Teruo Nishida

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.

Electron-microscopic studies on the presence of gap junctions between corneal fibroblasts in rabbits

SummaryCorneal fibroblasts, major cellular components of the corneal stroma, are loosely arrayed between collagen lamellae. They play an important role in the metabolic and physiological homeostasis mechanisms by which the cornea is kept transparent. This paper deals with the demonstration of the gap junctions between the corneal fibroblasts of rabbits by transmission electron microscopy of thin sections and of freeze-fracture specimens. Under the transmission electron microscope, the corneal fibroblasts are seen between the lamellae of collagen fibers of the corneal stroma. Their long cytoplasmic processes are in contact with those of neighboring fibroblasts. Typical gap junctions are found between these cytoplasmic processes. In the freeze-fracture images, intramembrane particles with a diameter of 10.3 nm form polygonal aggregates on P faces. These findings suggest that corneal fibroblasts, coupled with each other, might function synchronously through gap junctions responsible for metabolic activities essential for the maintenance of corneal transparency.

Transforming growth factor-β 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-β (TGF-β) 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-β 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-β into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-β itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured ...

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

SummaryCorneal 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.

Uptake of india ink particles and latex beads by corneal fibroblasts.

SummaryThe fate of India ink particles and polystyrene latex beads injected into the corneal stroma of rabbits was studied by the naked eye, light microscopy, and electron microscopy. All the injected ink particles or latex beads were unchanged in shape, size, and number for at least 6 months. India ink particles and latex beads were endocytosed by the corneal fibroblasts within 3–4 days after injection. Numerous ink particles were packed into vacuoles, 0.5–10 μm in diameter, which occupy a large volume of the cytoplasm of the cell body and processes of fibroblasts in and near the injected area. Each latex bead, 0.72 μm in diameter, is usually enclosed in one vesicle, and a large number of vesicles are distributed throughout the cytoplasm. In corneal tissue removed 10 min after injection of India ink and cultured for 3 or 7 days, uptake of many ink particles by the fibroblasts was seen. By this experiment, the contribution of the blood-derived cells was completely excluded, and it is more distinctly shown that the corneal fibroblast has a strong endocytotic activity.The uptake and long-term storage of ink particles and latex beads by the corneal fibroblast are reactions that protect the organ without inflammation from the injury and harm by non-toxic foreign materials.

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

SummaryThe 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.

Effects of antimicrobials on corneal epithelial migration

The slowed healing rates observed by some investigators may be caused by vehicles or preservatives in the antimicrobials preparations tested. To determine whether antimicrobials directly inhibit corneal epithelial wound healing, we cultured blocks of the rabbit cornea in media containing various concentrations of antibiotics or antimicrobials (at 1, 10, or 100 micrograms/ml); after 24 hours, we measured the distance of epithelium that had migrated down the side of each block. The higher concentrations of fluoroquinolones (ofloxacin; 74 +/- 5.8% of control at 100 micrograms/ml, p < 0.05, ciprofloxacin; 4.4 +/- 1.5% of control at 100 micrograms/ml, p < 0.01, or norfloxacin; 71 +/- 7.0% at 10 mu g/ml, p < 0.01, and 1.5 +/- 0.4% of control at 100 mu g/ml, p < 0.01) and the highest concentrations of peptides (polymyxin B; 64 +/- 3.0% of control at 100 micrograms/ml, p < 0.01, or colistin; 67 +/- 5.7% of control at 100 micrograms/ml, p < 0.01) or fosfomycin (79 +/- 6.2% of control at 100 micrograms/ml, p < 0.05) had an inhibitory effect on corneal epithelial migration. Among aminoglycosides tested, sisomicin (85 +/- 10.0% of control, not significant), dibekacin (76 +/- 11.6% of control, p < 0.05) and streptomycin (77 +/- 9.4% of control, not significant) were inhibitory at 100 micrograms/ml, but tobramycin had no effect. Penicillins (aspoxicillin, sulbenicillin or ampicillin), cephalosporins (cefmenoxime or cefminox), oxytetracycline, erythromycin and chloramphenicol did not affect epithelial migration at all. These results demonstrate that some antimicrobials are inhibitory at high concentrations, but penicillins, cephalosporins, oxytetracycline, erythromycin or chloramphenicol has no inhibitory effect on corneal epithelial migration.

Actin organization in migrating corneal epithelium of rabbits in situ

We have found previously that fibronectin enhances the migration of rabbit corneal epithelium both in vitro and in vivo. In this paper we report a change of actin localization in migrating corneal epithelium as determined by immunofluorescent microscopy. Rabbit cornea was cut into small blocks and cultured in TC-199 medium. In the normal cornea, actin was detected as diffuse fluorescence at each epithelial layer. After 8 hr of cultivation epithelial cells had not started to migrate significantly, but actin had accumulated at the cell membrane. After 24 hr, epithelial migration had begun, and actin-specific fluorescence was detected mainly in the basal cell layer at the leading edge. When fibronectin or epidermal growth factor was added to the culture medium, epithelial migration began 8 hr after initiation of culture, and at 24 hr actin-specific fluorescence at the basal side of the migrating epithelial cells appeared stronger than that of a control group cultured in TC-199 unsupplemented medium. At the same time, fibronectin-specific fluorescence was more intense beneath the migrating epithelial cells. It is known that fibronectin has an affinity to collagen, and thus it might coat the cut surface of the stroma. Epithelial cells may attach then to the stroma via coated fibronectin. When a large quantity of exogenous fibronectin is added or when fibronectin is synthesized by the addition of epidermal growth factor, it may further stimulate the organization of intracellular actin from globular form (G-actin) to fibrilar form (F-actin). As a result, the change of intracellular localization and appearance of organized actin molecule might lead to cellular migration.