Toshinari Funaki

Adhesion, migration, and proliferation of cultured human corneal endothelial cells by laminin-5.

PURPOSE To investigate the expression of laminin-5 (LM5) and its receptors by human corneal endothelial cells (HCECs) and whether recombinant human LM5 influences adhesion, proliferation, and migration of cultured HCECs. METHODS The expression of LM5 and its receptors was examined in human donor corneas by immunohistochemistry, reverse transcription-polymerase chain reaction, and flow cytometry. HCECs cultured under serum-free conditions were used for analysis of the biological effects of LM5. Changes in HCEC adhesion and proliferation due to LM5 were evaluated by counting the number of cells. HCEC migration was assessed by quantifying the percentage of wound closure in the wound-healing assay with an image-processing and -analysis software program. RESULTS Adult HCECs expressed the LM5 receptor α3β1 integrin, but not LM5 itself. Significantly more cells became adherent to recombinant LM5 (1.0 μg/mL)-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by LM5 (1.0 μg/mL) and soluble LM5 (20 ng/mL and 50 ng/mL) in the cell proliferation assay. A significantly higher percentage of wound closure was obtained with medium containing soluble LM5 than with control medium in the wound-healing assay. CONCLUSIONS HCECs express the LM5 receptor α3β1 integrin. Recombinant LM5 promotes adhesion, migration, and moderate proliferation of cultured HCECs. It may be a critical factor in promoting HCEC culture and may contribute to the practical use of tissue-engineered HCECs.

Mast Cell Chymase Decreases the Barrier Function and Inhibits the Migration of Corneal Epithelial Cells

Purpose: We investigated the in vitro effects of human mast cell chymase on corneal epithelial cells. Methods: Human corneal epithelial cells were incubated with human chymase at activity levels that were likely to exist in the tears of patients with vernal keratoconjunctivitis. Results: Incubation of chymase resulted in a decrease of barrier function of corneal epithelium. Occludin protein was cleaved by chymase. In the wound assay, incubation with chymase resulted in an inhibition of cell migration. Conclusion: Human chymase causes the proteolysis of occludin and fibronectin, resulting in a decrease of barrier function and inhibition of the migration of corneal epithelial cells.

Perlecan-Deficient Mutation Impairs Corneal Epithelial Structure

PURPOSE To elucidate the role of perlecan (Hspg2), a large multidomain heparan sulfate proteoglycan expressed in the basement membrane, in the structure of the corneal epithelium. METHODS A previously developed perlecan-deficient (Hspg2⁻/⁻-Tg) mouse model was used. Histologic analysis of their corneas was performed by light and transmission electron microscopy. The localization of perlecan in the corneas of wild-type (WT) mice and Hspg2⁻/⁻-Tg mice was examined by immunohistochemistry. The effects of perlecan deficiency on corneal epithelial structure was analyzed with respect to the expression of corneal epithelial proliferation and differentiation markers, such as Ki67, cytokeratin12 (K12), connexin43 (Cx43), Notch1, and Pax6 by immunohistochemistry and real-time polymerase chain reaction (PCR). RESULTS The Hspg2⁻/⁻-Tg mice had microphthalmos and a thinner corneal epithelium compared with that of the WT mice. Perlecan was localized in the corneal epithelial basement membrane in the WT mice, but not in the Hspg2⁻/⁻-Tg mice. The Hspg2⁻/⁻-Tg corneal epithelium exhibited thinner wing cell layers and a decreased number of Ki67-positive cells, but no dead cells, compared with the WT corneal epithelium. Immunohistochemistry and real-time PCR analysis revealed a significantly decreased expression of corneal epithelial differentiation markers such as K12, Cx43, Notch1, and Pax6 in Hspg2⁻/⁻-Tg mice, compared with those of the WT mice. CONCLUSIONS The findings of this study highlight a strong correlation between the presence of perlecan in the basement membrane and the structure of corneal epithelium and that the perlecan-deficient mutation impairs corneal epithelial structure.

Tear chymase in vernal keratoconjunctivitis.

Purpose. To determine the levels of mast cell chymase and tryptase activity in the tears of patients with vernal keratoconjunctivitis (VKC). Methods. Subjects were 38 VKC patients and 18 healthy controls whose chymase and tryptase activity in tears was measured by enzyme assay. VKC severity was quantified based on the following clinical signs: papillary hypertrophy, conjunctival hyperemia, edema, punctate keratitis, Trantas dots, and mucus production. Of the 38 VKC patients, the degree of disease severity was mild in 13, moderate in 18, and severe in 7. Results. Mean chymase activity and standard deviation in tears was 0.23 ± 0.07 mU in mild VKC, 0.68 ± 0.22 mU in moderate VKC, 1.91 ± 0.71 mU in severe VKC, and 0.11 ± 0.05 mU in healthy controls. The increase in all VKC stages was statistically significant compared to that in healthy control. The degree of chymase activity in tears correlated significantly with VKC severity (r = 0.9245, p < 0.001). High tryptase activity was also detected in the tears of VKC patients, although increased tryptase activity in tears did not correlate with disease severity (r = 0.1999). Conclusions. Chymase activity in tears may thus be a sensitive marker for determining the severity of VKC.

Smad7 Suppresses the Inhibitory Effect of TGF-β2 on Corneal Endothelial Cell Proliferation and Accelerates Corneal Endothelial Wound Closure In Vitro

Purpose. The inhibitory activity of transforming growth factor-&bgr;2 (TGF-&bgr;2) on corneal endothelial cell proliferation is thought to be a cause of the limited regenerative capacity of corneal endothelial cells that may be related to impaired corneal transparency when many corneal endothelial cells are lost due to various stresses. We determined whether Smad7, an intracellular antagonist of TGF-&bgr; signaling, regulated the inhibitory activity of TGF-&bgr;2 or aqueous humor on corneal endothelial cell proliferation. Methods. The effect of Smad7 on TGF-&bgr;2– or aqueous humor–mediated inhibition of corneal endothelial cell proliferation was evaluated using thymidine uptake assay with cultured rabbit corneal endothelial cells infected with adenovirus carrying Smad7. Expression of Smad or cell cycle–related proteins was detected by immunoblotting. In addition, a small scrape wound was made across a monolayer of Smad7-expressing cultured rabbit corneal endothelial cells to examine the effect of Smad7 on the wound-healing process in vitro. Results. Overexpression of Smad7 abolished the inhibitory effect of TGF-&bgr;2 or aqueous humor on the proliferation of cultured rabbit corneal endothelial cells associated with the inhibition of phosphorylation of Smad2 and downregulation of p27kip1. Smad7-overexpressing cultured rabbit corneal endothelial cells exhibited shorter wound closure time in the presence of aqueous humor than LacZ-expressing cells. Conclusion. Overexpression of Smad7 suppressed the inhibitory effect of TGF-&bgr;2 or aqueous humor on corneal endothelial cell proliferation and accelerated corneal endothelial wound closure in vitro. Modification of Smad7 expression in corneal endothelial cells may thus have applicability in the treatment of wounded corneal endothelium.

Corneal Abnormalities in the NC/Nga Mouse: An Atopic Dermatitis Model

Purpose: To study corneal abnormalities in the NC/Nga mouse, which is an animal model of atopic dermatitis. Methods: To study histopathologic changes of the eyelid, conjunctiva, and cornea, we extracted the eyeballs together with upper and lower eyelids and fixed them for examination by light and electron microscopy or snap-froze them for immunohistochemistry. Transferase mediated-dUTP digoxigenin nick-end labeling staining was performed to detect apoptotic cells. In order to assess eye scratching behavior and the effect of tacroliums hydrate ointment, we made video recordings. Results: Mice kept in a conventional room suffered from various grades of blepharoconjunctivitis and scratched their eyes furiously. Tacrolimus hydrate ointment reduced their eye-scratching behavior. Histopathologic study of the eyelid and conjunctiva showed that this blepharoconjunctivitis was caused by allergic inflammation. Mice with severe blepharoconjunctivitis showed thinning of the corneal epithelium, an irregular interface between the epithelium and stroma, subepithelial deposition of materials, and neovascularization of the stroma. Their corneas were cone shaped. Many transferase mediated-dUTP digoxigenin nick-end labeling-positive cells were recognized among superficial epithelial cells and keratocytes. Conclusions: NC/Nga mice are a useful animal model of atopic blepharoconjunctivitis. Various corneal disorders in these mice may depend on their eye-scratching behavior.

Establishment of a Human Corneal Epithelial Cell Line Lacking the Functional TACSTD2 Gene as an In Vitro Model for Gelatinous Drop-Like Dystrophy

PURPOSE Gelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model. METHODS A corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed. RESULTS The immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction-related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy. CONCLUSIONS We established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.

Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty

PurposeTo determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor β (TGF-β), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model.MethodsRabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flagtagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas.ResultsTransplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction.ConclusionsEx vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.

Genetic characteristics of Haemophilus influenzae and Streptococcus pneumoniae isolated from children with conjunctivitis-otitis media syndrome

Acute conjunctivitis is the most common ocular disorders among children and frequently concomitant with acute otitis media (AOM) as conjunctivitis-otitis syndrome. In this study, we evaluated prevalence of causative pathogens and PCR-based genotypes of Haemophilus influenzae and Streptococcus pneumoniae among children with conjunctivitis-otitis media syndrome. Nontypeable H. influenzae (NTHi) is identified most often at 61.8% in conjunctiva exudates followed by S. pneumoniae at 28.2% and Moraxella catarrhalis at 19.1%. Genetic β-lactamase nonproducing ampicillin resistant (gBLNAR) strains of NTHi and genetic penicillin resistant S. pneumoniae (gPRSP) were identified at 72.1% and at 74.2% among conjunctiva isolates by polymerase chain reaction (PCR), respectively. Pneumococcal strains having either ermB or mefE genes were identified at 93.5% among conjunctiva isolates. The restriction fragment of patterns of 89.7% pairs of H. influenzae isolates and 100% pairs of pneumococcal isolates from conjunctiva exudates, middle ear fluids (MEFs) and nasopharyngeal swabs were identical. In contrast to the previous reports, most prevalent strains from conjunctivitis-otitis media syndrome was BLNAR H. influenzae in this study. The causative pathogen responsible for acute conjunctivitis will be originated from the nasopharynx. In the absence of MEFs one can possibly rely on the nasopharyngeal culture to guide an appropriate treatment.

Role of TGF-β in tissue eosinophilia associated with vernal keratoconjunctivitis

To determine the role of TGF-β(1) in the tissue eosinophilia associated with vernal keratoconjunctivitis (VKC), we investigated the immunohistochemical expression of TGF-β(1) and TGF-β(1)-related proteins in giant papillae obtained from VKC patients. We also investigated the effect of TGF-β(1) on production of eotaxin by cultured conjunctival and corneal fibroblasts using ELISA. Finally, the effects of glucocorticoids, cyclosporine, and tacrolimus on eotaxin production by corneal fibroblasts were assessed. Our investigations revealed that eosinophils expressing TGF-β(1) and TGF-β(1)-related proteins (such as phosphorylated Smad2, integrin αvβ(6), α-smooth muscle actin, type I procollagen, and tenascin-C) were expressed in the giant papillae. TGF-β(1) and IL-4/IL-13 caused a synergistic increase of eotaxin production in cultured conjunctival and corneal fibroblasts. This effect of TGF-β(1) and IL-4/IL-13 was inhibited by glucocorticoids, but neither by cyclosporine nor by tacrolimus. In conclusion, TGF-β(1) has an important role in the tissue eosinophilia associated with VKC.