Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line.

PURPOSE: To present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line. METHODS: iHCjECs were developed to induce Simian Virus 40 large T-antigen (SV40LT) by incorporating lentivirus in a tetracycline (Tet)-regulated gene-expression system into primary cultures of human conjunctival epithelial cells. The population doubling time and morphology of the iHCjECs were analyzed. The expressions of CK13, CK19, CK12, and MUC1, MUC4, MUC16, and MUC5AC were determined by real time PCR and immunohistochemically under different culture conditions. The organotypic culture model in which iHCjECs were cultured on rabbit conjunctival fibroblast-embedded collagen gel was used to characterize the iHCjECs. RESULTS: The iHCjECs cultured with doxycycline (Dox) continued to proliferate for at least 20 passages and had a cobblestone-like appearance. The expressions of CK13 and CK19 but not CK12 were detected in the iHCjECs, and the expression of CK13 increased in culture media lacking Dox (Dox-). The expressions of MUC1, MUC4, MUC16, and MUC5AC were detected in iHCjECs, and a relatively strong immunostaining of MUC5AC was detected with Dox(-) added 5% FBS. Stratified iHCjECs were observed in organotypic culture at 5 days. CONCLUSION: The iHCjECs had high proliferation rates and abilities to control the differentiation potency to control the expression of SV40 LT-antigen with Tet-regulated gene-expression system. They are able to express the mucin gene repertoire of their native epithelia. The iHCjECs can be a useful experimental cell line to study conjunctival epithelial cell characteristics and for pathophysiological and toxicological studies.

In Vivo Confocal Microscopic Observations of Vortex Keratopathy in Patients with Amiodarone-Induced Keratopathy and Fabry Disease.

PURPOSE: To compare the morphology of two types of vortex keratopathy: amiodarone-induced keratopathy and the Fabry disease-associated keratopathy. PATIENTS AND METHODS: Eight patients who were receiving oral amiodarone therapy and 3 patients with Fabry disease, a mother and her 2 daughters, were examined by slit-lamp biomicroscopy and in vivo confocal microscopy (IVCM) regularly. RESULTS: Amiodarone-induced keratopathy developed in 7 of the 8 patients, and it was detected as early as 7 days by IVCM and 14 days by slit-lamp biomicroscopy. The in vivo confocal microscopic images showed a clustering of corneal epithelial cells with a highly reflective cytoplasm in both types of keratopathy. In the amiodarone-induced keratopathy, the highly reflective epithelial cells were first found at the center of the cornea and then spread to the periphery with increasing time on amiodarone. In Fabry disease, the highly reflective epithelial cells were consistently observed extending from the limbus to the central cornea. CONCLUSION: These findings suggest that the corneal epithelial cells most likely endocytose amiodarone from the tear film in the amiodarone-induced keratopathy. In Fabry disease, globotriaosylceramide deposits are taken up by the lysosomes of the limbal epithelial stem cells, and they differentiate and migrate to the center of the cornea to form the whorl pattern.

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

Purpose: We created a novel inducible mouse line Keratocan-rtTA (KeraRT) that allows specific genetic modification in corneal keratocytes and tenocytes during development and in adults. Methods: A gene-targeting vector (Kera- IRES2-rtTA3) was constructed and inserted right after the termination codon of the mouse Kera allele via gene targeting techniques. The resulting KeraRT mouse was crossed to tet-O-Hist1H2B-EGFP (TH2B-EGFP) to obtain KeraRT/TH2B-EGFP compound transgenic mice, in which cells expressing Kera are labeled with green fluorescence protein (GFP) by doxycycline (Dox) induction. The expression patterns of GFP and endogenous Kera were examined in KeraRT/TH2B-EGFP. Moreover, KeraRT was bred with tet-O-TGF-α to generate a double transgenic mouse, KeraRT/tet-O-TGF-α, to overexpress TGF-α in corneal keratocytes upon Dox induction. Results: Strong GFP-labeled cells were detected in corneal stroma, limbs, and tail when KeraRT/TH2B-EGFP mice were fed Dox chow. There was no GFP in any single transgenic KeraRT or TH2B-EGFP mouse. Histological analysis showed that GFP in the cornea was limited to stromal keratocytes of KeraRT/TH2B-EGFP, which is consistent with Kera expression. Induction of GFP occurred in 24 hours and reached a plateau by 7 days after Dox induction. GFP could be detected 3-months after induction of KeraRT/TH2B-EGFP. Ectopic expression of TGF-α in corneal keratocytes caused hyperplasia in the corneal epithelium and stroma. Conclusions: The novel Dox inducible KeraRT driver mouse line is a useful genetic tool for gene manipulation and elucidating gene functions in corneal stroma and tendons of limbs and tail during embryonic development, homeostasis and pathogenesis.

In vivo confocal microscopic observations of eyes diagnosed with posterior corneal vesicles.

PURPOSE: To determine the morphological characteristics of the cornea in eyes with posterior corneal vesicles (PCV) determined by in vivo confocal microscopy (IVCM). In addition, to compare the morphological characteristics of the corneas of eyes with PCV to those of eyes with posterior polymorphous corneal dystrophy (PPCD). METHODS: Three males and three females aged 17-75 years who were diagnosed with PCV by slit-lamp biomicroscopic findings were studied. The slit-lamp findings, specular microscopic images, corneal endothelial cell densities (CDs), central corneal thickness, and IVCM images were evaluated. Three cases that had been diagnosed with PPCD were also evaluated, and the findings were compared to those in eyes with PCV. RESULTS: All six cases of PCV had unilateral lesions of Descemet's membrane and the corneal endothelium. Slit-lamp biomicroscopy showed broad, band-shaped lesions in five cases and multiple vesicle lesions in one case of PCV. The IVCM images of the six cases had a common appearance of the corneal endothelial cells in the affected areas showing iso-reflective nuclei and low reflective cell membranes as seen in normal corneal endothelial cells. The CDs in the affected eyes were about one-half of those of the not affected eyes. The IVCM images of the corneas of patients with PPCD showed polymorphic cells with hyperreflective nuclei, which are characteristics of epithelial cells. CONCLUSIONS: The IVCM images demonstrated that the corneal endothelial cells of patients with PCV have normal morphology. These findings indicate that PCV is a distinct clinical entity different from PPCD.

Horizontal intracorneal swirling water migration indicative of corneal endothelial function.

PURPOSE: To test our hypothesis about whether there is water migration in the horizontal corneal plane and investigate its developmental mechanism. METHODS: A fluorescein solution was intrastromally injected into normal and edematous corneas of rabbits, and the movement of the fluorescein solution was observed and recorded over time. RESULTS: In normal corneas, the water flow was characterized by a swirling movement from the center to the periphery in the stroma. The fluorescein solution ultimately spread and occupied the entire cornea, indicating horizontal intracorneal swirling of water. In contrast, when the corneal endothelia were injured by intracameral injection of a preservative to create corneal edema, no water migration occurred, suggesting that the integrity of the corneal endothelial function is essential for water migration. The water migration stopped with injection of a sodium-potassium pump inhibitor, indicating that the enzyme is necessary for physiologic water migration in the cornea. With recovery of corneal endothelial function, the water migration began, and focal edema remained in the periphery with no water migration in this edematous area. CONCLUSIONS: We report for the first time the presence of horizontal water migration in the cornea in a swirling pattern (i.e., intracorneal swirling migration of water, generated by the pump function in the corneal endothelial cells), which may supplement the conventional concept of development of corneal edema in the vertical plane. This dynamic water circulatory system may be involved in increasing the efficiency of the water transfer in the entire cornea.

Pseudomonas aeruginosa MucD protease mediates keratitis by inhibiting neutrophil recruitment and promoting bacterial survival.

PURPOSE: Pseudomonas aeruginosa is a leading pathogen of blinding keratitis worldwide. In this study, the role of the serine protease in the pathogenesis of P. aeruginosa keratitis in the mouse cornea was investigated by comparing the parent and rescue strains. METHODS: Cornea of C57BL/6 mice were infected with P. aeruginosa strain PAO1, serine protease (MucD protease or PA3535) mutants (ΔmucD or ΔPA3535), or a complemented strain. Corneal virulence was evaluated by determining clinical scores and bacterial enumeration. A myeloperoxidase assay was performed to determine the number of polymorphonuclear (PMN) cells infiltrating the cornea. An ELISA was used to quantify inflammatory cytokines and chemokines in the cornea. RESULTS: The clinical score and bacterial numbers in eyes infected with ΔmucD were significantly lower than in those infected with PAO1, ΔPA3535, or the MucD rescue strain after 48 hours (P < 0.001). A larger number of infiltrating PMN cells was observed in eyes infected with ΔmucD at 12 and 24 hours, compared with eyes infected with PAO1. IL-1ß, KC, and MIP2 levels were higher in eyes infected with ΔmucD than in those infected with PAO1 after 12 hours. CONCLUSIONS: The MucD protease suppressed IL-1ß, KC, and MIP2 during the early stages of the infection and inhibited neutrophil recruitment in the cornea. Therefore, the MucD protease contributes significantly to the pathogenesis of keratitis. MucD protease plays a critical role in the establishment of Pseudomonas aeruginosa keratitis by facilitating evasion of the immune response.

The "replacement hypothesis": corneal stem cell origin epithelia are replaced by limbal stem cell origin epithelia in mouse cornea during maturation.

PURPOSE: Previous studies have shown that the stem cells of corneal epithelia are located at the limbal basal layer. Limbal stem cells are believed to be the source of corneal epithelial cell proliferation and differentiation. This study tested the replacement hypothesis, which suggests that corneal stem cell origin epithelia may be replaced by limbal stem cell origin epithelia after 2 weeks of age in mice. METHODS: The cytokeratin 12 expression pattern in the cornea was examined using K12 IRES-Cre and Cre reporter mice. RESULTS: Before 2 weeks of age, K12 expression in corneal epithelia showed a mosaic pattern. After 2 weeks of age, centripetal K12 IRES-Cre expression gradually elongated from the limbal area. Around 12 weeks of age, the mosaic expression pattern disappeared from the center of the cornea. Temporal and spatial observations of K12 IRES-Cre expression patterns suggested that the mosaic pattern cells proliferated and amassed at the same position from day 15.5 of the embryonic stage at the latest. CONCLUSIONS: Therefore, these cells were considered corneal stem cell origin epithelia. In contrast, centripetal pattern cell populations were considered limbal stem cell origin epithelia because they originated from the limbal area and moved to the center of the cornea. These observations suggest that corneal stem cell origin epithelia are replaced by limbal stem cell origin epithelia after 2 weeks of age in mice.

Effect of photodynamic therapy with methylene blue on Acanthamoeba in vitro.

PURPOSE: To evaluate the disinfectant effect of methylene blue (MB)-mediated photodynamic therapy (PDT) on a pathogenic strain of Acanthamoeba. METHODS: Acanthamoeba castellanii (ATCC 50370) used in this study were treated under one of four experimental conditions: light irradiation and incubation in MB (L+M+), light irradiation and incubation in physiologic solution (L+M-), incubation in MB only (L-M+), and incubation in physiologic solution (L-M-). M+ trophozoites were incubated in either 0.25 or 0.5 mM MB for 10 minutes. L+ organisms were irradiated for 30 minutes following incubation in solution. A halogen lamp (660 ± 10 nm) with a maximum output of 6 mW/cm(2) was used as the PDT light source. After treatment, antiacanthamoeba activity was evaluated by checking the respiratory activity of the amoeba with 5-cyano-2,3-tetrazolium chloride (CTC) staining. We also determined whether the effect of PDT with MB had been retained or augmented when it was performed in combination with conventional antiamoebic agents. RESULTS: MB-PDT suppressed the respiratory activity of trophozoites in an MB-concentration-dependent manner at total light doses of 10.8 J/cm(2). The respiratory activity of each group as a percentage of that of L-M- is as follows: L+M+ 11.6% (0.5 mM), 60.9% (0.25 mM); L-M+ 116.5% (0.5 mM), 105.5% (0.25 mM); L+M- 107.6%; and L-M- 106.3%. (L+M+ versus L-M- P < 0.05). MB-PDT had a synergistic effect when used in combination with polyhexamethylene biguanide (PHMB) or amphotericin B, but not with voriconazole. CONCLUSIONS: MB-PDT is effective against Acanthamoeba in vitro and has synergistic effects with PHMB and amphotericin B.

Involvement of stem cell factor and c-kit in corneal wound healing in mice.

PURPOSE: To study the roles played by stem cell factor (SCF) and SCF receptor c-kit in wound healing of corneal epithelial cells. METHODS: A 2 mm corneal epithelial wound was made in control (WBB6F1(+/+)), SCF (Sl/Sl(d))-, and c-kit (W/W(v)) mutant mice, and the speed of wound healing, 5-bromo-2'-deoxyuridine (BrdU) incorporation, and scanning electron microscopic (SEM) morphology of the corneas were examined. The incorporation of BrdU and the degree of cell attachment in cultured mouse corneal epithelial cells (MCECs) isolated from WBB6F1(+/+), Sl/Sl(d), and W/W(v) mice were examined. Cultured immortalized human corneal epithelial cells (HCECs) were examined by a cell attachment assay after their exposure to anti-SCF antibodies, tyrosine kinase inhibitor (genistein), and competitive Arg-Gly-Asp (RGD) peptide, as well as on cultures treated with extracellular matrix. RESULTS: The speed of corneal wound healing was slower in Sl/Sl(d) and W/W(v) mice than in controls (p<0.01) and the speed of healing in Sl/Sl(d) mice recovered after topical application of SCF (8 ng/ml). No significant difference was found in the BrdU incorporation assay either in vivo or in vitro. Loosened epithelial cells were detected at wound margins in W/W(v) mice by SEM. The cell attachment rate was increased by 157% in cells from WBB6F1(+/+) and 252% in Sl/Sl(d) MCECs by recombinant mouse SCF; however, no significant difference was found in W/W(v) MCECs. Anti-SCF antibodies (Ab), genistein, and RGD peptide reduced the percentage of attached HCECs. Anti-SCF Ab inhibited the attachment of HCECs on fibronectin, laminin, or type IV collagen coated dishes. CONCLUSIONS: These findings indicate that the SCF/c-kit system may play a role in corneal wound healing through epithelial cell attachment.

Important role of epiregulin in inflammatory responses during corneal epithelial wound healing.

PURPOSE: To investigate the role played by epiregulin in corneal epithelial wound healing in vivo in epiregulin-knockout (KO) mice and cultured mouse corneal epithelial cells (MCECs). METHODS: A 2-mm diameter central epithelial wound was created in epiregulin-KO and wild-type (WT) mouse corneas. The size of the unhealed area and the epithelial cell proliferation and migration were examined. Myeloperoxidase assay was performed to determine the number of polymorphonuclear (PMN) cells infiltrating corneal stroma. Real-time PCR was used to determine expression of the mRNA of inflammatory cytokines in the corneal epithelial cells. Expression of chemokine (C-X-C motif) ligand 2 (CXCL2) response to IL-1ß was examined in MCECs with or without recombinant mouse epiregulin. Repetitive injuries were created to determine the effect of inflammation in healing in epiregulin-KO mice. RESULTS: After a single injury, corneal epithelial wound healing and cell migration and proliferation were unimpaired. However, corneal opacities and a larger number of infiltrating PMN cells were observed in epiregulin-KO mice. Expression levels of IL-1ß, IL-6, CXCL1, and CXCL2 were higher in epiregulin-KO than in WT corneal epithelia cells. The addition of epiregulin significantly reduced the expression of CXCL2 in response to IL-1ß in MCECs. In response to repetitive injuries, a significant delay in healing and more severe opacities were observed in epiregulin-KO mice than in WT mice. CONCLUSIONS: Our results indicate that during wound healing, epiregulin may regulate the expression of cytokines and chemokines to reduce an excessive accumulation of PMN cells, which will cause corneal opacity and persistent epithelial defects.

Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype.

It remains elusive as to what bone marrow (BM) cell types infiltrate into injured and/or diseased tissues and subsequently differentiate to assume the phenotype of residential cells, for example, neurons, cardiac myocytes, keratocytes, etc., to repair damaged tissue. Here, we examined the possibility of whether BM cell invasion via circulation into uninjured and injured corneas could assume a keratocyte phenotype, using chimeric mice generated by transplantation of enhanced green fluorescent protein (EGFP)(+) BM cells into keratocan null (Kera(-/-)) and lumican null (Lum(-/-)) mice. EGFP(+) BM cells assumed dendritic cell morphology, but failed to synthesize corneal-specific keratan sulfate proteoglycans, that is KS-lumican and KS-keratocan. In contrast, some EGFP(+) BM cells introduced by intrastromal transplantation assumed keratocyte phenotypes. Furthermore, BM cells were isolated from Kera-Cre/ZEG mice, a double transgenic mouse line in which cells expressing keratocan become EGFP(+) due to the synthesis of Cre driven by keratocan promoter. Three days after corneal and conjunctival transplantations of such BM cells into Kera(-/-) mice, green keratocan positive cells were found in the cornea, but not in conjunctiva. It is worthy to note that transplanted BM cells were rejected in 4 weeks. MSC isolated from BM were used to examine if BM mesenchymal stem cells (BM-MSC) could assume keratocyte phenotype. When BM-MSC were intrastromal-transplanted into Kera(-/-) mice, they survived in the cornea without any immune and inflammatory responses and expressed keratocan in Kera(-/-) mice. These observations suggest that corneal intrastromal transplantation of BM-MSC may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes.

Crosstalk between TGF-beta and MAPK signaling during corneal wound healing.

PURPOSE: The aim of this study was to elucidate the mechanisms governing epithelial cell migration and proliferation during wound healing. METHODS: The authors used wound healing of mouse corneal epithelium to examine the role TGF-ß signaling plays during the healing process. To achieve this goal, they used transgenic mice in which the TGF-ß receptor type II (Tbr2) was conditionally ablated from the corneal epithelium. Epithelium debridement wounds were made, followed by the assessment of cell migration, proliferation, and immunostaining of various signaling pathway components. RESULTS: The authors showed that in the absence of TGF-ß signaling corneal epithelial wound healing is delayed by 48 hours; this corresponds to a delay in p38MAPK activation. Despite the delayed p38MAPK activation, ATF2, a substrate of p38MAPK, is still phosphorylated, leading to the suppression of cell proliferation at the leading edge of the wound. These data provide evidence that in the absence of TGF-ß signaling, the suppression of cell proliferation during the early stages of wound healing is maintained through the JNK activation of ATF2. CONCLUSIONS; Together the data presented here demonstrate the importance of the TGF-ß and MAPK signaling pathways in corneal epithelial wound healing.

Lumican is required for neutrophil extravasation following corneal injury and wound healing.

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

Corneal epithelial wound healing impaired in keratinocyte-specific HB-EGF-deficient mice in vivo and in vitro.

PURPOSE: To study the role played by HB-EGF in corneal epithelial wound healing. METHODS: A 2-mm corneal epithelial wound was created in keratinocyte-specific, HB-EGF-deficient mice--HB(lox/lox):K-5Cre (HB(-/-))--and the speed of wound healing was compared with that in wild-type (WT) mice. Cultured confluent mouse corneal epithelial cells (MCECs) from WT and HB(-/-) mice were scraped, and the bare area was measured. The proliferation of MCECs was determined by BrdU incorporation. The degree of attachment of WT and HB(-/-) MCECs was also determined. The mRNA expression of EGF family and cell adhesion molecules was determined by real-time PCR. RESULTS: Corneal epithelial wound healing was significantly delayed in HB(-/-) mice, and the expression of HB-EGF was detected at the leading edge of the wound in HB(lox/+):K5-Cre (HB(+/-)) mice by the presence of lacZ staining. The wound closure was significantly impaired in HB(-/-) MCECs and was improved by adding HB-EGF. The number of BrdU-positive MCECs of WT and HB(-/-) mice was not significantly different, and both increased to different degrees when HB-EGF was added. The adhesion of isolated HB(-/-) MCECs was lower than that of WT MCECs, but the degree of adhesion was restored by adding HB-EGF. The expression of epiregulin was upregulated in HB(-/-) MCECs, and α6- and ß1-integrin were upregulated by adding HB-EGF. CONCLUSIONS: HB-EGF plays an important role in corneal epithelial cell healing by enhancing cellular attachment and part of cell proliferation.

Monoallelic expression of Krt12 gene during corneal-type epithelium differentiation of limbal stem cells.

PURPOSE: The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation. METHODS: Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. RESULTS: Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. CONCLUSIONS: These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.

Promiscuous recombination of LoxP alleles during gametogenesis in cornea Cre driver mice.

PURPOSE: To examine whether promiscuous Cre/LoxP recombination happens during gametogenesis in double transgenic mice carrying LoxP modified alleles and Cre transgene driven by tissue-specific promoter outside the gonads of adult mice. METHODS: Cre driver mice were crossbred with reporter mouse lines (e.g., ZEG and Rosa26R) to obtain Cre/ZEG and Cre/Rosa26R double transgenic mice. The frequency of promiscuous LoxP/Cre recombination was determined by the expression of second reporter genes in the offspring of double transgenic mice. RESULTS: The frequency of promiscuous LoxP/Cre recombination varied in different lines of Cre driver mice and in the sex of the same driver mice with higher penetrance in male than in female double transgenic mice. Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis. Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion. CONCLUSIONS: Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.

Excess FGF-7 in corneal epithelium causes corneal intraepithelial neoplasia in young mice and epithelium hyperplasia in adult mice.

We hypothesized that human ocular surface squamous neoplasia (OSSN) may result from the continuous growth stimulation of corneal epithelial progenitor cells. In the present study, we analyzed the effects of excess fibroblast growth factor-7 (FGF-7) on both the proliferation and differentiation of corneal epithelium in a novel Krt12-rtTA/tet-O-FGF-7 double transgenic mouse model in which cornea-specific FGF-7 overexpression is achieved by doxycycline (Dox) treatment. When such adult mice were exposed to Dox, they exhibited epithelial hyperplasia with increases in phospho-extracellular signal-regulated kinase 1/2-, nuclear beta-catenin-, and 5-bromo-2'-deoxyuridine-labeled cells and altered keratin (K) 14 (K14) expression pattern, a normal K12 expression pattern, and the normal absence of K10. Hyperplasia of the adult cornea was fully reversible 2 weeks after the removal of Dox from chow. In contrast, double transgenic embryos that were exposed to Dox from embryonic day 0.5 to postnatal day 21 developed papillomatous tumors in the cornea, resembling human OSSN, and ectopic gland-like structures in the limbus, accompanied by the down-regulation of K12 and the up-regulation of K14, Pax6, and p63. These epithelial anomalies observed in young experimental mice were not fully resolved after the termination of Dox induction. Taken together, Krt12-rtTA/tet-O-FGF-7 mice may be a suitable animal model for the study of the molecular and cellular mechanisms of human OSSN.

Presence of adipose differentiation-related protein in rat meibomian gland cells.

Adipose differentiation-related protein (ADRP) is an intrinsic lipid storage protein found in lipid droplets of different type of cells. ADRP has been recognized to be a specific marker of lipid accumulation and a marker of differentiated adipocytes. The purpose of this study was to determine whether ADRP was present in the cells of the meibomian gland. The expression of the mRNA of ADRP was determined by RT-PCR and Northern blot analysis of the meibomian gland and other rat tissues. A newly generated polyclonal antibody against rat ADRP was used for Western blot analysis and immunohistochemical staining to determine whether ADRP was expressed in the rat meibomian gland. Meibomian gland acinar cells were isolated to determine when ADRP was expressed during cell differentiation in vitro. Northern blot analysis and Western analysis showed that ADRP was expressed in the meibomian gland. Immunoreactivity to ADRP was observed in the lobules of acinar cells in the meibomian gland, and was preferentially located adjacent the vacuolated cytoplasm. In culture, the meibocytes began to store lipid droplets in the cytoplasm as they became confluent, and the immunoreactivity for ADRP was found at the margins of the oil droplets. Our results suggest that ADRP can serve as a new marker for the identification of differentiated meibocytes containing lipid droplets.

Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth.

PURPOSE: To determine the kinetics of corneal epithelial maturation during embryonic development and postnatal growth. METHODS: Expression patterns of keratin (K)12 and K14 were determined in mouse embryos (embryonic days [E]15.5-19.5), corneas of postnatal day (P)0 to 10 months, and healing corneas after epithelial debridement in P30 and P90 mice. The expression of alkaline phosphatase (AP) was determined during postnatal growth and healing of epithelial debridement of Krt12(Cre/Cre)/ZAP bitransgenic mice. RESULTS: During embryonic development, K12 expression by corneal peridermal epithelium commenced at E15.5. In the period from E15.5 to P10, the expression of K12 was restricted to the suprabasal and/or superficial cells of the corneal epithelium, whereas the K14 expression was restricted to the basal cells. After P30, K12 expression was sporadically detected in the basal corneal epithelium, and the number of K12-positive basal cells increased as the mice grew older. The number of K14-positive cells that coexpressed K12 increased with age and reached a plateau after P180. Healing of the debrided epithelium facilitated the increase in K14-positive cells that coexpressed K12. Many basal cells of Krt12(Cre/Cre)/ZAP mice remained undifferentiated and expressed LacZ at P15, and they then differentiated to express Cre, which leads to excision of LacZ and AP expression. CONCLUSIONS: In the mouse, the corneal epithelium does not become fully mature until 3 to 6 months after birth, in that a significant number of corneal basal epithelial cells of young mice (

Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice.

PURPOSE: To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. METHODS: A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. RESULTS: Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. CONCLUSIONS: A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.