Differential rapid adhesion of bovine ocular surface epithelial cells to laminin isoforms.

PURPOSE. To examine the ability of bovine corneal and conjunctival epithelial cells to adhere to different types of exogenous laminin preparations. METHODS. The ability of bovine corneal and conjunctival epithelial cells in primary culture to attach to laminin isolated from human placenta or from mouse EHS tumor was measured using a short-term colorimetric adhesion assay. Focal adhesion formation in response to interaction with laminins was determined by immunofluorescence microscopy using antibodies to vinculin and morphometric analysis. The influence of laminin on the secretion of adhesion complex proteins by bovine corneal epithelial cells in culture was analyzed using immunofluorescence microscopy. RESULTS. In short-term assays, primary bovine corneal epithelial cells demonstrate rapid and efficient adhesion to placental laminin, and significantly more cells contain focal adhesions, compared to those incubated on EHS laminin. In contrast, primary bovine conjunctival epithelial cells adhere equally well to placental and EHS laminin over a range of substrate concentrations. Additionally, the percentage of cells containing focal adhesions is not significantly different. In primary bovine corneal epithelium, the deposition of collagen type IV and collagen type VII into extracellular network-like structures is inhibited in cells cultured on placental laminin compared to cells cultured on EHS laminin. CONCLUSIONS. In vitro, bovine corneal epithelial cells attach more rapidly and efficiently to exogenous placental laminin compared to EHS laminin. However, this isoform inhibits the ready formation of adhesion complex-like structures in culture. The laminin isoform found in human placental preparations may therefore modulate corneal epithelial cell motility as opposed to permanent adhesion.     

Evidence for differential signaling in human conjunctival epithelial cells adherent to laminin isoforms

Both the laminin composition of the basement membrane and the keratin intermediate filament composition of the epithelial cell differs between cornea and conjunctiva, suggesting that at least some aspects of ocular surface epithelial cell differentiation may be regulated by extracellular matrix. The purpose of this study was to analyse the role of beta1 integrin in intracellular signaling pathways in human conjunctival epithelial cells adherent to laminin. In addition, the purpose was to compare the phosphorylation kinetics of signaling intermediates in cells adherent to different laminin isoforms. Cell adhesion assays, integrin clustering experiments, and integrin function blocking experiments demonstrated that beta1 but not beta4 integrin mediated human conjunctival epithelial cell adhesion to placental laminin isoforms (laminin-10/11) and induced focal adhesion kinase (FAK) tyrosine phosphorylation. Western blot analysis of cell lysates adherent to placental laminin showed that the tyrosine phosphorylation of p130Cas and FAK was maximally above constitutive levels after 60 min. In cells adherent to EHS laminin (laminin-1), the tyrosine phosphorylation kinetics of tensin, p130Cas, FAK and unknown proteins of 138 kDa and 110 kDa were similar, and peaked above constitutive levels after 30 min. Tyrosine phosphorylation of a 70 kDa protein was induced by cell adhesion to EHS laminin after 5 min, and phosphorylation peaked at 15 min. In contrast, the tyrosine phosphorylation of the 70 kDa protein was undetected in cells adherent to placental laminin. Erk-1 phosphorylation and activation was not differentially modulated by conjunctival epithelial cell adhesion to laminins. However, phosphorylation and activation kinetics of Erk-2 in cells adherent to placental laminin was similar to that observed for FAK and p130Cas. Erk-2 phosphorylation and activation was essentially undetectable in cells adherent to EHS laminin. These observations suggest that human conjunctival epithelial cell adhesion to different laminin isoforms activates different intracellular signaling pathways, and provides support for the hypothesis that extracellular matrix molecules can modulate ocular surface epithelial cell differentiation via alternate signaling pathways.     

Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha(2), alpha(3), alpha(6), beta(1)and beta(4)subunits, integrins alpha(6)and beta(4)being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha 3, beta 3 and gamma 2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in colocalization with beta(4)integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin beta(1)subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin alpha-subunits inhibited the adhesion. Integrin alpha(3)beta(1)complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex alpha(3)beta(1), as well as laminin alpha(3)chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha(3)beta(1)complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin alpha subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes.     

The functions of exogenous and endogenous laminin-5 on corneal epithelial cells

Recent evidence suggests that the basement membrane not only separates basal cells from Bowman's layer, but also has a crucial role in the proliferation, differentiation and migration of corneal epithelial cells. The basement membrane is composed of a mixture of matrix components including collagens, laminins and heparan sulfate proteoglycans. In these extracellular matrixes, laminin is a major component of the basement membrane. Of 11 laminin isoformes, laminin-5 is a variant, composed of three nonidentical subunits alpha3, beta3, gamma2 and is a major component of the corneal basement membrane. However, little is known about the interactions of laminin-5 with corneal epithelial cells. In this study, we investigated the functions of laminin-5 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-5 on HCE cells. Laminin-5 is synthesized initially as a 490 kDa molecule that undergoes specific processing to cleavaged isoforms after being secreted. The alpha3 subunit is processed from 200-190 kDa to 160 kDa/145 kDa. The gamma2 subunit is processed from 150 kDa to 105 kDa/80 kDa. The beta3 subunit (140 kDa) is not processed. Exogenously added laminin-5 (soluble form) in this study was purified from a serum-free, conditioned medium of a human gastric carcinoma cell line STKM-I. This soluble laminin is a processed isoform containing alpha3 (160 kDa), beta3 (140 kDa) and gamma2 (105 kDa) chains. On the other hand, immunocytochemical analysis showed that HCE cells themselves secreted laminin-5 endogenously. Western blotting analysis revealed that HCE cells initially produced unprocessed isoform containing 190 kDa alpha3, 140 kDa beta3 and 150 kDa gamma2 chains and that after being secreted, the alpha3 chain was processed to 160 kDa/145 kDa and the gamma2 chain was processed to 105 kDa.Initially we investigated the functions of exogenous (processed) laminin-5 on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion via alpha3beta1 integrin, cell spreading, assembly of hemidesmosomes and mildly inhibited cell migration. Next we estimated the effect of endogenous (unprocessed) laminin-5 on HCE cells. Using an anti laminin-5 monoclonal antibody (mAb) or anti integrin alpha3beta1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin alpha3beta1 and alpha6beta4 were expressed in HCE cells. These integrins are receptors of laminin-5. But, anti integrin alpha6beta4 mAbs did not have any blocking ability against cell migration. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via alpha3beta1 integrin.In conclusion, structural differences between exogenous (processed) and endogenous (unprocessed) laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration. In the future, processed soluble laminin-5 could be a useful drug for the prevention of recurrent corneal erosion, and unprocessed soluble laminin-5 could be applied for the treatment of prolonged corneal epithelial defects.     

整合素在真菌与角膜上皮细胞黏附过程中的表达

目的 探讨不同真菌菌种与人角膜上皮细胞黏附过程中整合素的表达及其作用机制.方法 实验研究.体外培养人角膜上皮细胞系,建立茄病镰刀菌(CGMCC 3.1829)和烟曲霉菌(CGMCC 3.0772)与角膜上皮细胞黏附的体外模型.茄病镰刀菌或烟曲霉菌与人角膜上皮细胞共孵育后,用无菌的磷酸盐缓冲溶液冲洗掉未黏附的真菌.提取细胞的总RNA,反转录为cDNA后采用实时荧光定量聚合酶链反应(RT-PCR)检测不同时间点上不同真菌与角膜上皮细胞黏附的14种整合素分子mRNA水平的表达.各时间点之间基因表达差异的比较采用单因素方差分析.结果 在烟曲霉菌与人角膜上皮细胞黏附的过程中,随黏附时间延长,整合素家族白细胞黏附受体组成员编码基因整合素αL(ITGAL)、整合素α型(ITGAM)、整合素αX(ITGAX)及整合素β2(ITGB2)的表达显著上调.其中ITGAL的表达最高上调2倍(F=29.39,P<0.01),ITGAM的表达最高上调4倍(F=20.26,P<0.01),ITGAX的表达最高上调2.5倍(F=2.51,P<0.05),ITGB2的表达最高上调3.4倍(F=3.923,P<0.05).而在茄病镰刀菌与人角膜上皮细胞黏附的过程中,此14种整合素的表达未见显著差异.结论 整合素家族白细胞黏附受体组(β2组)成员αLβ2、αMβ2及αXβ2均参与烟曲霉菌与角膜上皮细胞的黏附;未见茄病镰刀菌与角膜上皮细胞的黏附过程中整合素表达的差异.整合素介导的黏附因菌种而异.     

Production of fibronectin and tenascin isoforms and their role in the adhesion of human immortalized corneal epithelial cells

PURPOSE: To study the production and deposition of fibronectin (Fn) isoforms and tenascin-C (Tn-C) by immortalized human corneal epithelial (HCE) cells and their integrin-dependent adhesion characteristics. METHODS: Indirect immunofluorescence with isoform-specific monoclonal antibodies (mAbs) was used to study extracellular matrix (ECM) protein composition and their integrin receptors in HCE cells. The synthesis of proteins was studied by Western blot analysis and adhesion by quantitative adhesion assay. RESULTS: HCE cells deposited fibrillar matrix containing extradomain EDA-Fn and sparser deposits of Onc-Fn, whereas Tn-C was deposited diffusely. EDA-Fn was present both in ECM and in culture medium, whereas Tn-C was present only in ECM. Fn-binding integrin (Int) alpha(5) subunit was present in subconfluent cells in focal adhesions (FAs) and matrix adhesions, whereas Int alpha(v)beta(5) was present in FAs in sparse cultures and as ringlike structures in denser cultures. Int alpha(v)beta(6) was colocalized with Int alpha(5) in FAs, only in cells adhering to growth substratum coated with Fn or Fn/Tn-C. Ints alpha(5)beta(1) and alpha(v)beta(6) mediated adhesion to Fn and Int alpha(v)beta(5) mediated adhesion to Vn, and both were inhibited by RGD peptide. The cells failed to adhere to Tn-C but adhered to Fn/Tn-C and were then more efficiently inhibited by the function-blocking integrin mAbs and RGD peptide. CONCLUSIONS: The results suggest corneal epithelial cells as the possible source for Fn isoforms and Tn-C in wound healing and pathologic conditions. The presence of Tn-C only in ECM suggest a vectorial deposition and adhesion experiments also indicate a role for Tn-C in Fn functioning.     

Adhesion structures of amniotic membranes integrated into human corneas

PURPOSE: The aim of this study was to investigate the structural relationship between integrated amniotic membrane (AM) and corneal tissues in various integration patterns, focusing on adhesion structures along the interface. METHODS: Fourteen eyes of 14 patients (age, 65.8 +/- 13.5 years) underwent penetrating keratoplasty (PKP) 19.3 +/- 20.7 weeks after cryopreserved human AM transplantation (AMT). The corneal buttons (after PKP) and the corresponding original AM (before AMT) were examined with the use of transmission electron microscopy (TEM) and immunohistochemistry for integrin beta4, type VII collagen, and laminin. Main outcome measures included thickness of the corneal epithelium and AM, density of the epithelial desmosomes and hemidesmosomes, continuity, and thickness of the epithelial basement membrane. RESULTS: Integrated AM was found by slit lamp in only 2 of 14 patients, but histology and TEM revealed AM integration in 11 of 14 patients up to 77 weeks after AMT. No amniotic epithelial cell was detected in any cornea with integrated AM stroma. Three basic patterns of integration could be described: subepithelial, intraepithelial, and intrastromal. Hemidesmosomes anchored the corneal epithelial cells to the AM at a density up to 165.3 +/- 22.9 per 100 microm cell membrane length. Discontinuous basement membrane segments 17.2 +/- 4.9 nm thick could be detected. Desmosomes among recovered corneal epithelial cells were found at a density of 21.2 +/- 5.3 per 10 microm cell membrane length. CONCLUSIONS: The AM stroma can integrate into the host corneal tissue. Integration is associated with the formation of adhesion structures such as hemidesmosomes and desmosomes, which provide anchoring and stability of the regenerating corneal epithelium. The presence of integrated AM and adhesion structures with host corneal tissue supports the clinical experience obtained with AMT in ocular surface disease.     

The lack of extracellular laminin beta2 chain deposition correlates to the loss of conjunctival epithelial keratin K4 localization in culture

PURPOSE: To examine the effects of external modulation of epithelial-mesenchymal interaction on conjunctival epithelial cell differentiation characteristics. METHODS: Keratin K4 and laminin beta2 chain protein localization was examined in an organotypic model which facilitates the comparison of differentiation characteristics of conjunctival epithelium interacting with conjunctival basement membrane or corneal basement membrane. In addition, keratin K4 and laminin beta2 chain localization was examined in primary cultures of conjunctival epithelial cells and fibroblasts. The synthesis and secretion of laminin beta2 chain by conjunctival fibroblasts in culture was determined by western blot analysis and immunoprecipitation. The ability of conjunctival epithelium to respond to exogenous laminin beta2 chain was assayed by culturing epithelial cells on a laminin matrix isolated from human placenta. RESULTS: In culture, conjunctival fibroblasts synthesize and secrete laminin beta2 chain but do not deposit this chain into an extracellular matrix substrate or basement membrane-like structure. The lack of extracellular deposition of this chain correlates to the gradual loss of keratin K4 protein in conjunctival epithelial cell culture. Conjunctival epithelium remains responsive to laminin beta2 chain in vitro because keratin K4 localization can be rescued in these cells by culture on a substrate of exogenous placental laminin. CONCLUSIONS: In vitro, alterations in native conjunctival epithelial-mesenchymal interactions results in aberrant basement membrane laminin isoform composition. This, in turn, leads to the loss of adult epithelial cell phenotype characteristics, suggesting that at least some aspects of conjunctival epithelial cell differentiation are regulated by the extracellular matrix.     

Propagation and phenotypic preservation of rabbit limbal epithelial cells on amniotic membrane

PURPOSE: To describe the phenotypic characteristics of a limbal epithelial cell sheet outgrowth from a limbal explant cultured on amniotic membrane. METHOD: Immunofluorescent staining and confocal microscopy were used to examine the expressions of p63, Ki-67, keratins 3 and 14, connexin 43, and the integrin alpha6/beta4 and alpha3/beta1 subunits in corneal and limbal tissues in a limbal explant and epithelial outgrowth cultured for 2 weeks on amniotic membrane. RESULTS: The expression patterns of p63, Ki-67, keratins, integrins, and connexin 43 in a limbal explant with an epithelial outgrowth cultured for 2 weeks on amniotic membrane resembled those in freshly prepared limbus. Moreover, the distribution of integrin subunits in positive cells of the limbal explant and its epithelial outgrowth was similar to that of the corneal epithelial cells during wound repair. CONCLUSIONS: The epithelial cell sheet grown from a limbal explant on amniotic membrane exhibited a phenotype similar to that of the limbus, suggesting that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells.     

Modulation of corneal epithelial cell functions by the neutrophil-derived inflammatory mediator CAP37

PURPOSE: To investigate the effect of CAP37, an inflammatory mediator in neutrophils, on three important events in corneal wound healing: proliferation, migration, and adhesion. METHODS: Immortalized human corneal epithelial cells (HCEC) were treated with CAP37, and its effects on migration and proliferation were measured using the modified Boyden chemotaxis chamber and the proliferation assays (CyQUANT; Molecular Probes, Eugene, OR), respectively. Effects on adhesion were determined by measuring upregulation of adhesion molecules belonging to the selectin, integrin, and immunoglobulin superfamily using RT-PCR and flow cytometry. RESULTS: CAP37 promoted proliferation of HCEC in a time- and dose-dependent fashion. CAP37 was maximally chemotactic for HCEC over a range of 1.3 x 10(-8) to 5.2 x 10(-8) M. CAP37 upregulated intercellular adhesion molecule (ICAM)-1, platelet endothelial cell adhesion molecule (PECAM)-1, and integrin molecules alpha3 (CD49c) and beta1 (CD29). Data on migration and ICAM-1 and PECAM-1 upregulation were corroborated using primary human corneal epithelial cells. CONCLUSIONS: CAP37 modulated corneal epithelial cell proliferation and migration and upregulated adhesion molecules involved in leukocyte-epithelial and epithelial-extracellular matrix interactions.     

Plasminogen activator inhibitor-1 (PAI-1) stimulates human corneal epithelial cell adhesion and migration in vitro

In addition to its role as an inhibitor of urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) is hypothesized to regulate epithelial cell adhesion and migration. We have previously reported that PAI-1 may be an important regulatory factor of the uPA system in cornea. The purpose of this study was to extend those observations by determining the effect of exogenous PAI-1 on the migration and adhesion of human corneal epithelial cells (HCEC) in vitro. The expression of PAI-1 in non-transformed early passage HCEC was confirmed by immunofluorescence microscopy and Western blot analysis. Colorimetric assays coupled with function-inhibiting antibody studies using the matrix assembled in situ by cultured cells demonstrate that immobilized PAI-1 serves as an efficient substrate for HCEC adhesion. HCEC attachment to PAI-1 is comparable to that of laminin-10, a known strong adhesion protein for epithelial cells. In addition to serving as an adhesion substrate, PAI-1 also functions as a chemotactic agent for corneal epithelium. Additionally it promotes the random migration of HCEC, from an initial cell cluster, along a culture substrate. Our results in corneal epithelium are consistent with reports from other investigators showing that PAI-1 facilitates both epithelial adhesion and migration. From our studies we conclude that PAI-1 may play a dual role in corneal wound healing. Initially PAI-1 may function to stimulate migration and facilitate the reepithelialization of the wound bed. Post-reepithelization, PAI-1 may ensure corneal epithelial cell adhesion to matrix to promote successful wound healing.     

Cooperation of isoforms of laminin-332 and tenascin-CL during early adhesion and spreading of immortalized human corneal epithelial cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. We have studied the early adhesion process of immortalized human corneal epithelial (HCE) cells and show by field emission scanning electron microscopy (FESEM) that the cells first adhere via foot-like process to the growth substratum and later present lamellar spreading. During early adhesion indirect immunofluorescence showed that the cells codeposited laminin (Lm) -332 and the large subunit of tenascin-C (Tn-CL) as a demarcated plaque beneath the cells. Instead, unprocessed Lm-332 (alpha 3'32) was found in a wider area in cells showing lamellar spreading and was also prominently expressed in the cytoplasm of the migrating marginal cells in the in vitro wounded HCE cultures. Confocal laser scanning microscopy (CLSM) showed that the Golgi apparatus was located to the vicinity of the Lm-332/Tn-CL-containing adhesion plaque and accordingly treatment of the cells with demecolcine, dispersing the Golgi apparatus, prevented the formation of plaques. This suggests that formation of the adhesion plaque depends on a direct vectorial secretion of Lm-332 and Tn-CL to the culture substratum. Instead, cytochalasin B treatment disrupted microfilaments and arborized the cells but did not affect the deposition of Tn-CL/Lm-332 as a plaque beneath the cells. The suggestion was supported by immunoprecipitation experiments which showed that Tn-CL and Lm alpha 3' chain were found in cell-free matrices on the culture substratum of spreading cells but not at all (Tn-CL) or much less (Lm-332) in the culture medium. Quantitative cell adhesion experiments showed that HCE cells did not adhere to plain Tn-C coat and that integrin (Int) alpha(3)beta(1) mediated the adhesion of HCE cells to purified Lm-332 and to Lm-332/Tn-C while Int beta4 did not mediate adhesion to these proteins. Taken together, our data suggest that Lm-332 and Tn-CL cooperate in early adhesion process of HCE cells. Furthermore, the results show that Lm-3'32 isoform functions in the spreading of the cells beyond the early adhesion stage and appears to emerge into HCE cells starting to migrate in experimental wounds.     

Insulin-like growth factor-1 induces migration and expression of laminin-5 in cultured human corneal epithelial cells

PURPOSE: The effects of insulin-like growth factor (IGF)-1 on laminin (Ln)-5 and the associated integrins during in vitro HCEC migration were examined. Also investigated were the effects of IGF-1 on the migration of human corneal epithelial cells (HCECs). METHODS: HCEC migration was examined by wound-healing and chemoattraction assays. For migration inhibition assays, HCECs were pretreated with inhibitors of the IGF-1 receptor (alphaIR3), the PI3-K/AKT pathway (LY294002), and the MEK-ERK pathway (PD98059). The expression levels of Ln-5 and fibronectin (Fn) were determined by Western blot analysis, and the expression levels of the beta1 and alpha3 integrins were determined by confocal microscopy and Western blot analysis. The migration inhibition with anti-integrin alpha3 and beta1 antibodies was also determined. RESULTS: HCEC migration was significantly increased in the presence of IGF-1 and Ln-5. IGF-1 enhanced the production of Ln-5 in both a dose- and time-dependent manner, and this upregulation was blocked by pretreatment with alphaIR3 or LY294002. IGF-1 treatment upregulated expression of beta1 integrin, but not alpha3 integrin. The HCEC migration facilitated by IGF-1 was inhibited with the anti-integrin antibody for beta1. However, there was no cross-talk between Ln-5 and integrin beta1 production. CONCLUSIONS: The results reveal that IGF-1 induces HCEC migration through the independent production of Ln-5 and beta1 integrin, which are directed at least in part by activation of the PI3-K/AKT pathway, but are not affected by the MEK-ERK pathway.     

Internalization is essential for the antiapoptotic effects of exogenous thymosin beta-4 on human corneal epithelial cells

PURPOSE: Exogenous thymosin beta-4 (Tbeta(4)) has been shown to inhibit the apoptosis in nontransformed human corneal epithelial cells that is triggered by ethanol. The purpose of this study is to examine whether exogenous Tbeta(4) protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H(2)O(2)) and to elucidate its mechanism of action. METHODS: HCE-T cells were incubated without or with the recombinant histidine-tagged Tbeta(4) produced by Escherichia coli before the addition of FasL or H(2)O(2). Cell viability was determined by MTT or MTS assay, and activation of caspase-8, -9, and -3 was examined by colorimetric and fluorescent substrate cleavage assays. The internalization of exogenous Tbeta(4) in HCE-T cells was analyzed by immunofluorescence staining. Cytochalasin D, an actin depolymerization agent, was added to examine whether the actin cytoskeleton is involved in Tbeta(4) entry and whether the internalization of this peptide is crucial for its cytoprotection. RESULTS: The death of HCE-T cells induced by both FasL and H(2)O(2) was dramatically reduced by the recombinant Tbeta(4) pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H(2)O(2)-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tbeta(4). Of note, internalization of this G-actin-sequestering peptide into HCE-T cells was found to be essential in cell death prevention, in that disruption of the cellular entry of Tbeta(4) by cytochalasin D abrogated its cytoprotective effects. CONCLUSIONS: This is the first report to demonstrate that the internalization of exogenous Tbeta(4) is essential for its antiapoptotic activity in human corneal epithelial cells.     

Prolonged exposure to high glucose impaired cellular behavior of normal human corneal epithelial cells

PURPOSE: To determine the effect of prolonged exposure to high glucose on cellular behavior of normal human corneal epithelial cells (HCEC). METHODS: HCEC were cultured in medium under normal or high glucose conditions for 14 days. Proliferation was evaluated by direct cell counting and [(3)H]thymidine incorporation. Cell cycle analysis was performed using flow cytometry. The ability of HCEC to attach to type I collagen was evaluated using a short-term colorimetric adhesion assay. The effect of high glucose on the expression of integrin alpha(3)beta(1) was also evaluated using flow cytometry. RESULTS: Cell number and [(3)H]thymidine incorporation under high glucose conditions decreased compared with those under normal glucose conditions. The cells exposed to high glucose were G(0)/G(1) than untreated cells. The adhesion ability of HCEC under high glucose conditions decreased compared to normal glucose conditions. Expression of integrin alpha( 3)beta(1) was down-regulated under high glucose conditions. CONCLUSIONS: High glucose had deleterious effects on cellular behavior of HCEC, which might cause delayed corneal epithelial wound healing in diabetic keratopathy.     

Human corneal epithelial cells express functional PAR-1 and PAR-2

PURPOSE: The objective of this study was to examine whether HCECs express functional proteinase-activated receptor (PAR)-1 and -2 and evaluate the effects of receptor activation on corneal epithelial cell proinflammatory cytokine production. METHODS: Expression of PAR-1 and -2 mRNAs was determined by RT-PCR in cultured primary human corneal epithelial cells (HCECs) and the human corneal epithelial cell line HCE-T. Localization of PAR-1 and -2 in whole normal human corneas was determined by immunofluorescence with PAR-1 and -2 antibodies. The functional competence of PAR-1 and -2 in corneal epithelial cells was assessed by measuring the rapid induction of intracellular [Ca(2+)] in response to thrombin, trypsin, and specific receptor-activating peptides derived from the tethered ligands of the PAR receptors. HCE-T expression of cytokines (IL-6, IL-8, and TNFalpha) in response to activation of PAR-1 and -2 was measured by quantitative RT-PCR and ELISA. RESULTS: Functional PAR-1 and -2 were expressed in both HCECs and HCE-T cells. Immunoreactivity for PAR-1 and -2 was detected in the outer epithelial layer of the cornea in whole human corneal sections. Activation of PAR-1 and -2 led to upregulation in HCE-T cells of both expression of mRNA and secretion of the proinflammatory cytokines IL-6, IL-8, and TNFalpha. CONCLUSIONS: The results show for the first time that functional PAR-1 and -2 are present in human cornea. Activation of these receptors results in the production of various corneal epithelial cell proinflammatory cytokines. These observations indicate that PAR-1 and -2 may play an important role in modulating corneal inflammatory and wound-healing responses. These receptors may be useful therapeutic targets in several corneal disease processes.     

人角膜上皮干细胞的识别

目的 探讨人角膜上皮干细胞的分子标记。方法 对人角膜和角膜缘部位行组织学检查以分析角膜缘解剖结构。对人角膜切片和整个角膜组织行免疫组织化学染色以检测中央角膜和角膜缘部位未分化标记,如核蛋白p63、乳腺癌抵抗蛋白（ABCG2,BCRP1）、烯醇化酶α、整合素拍、胡及β1、表皮生长因子受体（EGFR）、细胞角蛋白19（CK19）、14（CK14）及转铁蛋白受体（CDT1）的表达,经荧光显微镜和激光扫描共焦显微镜观察。对角膜中部和角膜缘上皮细胞的mRNA进行半定量逆转录聚合酶链反应（RT—PCR）和原位杂交以检测其相关基因的表达。结果 角膜缘部位横向切片显示角膜缘上皮细胞为乳头放射状排列,对应于Vogt栅栏环境。未分化标记整合素β1、EGFR、烯醇化酶α及CK19在角膜缘基底细胞胞质染色较表层细胞更强;p63、ABCG2、整合素胡蛋白仅见于角膜缘基底部上皮细胞。激光扫描共焦显微镜观察和RT—PCR结果显示角膜缘表达p63、ABCG2、整合素胡蛋白及mRNA。原位杂交显示p63仅表达于角膜缘基底层细胞。结论 角膜缘上皮呈乳头放射状排列,角膜缘干细胞群具有复合标记：p63表达于细胞核、ABCG2表达于胞质、整合素胡表达于胞膜。采用这些标记复合体,可将角膜缘干细胞群与其他上皮细胞区分。     

Laminin-5 is a component of preserved amniotic membrane

PURPOSE: To determine if laminin-5 is retained in the matrix of cryopreserved human amniotic membrane tissue prepared for ocular surgeries. METHODS: Amniotic membrane was solubilized in urea/SDS buffer. Constituent proteins were resolved by SDS-PAGE and laminin-5 content was determined by Western blot analysis using a panel of antibodies directed against the alpha3, beta3 or gamma2 chains of the molecule. Human corneal epithelial cells were seeded on amniotic membrane and cultured in the presence or absence of EGF. The cell-membrane construct was examined for laminin-5 content using Western blot analysis and immunofluorescence microscopy. RESULTS: In preserved amniotic membrane the laminin-5 alpha3 chain is present in both the unprocessed (190-kDa) and processed (160-kDa) forms. The beta3 chain is found in the 145-kDa form. The gamma2 chain appears to be predominantly in the processed (105-kDa) form. Very little of the unprocessed form of the gamma2 chain (155-kDa) could be detected using immunoblot analysis. A similar distribution of laminin-5 was also present in extracts of corneal epithelial cells cultured on amniotic membrane. Immunofluorescence analysis of cells cultured on the membrane demonstrated polarization of laminin-5 at the cell-membrane interface. CONCLUSIONS: The presence of both the unprocessed and processed forms of laminin-5 alpha3 and gamma2 chains in preserved human amniotic membrane suggests that when used as a substrate in ocular surgeries, this membrane may be capable of promoting corneal epithelial cell motility and adhesion. Regulation of the motile or adhesive function may lie with factors secreted by the corneal epithelium that populates the membrane following surgery.