[Show abstract][Hide abstract] ABSTRACT: Periostin is a non-structural matricellular protein. Little is known about periostin in human limbal stem cells (LSCs). This study was to explore the unique expression pattern and functional role of periostin in maintaining the properties of human LSCs. Fresh donor corneal tissues were used to make cryosections for evaluation of periostin expression on ex vivo tissues. Primary human limbal epithelial cells (HLECs) were generated from limbal explant culture. In vitro culture models for proliferation and epithelial regeneration were performed to explore functional role of periostin in LSCs. The mRNA expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), and the protein production and localization were detected by immunofluorescent staining and Western blot analysis. Periostin protein was found to be exclusively immunolocalized in the basal layer of human limbal epithelium. Periostin localization was well matched with nuclear factor p63, but not with corneal epithelial differentiation marker Keratin 3. Periostin transcripts was also highly expressed in limbal than corneal epithelium. In primary HLECs, periostin expression at mRNA and protein levels was significantly higher in 50% and 70% confluent cultures at exponential growth stage than in 100% confluent cultures at slow growth or quiescent condition. This expression pattern was similar to other stem/progenitor cell markers (p63, integrin β1 and TCF4). Periostin expression at transcripts, protein and immunoreactivity levels increased significantly during epithelial regeneration in wound healing process, especially in 16-24 hours at wound edge, which was accompanied by similar upregulation and activation of p63, integrin β1 and TCF4. Our findings demonstrated that periostin is exclusively produced by limbal basal epithelium and co-localized with p63, where limbal stem cells reside. Periostin promotes HLEC proliferation and regeneration with accompanied activation of stem/progenitor cell markers p63, integrin β1 and TCF4, suggesting its novel role in maintaining the phenotype and functional properties of LSC.
[Show abstract][Hide abstract] ABSTRACT: There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.
[Show abstract][Hide abstract] ABSTRACT: To examine the α-Gal gene expression and distribution in the different species/genus and developing phase animal ocular surface tissue.
α-Gal binding assay were carried out on various animal eye sections. Photograph, slit-lamp observation on various eye showed normal corneal transparence.
A strong α-Gal expression in invertebrates and some vertebrates ocular tissue, but no α-Gal binding in birds, fish and mammal. α-Gal expression change in the development of mice ocular surface tissue (except sclera) and display genus dependency in the different murine ocular surface tissue.
This study identified specific α-Gal epitopes binding area in the ocular surface of several species and may solve the problem that naive ocular surface may be used as natural α-Gal gene knockout model/high risk immunologic rejection model or ocular surface scaffold material.
[Show abstract][Hide abstract] ABSTRACT: There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4) in a 35-mm dish (9.6 cm(2)) grew to confluence (about 1.87-2.41 × 10(6) cells) in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.
[Show abstract][Hide abstract] ABSTRACT: TCF4, a key transcription factor of Wnt signaling system, has been recently found to be essential for maintaining stem cells. However, its signaling pathway is not well elucidated. This study was to explore the functional roles and signaling pathway of TCF4 in maintaining adult stem cell properties using human corneal epithelial stem cells as a model. With immunofluorescent staining and real-time polymerase chain reaction, we observed that TCF4 was exclusively expressed in the basal layer of human limbal epithelium where corneal epithelial stem cells reside. TCF4 was found to be well colocalized with ABCG2 and p63, two recognized epithelial stem/progenitor cell markers. Using in vitro culture models of primary human corneal epithelial cells, we revealed that TCF4 mRNA and protein were upregulated by cells in exponential growth stage, and RNA interference by small interfering RNA-TCF4 (10-50 nM) transfection blocked TCF4 signaling and suppressed cell proliferation as measured by WST-1 assay. TCF4 silence was found to be accompanied by downregulated proliferation-associated factors p63 and survivin, as well as upregulated cyclin-dependent kinase inhibitor 1C (p57). By creating a wound healing model in vitro, we identified upregulation and activation of β-catenin/TCF4 with their protein translocation from cytoplasm to nuclei, as evaluated by reverse transcription-quantitative real-time polymerase chain reaction, immunostaining, and Western blotting. Upregulated p63/survivin and downregulated p57 were further identified to be TCF4 downstream molecules that promote cell migration and proliferation in wound healing process. These findings demonstrate that transcription factor TCF4 plays an important role in determining or maintaining the phenotype and functional properties of human corneal epithelial stem cells.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the clinical effect and complications of two different filling materials (aerocyst urethral catheter and expansion sponges) applying in external dacryocystorhinostomy (EXT-DCR) and compare their advantages and disadvantages.
A retrospective study was made in the period from April, 1 2000 to April, 1 2005. Totally 180 patients (240 eyes) underwent the EX-DCR using different filling materials and divided into three groups randomly: negative control groups (group 1), expansion sponges group (group 2) and aerocyst urethral catheter group (group 3). The gender, etiology, clinical findings, surgical technique, filling materials, the condition of ocular surface and complications were analyzed. Filling materials were removed during day 7. Postoperative success was determined by lacrimal patency to irrigation, a positive dye test, hemorrhage and errhysis conditions after extubation and subjective resolution of epiphora and liquor puris.
During a mean follow-up of 5.14±1.69 years, the success rate were 73.7% (group 1), 86.5% (group 2), 98.7% (group 3) in three groups. There was significant statistical difference among three groups in the surgical success rate and the operative complications (including hemorrhage, errhysis, periorbital ecchymosis after extubation)(P<0.05).
EXT-DCR with aerocyst urethral cathete intraoperatively have higher success rate, fewer operative complications and a high patient satisfaction ,and can be used to simplify and speed up traditional EXT-DCR.
[Show abstract][Hide abstract] ABSTRACT: Allergic diseases affect a large population. Pollen, an ubiquitous allergen, is the trigger of seasonal rhinitis, conjunctivitis, and asthma, as well as an exacerbating factor of atopic dermatitis. However, the underlying mechanism by which pollen induces thymic stromal lymphopoietin (TSLP)-triggered allergic inflammation through epithelial innate immunity is largely unknown.
We sought to explore whether short ragweed (SRW) pollen induces TSLP/OX40 ligand (OX40L)/OX40 signaling through Toll-like receptor (TLR) 4-dependent pathways in patients with allergic disease.
Three models were used for this study, a well-characterized murine model of allergic conjunctivitis induced by SRW pollen, a topical challenge model on the murine ocular surface, and a culture model of primary human corneal epithelium exposed to aqueous extract of defatted SRW pollen (SRWe).
The topical challenges with SRW pollen generated typical allergic conjunctivitis in BALB/c mice. Clinical signs, stimulated TSLP/OX40L/OX40 signaling, and T(H)2 cytokine levels in the ocular mucosa and draining cervical lymph nodes were significantly reduced or eliminated in TLR4-deficient (Tlr4-d) or myeloid differentiation primary response gene 88 (MyD88) knockout (MyD88(-/-)) mice compared with those seen in their wild-type littermates. SRWe stimulated TSLP production by ocular epithelia in wild-type but not Tlr4-d or MyD88(-/-) mice. SRWe-stimulated TSLP was blocked by TLR4 antibody and nuclear factor κB inhibitor in murine and human corneal epithelia.
For the first time, we have shown that SRW pollen, acting as a functional TLR4 agonist, initiates TLR4-dependent TSLP/OX40L/OX40 signaling, which triggers T(H)2-dominant allergic inflammation. These findings shed light on the understanding of mucosal epithelial innate immunity and create new therapeutic targets to cure allergic diseases.
Full-text · Article · Aug 2011 · The Journal of allergy and clinical immunology
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: To evaluate the expression of Pax6 in pterygia epithelium. METHODS: Fifteen patients (15 eyes) with pterygium who underwent simple excision were enrolled in this study. Pax6, K10, K19 and MUC5AC immunostaining were performed in pterygia tissue and normal conjunctiva. RESULTS: A decline or absence of Pax6 expression and K19 keratin and MUC5AC but increased of K10 expression with epidermal differentiation was observed in pterygia epithelium, in comparison with the normal conjunctival tissue. CONCLUSIONS: These results indicate that down-regulation of Pax6 is associated with abnormal differentiation of the epithelial cells in pterygium.
No preview · Article · Aug 2010 · Yan ke xue bao = Eye science / "Yan ke xue bao" bian ji bu