Yueping Zhou

Netrin-1 Simultaneously Suppresses Corneal Inflammation and Neovascularization

PURPOSE To investigate the effect of netrin-1 on alkali burn-induced corneal inflammation and neovascularization. METHODS The expression of netrin-1 and its receptors UNC5A, UNC5B, UNC5C, UNC5D, adenosine 2b receptor (A2BAR), deleted in colorectal cancer (DCC), and neogenin in normal and alkali-burned rat cornea were determined by RT-PCR and/or Western blot analysis, or immunostaining. Topical netrin-1 protein was applied to treat rat corneal alkali-burn injury for 14 consecutive days, started right after the injury or 10 days postinjury. Corneal inflammation and neovascularization were observed under slit lamp microscope. The apoptosis of corneal cells was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Corneal inflammatory cell infiltration was evaluated by immunostaining of anti-PMN and anti-ED1 antibodies. The expression of epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), and pigment epithelium-derived factor (PEDF) in rat cornea was determined by Western blot analysis. RESULTS Netrin-1 and its receptor UNC5B were expressed in normal rat corneal epithelium and stromal cells, and their expression decreased after corneal alkali burn. Exogenous netrin-1 administered on rat ocular surfaces resolved alkali burn-induced corneal inflammation, and also suppressed corneal neovascularization. Furthermore, netrin-1 could reverse neovascularization in alkali-burned cornea. The authors found that netrin-1 executed the functions through various mechanisms, including upregulating EGF expression, accelerating epithelial wound healing, inhibiting neutrophil and macrophage infiltration, reducing corneal cell apoptosis, and restoring the equilibrium of VEGF and PEDF in the wounded cornea. CONCLUSIONS Netrin-1 could dampen inflammation, inhibit, and reverse neovascularization in alkali-burned cornea.

Anti-Angiogenic and Anti-Inflammatory Effects of SERPINA3K on Corneal Injury

SERPINA3K is a member of the serine proteinase inhibitor (SERPIN) family. Here we evaluated the therapeutic effects of SERPINA3K on neovascularization and inflammation in a rat cornea alkali burn model that is commonly employed to study corneal wounding. Topical treatment of the injured rat cornea with SERPINA3K (20 µg/eye/day) for 7 days significantly decreased the neovascular area, compared with the groups treated with BSA or PBS. The SERPINA3K treatment also ameliorated the corneal inflammation as evaluated by the inflammatory index. Furthermore, SERPINA3K enhanced the recovery of corneal epithelium after the alkali injury. Toward the mechanism of action, SERPINA3K down-regulated the expression of the pro-angiogenic and pro-inflammatory factors, vascular endothelial growth factor and tumor necrosis factor-α and up-regulated the expression of the anti-angiogenic factor, pigment epithelium-derived factor. SERPINA3K specifically inhibited growth of vascular endothelial cells. Meanwhile, SERPINA3K significantly up-regulated the expression of EGFR in the corneal epithelium. These findings suggest that SERPINA3K has therapeutic potential for corneal inflammation and NV.

Importin 13 Serves as a Potential Marker for Corneal Epithelial Progenitor Cells

Importin13 (IPO13), the newest member of importin‐β family discovered recently, is a unique nucleus‐cytoplasm bidirectional transport receptor protein. In this study, IPO13 expression in human corneal tissue, limbal epithelial primary explant and clonal culture was evaluated by immunostaining and reverse‐transcription polymerase chain reasgon. IPO13 function was evaluated in the corneal epithelial culture treated with IPO13 inhibitor, or fetal bovine serum (FBS)‐containing Dulbeccos modified Eagles medium (DMEM) medium by colony‐forming efficiency, clone growth capacity, MTT, immunostaining, and Western blotting assay. IPO13 protein was expressed mainly in nuclei of limbal epithelial basal cells, but not in the other cell layers of limbus and full thickness of corneal epithelia. IPO13 was expressed in the majority of epithelial cells in early‐stage clones and in the margin of late‐stage clones. IPO13 was positively expressed in mouse TKE2 progenitor cells cultured in keratinocyte serum‐free defined medium, while it became negative in FBS‐containing DMEM, which promoted TKE2 cell differentiation. In the presence of IPO13 inhibitor, IPO13 expression and the proliferative capacity decreased in human limbal epithelial clones and mouse TKE2 cells, which were accompanied with the cell differentiation. In conclusion, our findings demonstrate for the first time that IPO13 is uniquely expressed by human limbal basal epithelial cells, and plays an important role in maintaining the phenotype, high proliferative potential, and less differentiation of corneal epithelial progenitor cells, suggesting that IPO13 could serve as a novel potential marker for corneal epithelial progenitor cells. STEM CELLS 2009;27:2516–2526

Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway

Kallistatin is a member of the serine proteinase inhibitor superfamily. Kallistatin levels have been shown to be decreased in the vitreous while increased in the circulation of patients with diabetic retinopathy (DR). Overactivation of the Wnt pathway is known to play pathogenic roles in DR. To investigate the role of kallistatin in DR and in Wnt pathway activation, we generated kallistatin transgenic (kallistatin-TG) mice overexpressing kallistatin in multiple tissues including the retina. In the oxygen-induced retinopathy (OIR) model, kallistatin overexpression attenuated ischemia-induced retinal neovascularization. In diabetic kallistatin-TG mice, kallistatin overexpression ameliorated retinal vascular leakage, leukostasis, and overexpression of vascular endothelial growth factor and intracellular adhesion molecule. Furthermore, kallistatin overexpression also suppressed Wnt pathway activation in the retinas of the OIR and diabetic models. In diabetic Wnt reporter (BAT-gal) mice, kallistatin overexpression suppressed retinal Wnt reporter activity. In cultured retinal cells, kallistatin blocked Wnt pathway activation induced by high glucose and by Wnt ligand. Coprecipitation and ligand-binding assays both showed that kallistatin binds to a Wnt coreceptor LRP6 with high affinity (Kd = 4.5 nmol/L). These observations suggest that kallistatin is an endogenous antagonist of LRP6 and inhibitor of Wnt signaling. The blockade of Wnt signaling may represent a mechanism for its antiangiogenic and antineuroinflammatory effects.

Therapeutic Effects of Epidermal Growth Factor on Benzalkonium Chloride–Induced Dry Eye in a Mouse Model

PURPOSE To investigate the therapeutic effects and possible mechanisms of epidermal growth factor (EGF) on the mouse dry eye model induced by benzalkonium chloride (BAC). METHODS The eye drop containing EGF was topically administered (3 ng per day) on a BAC-induced dry eye model. The following clinical indications of dry eye were evaluated on Days 2, 4, and 6: tear break-up time (BUT), corneal fluorescein staining, inflammatory index, and tear volume. Global specimens were collected on Day 6 and then the following examinations were performed: histologic investigation, TUNEL assay to measure the dead cells, periodic acid-schiff (PAS) assay to detect goblet cells, and immunostaining of antibodies of Ki-67, EGF receptor (EGFR), and MUC1 in the corneas. The levels of EGFR and p-ERK of the corneas were also measured by Western blot analysis. RESULTS EGF resulted in longer BUTs on Days 2 and 6, lower fluorescein staining scores on Days 4 and 6, while no significant changes in inflammatory index or tear volume. EGF induced higher EGFR expression in corneal tissues by immunofluorescent staining and Western blot analysis. EGF also upregulated p-ERK, increased Ki-67 positive cells, and decreased TUNEL positive cells. In addition, EGF significantly increased the goblet cells number and MUC1 expression in the epithelium. CONCLUSIONS Topical application of EGF presented clinical improvements on dry eye by stabilizing the tear film and maintaining the integrity of epithelium. The results indicate that EGF has potential as a therapeutic agent in clinical treatment of dry eye.

SERPINA3K Protects against Oxidative Stress via Modulating ROS Generation/Degradation and KEAP1-NRF2 Pathway in the Corneal Epithelium

PURPOSE We recently reported that SERPINA3K (SA3K), a member of the serine proteinase inhibitor (SERPIN) family, has antiangiogenic and anti-inflammatory activities. Here we investigated the antioxidant effects of SA3K in the corneal epithelium and the mechanism underlying its action. METHODS We established the oxidative stress models induced by hydrogen peroxide (H₂O₂) in cultured human corneal epithelial (HCE) cells and in rat corneal epithelium in vivo. Cell viability, flow cytometry, and TUNEL analysis were conducted to detect viable cells and cell death; reactive oxygen species (ROS) and 3-Nitrotyrosine fluorescent assay was applied to measure ROS levels. Activity assay, immunostaining, Western blot, and quantitative RT-PCR were performed to analyze the factors of the ROS generation/degradation system and pathway. RESULTS SA3K protected the HCE cells from H₂O₂-induced oxidative stress in a dose- and time-dependent manner. SA3K also significantly reduced the production of ROS. Regarding the mechanism underlying these effects, SA3K downregulated ROS generation by inhibiting NOX4 and upregulated ROS degradation by increasing the activity of superoxide dismutases and catalase. Furthermore, H₂O₂ induced activation of the Kelch-like ECH-associated protein 1 (KEAP1)/NF-E2-related factor-2 (NRF2) pathway, while SA3K inhibited H₂O₂-induced activation of KEAP1 and NRF2 and their downstream factors, including NAD(P)H quinone oxidoreductase and glutathione S-transferase. In the H₂O₂-induced rat corneal epithelium, SA3K alleviated the oxidative stress and downregulated NOX4 and NRF2. CONCLUSIONS Collectively, SA3K protects against oxidative stress by targeting the ROS generation/degradation system and modulating the KEAP1-NRF2 signaling pathway.

Down-Regulation of MicroRNA-184 Is Associated With Corneal Neovascularization.

PURPOSE Although microRNA-184 (miR-184) is abundantly expressed in the corneas, the role of miR-184 in corneal neovascularization remains unknown. Here we investigated the association between miR-184 expression and corneal neovascularization. METHODS Quantitative real-time PCR assay was conducted to detect the expression of miR-184 and its potential target genes in the corneal epithelium of rats with corneal suture-induced neovascularization. MicroRNA-184 was also applied topically to the suture rats. Mimic and inhibitor of miR-184 were transfected into the cultured human umbilical vein endothelial cells (HUVECs), human corneal epithelial (HCE) cells, and simian choroidal endothelial cells (RF/6A). The following experiments were performed to evaluate the effects of miR-184 in these transfected cells: cell proliferation by cell viability assay, cell migration by a scratch wound test, VEGF-induced tube formation, and VEGF and β-catenin levels by Western blot analysis. RESULTS The expression of miR-184 was significantly reduced, whereas the gene expression of frizzled-4, a receptor of the Wnt pathway, was up-regulated in the corneal epithelium of corneal suture rats. The corneal neovascularization induced by suture was ameliorated by topical administration of miR-184. In the cells transfected with mimic and inhibitor of miR-184, miR-184 significantly suppressed the cell proliferation and cell migration of HUVECs, miR-184 down-regulated VEGF, and β-catenin expression in HUVECs and HCE cells. Furthermore, miR-184 inhibited the tube formation of RF/6A cells. CONCLUSIONS Down-regulation of miR-184 is associated with up-regulation of VEGF and Wnt/β-catenin expression as well as corneal neovascularization, indicating that miR-184 negatively regulates corneal neovascularization.

SERPINA3K Plays Antioxidant Roles in Cultured Pterygial Epithelial Cells through Regulating ROS System

We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM). The cultured pterygial epithelial cells (PECs) were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR) assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4), which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(P)H dehydrogenase (quinone 1) (NQO1), NF-E2–related factor-2 (NRF2) and superoxide dismutases (SOD2). Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: β-catenin, nonphospho-β-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6). We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.

Plasma and vitreous fluid levels of Dickkopf-1 in patients with diabetic retinopathy

PurposeDickkopf-1 (DKK-1) is a secreted inhibitor of the Wnt/β-catenin signaling pathway, which plays a pathogenic role in diabetic retinopathy (DR). We aimed to investigate whether DKK-1 levels in the plasma and the vitreous are associated with DR in type 2 diabetes mellitus (DM) patients.MethodsCase–control study: plasma samples were collected from 125 type 2 DM including 81 DR (29 non-proliferative DR (NPDR) and 52 proliferative DR (PDR)), 44 non-DR patients (NDR), and 100 non-diabetic controls. Undiluted vitreous fluid samples were obtained from 30 PDR and 25 non-diabetic patients. DKK-1 concentrations in samples were determined using enzyme-linked immunosorbent assay. Variables were compared with the Kruskal–Wallis H test, Mann–Whitney U-test, and χ2-test, when appropriate.ResultsPlasma DKK-1 levels were significantly lower in DR patients (median: 465.77 pg/ml, range: 137.11–1190.31) than in non-diabetic controls (656.83 pg/ml, 171.63–1795.08; P<0.001) and NDR patients (693.04 pg/ml, 305.43–1218.35; P<0.001). Furthermore, DKK-1 levels were lower in PDR patients (425.21 pg/ml, 137.10–1077.32) compared with NPDR patients (594.86 pg/ml, 256.36–1393.27; P=0.003). Vitreous absolute DKK-1 levels in PDR patients (259.04 pg/ml, 104.44–596.96) were higher than in non-diabetic controls (138.26 pg/ml, 18.69–239.52; P<0.001). After normalizing by total vitreous protein concentrations, however, there was no significant difference between the groups. DKK-1 levels in vitreous were lower than those in plasma in both groups (P<0.001 for controls; P=0.002 for PDR patients).ConclusionsDecreased plasma DKK-1 levels, which may contribute to the Wnt pathway activation, are associated with the presence and progression of DR, and have potential to become a biomarker for DR.

The oxidant role of 4-Hydroxynonenal in corneal epithelium

4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in tissues and is reported to play pathogenic roles in eye diseases. Here we investigated the association between 4-HNE and oxidative stress in the corneal epithelium. 4-HNE suppressed the cell viability of human corneal epithelial cells (HCE) in a concentration dependent manner. 4-HNE significantly increased the level of 3-Nitrotyrosine (3-NT), a marker of oxidative stress, in HCE cells and corneal epithelium of rats by immunofluorescent staining and Western blot analysis. To its underlying mechanistic on ROS system, 4-HNE elevated the ROS generation enzyme NADPH oxidase 4 (NOX4) and induced the activation of NF-E2–related factor-2 (NRF2) and its downstream effectors: NAD(P)H dehydrogenase (quinone 1) (NQO1) and glutathione S-transferase P (GSTP). Furthermore, N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the inhibitory and oxidant effects of 4-HNE on the corneal epithelial cells. In conclusion, 4-HNE plays an oxidant role in the corneal epithelium and this work provides a new strategy for the pathogenesis and treatment of corneal diseases.