Glucocorticoid Receptor Signaling Is Critical for Mouse Corneal Development, Inhibition of Inflammatory Response, and Neovascularization of the Cornea.

The cornea protects the interior of the eye from external agents such as bacteria, viruses, and debris. Synthetic glucocorticoids are widely prescribed in the treatment of ocular infections and disorders. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR); however, the molecular and physiological functions of GR signaling in the cornea are poorly understood. This study found that treatment of mice with glucocorticoid eye drops led to a profound regulation of the corneal transcriptome. These glucocorticoid-regulated genes were associated with multiple biological functions, including the immune response. To understand the direct role of GR signaling in the cornea, mice with conditional knockout of GRs in the corneal epithelium were generated. Mice lacking corneal GRs exhibited microphthalmia, loss of pupils, a deformed and opaque lens, and mislocalization of key structural proteins within the corneal epithelial layers. Global transcriptomic approaches revealed that loss of GR signaling in the cornea also resulted in the dysregulation of a large cohort of genes strongly associated with an enhanced inflammatory response. Finally, corneal GR signaling was required for preventing neovascularization of blood and lymphatic vessels and thereby immune cell infiltration of the cornea. These results reveal that corneal GR signaling plays a critical role in ocular development and in maintaining the homeostasis of the eye.

Bone morphogenetic protein 4 inhibits corneal neovascularization by blocking NETs-induced disruption to corneal epithelial barrier.

Corneal neovascularization (CoNV) is the second leading cause of visual impairment worldwide, and current drugs have certain limitations. Inflammatory response is the core pathological process of CoNV. Neutrophil extracellular traps (NETs) are generated after neutrophil activation, which promotes neovascularization. Prior studies demonstrated that bone morphogenetic protein 4 (BMP4) could significantly reduce inflammation and CoNV formation, its exact molecular mechanism remains unclear. Therefore, we stimulated human peripheral blood neutrophils with phorbol myristate acetate (PMA) or deoxyribonuclease I (DNase I) to induce or inhibit NETs formation. By using corneal sutures and subconjunctival injections of NETs or DNase I, rat CoNV models were established. Compared with the suture group, NETs formation and inflammatory cell infiltration in the corneal stroma were significantly increased, corneal edema was aggravated, and the length, area and diameter of CoNV were significantly enhanced in the NETs group. Furthermore, by curetting the corneal epithelial apical junctional complexes (AJCs), a crucial component in preserving the function of the corneal epithelial barrier, we discovered that the damage of AJCs had a significant role in inducing CoNV formation. NETs could induce CoNV formation by injuring corneal epithelial AJCs. Finally, by comparing the aforementioned indicators after the intervention of BMP4, BMP4 inhibitor Noggin and NADPH oxidase (NOX) inhibitor, we finally demonstrated that BMP4 could inhibit NETs-induced inflammation and corneal epithelial AJC injury, repair corneal epithelial barrier function and eventually inhibit CoNV formation by blocking NOX-2-dependent NETs formation.

Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1.

Background: Mesenchymal stem/stromal cells (MSCs) maintain tissue homeostasis in response to microenvironmental perturbations. Toll-like receptors (TLRs) are key sensors for exogenous and endogenous signals produced during injury. In this study, we aimed to investigate whether TLRs affect the homeostatic functions of MSCs after injury. Methods: We examined the expression of TLR2, TLR3 and TLR4 in MSCs, and analyzed the functional significance of TLR2 activation using single-cell RNA sequencing. Additionally, we investigated the effects and mechanisms of TLR2 and its downstream activation in MSCs on the MSCs themselves, on monocytes/macrophages, and in a mouse model of sterile injury-induced inflammatory corneal angiogenesis. Results: MSCs expressed TLR2, which was upregulated by monocytes/macrophages. Activation of TLR2 in MSCs promoted their immunoregulatory and angiostatic functions in monocytes/macrophages and in mice with inflammatory corneal angiogenesis, whereas TLR2 inhibition attenuated these functions. Single-cell RNA sequencing revealed AKR1C1, a gene encoding aldo-keto reductase family 1 member C1, as the most significantly inducible gene in MSCs upon TLR2 stimulation, though its stimulation did not affect cell compositions. AKR1C1 protected MSCs against ferroptosis, increased secretion of anti-inflammatory cytokines, and enhanced their ability to drive monocytes/macrophages towards immunoregulatory phenotypes, leading to the amelioration of inflammatory corneal neovascularization in mice. Conclusion: Our findings suggest that activation of TLR2-AKR1C1 signaling in MSCs serves as an important pathway for the survival and homeostatic activities of MSCs during injury.

Inhibition of Experimental Corneal Neovascularization by the Tight Junction Protein ZO-1.

Purpose: To explore the effects of the tight junction protein zonula occludens 1 (ZO-1) on experimental corneal neovascularization (CNV). Methods: CNV models were established in the left eyes of BALB/c mice using NaOH. Anti-ZO-1 neutralizing antibody was topically applied to the burnt corneas after modeling thrice a day for 1 week. CD31 expression was analyzed to calculate the ratio of CNV number to area using a corneal whole-mount fluorescent immunohistochemical assay. Messenger ribonucleic acid (mRNA) and protein expression levels of ZO-1, vascular endothelial growth factor (VEGF), interleukin (IL)-1ß, IL-6, IL-8, IL-18, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), phosphorylated protein kinase C (pPKC), and clusterin in burned corneas were detected by reverse transcriptase polymerase chain reaction (PCR) and western blot analyses. Infiltration of neutrophils, macrophages, and progenitor cells was examined by flow cytometry. Results: CNV was obviously greater in 45 s than in 15 s alkali injury group. In another experiment, CNV was obviously greater in the ZO-1 antibody group than in the vehicle-treated group. Corneal mRNA and protein expression levels of VEGF, IL-1ß, IL-6, IL-8, IL-18, and MCP-1 were significantly higher in the ZO-1 antibody group than in the control group. Infiltration of neutrophils, macrophages, and progenitor cells was significantly greater in the ZO-1 antibody group than in the control group. TNF-α expression was much higher in 45 s than in 15 s alkali injury group. However, protein expression of pPKC and clusterin was much lower in 45 s than in 15 s alkali injury group. Conclusions: Anti-ZO-1 neutralizing antibody-treated mice exhibited enhanced alkali-induced CNV through enhanced intracorneal infiltration of progenitor and inflammatory cells.

Cannabidiol alleviates suture-induced corneal pathological angiogenesis and inflammation by inducing myeloid-derived suppressor cells.

BACKGROUND: Currently, no perfect treatment for neovascularization and lymphangiogenesis exist, and each treatment method has its complications and side effects. This study aimed to investigate the anti-angiogenic and anti-inflammatory effects of cannabidiol and its mechanism of action. METHOD: An in vivo corneal neovascularization (CNV) model was established using the suture method to investigate the inhibitory effects of CBD on suture-induced corneal inflammation, pathological blood vessel formation, and lymphangiogenesis. Additionally, the impact of CBD on immune cells was studied. In vitro methodologies, including cell sorting and co-culture, were employed to elucidate its mechanism of action. RESULTS: Compared with the CNV group, CBD can inhibit CNV, lymphangiogenesis, and inflammation induced via the suture method. In addition, CBD specifically induced CD45+CD11b+Gr-1+ cell upregulation, which significantly inhibited the proliferation of CD4+ T lymphocytes in vitro and exhibited a CD31+ phenotype, proving that they were myeloid-derived suppressor cells (MDSCs). We administered anti-Gr-1 to mice to eliminate MDSCs in vivo and found that anti-Gr-1 partially reversed the anti-inflammatory and angiogenic effects of CBD. Furthermore, we found that compared with MDSCs in the normal group, CBD-induced MDSCs overexpress peroxisome proliferator-activated receptor-gamma (PPAR-γ). Administering PPAR-γ inhibitor in mice almost reversed the induction of MDSCs by CBD, demonstrating the role of PPAR-γ in the function of CBD. CONCLUSION: This study indicates that CBD may induce MDSCs upregulation by activating the nuclear receptor PPAR-γ, exerting anti-inflammatory, antiangiogenic, and lymphangiogenic effects, and revealing potential therapeutic targets for corneal neovascularization and lymphangiogenesis.

Inhibiting PRMT1 protects against CoNV by regulating macrophages through the FGF2/PI3K/Akt pathway.

Corneal neovascularization (CoNV) is predominantly initiated by inflammatory processes, resulting in aberrant vascular proliferation and consequent visual impairment. Existing therapeutic interventions for CoNV demonstrate limited efficacy and potential for adverse reactions. Protein arginine methyltransferase 1 (PRMT1) is associated with the regulation of inflammation and M2 macrophage polarization. Nevertheless, the precise mechanism by which PRMT1 operates in CoNV remains uncertain. This study explored the impact of PRMT1 inhibition in a murine model of CoNV induced by alkali burn. Our findings indicated a direct relationship between PRMT1 levels and corneal damage. Moreover, our observations indicated an increase in fibroblast growth factor 2 (FGF2) expression in CoNV, which was reduced after treatment with a PRMT1 inhibitor. The inhibition of PRMT1 alleviated both corneal injury and CoNV, as evidenced by decreased corneal opacity and neovascularization. Immunofluorescence analysis and evaluation of inflammatory factor expression demonstrated that PRMT1 inhibition attenuated M2 macrophage polarization, a phenomenon that was reversed by the administration of recombinant FGF2 protein. These results were confirmed through experimentation on Human Umbilical Vein Endothelial Cells (HUVECs) and Mouse leukemia cells of monocyte macrophage cells (RAW264.7). Furthermore, it was established that FGF2 played a role in PI3K/Akt signal transduction, a critical regulatory pathway for M2 macrophage polarization. Importantly, the activity of this pathway was found to be suppressed by PRMT1 inhibitors. Mechanistically, PRMT1 was shown to promote M2 macrophage polarization, thereby contributing to CoNV, through the FGF2/PI3K/Akt pathway. Therefore, targeting PRMT1 may offer a promising therapeutic approach.

Efficacy and safety of combined UV-light corneal crosslinking and fine-needle diathermy to regress pathological murine corneal (lymph)angiogenesis in vivo.

PURPOSE: To compare safety and efficacy of isolated and combined UV-light corneal crosslinking (CXL) and fine-needle diathermy (FND) to regress pathological corneal vessels in vivo. METHODS: Mice with inflamed and pathologically vascularized corneas received CXL or FND as monotherapy or a combination of both treatments. Corneal pathological blood and lymphatic vessels, immune cells and the morphology of anterior segment structures were evaluated. RESULTS: All three approaches were able to regress blood and lymphatic vessels in mice. A comparative analysis of the three methods revealed that the FND monotherapy and the CXL + FND combination were significantly more effective than the CXL monotherapy, one and 2 weeks after therapy and especially in regressing lymphatic vessels. Furthermore, the combination therapy induced significantly less immune cell recruitment compared to the monotherapies. All three methods were safe to use in regards of corneal integrity. CONCLUSIONS: A combination of FND and CXL led to regression of pathological corneal lymphatic and blood vessels and reduced the infiltration of immune cells into inflamed murine corneas. This approach offers a new effective, safe and clinically usable strategy to treat eyes with mature pathological blood vessels and even more so for lymphatic vessels, for example prior to high-risk corneal transplantation.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Purpose: Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Methods: Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Results: Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10 days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Conclusions: Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

DZ2002 alleviates corneal angiogenesis and inflammation in rodent models of dry eye disease via regulating STAT3-PI3K-Akt-NF-κB pathway.

Dry eye disease (DED) is a prevalent ocular disorder with a multifactorial etiology. The pre-angiogenic and pre-inflammatory milieu of the ocular surface plays a critical role in its pathogenesis. DZ2002 is a reversible type III S-adenosyl-L-homocysteine hydrolase (SAHH) inhibitor, which has shown excellent anti-inflammatory and immunosuppressive activities in vivo and in vitro. In this study, we evaluated the therapeutic potential of DZ2002 in rodent models of DED. SCOP-induced dry eye models were established in female rats and mice, while BAC-induced dry eye model was established in female rats. DZ2002 was administered as eye drops (0.25%, 1%) four times daily (20 µL per eye) for 7 or 14 consecutive days. We showed that topical application of DZ2002 concentration-dependently reduced corneal neovascularization and corneal opacity, as well as alleviated conjunctival irritation in both DED models. Furthermore, we observed that DZ2002 treatment decreased the expression of genes associated with angiogenesis and the levels of inflammation in the cornea and conjunctiva. Moreover, DZ2002 treatment in the BAC-induced DED model abolished the activation of the STAT3-PI3K-Akt-NF-κB pathways in corneal tissues. We also found that DZ2002 significantly inhibited the proliferation, migration, and tube formation of human umbilical endothelial cells (HUVECs) while downregulating the activation of the STAT3-PI3K-Akt-NF-κB pathway. These results suggest that DZ2002 exerts a therapeutic effect on corneal angiogenesis in DED, potentially by preventing the upregulation of the STAT3-PI3K-Akt-NF-κB pathways. Collectively, DZ2002 is a promising candidate for ophthalmic therapy, particularly in treating DED.

Predicting clinical outcome of sulfur mustard induced ocular injury using machine learning model.

The sight-threatening sulfur mustard (SM) induced ocular injury presents specific symptoms in each clinical stage. The acute injury develops in all exposed eyes and may heal or deteriorate into chronic late pathology. Early detection of eyes at risk of developing late pathology may assist in providing unique monitoring and specific treatments only to relevant cases. In this study, we evaluated a machine-learning (ML) model for predicting the development of SM-induced late pathology based on clinical data of the acute phase in the rabbit model. Clinical data from 166 rabbit eyes exposed to SM vapor was used retrospectively. The data included a comprehensive clinical evaluation of the cornea, eyelids and conjunctiva using a semi-quantitative clinical score. A random forest classifier ML model, was trained to predict the development of corneal neovascularization four weeks post-ocular exposure to SM vapor using clinical scores recorded three weeks earlier. The overall accuracy in predicting the clinical outcome of SM-induced ocular injury was 73%. The accuracy in identifying eyes at risk of developing corneal neovascularization and future healed eyes was 75% and 59%, respectively. The most important parameters for accurate prediction were conjunctival secretion and corneal opacity at 1w and corneal erosions at 72 h post-exposure. Predicting the clinical outcome of SM-induced ocular injury based on the acute injury parameters using ML is demonstrated for the first time. Although the prediction accuracy was limited, probably due to the small dataset, it pointed out towards various parameters during the acute injury that are important for predicting SM-induced late pathology and revealing possible pathological mechanisms.

Up-to-date molecular medicine strategies for management of ocular surface neovascularization.

Ocular surface neovascularization and its resulting pathological changes significantly alter corneal refraction and obstruct the light path to the retina, and hence is a major cause of vision loss. Various factors such as infection, irritation, trauma, dry eye, and ocular surface surgery trigger neovascularization via angiogenesis and lymphangiogenesis dependent on VEGF-related and alternative mechanisms. Recent advances in antiangiogenic drugs, nanotechnology, gene therapy, surgical equipment and techniques, animal models, and drug delivery strategies have provided a range of novel therapeutic options for the treatment of ocular surface neovascularization. In this review article, we comprehensively discuss the etiology and mechanisms of corneal neovascularization and other types of ocular surface neovascularization, as well as emerging animal models and drug delivery strategies that facilitate its management.

A Mouse Model for Corneal Neovascularization by Alkali Burn.

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus and adversely affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection.

BACKGROUND: Corneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N6-methyladenosine (m6A) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and m6A in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo. METHODS: A PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA m6A abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo. RESULTS: METTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased m6A levels. METTL3 knockdown or inhibition by STM2457 further reduced m6A levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an m6A-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo. CONCLUSION: Our findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.

Effect of Conbercept on Corneal Neovascularization in a Rabbit Model.

OBJECTIVE: To study the efficacy of Conbercept for the treatment of corneal neovascularization (NV) in a rabbit model. METHODS: NV was induced by placing sutures. Eight rabbits were used as a control. The other 136 rabbits were randomly divided into two equal groups, and 68 rabbits in each group were divided into four subgroups and given different treatments. Time-course photographs, histological examination, and enzyme-linked immunoassay ELISA analysis for vascular endothelial growth factor were performed at weeks 1, 2, and 3 after injection placement. RESULTS: At weeks 1, 2, and 3 after injection placement, there was less expression of corneal NV and VEGF in the conbercept-treated groups than in the saline-treated control groups and less corneal NV and VEGF were expressed in the early treatment group than in the late treatment group. At weeks 2 and 3 after injection, there were fewer corneal NV (length and area) in the early intrastromal injection group with conbercept than in the early subconjunctival injection group with conbercept and a smaller diameter of corneal NV than in the late intrastromal injection group treated with conbercept. Histological examination showed a smaller diameter of corneal NV in all eyes in conbercept-treated groups 1 w after injection than before injection. Treatment with subconjunctival injection with conbercept led to a larger diameter at weeks 2 and 3 than at week 1. CONCLUSIONS: Subconjunctival and intrastromal administrations of conbercept effectively inhibit corneal NV in rabbits, and the latter has the better effect. The effect is the best in the group with cornea intrastromal injection of conbercept 1 w after suture. Early administration of conbercept may successfully inhibit corneal NV in an animal model.

Effect of adalimumab on experimental corneal neovascularization model.

PURPOSE: To evaluate the efficacy of adalimumab (ADA) on inhibition of experimental corneal neovascularization (CNV) and compare the outcomes with bevacizumab (BEVA). METHODS: Twenty-four female Winstar rats (48 eyes) were used. Silver/Potassium Nitrate sticks were used for creating CNV. Forty-eight eyes of the rats were separated into 6 groups. The eyes which only NaCl was injected subconjunctivally (SC) formed Group-1. The eyes which CNV was created and NaCl, BEVA (2.5 mg/0.05 mL), ADA (2.5 mg/0.05 mL), respectively, were injected SC formed group-2, 3 and 4. The eyes which only BEVA and ADA, respectively, were injected SC formed group-5 and 6. Five days later the animals were sacrificed. Hematoxylin and eosin staining, Masson trichrome staining, Vascular endothelial growth factor (VEGF), and Platelet-derived growth factor (PDGF) antibodies were performed. RESULTS: Histochemical results showed that there was no histopathological finding in group-1, 5, and 6. Collagen fiber irregularity was observed in group-2 and there was a significant improvement in collagen fiber irregularity in group-3 and 4. Collagen fiber proliferation was higher in group-2 than in group-3 and 4. VEGF and PDGF stainings were not observed in group-1, 5, and 6. VEGF and PDGF stainings were observed in group-2 and significantly decreased in group-3 and 4 compared to group-2. ADA was found to be superior to BEVA in terms of decreasing VEGF staining. CONCLUSION: Both BEVA and ADA were effective in inhibiting CNV. Subconjunctival ADA seems to be more effective than BEVA in terms of inhibiting VEGF expression. Further experimental studies about ADA and BEVA are needed.

WTAP promotes macrophage recruitment and increases VEGF secretion via N6-methyladenosine modification in corneal neovascularization.

BACKGROUND: Corneal neovascularization (CNV) can be caused by chemical burns. Macrophages are involved in angiogenesis and lymphangiogenesis during CNV. The aim of this study was to investigate whether Wilms' tumor 1-associated protein (WTAP) is involved in macrophage recruitment and VEGF secretion via N6-methyladenosine (m6A) modification. METHODS: A CNV mouse model was established by corneal alkali burn. Tumor necrosis factor alpha (TNF-α) was used to stimulate vascular endothelial cells. m6A immunoprecipitation qPCR was used to determine the enrichment of m6A levels in mRNAs. The H3K9me3 enrichment in the promoter region of CC motif chemokine ligand 2 (CCL2) was detected by chromatin immunoprecipitation assay. The WTAP inhibition in vivo was performed using the adeno-associated virus. RESULTS: In the alkali burn corneal tissues, angiogenesis and lymphangiogenesis were promoted as CD31 and LYVE-1 expressions were elevated, and the number of macrophages as well as WTAP expression were increased. Under the TNF-α stimulation, WTAP promoted the recruitment of endothelial cells to macrophages by promoting CCL2 secretion. Mechanistically, WTAP affected the enrichment of H3K9me3 at the CCL2 promoter by regulating the m6A level of SUV39H1 mRNA. The in vivo experiment showed that VEGFA/C/D secretion of macrophages was reduced after WTAP interference. Mechanistically, WTAP regulated the translational efficiency of HIF-1α via m6A modification. CONCLUSION: WTAP affected macrophage recruitment to endothelial cells via regulation of H3K9me3-mediated CCL2 transcription. WTAP also affected macrophage secretion of VEGFA/C/D via m6A-mediated translation regulation of HIF-1α. Both pathways were involved in the WTAP regulation of angiogenesis and lymphangiogenesis during CNV.

Lymphangiogenesis Guidance Mechanisms and Therapeutic Implications in Pathological States of the Cornea.

Corneal lymphangiogenesis is one component of the neovascularization observed in several inflammatory pathologies of the cornea including dry eye disease and corneal graft rejection. Following injury, corneal (lymph)angiogenic privilege is impaired, allowing ingrowth of blood and lymphatic vessels into the previously avascular cornea. While the mechanisms underlying pathological corneal hemangiogenesis have been well described, knowledge of the lymphangiogenesis guidance mechanisms in the cornea is relatively scarce. Various signaling pathways are involved in lymphangiogenesis guidance in general, each influencing one or multiple stages of lymphatic vessel development. Most endogenous factors that guide corneal lymphatic vessel growth or regression act via the vascular endothelial growth factor C signaling pathway, a central regulator of lymphangiogenesis. Several exogenous factors have recently been repurposed and shown to regulate corneal lymphangiogenesis, uncovering unique signaling pathways not previously known to influence lymphatic vessel guidance. A strong understanding of the relevant lymphangiogenesis guidance mechanisms can facilitate the development of targeted anti-lymphangiogenic therapeutics for corneal pathologies. In this review, we examine the current knowledge of lymphatic guidance cues, their regulation of inflammatory states in the cornea, and recently discovered anti-lymphangiogenic therapeutic modalities.

Evaluation of the Algerbrush II rotating burr as a tool for inducing ocular surface failure in a mouse model.

Purpose: The Algerbrush II has been widely used to induce corneal and limbal injuries in animal models. The extent of injury varies with the duration of exposure, pressure from the placement of the burr, and the size of the burr. However, no study has explored the correlation between the duration of exposure and the severity of injury in mouse model with corneal and limbal stem cell deficiency (LSCD) induced using the Algerbrush II. Therefore, this study aimed to evaluate the variations in the severity of corneal and limbal injury with different durations of the Algerbrush II application. Methods: The entire cornea and limbus of C57BL/6 mice were injured for 30-45 s, 60-75 s, 90-120 s, and 3-4 min. Photography and slit-lamp examination was performed on days 0, 2, 4, and 7, followed by hematoxylin & eosin, periodic acid-Schiff, and immunohistochemical staining. Statistical analysis was performed using one way ANOVA analysis. Results: A duration of 30-45 s of injury was found to be sufficient to induce superficial corneal and limbal epithelial debridement and re-epithelialization was completed in all eyes by day 7; however, clinical signs of LSCD were not observed in all mice. Increasing the exposure time to 90-120 s resulted in central 2+ corneal opacity with limbal and paracentral corneal neovascularization. All eyes injured for 3-4 min displayed clinical signs of LSCD, such as persistent epithelial defects on day 7 after the injury, central corneal neovascularization, and 2.2+ diffuse corneal opacity. Histological signs of LSCD, including goblet cell metaplasia and K13 expression on the corneal surface, were observed in all injured eyes. Conclusions: Our findings suggest that the duration of injury is an important factor influencing the severity of LSCD in a murine model of injury. A 1-mm rotating burr was found to be more effective for keratectomy and pigment release, whereas a 0.5-mm burr was more suitable for corneal epithelial debridement.

The protective effect of URP20 on ocular Staphylococcus aureus and Escherichia coli infection in rats.

BACKGROUND: Infectious keratitis, a medical emergency with acute and rapid disease progression may lead to severe visual impairment and even blindness. Herein, an antimicrobial polypeptide from Crassostrea hongkongensis, named URP20, was evaluated for its therapeutic efficacy against keratitis caused by Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infection in rats, respectively. METHODS: A needle was used to scratch the surface of the eyeballs of rats and infect them with S. aureus and E.coli to construct a keratitis model. The two models were treated by giving 100 µL 100 µM URP20 drops. Positive drugs for S. aureus and E. coli infection were cefazolin eye drops and tobramycin eye drops, respectively. For the curative effect, the formation of blood vessels in the fundus was observed by a slit lamp (the third day). At the end of the experiment, the condition of the injured eye was photographed by cobalt blue light using 5 µL of 1% sodium fluorescein. The pathological damage to corneal tissues was assessed using hematoxylin-eosin staining, and the expression level of vascular endothelial growth factor (VEGF) was detected by immunohistochemistry. RESULTS: URP20 alleviated the symptoms of corneal neovascularization as observed by slit lamp and cobalt blue lamp. The activity of S. aureus and E.coli is inhibited by URP20 to protect corneal epithelial cells and reduce corneal stromal bacterial invasion. It also prevented corneal thickening and inhibited neovascularization by reducing VEGF expression at the cornea. CONCLUSION: URP20 can effectively inhibit keratitis caused by E.coli as well as S. aureus in rats, as reflected by the inhibition of corneal neovascularization and the reduction in bacterial damage to the cornea.