditioned medium (WCM) were determined by Western blotting and a luciferase-based promoter assay. Cytotoxicities of these antibodies were evaluated by MTT assay. RESULTS: H1L1 reduced the area of corneal NV and opacity, similar to Mab2F1. Both Mab2F1 and H1L1 down-regulated the overexpression of angiogenic and inflammatory factors including VEGF, TNF-α and ICAM-1, and blocked the aberrant activation of the Wnt/ß-catenin pathway as shown by down-regulation of phosphorylated LRP6, total LRP6 and non-phosphorylated ß-catenin in the cornea of the NV model and cultured HCE cells exposed to WCM. Both antibodies also inhibited the transcriptional activity of ß-catenin induced by WCM in HCE cells. No toxic effects of the antibodies were observed in cultured HCE cells. CONCLUSIONS: H1L1 exhibits anti-angiogenic activities through blocking the Wnt/ß-catenin pathway.

Successful single treatment with ziv-aflibercept for existing corneal neovascularization following ocular chemical insult in the rabbit model.

PURPOSE: To evaluate the efficacy of ziv-aflibercept as a treatment for established corneal neovascularization (NV) and to compare its efficacy to that of bevacizumab following ocular chemical insult of sulfur mustard (SM) in the rabbit model. METHODS: Chemical SM burn was induced in the right eye of NZW rabbits by vapor exposure. Ziv-aflibercept (2 mg) was applied once to neovascularized eyes by subconjunctival injection while subconjunctival bevacizumab (5 mg) was administered twice a week, for 3 weeks. Non-treated exposed eyes served as a control. A clinical follow-up employed by slit-lamp microscope, was performed up to 12 weeks following exposure and digital photographs of the cornea were taken for measurement of blood vessels length using the image analysis software. Eyes were taken for histological evaluation 2, 4 and 8 weeks following treatment for general morphology and for visualization of NV, using H&E and Masson Trichrome stainings, while conjunctival goblet cell density was determined by PAS staining. RESULTS: Corneal NV developed, starting as early as two weeks after exposure. A single subconjunctival treatment of ziv-aflibercept at 4 weeks post exposure, significantly reduced the extent of existing NV already one week following injection, an effect which lasted for at least 8 weeks following treatment, while NV in the non-treated exposed eyes continued to advance. The extensive reduction in corneal NV in the ziv-aflibercept treated group was confirmed by histological evaluation. Bevacizumab multiple treatment showed a benefit in NV reduction, but to a lesser extent compared to the ziv-aflibercept treatment. Finally, ziv-aflibercept increased the density of conjunctival goblet cells as compared to the exposed non-treated group. CONCLUSIONS: Subconjunctival ziv-aflibercept single treatment presented a highly efficient long-term therapeutic benefit in reducing existing corneal NV, following ocular sulfur mustard exposure. These findings show the robust anti-angiogenic efficacy of ziv-aflibercept and demonstrate the advantage of this treatment over the other anti-angiogenic therapies in ameliorating corneal NV and protecting the ocular surface.

B-cell leukemia/lymphoma 10 promotes angiogenesis in an experimental corneal neovascularization model.

PURPOSE: Corneal neovascularization (CrNV) arises from many causes including corneal inflammatory, infectious, or traumatic insult, and frequently leads to impaired vision. This study seeks to determine the role of B-cell leukemia/lymphoma 10 (BCL-10) in the development of experimental CrNV. METHODS: Corneas from BCL-10 knockout (KO) mice and wild-type (WT) mice were burned by sodium hydroxide (NaOH) to create the CrNV model and neovascular formation in the corneas was assessed 2 weeks later. Intracorneal macrophage accumulation and the expression of angiogenic factors were quantified by flow cytometric analysis (FCM) and real-time PCR, respectively. RESULTS: The amount of CrNV was determined 2 weeks after alkali burn. Compared to WT mice, the amount of CrNV in BCL-10 KO mice was significantly decreased. FCM revealed that F4/80-positive macrophages were markedly decreased in BCL-10 KO mice compared with WT mice. Reverse transcription PCR showed that the mRNA expression levels of intracorneal vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF) and monocyte chemotactic protein 1 were reduced in BCL-10 KO mice compared with WT mice. CONCLUSION: BCL-10 KO mice exhibited reduced alkali-induced CrNV by suppressing intracorneal macrophage infiltration, which subsequently led to decreased VEGF-A and bFGF expression, suggesting that BCL-10 may become a potential clinical intervening target of CrNV.

Effects of sunitinib and bevacizumab on VEGF and miRNA levels on corneal neovascularization.

AIM: To evaluate the effects of sunitinib (0.5 mg/ml) and bevacizumab (5 mg/ml) on VEGF-A, VEGFR-2 and microRNA (miRNA) levels on corneal neovascularization (CNV). METHODS: In this study, CNV was induced by silver nitrate application to the cornea, and 40 Albino male rats were equally divided into four subgroups: Group 1 (sunitinib): After silver nitrate application to the cornea, 0.5 mg/ml sunitinib eyedrop was administered twice daily for two weeks (n = 10). Group 2 (bevacizumab): After silver nitrate application to the cornea, 5 mg/ml bevacizumab eyedrop was administered twice daily for two weeks (n = 10). Group 3 (control): After silver nitrate application to the cornea, normal saline eyedrop was administered twice daily for two weeks (n = 10). Group 4 (vehicle): After silver nitrate application to the cornea, 1% DMSO eyedrop was administered twice daily for two weeks (n = 10). After two weeks from the silver nitrate application, corneas were evaluated by hand-held biomicroscope for their vascularization status. Then, corneas were excised and the expression levels of VEGFR-2, VEGF-A and the common miRNA markers for neovascularization (miR-15 b, miR-16, miR-23a, miR-126, miR-188, miR-210, miR-221, miR-222, miR-410 and miR-423) were evaluated by real-time PCR. RESULTS: It was seen that the CNV was decreased in sunitinib- and bevacizumab-administered groups compared to the control and DMSO groups. Also, in comparison with the control group; VEGF-A expression was downregulated by nearly 0.75 times in sunitinib group and nearly 0.52 times in bevacizumab group. VEGFR-2 expression was downregulated by 0.89 times in sunitinib group and 0.68 times in bevacizumab group, compared to the control group. miR-15 b, miR-16 and miR-126 levels were statistically lower in sunitinib and bevacizumab groups, but miR-188 and miR-410 levels were two-fold higher compared to the control group. The miR-210 level was found higher only in sunitinib group compared to the control group. There were no statistically significant changes in miR-23a, miR-221, miR-222 and miR-423 levels among the groups. CONCLUSION: Topical application of bevacizumab (5 mg/ml) and sunitinib (0.5 mg/ml) decreases the levels of VEGFR-2 and VEGF-A in CNV. Further studies are needed for detailed analysis of genes which are targeted by up- or downregulated miRNAs in this study.

Histopathological and Molecular Changes in the Rabbit Cornea From Arsenical Vesicant Lewisite Exposure.

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury.

Chronic pesticide exposure and consequential keratectasia & corneal neovascularisation.

Ocular toxicity as a consequence of chronic pesticide exposure is one of the health hazards caused due to extended exposure to pesticides. The cornea, due to its position as the outer ocular layer and its role in protecting the internal layers of the eye; is gravely affected by this xenobiotic insult to the eye, leading to ocular irritation and damage to normal vision. The deleterious effects of chronic pesticide exposure on the various corneal layers and the ocular risks involved therein, were explored by mimicking the on-field scenario. Cytological, histological and flowcytometric parameters were taken into consideration to determine the enhanced risk of corneal neovascularisation and keratectasia, specifically, keratoconus. Chronic exposure to pesticides leads to heightened ocular morbidity wherein there were visible pathophysiological changes to the ocular surface. The cornea was found to be adversely affected with visible protuberance in a cone-like shape, characteristic of keratoconus in a majority of the experimental animals. Further analyses revealed a detrimental impact on all the corneal layers and an amplified expression of inflammation markers such as TNF-α, VCAM-1 and ICAM-1. Additionally, it was found that post pesticide exposure, the corneal surface developed hypoxia, leading to a significant increase of angiogenesis promoting factors and consequential neovascularisation. Apart from ocular toxicity, chronic exposure to pesticides significantly increases the risks of keratectasia and corneal neovascularisation; disorders which lead to diminished vision and if untreated, blindness.