Fujian Provincial Key Laboratory of Ophthalmology and Visual Science

Xiamen, China

Fujian Provincial Key Laboratory of Ophthalmology and Visual Science

Xiamen, China
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Han Y.,Xiamen University | Han Y.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Han Y.,Nanchang University | Shao Y.,Xiamen University | And 19 more authors.
Investigative Ophthalmology and Visual Science | Year: 2012

PURPOSE. To investigate the effect of netrin-1 on alkali burninduced 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 burninduced 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. © 2012 The Association for Research in Vision and Ophthalmology, Inc.


Tan Y.,Xiamen University | Tan Y.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Tan Y.,Shantou University | Tan Y.,Nanhua University | And 13 more authors.
American Journal of Physiology - Cell Physiology | Year: 2011

Squamous metaplasia is a common pathological process that occurs in the ocular surface epithelium. At present, there is no effective treatment for this abnormality. In the current study, we established an ex vivo conjunctival squamous metaplasia model by culturing human conjunctival tissues at an air-liquid interface for durations of up to 12 days. We then investigated the effects of amniotic membrane (AM) on squamous metaplasia through coculture of conjunctival tissues with AM or AM extract. We found that metaplasia features such as hyperproliferation and abnormal epidermal differentiation of conjunctival epithelium could be inhibited by AM or its extract. In addition, existing squamous metaplasia of conjunctival epithelium could be reversed to a nearly normal phenotype by AM. The mechanism by which AM prevents squamous metaplasia may involve downregulation of p38 mitogen-activated protein kinase and Wnt signaling pathways, which were activated in conjunctival explants cultured with an airlift technique. In conclusion, AM can inhibit and reverse squamous metaplasia of conjunctival epithelium. This finding may shed new light on prevention and treatment of diseases that involve epithelial squamous metaplasia. © 2011 the American Physiological Society.


Lin Z.,Xiamen University | Zhou Y.,Xiamen University | Zhou Y.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Wang Y.,Xiamen University | And 13 more authors.
Investigative Ophthalmology and Visual Science | Year: 2014

PURPOSE. To investigate the effects and possible mechanisms of serine protease inhibitor A3K (SERPINA3K) on the formation of ocular surface squamous metaplasia in a mouse dry eye model induced by topical benzalkonium chloride (BAC).METHODS. The eye drops containing SERPINA3K were topically administered during the induction of BAC-induced dry eye. The clinical indications of dry eye were evaluated on day (D)16, including tear break-up time (BUT), tear volume, corneal fluorescein staining, and inflammatory index. Global specimens were collected on D16 and the following examinations were performed: histologic investigation, immunostaining of cytokeratin 10 (K10), p63 and Ki67 in the cornea, and Western blot analysis of tumor necrosis factor-α (TNF-α).RESULTS. Serine protease inhibitor A3K suppressed the formation of BAC-induced dry eye, presenting with longer BUTs, lower corneal fluorescein staining scores, and inflammatory index, while no significant changes in tear volume. It also reduced the severity of abnormal differentiation and proliferation on ocular surface with lower expressions of K10, p63, and Ki67, and retained the number of goblet cells in the conjunctival fornix. Serine protease inhibitor A3K significantly decreased the levels of TNF-α in the cornea.CONCLUSIONS. Topical application of SERPINA3K ameliorated the severity of ocular surface squamous metaplasia and suppressed the formation of BAC-induced dry eye. © 2014 The Association for Research in Vision and Ophthalmology, Inc.


Li C.,Xiamen University | Li C.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Dong N.,Xiamen University | Dong N.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | And 13 more authors.
Investigative Ophthalmology and Visual Science | Year: 2013

PURPOSE. We investigated the efficacy of low-temperature airlift preservation of human corneal limbal tissue for ex vivo expansion and allograft keratolimbal transplantation. METHODS. Human limbal tissue either was submerged or airlifted in Optisol-GS medium and preserved at 48C for up to eight days. Hematoxylin and eosin, and E-cadherin staining was performed to investigate epithelial structure and cell-cell junction. Epithelial cell differentiation and proliferation were studied using the biomarkers, such as K10, K12, K14, Ki67, and p63. Cell apoptosis was detected with the TUNEL assay. The epithelial progenitor cell pool was evaluated by clonal culture of epithelial cells on 3T3 feeder layers. For clinical application, keratolimbal transplantation was performed in three patients with partial limbal stem cell deficiency, using limbal tissues preserved under the airlift manner. Pre- and postoperative evaluations were conducted by slit-lamp microscopy and fluorescein staining. RESULTS. After eight days, intact epithelia with strong cell-cell junctions were retained only in airlifted tissues. Airlifting maintained a normal corneal differentiation pattern, along with low proliferation activity and increased proliferation potential, but little apoptosis. Epithelial cells harvested from the airlift preservation for up to eight days exhibited stable clonogenicity. Limbal tissues preserved under the airlift manner successfully reconstructed corneal and limbal surfaces in partial limbal stem cell-deficient patients. CONCLUSIONS. Limbal tissues preserved under hypothermic airlift conditions maintain the intact structure, normal phenotype, high viability, and stem cell pool of limbal epithelia. Such a method may be used in eye bank tissue processing and corneal epithelial tissue engineering. © 2013 The Association for Research in Vision and Ophthalmology, Inc.


Li J.,Xiamen University | Li J.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Li C.,Xiamen University | Li C.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | And 12 more authors.
Investigative Ophthalmology and Visual Science | Year: 2016

PURPOSE. Squamous metaplasia is a common pathologic condition in ocular surface diseases for which there is no therapeutic medication in clinic. In this study, we investigated the effect of a small molecule, APR-246/PRIMA-1Met, on squamous metaplasia in human conjunctival epithelium. METHODS. Human conjunctival explants were cultured for up to 12 days under airlifting conditions. Epithelial cell differentiation and proliferation were assessed by Cytokeratin 10 (K10), K14, K19, Pax6, MUC5AC, and p63 immunostaining patterns. β-catenin and TCF-4 immunofluorescent staining and real-time PCR characterized Wnt signaling pathway involvement. Pterygium clinical samples were cultured under airlifting conditions with or without APR-246 for 4 days. p63, K10, β-catenin, and TCF-4 expression in pterygial epithelium was determined by immunofluorescent staining and real-time PCR. RESULTS. Airlift conjunctival explants resulted in increased stratification and intrastromal epithelial invagination. Such pathology was accompanied by increases in K10, K14, and p63 expression, whereas K19 and Pax6 levels declined when compared to those in freshly isolated tissue. On the other hand, APR-246 reversed all of these declines in K10, K14, and p63 expression. Furthermore, K19 and Pax6 increased along with rises in goblet cell density. These effects of APR-246 were accompanied by near restoration of normal conjunctival epithelial histology. APR-246 also reversed squamous metaplasia in pterygial epithelium that had developed after 4 days in ex vivo culture. CONCLUSIONS. Reductions in squamous metaplasia induced by APR-246 suggest it may provide a novel therapeutic approach in different squamous metaplasia–associated ocular surface diseases. © 2016, Association for Research in Vision and Ophthalmology Inc. All rights reserved.


Lin Z.,Sun Yat Sen University | Lin Z.,Xiamen University | Lin Z.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Liu X.,Xiamen University | And 11 more authors.
Molecular Vision | Year: 2011

Purpose: To develop a dry eye model of mouse induced by topical administration of benzalkonium chloride (BAC) and investigate the possible mechanisms. Methods: BAC at concentration of 0.2% was applied to the mouse ocular surface for 7 days. Phenol red thread tear test, tear break-up time (BUT) test, corneal inflammatory index scoring, fluorescein and rose bengal test were performed to evaluate the toxic effects of BAC on the ocular surface. Global specimens were collected on day (D) 7 and labeled with a series of antibodies including cytokeratin 10 (K10) and mucin 5AC (MUC5AC). Apoptosis of ocular surface epithelium was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Histologic analysis and transmission electron microscopy (TEM) were performed on D7. Results: BAC at a concentration of 0.2% successfully induced a dry eye condition with decreased tear volume and BUTs, increased corneal fluorescein and rose bengal scores. The Inflammatory index was increased in accompanyment with higher tumor necrosis factor-α (TNF-α) expression and more inflammatory infiltration in the cornea. Immunolabeling revealed positive K10 expression in BAC-treated corneal epithelium and fewer MUC5AC-positive cells in the BACtreated conjunctival fornix. TUNEL assay showed more apoptotic cells in the corneal basal epithelium. TEM showed that the size and intervals of the microvillis were both reduced in the corneal epithelium. Conclusions: Topical administration of 0.2% BAC in mouse induces changes resembling that of dry eye syndrome in humans, and thus, represents a novel model of dry eye. © 2011 Molecular Vision.


Li C.,Xiamen University | Li C.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Dong F.,Xiamen University | Dong F.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | And 14 more authors.
American Journal of Pathology | Year: 2013

Endothelial-to-mesenchymal transition (EnMT) is a cell transformation process involved in both morphogenesis and pathogenesis. EnMT of corneal endothelial cells happens after endothelial injury and during ex vivo culture. Previous studies have shown that the transforming growth factor-β signaling pathway is involved in this transition. In this study, we found that rat corneal endothelial cells could spontaneously undergo EnMT during ex vivo culture. This change in rat corneal endothelial cells was associated with Notch signaling pathway activation after the first passage, which was blocked by the Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). This inhibitor also prevented transforming growth factor β1-, β2-, and β3-induced EnMT and reversed transformed rat corneal endothelial cells to a normal phenotype. Furthermore, DAPT treatment blocked retrocorneal membrane formation in a rat corneal endothelium damage model. Our study indicates that the Notch signaling pathway is involved in the corneal EnMT process, which may be a novel therapeutic target for treating corneal endothelial fibrogenic disorders. © 2013 American Society for Investigative Pathology.


Lin Z.,Xiamen University | He H.,Xiamen University | He H.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Zhou T.,Xiamen University | And 8 more authors.
Investigative Ophthalmology and Visual Science | Year: 2013

Purpose. To develop a mouse model of limbal stem cell deficiency (LSCD) by topical administration of benzalkonium chloride (BAC). Methods. BAC solutions (0%-0.5%) were applied to the mouse ocular surface for 4 weeks. Corneal neovascularization, inflammation, and epithelial status were observed under slit-lamp microscope. The eyeball and ocular surface tissues were collected at 4 and 12 weeks and labeled with a series of antibodies. Limbal structure was evaluated by light and transmission electron microscopy (TEM). Corneal impression cytology was performed at 12 weeks, and specimens were labeled with periodic acid Schiff (PAS) reagents. Results. BAC (0.5%) four times per day for 28 days successfully induced the typical manifestations of LSCD, including corneal neovascularization, severe inflammation in the stroma, and diffuse epithelial defect (P < 0.001). Conjunctival epithelium markers K19 and K13 were positive on the corneal surface. Expression of the putative limbal stem cell markers P63 and ABCG2 was abolished in the limbal epithelium. β-catenin was negative in the basal layer. TEM revealed the irregular basement membrane and the loss of stem cell-specific ultrastructure in the limbal basal epithelium. In the 0.5% BAC group, goblet cells could not be observed on day 28 but emerged after the cessation of BAC, and remained over the cornea after 8 weeks. K13-positive cells were still present over the cornea with the loss of K12. Conclusions. Topical administration of BAC at high concentration and frequency in mouse induces ocular surface changes resembling those of LSCD in humans, representing a novel model of LSCD. © 2013 The Association for Research in Vision and Ophthalmology, Inc.


Li C.,Xiamen University | Li C.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | Yin T.,Xiamen University | Yin T.,Fujian Provincial Key Laboratory of Ophthalmology and Visual Science | And 14 more authors.
Journal of Cellular Physiology | Year: 2011

Oxygen concentration has been shown to be crucial in the proliferation and differentiation of various types of cells, while the impact of oxygen tension on the lineage commitment of epithelial cells remains elusive. In this study, we investigated the effect of hypoxia on the differentiation of corneal limbal epithelium using an ex vivo squamous metaplasia model. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of human corneal limbal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The Notch signaling pathway was activated in hypoxic cultures, whereas the p38 MAPK signaling pathway was downregulated. The addition of Notch inhibitor under hypoxic conditions restored the activation of p38 MAPK and resulted in the recidivation of limbal epithelial cells to epidermal-like differentiation. Moreover, the epidermal-like differentiation of rabbit limbal epithelial cells was also blocked under hypoxic conditions in corneal epithelial cell sheets engineered ex vivo. We concluded that hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal limbal epithelial cells. Hypoxia coupled with air exposure can be used in the tissue engineering of corneal limbal epithelium. © 2010 Wiley-Liss, Inc.

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