Kosuke Abe

Slug Is Upregulated during Wound Healing and Regulates Cellular Phenotypes in Corneal Epithelial Cells

PURPOSE The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. METHODS Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. RESULTS Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. CONCLUSIONS It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.

The cytokine regulation of SPARC production by rabbit corneal epithelial cells and fibroblasts in vitro.

Objective SPARC (osteonectin/BM40) is detected in the corneal stroma during the wound-healing process. To understand the metabolism of SPARC in the cornea, we investigated the effects of cytokines and growth factors on SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. Methods Rabbit corneal epithelial cells or fibroblasts were cultured for 3 days with serum-containing minimal essential medium (MEM), then subcultured for 3 days on serum-free MEM with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-&bgr; (TGF-&bgr;), or interleukin-1&bgr; (IL-1&bgr;). SPARC concentration in the medium was measured by the ELISA method using anti-SPARC monoclonal antibody. Results The concentration of SPARC in the conditioned medium of the epithelial cells depended on either cell numbers or cultivation periods. When EGF was added to the medium, the amount of SPARC in the medium decreased. The addition of IL-1&bgr;, PDGF, or TGF-&bgr; did not affect SPARC synthesis by the epithelial cells. The production of SPARC by rabbit corneal fibroblasts was low compared with that by epithelial cells. However, the synthesis of SPARC by corneal fibroblasts was significantly enhanced by the addition of TGF-&bgr;. The addition of IL-1&bgr;, PDGF, or EGF slightly increased SPARC synthesis by corneal fibroblasts. Conclusions Cytokines and growth factors modulate SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. These results suggest that cytokines and growth factors modulate cell–matrix interaction in corneal wound healing, possibly by regulating SPARC synthesis.

Regulatory Mechanism of Procollagenase Synthesis by Keratocytes

Keratocytes play an important role in collagen metabolism in the corneal stroma. It has been reported that various cellular functions of keratocytes are regulated by extracellular matrices and cytokines. To understand the regulatory mechanism of collagenase synthesis by keratocytes, this study investigated the effects of extracellular collagen and cytokines on procollagenase synthesis by keratocytes. To perform this evaluation, subcultured human keratocytes were embedded in a type I collagen matrix. The cells were cultured for 24 hours with unsupplemented MEM. Various cytokines (IL-lβ, EGF, TGF-β, PDGF) were added to the medium. The amount of procollagenase in the medium was estimated by ELISA using a monoclonal antibody against human procollagenase. Results reveal that when keratocytes were cultured in a collagen matrix, the amount of procollagenase in the medium depended on either the incubation period or the number of cells. Furthermore, the amount of enzyme increased in proportion to the concentration of extracellular collagen. The amount of enzyme in the medium also increased by the addition of heat-denatured collagen. The addition of either IL-lβ, EGF, or PDGF stimulated procollagenase synthesis by the cells. On the other hand, enzyme synthesis was inhibited by the addition of TGF-β. The present results seem to indicate that procollagenase synthesis by keratocytes is regulated by extracellular collagen and various kinds of cytokines.

Optical coherence tomographic findings at the fixation point in a case of bilateral congenital macular coloboma

Background Congenital macular coloboma is a rare ocular disease that consists of atrophic lesions in the macula with well-circumscribed borders. We report the findings of spectral domain optical coherence tomography (SD-OCT) at the fixation point in a case of bilateral macular coloboma. Case report The subject is a 4-year-old boy. He visited our hospital at age 1 year and 4 months for the evaluation of strabismus. The fundus examination of both eyes showed round-shaped sharply-demarcated atrophic lesions involving the macula with large choroidal vessels and bared sclera at the base. Immunologic tests including toxoplasmosis, rubella, varicella, herpes virus, and human T-cell leukemia virus were all negative. At age 4 years and 1 month, cycloplegic refraction showed insignificant refractive errors and his best corrected visual acuity was 0.6 bilaterally. The SD-OCT showed a crater-like depression accompanying atrophic neurosensory retina, and the absence of retinal pigment epithelium and choroid. Examination of the fixation behavior by visuscope showed steady fixation with an area 0.5° nasal to the nasal edge of the atrophic lesion bilaterally. The SD-OCT findings at fixation area showed remaining normal retinal structures involving inner segment-outer segment (IS/OS) junction line. Conclusion The findings of SD-OCT have been shown to be useful in the diagnosis of macular coloboma. In the fixation point, the structure of retina and choroid were well preserved.

Role of fusional convergence amplitude in postoperative phoria maintenance in children with intermittent exotropia

PurposeTo examine the role of fusional convergence amplitude in postoperative phoria maintenance in childhood intermittent exotropia [X(T)].MethodsThe medical records of 29 children aged 15 years or younger (mean age, 10.8 ± 2.4 years) and treated with monocular recession-resection for X(T) were reviewed retrospectively. The patients’ fusional convergence amplitude (break point/total amplitudes), physiologic diplopia, and phoria maintenance (presence/absence of phoria maintenance and ability to maintain phoria) were assessed. The presence of phoria maintenance was confirmed by a cover test, and the ability to maintain phoria was quantified using the Bagolini red filter bar. Correlations of the amplitude size with the presence and ability of phoria maintenance were investigated.ResultsA significant correlation was seen between fusional amplitude (break point/total) and ability to maintain phoria at near and at far (break point: P < .05 at near/P < .01 at far; total: P < .05 at near/far). Neither the break point amplitude nor the total amplitude significantly differed between the patients with phoria maintenance and those without it (break point: P = .71 at near, P = .29 at far; total: P = .98 at near, P = .85 at far). Phoria maintenance correlated with the suppression of physiologic diplopia during phoria (P < .01). The deviation angle did not significantly correlate with fusional amplitude either at near (P = .58) or at far (P = .27).ConclusionsIn childhood X(T), fusional amplitude plays a role in enforcing the patient’s ability to maintain phoria. However, sufficient fusional amplitude does not guarantee fully functioning fusion if suppression is present during phoria.

Influence of Test Distance on Stereoacuity in Intermittent Exotropia

ABSTRACT Aims: To investigate influence of test distance on stereoacuity in intermittent exotropia (X[T]) using the same test conditions for both near and far distances. Methods: Subjects were 38 consecutive patients with X(T). All the patients were between ages 6 and 15 years and had decimal visual acuity of 1.0 or better. Another inclusion criterion was presence of phoric condition at near and far distances. Stereoacuity was measured at a near distance of 40 cm and at a far distance of 5 m. The following test conditions were used for both test distances: separation of the two eyes using polarized glasses, and a target with a random dot pattern. All the stereograms had the same subtended angle of 2.5º, and binocular disparity of 480, 240, 120, and 60 arcsec. We used two stereogram types with crossed and uncrossed disparities. Results: Far stereoacuity of 38 subjects measured with the crossed disparity was significantly worse than near stereoacuity (P<0.05, Wilcoxon signed-ranks test), although 30 (78.9%) of the 38 subjects showed no differences in stereopsis between the near and far distances. Far stereoacuity of 38 cases measured with the uncrossed disparity was significantly worse than at near (P<0.05, Wilcoxon signed-ranks test), although 20 (52.6%) of the 38 subjects showed no differences between stereoacuity at near and far. In comparison of stereoacuity with crossed disparity and uncrossed disparity, stereoacuity with crossed disparity was significantly better than that with uncrossed disparity both at near and far (P<0.05, Wilcoxon signed-ranks test). Conclusions: Stereoacuity in X(T) was different according to test distance when measured controlling subtended angle of stereogram at both distances. Far stereoacuity was significantly worse than near stereoacuity when measured using test targets with both crossed and uncrossed disparities. Additionally, stereoacuity measured with crossed disparity was better than that with uncrossed disparity at both distances.