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T Otori

Publications and source records attributed to T Otori.

At least 37 records · Page 2Linked to original sources
PubMed1992

Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.

Animals↗
PubMed1992

Coordinated appearance of beta 1 integrins and fibronectin during corneal wound healing.

Fibronectin, which appears at wound sites, serves as a temporary matrix for the epithelial migration involved in healing. Cellular responses to fibronectin have not yet been elucidated. We visualized beta 1 integrins, fibronectin, laminin, and collagen type IV by immunofluorescence techniques at various intervals after single, nonpenetrating incision in the corneas of rats to investigate the chronologic changes in the localization of these proteins during corneal epithelial wound healing. In unwounded corneas beta 1 integrins were found on basal cells of the corneal epithelium and fibronectin was found in the corneal subepithelial region, at Descemet's membrane, and in streaks between collagen fibers of the stroma. Both laminin and collagen type IV were found in the subepithelial region and Descemet's membrane. Immediately after the incision, fibronectin was visible on the surface of the V-shaped defect of the stroma; epithelial cell expressed beta 1 integrins began to migrate over the defect and to fill it. When healing was almost completed, beta 1 integrin staining of epithelial cells diminished, except for in basal cells. Staining of fibronectin diminished at the interface between the new epithelium and the stroma. Laminin and collagen type IV were not seen between the migrating epithelial cells and the underlying stroma until epithelial cells completely covered the defect. Appearance and disappearance of beta 1 integrins and fibronectin are thus well coordinated during corneal epithelial wound healing process, suggesting an active involvement of beta 1 integrins and fibronectin.

Animals↗
PubMed1992

Epidermal growth factor stimulates corneal epithelial cell attachment to fibronectin through a fibronectin receptor system.

Epidermal growth factor (EGF) stimulates corneal epithelial migration in vivo and in vitro. Antibody against fibronectin inhibits this effect in vitro, suggesting that a fibronectin-dependent mechanism in involved. To elucidate the action of EGF, we placed rabbit corneal epithelial cells, preincubated in the absence or presence of EGF (10 ng/ml), into wells coated with fibronectin. After 45 minutes of incubation, the numbers of cells attached to the wells were counted. Preincubation with EGF for 6 hr was not effective, but preincubation for 9 hr significantly increased the numbers of cells attached to the wells. These numbers were not increased further by additional preincubation. When concentrations of EGF were reduced, numbers of attached cells decreased proportionally, but remained significantly higher than the numbers obtained with cells not exposed to EGF. The EGF-stimulated attachment to the fibronectin matrix was inhibited in a dose-dependent fashion by antifibronectin IgG and by GRGDSP, a synthetic peptide that mimics the amino acid sequence of the cell-binding domain of fibronectin. The authors conclude that a fibronectin/fibronectin receptor system mediates EGF-induced stimulation of cellular attachment. These findings suggest that EGF may increase the expression of fibronectin receptors.

Amino Acid Sequence↗
PubMed1992

Increased lysosomal enzyme activity of keratocytes after endocytosis of foreign particles.

It has been reported that keratocytes endocytose foreign particles both in vitro and in vivo, suggesting the active participation of keratocytes in corneal wound healing and host defense mechanism. To understand the cellular response and the fate of engulfed foreign particles, we investigated the changes in the lysosomal enzyme activities after keratocytes endocytosed latex beads or glutaraldehyde-fixed Staphylococcus aureus. Acid phosphatase activity in the cells endocytosing either latex beads or fixed bacteria increased significantly as compared with that in the control over the period of 12 hours. Changes in acid phosphatase activity depended on both the incubation periods and the amount of latex beads or bacteria added. The activity reached a plateau after 6 hours of incubation. When keratocytes endocytosed latex beads, the activity of N-acetyl-beta-D-glucosaminidase in the cells showed an increase similar to that of acid phosphatase. However, the N-acetyl-glucosaminidase activity in the cells endocytosing fixed bacteria did not increase significantly. These findings suggest that endocytosing keratocytes increase degrading enzyme activities in the cells to break down engulfed foreign particles. It is also suggested that the intracellular response is varied according to the character of the engulfed materials.

Acetylglucosaminidase↗
PubMed1991

Expression of fibronectin receptor (integrin) in the uterus of rats in relation to the estrous cycle.

The expression and localization of fibronectin receptor (integrin), fibronectin, laminin and collagen type IV in the endometrium of the rat uterus during each period of the estrous cycle were investigated by immunofluorescent microscopy. Fibronectin receptor was observed at the epithelial cells of the endometrium and at vascular endothelial cells. At proestrus, when epithelial cells actively migrate, fibronectin receptor was observed at the basal and lateral epithelial cell surfaces. During estrus, fibronectin receptor had begun to disappear, little fibronectin receptor was observed at metestrus or diestrus. No prominent changes in the localization of fibronectin (seen at the vascular endothelial cells and in the stroma) or of laminin and collagen type IV (seen at the muscles and at the basement membranes of the epithelial and vascular endothelial cells) were observed in relation to the estrous cycle. Thus, uterine epithelial cells, like epithelial cells of the healing cornea, increase their expression of fibronectin receptor during active migration, probably facilitating their attachment to stromal fibronectin. This fibronectin-fibronectin receptor mechanism may underlie epithelial repair, whether the defect results from physiological processes or from an insult.

Animals↗
PubMed1991

Hyaluronan stimulates corneal epithelial migration.

Hyaluronan (hyaluronic acid), well-known for its viscoelastic properties, is also recognized as a biological signal to cells. Using organ cultures of the rabbit cornea, we investigated the effects of hyaluronan on the migration of corneal epithelium. The addition of hyaluronan to the culture medium increased the length of the path of the corneal epithelial layer in a dose-dependent fashion. Other glycosaminoglycans (chondroitin, chondroitin sulphate, keratan sulphate and heparan sulphate) were also tried, but only hyaluronan exhibited a stimulatory effect on corneal epithelial migration. The effects of hyaluronan and fibronectin or epidermal growth factor (EGF) were additive; the addition of antisera against fibronectin or against EGF did not alter the stimulatory effect of hyaluronan. These results demonstrate that hyaluronan stimulates corneal epithelial migration by mechanism(s) different from those of fibronectin and EGF.

Animals↗
PubMed1991

Long-term storage of endocytosed latex beads in keratocytes in vivo.

Endocytosis by keratocytes (corneal fibroblasts) is an important part of the host defense system. To investigate the long-term fate of endocytosed materials, we injected polystyrene latex beads into the corneal stroma of four rabbits. The corneal stroma was observed under a transmission electron microscope 4 and 800 days after the injection. After 4 days, the beads were found not only between the collagen fibers of the stroma, but also in some keratocytes. After 800 days, no extracellular beads were seen, but endocytosed beads remained, surrounded by limiting membranes, in the cytoplasm of keratocytes. These observations demonstrate that keratocytes endocytose latex beads and store them for a long time, isolating these foreign materials from the corneal stroma. These observations suggest that keratocytes, like some other fibroblasts perform a noninflammatory and nonimmunological defense function.

Animals↗
PubMed1991

Requirement of microtubule assembly for initiation of EGF-stimulated corneal epithelial migration.

To understand the mechanisms of epidermal growth factor (EGF)-stimulated re-epithelialization, the roles of microtubules and microfilaments in epithelial migration were investigated using the organ culture of rabbit cornea. The action of EGF was also compared with that of fibronectin. The addition of either EGF or fibronectin increased the epithelial sheet migration. Nocodazole, an inhibitor of the microtubule assembly, alone did not affect the epithelial migration. Nocodazole antagonized the EGF-dependent increase of the epithelial migration when nocodazole coexisted during the period of delivery of the signal from EGF. However, the stimulatory action of fibronectin was observed even in the presence of nocodazole. On the other hand, cytochalasin B, inhibitor of the microfilament assembly, inhibited the epithelial migration in a dose-dependent manner regardless of whether EGF or fibronectin was present or not. These results indicated that the formation of microfilaments was essential for the corneal epithelial migration and that the stimulatory action of EGF on the corneal epithelial migration depended on the assembly of microtubules. Therefore, in corneal epithelial wound healing, the action of EGF requires the organization of both microfilaments and microtubules, while that of fibronectin requires the reorganization of the microfilaments.

Actin Cytoskeleton↗
PubMed1991

Effects of intraocular irrigating solutions on the spreading of rabbit corneal endothelial cells on extracellular matrices.

The effects of four commercially available irrigating solutions on the spreading of rabbit corneal endothelial cells on various extracellular matrices were studied. Cultured rabbit corneal endothelial cells, suspended in one of the following intraocular irrigating solutions, Opeguard MA, BSS, BSS Plus, lactated Ringer solution (Lactec) or physiological saline, were placed on uncoated tissue culture plates or on plates coated with extracellular matrices (fibronectin, laminin, collagen type I, or collagen type IV). The cell area was measured after 45 minutes' incubation. The cells spread on all of the extracellular matrices examined but not on the uncoated tissue culture plates. On the fibronectin or laminin matrix, the cell area was significantly greater with Opeguard MA or BSS Plus. On laminin and collagen type IV, the cell area was the greatest with Opeguard MA. On collagen type I, the cell area was significantly greater with Opeguard MA, BSS, or BSS Plus. These results demonstrated that the rabbit corneal endothelial cells responded to the extracellular matrices, and that Opeguard MA or BSS Plus provided more favorable conditions for the spreading of these cells. These results indicated that both Opeguard MA and BSS Plus might aid the spreading of corneal endothelial cells during wound-healing immediately after intraocular surgery.

Animals↗
PubMed1990

Differential modes of action of fibronectin and epidermal growth factor on rabbit corneal epithelial migration.

In order to clarify the roles of fibronectin (FN) and epidermal growth factor (EGF) in corneal wound healing, we cultured blocks of excised rabbit cornea for 24 hours in media containing one of these agents, then measured the length of the path of the epithelial layer that had migrated down the side of the block. Both FN and EGF stimulated epithelial migration significantly in a dose-dependent fashion. Responses to EGF involved a time lag of at least 12 hours before stimulation could be observed, but there was no lag-time for FN-stimulated migration. FN was maximally effective only if it was continuously present. In contrast, exposure to EGF for 6 hours did not stimulate epithelial migration, but exposure for 9 hours resulted in the same stimulatory effects as were observed after 24 hours' continuous exposure. Anti-FN antibody inhibited the FN- and EGF-stimulated migration of corneal epithelium. But anti-EGF antibody inhibited only EGF-stimulated migration and had no effect on FN-stimulated migration. These results indicate that, unlike FN, EGF need not be present, once the epithelial cells have recognized its signal. Furthermore, the stimulatory effect of EGF depended on FN, while that of FN was independent of EGF. The effects of EGF on migration of corneal epithelium may, therefore, be mediated by FN.

Animals↗
PubMed1990

Role of actin filaments and microtubules in the spreading of rabbit corneal epithelial cells on the fibronectin matrix.

The migration and spreading of the corneal epithelial cells adjacent to a wound is the first step in successful epithelial resurfacing. To understand the role of actin filaments and microtubules of the cytoskeletal system in the spreading of corneal epithelial cells, we plated rabbit corneal epithelial cells on a fibronectin matrix and studied the effects of cytochalasin B, which inhibits actin filaments assembly, and colchicine, which inhibits microtubules assembly, on the ability of individual cells to spread. Changes in the morphology of actin filaments and microtubules were also studied using immunofluorescent microscopy. The area of spread epithelial cells depended on the concentration of fibronectin used to coat the surface. In spread cells, stress fibers of actin filaments were evenly distributed throughout the cytoplasm, whereas microtubules were observed only at the perinuclear region. The presence of cytochalasin B during the cell attachment and spreading decreased the area of the spread cells more than did colchicine. However, once the epithelial cells were spread on a fibronectin matrix, cytochalasin B and colchicine each decreased the cell area only slightly, and to the same extent. These results indicate that formation of actin filaments is more important than formation of microtubules to the spreading of corneal epithelial cells.

Actins↗
PubMed1988

Dexamethasone inhibition of phagocytosis by corneal keratocytes in culture.

To investigate the effect of steroid hormone on phagocytosis by keratocytes, we investigated the phagocytic activities for latex beads by rabbit corneal keratocytes cultured in the presence and absence of dexamethasone. Culturing the corneal keratocytes with dexamethasone inhibited the phagocytic activity for latex beads in a dose-dependent fashion. The inhibitory effects depended on the duration of the incubation period of the cells with dexamethasone.

Animals↗
PubMed1988

Interactions of extracellular collagen and corneal fibroblasts: morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix.

Corneal fibroblasts, also known as keratocytes are surrounded by an extracellular matrix of collagen in vivo. To understand the physiology and pathology of these corneal fibroblasts, it is important to study their interactions with this extracellular matrix. We cultured rabbit corneal fibroblasts on tissue culture plastic dishes or in a hydrated collagen gel and compared the changes in morphology and mitotic activity. Corneal fibroblasts on plastic dishes were flattened and widely spread, whereas those in collagen gel became spindle-shaped with long processes. Examination with an electron microscope revealed that the corneal fibroblasts in collagen gel formed gap junctions with neighboring cells. Gap junctions were hardly ever observed between corneal fibroblasts cultured on plastic dishes. Corneal fibroblasts cultured in a collagen matrix showed much less incorporation of [3H]thymidine than did corneal fibroblasts cultured on plastic, and this incorporation decreased with increasing concentration of collagen. Our present results suggest that the morphologic and biochemical characteristics of corneal fibroblasts cultured in collagen gel are different from those cultured on plastic.

Animals↗
PubMed1988

Localization of fibronectin and actin in cultured rabbit keratocytes.

Migration of activated keratocytes toward the corneal stromal wound is one of the most important processes of successful healing. To understand the motility of keratocytes and the interaction of fibronectin and intracellular actin filaments, we cultured rabbit corneal keratocytes and studied dynamic movements of the cells by time-lapse cinematography. We also examined the changes in the localization of fibronectin and actin using double staining immunofluorescent microscopy. The cultured keratocytes first attached to the substratum in round globular shape and then spread with many extending processes. In the early stage of the cultivation, fibronectin was observed inside the cells. Later, fibronectin was observed outside the cells, suggesting formation of the extracellular matrix. When keratocytes spread, actin was observed as a stress fiber inside the cells. At the edge of the cellular processes, a close interaction between fibronectin and actin was observed. The present results demonstrated that cultured keratocytes had active motility and that there were close interactions between the extracellular fibronectin and intracellular actin filaments. The organization of fibrillar actin filaments (F-actin) might be affected by the binding of extracellular fibronectin to the cell surface receptor for fibronectin.

Actins↗
PubMed1988

A peptide from fibronectin cell-binding domain inhibits attachment of epithelial cells.

Fibronectin is thought to contribute to the adhesion of corneal epithelium during epithelial repair, presumably by mediating epithelial attachment to stroma during epithelial migration. To understand the mechanism of the interaction of fibronectin with corneal epithelial cells, the effects of intact plasma fibronectin and of the synthetic peptide GRGDS from the cell binding domain of fibronectin on the attachment of rabbit corneal epithelial cells have been examined. When dissociated epithelial cells were plated on fibronectin-coated culture dishes, the number of cells that attached increased in proportion to the concentration of fibronectin used for coating. Attachment of the cells was inhibited in a dose-dependent manner by the addition of GRGDS to the medium. When the structurally similar control peptide GRGES was added to the medium, no inhibition of epithelial attachment was observed. Our results demonstrate that corneal epithelial cells use an adhesive recognition system for fibronectin related to that characterized in fibroblastic cells. GRGDS thus presumably competes for receptors for fibronectin on epithelial cells of the cornea, resulting in the observed inhibition of attachment. These results identify a mechanism for corneal epithelial cell adhesion, compatible with that identified in other fibronectin-dependent systems.

Amino Acid Sequence↗