Shin Hatou

Functional corneal endothelium derived from corneal stroma stem cells of neural crest origin by retinoic acid and Wnt/β-catenin signaling.

Corneal endothelial dysfunction remains a major indication for corneal transplantation. Both corneal endothelial cells and stromal cells originate from the neural crest, but have distinct phenotypes and function in the adult cornea. We previously reported that stem cells isolated from the adult corneal stroma [cornea-derived precursors (COPs)] show characteristics of multipotent neural crest-derived stem cells. In this study, we report the induction of functional tissue-engineered corneal endothelium (TECE) from mouse and human COPs. TECE was engineered from Wnt1-Cre/Floxed EGFP mouse COPs in a medium containing retinoic acid and glycogen synthase kinase (GSK) 3β inhibitor (activator of Wnt/β-catenin signaling). The expression levels of major markers characterizing corneal endothelial function (Atp1a1, Slc4a4, Car2, Col4a2, Col8a2, and Cdh2) were significantly upregulated. Both retinoic acid and GSK 3β inhibitor upregulated the expression of Pitx2, a homeobox gene involved in the development of the anterior segment of the eye. GSK 3β inhibitor increased Atp1a1 expression and Na,K-ATPase pump activity of TECE, which was significantly higher than COPs or control 3T3 cells, and 2.6-fold higher than cultured mouse corneal endothelial cells. Mouse TECE transplanted into rabbit corneas maintained transparency and corneal thickness, whereas control corneas without TECE showed marked edema and increased corneal thickness. Furthermore, we successfully induced TECE from human COPs, and human TECE transplanted into rabbit corneas also maintained corneal transparency and thickness. This protocol enables efficient production of corneal endothelium from corneal stromal stem cells by direct induction, which may lead to a novel stem cell therapy for corneal endothelial dysfunction.

Deposition of lipid, protein, and secretory phospholipase A2 on hydrophilic contact lenses.

Purpose. Recent studies have shown that low tear phospholipid levels are associated with tear film instability in hydrophilic contact lens wearers. The concentration of secretory phospholipase A2 (sPLA2), the enzyme that hydrolyzes phospholipids, in tears is known to exceed the levels found in serum by four orders of magnitude. This study was performed to determine the levels of sPLA2 from the deposition on two different frequent-replacement contact lens materials. Methods. Polymacon and etafilcon A contact lenses worn for 2 weeks by 16 experienced contact lens wearers were used for the analysis. Total lipids were determined by the sulfo-phospho-vanillin reaction. Phospholipids in lipid extracts were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Total protein was measured by bicinchoninic acid analysis. Double-antibody sandwich enzyme-linked immunosorbent assay was used to determine sPLA2 concentrations. Results. Total lipid deposition was found to be greater in the polymacon group (66.3 ± 16.3 &mgr;g/lens) than in the etafilcon A group, although phospholipids were not detected in either group. The etafilcon A group had greater deposition of protein (3.7 ± 0.7 mg/lens) than the polymacon group had. The etafilcon A group deposited statistically significantly more group IIa sPLA2 (1.1 ± 0.3 &mgr;g /lens) than the polymacon group (0.07 ± 0.04 &mgr;g/lens) did (P<0.001). Conclusions. There was a significant difference in the lipid and protein deposition profiles in the two lenses tested. A significant amount of sPLA2 in the deposition on contact lenses may play a role in tear film instability in hydrophilic contact lens wearers.

Mutations in the quinolone resistance determining region in Staphylococcus epidermidis recovered from conjunctiva and their association with susceptibility to various fluoroquinolones

Background: Staphylococcus epidermidis is one of the prominent pathogens in ocular infection. The prevalence of mutations in the quinolone resistance determining region (QRDR) area in S epidermidis isolated from the ocular surface and its association with fluoroquinolone resistance has not been fully elucidated. Methods: Mutations in the QRDR of gyrA, gyrB, parC, and parE genes of 138 isolates of S epidermidis recovered from the human conjunctival flora were analysed. The minimal inhibitory concentrations (MICs) of four fluoroquinolones (levofloxacin, gatifloxacin, moxifloxacin and tosufloxacin) against these isolates were also determined using agar dilution methods. Results: The MIC90 values of levofloxacin, gatifloxacin, moxifloxacin and tosufloxacin were 3.13, 1.56, 0.78 and 3.13 μg/ml, respectively. The MIC values of all fluoroquinolones showed a bimodal distribution (susceptible strain and less susceptible strain). Mutations with amino acid substitution in the QRDR were present in 70 (50.7%) isolates. 19 different combinations of mutations were detected: 3 isolates (2.2%) had four mutations, 8 (5.8%) had three mutations, 43 (31.2%) had double mutations and 16 (11.6%) had single mutations. Isolates with mutations in the QRDR of both gyrA and parC (n = 53) were less susceptible to fluoroquinolones. Conclusions: The present findings show that approximately half the S epidermidis isolates from the normal human conjunctiva have mutation(s) in the QRDR. The presence of mutations in both gyrA and parC is strongly associated with reduced susceptibility to fluoroquinolones.

Role of Insulin in Regulation of Na-/K-Dependent ATPase Activity and Pump Function in Corneal Endothelial Cells

PURPOSE The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. METHODS Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. RESULTS Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. CONCLUSIONS These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

The effects of dexamethasone on the Na,K-ATPase activity and pump function of corneal endothelial cells.

Purpose: The Na+- and K+-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Methods: Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase α1-subunit. Results: Dexamethasone (1 or 10 μ M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase α1-subunit. Conclusions: Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase α1-subunits.

Turnover rate of tear-film lipid layer determined by fluorophotometry

Aim: This study was performed to independently assess the turnover rates of aqueous and lipid layers of the tear film. Methods: Two fluorescent dyes, fluorescein sodium and 5-dodecanoylaminofluorescein (DAF), which is a free-fatty-acid conjugate of fluorescein, were applied to the right eye of 12 healthy volunteers. Fluorescent intensity of the precorneal tear film was measured at the central cornea every minute for 10 min for fluorescein sodium, and every 5 min for 50 min for DAF. The turnover rate was calculated by plotting fluorescent intensity against time in a semilog plot and expressed as %/min. Results: Turnover rates of fluorescein sodium and DAF were 10.3 (SD 3.7)%/min and 0.93 (0.36)%/min, respectively. The turnover rate of DAF was significantly lower than that of fluorescein sodium (p<0.05, Mann–Whitney test). The turnover rate of DAF positively correlated with that of fluorescein sodium (r = 0.93, p<0.05). Conclusion: Our results indicate that the turnover of lipids in tears is much slower than the aqueous flow of tears, and that this lipid turnover is associated with the aqueous flow of tears in healthy adults.

Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland, Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.

Establishment of functioning human corneal endothelial cell line with high growth potential.

Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na+- and K+-dependent ATPase (Na+/K+-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7), THCEH (Cyclin) and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7) and THCEH (Cyclin). THCEH (Cyclin) expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na+/K+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7). This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.

Biochemical analyses of lipids deposited on silicone hydrogel lenses

Purpose This study was performed to determine the levels of lipids deposited on in vivo worn silicone hydrogel lenses.

Simultaneous deep anterior lamellar keratoplasty and limbal allograft in bilateral limbal stem cell deficiency

PurposeTo report the efficacy of simultaneous keratolimbal allograft (KLAL) surgery and deep anterior lamellar keratoplasty (DALK) for limbal stem cell deficiency (LSCD).MethodsWe conducted a retrospective, interventional case series of six consecutive eyes of five patients with LSCD and stromal opacity due to gelatinous drop-like dystrophy (two eyes), Stevens-Johnson syndrome (SJS, two eyes), or aniridia (two eyes). Only patients with normal lid anatomy and Schirmer test values greater than 3 mm were enrolled. DALK was performed by viscodissection followed by a thin, 360° KLAL designed by using an artificial anterior chamber. KLAL sutures were removed after 2 weeks.ResultsDALK and KLAL were successfully performed in all eyes, which were followed for an average of 17.2 ± 10.8 months. All eyes recovered a smooth corneal epithelium, although one SJS patient developed a persistent epithelial defect (PED) leading to opacification of the central cornea. Visual acuity improved by more than 2 lines in all eyes except that of the SJS patient with PED. No other complications were observed.ConclusionSimultaneous DALK and thin-section KLAL is an effective treatment for ocular surface disease in patients with residual tear function and normal lid anatomy.