Evidence that Nectin-3 is the soybean agglutinin binding protein on rat corneal endothelium cell surfaces.

The means by which the lectin soybean agglutinin (SBA) binds to the corneal endothelium cell surface following explantation into organ culture was investigated using Sprague-Dawley rats. SBA binding does not occur in freshly isolated and fixed rat corneal endothelium. However, after 48 h in organ culture, SBA binding occurs in a punctate pattern that clearly outlines all endothelial cells of the tissue monolayer. To determine what cell surface component was responsible for this binding, a series of experiments were employed that focused on the possibility that SBA bound to a nectin molecule(s). To this extent we performed a series of immunocytochemical localizations using antibodies against either nectin-2, nectin-3 or nectin-4. Of these, only nectin-3 bound to the endothelium in a manner that mimicked SBA binding. To further verify that nectin-3 bound SBA, displacement experiments employing non-labeled SBA were undertaken. Following a 48 h organ culture, tissues were fixed and incubated with SBA followed by exposure to nectin-3 antibody. No subsequent immunofluorescence could be detected, indicating that anti-nectin-3 binding was prevented. Likewise, when organ-cultured tissues were fixed and incubated in anti-nectin-3 antibody, followed by SBA exposure, no SBA binding could be detected. These results suggest that stresses accompanying explantation of the tissue into organ culture promote the appearance of nectin-3 around the cell periphery. The emergence of nectin-3 along the peripheral endothelial cell membrane in organ culture may imply a necessary role for this molecule in maintaining monolayer integrity and barrier function during either a pathologic condition, wound repair, or in organ storage.

Rat corneal endothelial cell migration during wound repair on the basement membrane depends more on the PI-3K pathway than the cdc-42 pathway or actin stress fibers.

Following a central transcorneal circular freeze injury, organ-cultured rat corneal endothelial cells surrounding the wound reorganize peripheral actin bands into stress fibers and migrate individually into the wound. To ascertain the significance of this rearrangement relative to morphological changes accompanying migration and wound repair, some tissues were incubated overnight in 4 µM TRITC-conjugated phalloidin to stabilize actin and prevent its reorganization. After a freeze injury to the endothelium tissues were histologically observed at 24 h post-wounding and demonstrated that despite a lack of actin organization, cells responding to the injury appeared morphologically similar to their control counterparts. Tissues cultivated in the presence of either cytochalasin B (CB), soybean agglutinin (SBA), or fluorouracil (FU) and also exhibited actin cytoskeletal disruption. Under these conditions, migration continued despite the absence of detectable stress fibers. For SBA-, CB-, and FU-treated tissues, wound repair did not significantly differ from control preparations although FU-treated tissues showed a slower repair. Electron micrographs confirmed an absence of stress fibers in migrating cells treated with any of these agents. Tissues were also treated with ML 141 and EY294002 to inhibit the cdc-42 and PI-3K pathways, respectively. While cell movement still occurred in the presence of ML 141, migration into the wound was greatly restricted in the presence of EY294002. These results indicate that rat corneal endothelial cell movement in situ does not require actin reorganization into stress fibers, but the functioning of the PI-3K pathway is indispensable for their migration along the basement membrane during wound repair.

Insulin and IGF-2 support rat corneal endothelial cell growth and wound repair in the organ cultured tissue.

The ability of insulin and IGF-2 to support wound repair in the organ-cultured rat corneal endothelium was investigated. Corneas given a circular transcorneal freeze injury, were explanted into organ cultures containing either insulin or IGF-2 and cultured up to72 h. Both factors increased [3H]-thymidine incorporation and mitotic levels compared to controls. Insulin's ability to mediate wound closure without serum was dependent on its continuous presence in the medium. PKC was also investigated in endothelial repair using the PKC promoter phorbol 12-myristate 13-acetate (PMA). Concentrations between 10-6 and 10-8 M, PMA failed to accelerate wound closure. When injured endothelia were cultured in the presence of insulin and the PKC inhibitor H-7, wound closure was also unaffected. These results indicate that insulin and IGF-2 stimulate cell growth in injured rat corneal endothelium and that insulin without the benefit of serum promotes wound closure in situ independent of the PKC pathway.

Fibronectin antibody labels corneal stromal collagen fibrils in situ along their length and circumference and demonstrates distinct staining along the cell and stromal interfaces of Descemet's membrane.

PURPOSE/AIM OF THE STUDY: An immunoperoxidase cytochemical study of fibronectin localization in the rat corneal stroma and Descemet's membrane was conducted following organ culture to determine whether stromal swelling allowed better primary antibody penetration into the normally tough fibrous corneal stroma. MATERIALS AND METHODS: Following 24 h organ culture, corneas were fixed in 4% paraformaldehyde, washed and stained overnight at 4 °C in anti-fibronectin followed by washing and incubation in an appropriate secondary antibody and exposure to protein A-HRP. Cytochemical processing was carried out in a DAB-containing medium followed by dehydration and Epon embedding. RESULTS: Observations of the stromal lamellae revealed the presence of a novel punctate staining pattern along the length of the collagen fibrils that extended around the fibril's circumference. Measurements on the peroxidase reaction product spacing indicated a periodicity of approximately 20.69 ± 3.57 nm along the fibril's length. Light microscopic immunocytochemistry revealed the presence of fibronectin staining occurred within the endothelial cell layer but only along the DM/stromal interface. Electron microscopic observations however, revealed that fibronectin staining occurred in distinct linear patterns along the length of both the endothelial and stromal DM interfaces. DISCUSSION: Results indicate that organ culture mediated swelling helps facilitate the penetration of primary antibody into the corneal stroma. Observations suggest a novel association exists between fibronectin and stromal collagen fibrils that helps to mediate the arrangement and organization of the stromal extracellular matrix. Results also definitively indicate that fibronectin is deposited along both DM interfaces suggesting that it plays a role in the adhesion of both the endothelial cell layer and stroma to Descemet's membrane to help maintain the tissue architecture within this region of the cornea.

The effects of soybean agglutinin binding on the corneal endothelium and the re-establishment of an intact monolayer following injury--a short review.

This short review summarizes the localization and effects of the plant lectin soybean agglutinin (SBA) on the injured and non-injured organ-cultured rat corneal endothelium. Although the tissue exists as a non-cycling monolayer on the posterior corneal surface a circular freeze injury promotes wound repair as cells initiate DNA synthesis, mitosis and migration. As a result, by 24 h post-injury, endothelial cells express a surface protein that binds SBA in a diffuse punctate pattern, which by 48 h after injury, becomes confined to the cell periphery. As healing proceeds, SBA binding dramatically declines, such that, only scattered binding is observed by 72 h after wounding. In non-injured organ-cultured endothelia, weak SBA binding appears 24 h after explanation but becomes prominently detected around the cell periphery by 48 h. Incubating injured or non-injured endothelia in SBA leads to alterations in their cellular appearance due to the fact that lectin exposure results in the disruption of the actin cytoskeleton. Although this does not affect migration, treatment with either SBA or N-acetylgalactosamine (the SBA binding sugar) does interfere with the reestablishment of cell-cell contact. It is postulated that the surface protein that binds SBA is expressed during conditions that are stressful to the tissue. During organ-culture the protein's appearance suggests a cellular response to explantation in order to enhance or maintain monolayer integrity. In wound repair its appearance may serve to establish preliminary cell-cell contact during the restoration of the endothelial monolayer.

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

5-Fluorouracil interferes with actin organization, stress fiber formation and cell migration in corneal endothelial cells during wound repair along the natural basement membrane.

Corneal endothelial cells respond to a circular freeze wound by undergoing actin cytoskeletal reorganization that is mainly characterized by the disappearance of circumferential microfilament bundles (CMBs) and the subsequent appearance of distinct stress fibers. This cytoskeletal rearrangement is associated with changes in cell shape as migrating cells lose their polyhedral appearance, spread out, and assume a stellate morphology with cell processes extending outward into the injured area. We report here that in the presence of low concentrations (0.01-0.l mM) of the anti-metabolite 5-fluorouracil (5-FU), characteristic actin organization becomes disrupted and migrating cells do not display elongated processes typical of control tissues and translocation into the injury zone is retarded, but not inhibited. Rhodamine phalloidin staining revealed no evidence of stress fiber formation. A higher concentration of 5-FU (1.0 mM) not only prevented formation of discernible stress fibers but also resulted in a more restricted cell movement during wound repair. That this was not a cytotoxic effect was demonstrated by transferring tissues back into standard medium allowing endothelia to reinitiate migration and undergo complete wound healing by 72 h post-transfer. Overnight incubation of endothelia in 4 muM phallacidin resulted in limited CMB disruption the extent of which was dependent on the 5-FU concentration. The effects of 5-FU on the actin cytoskeleton are reversible and by 24 h after placing treated endothelia into medium without 5-FU, actin begins to become re-established and by 48 h microfilament patterns in the tissue resemble those of non-treated endothelia. Similarly, when non-injured tissues are cultured in the presence of 5-FU for 24 h, subsequently injured and returned to standard medium, they exhibit no stress fibers when observed at 24 h post-wounding. However, by 48 h post-injury these cells now display stress fibers and extend processes into the wound area. Biochemical studies on isolated muscle actin demonstrated that actin polymerization is unaffected in the presence of either 0.01 or 1 mM 5-FU as determined by the F-actin sedimentation and falling ball viscosity techniques. Thus, the mechanism(s) by which 5-FU exerts its actions on the actin cytoskeleton appears to be one of an indirect nature.

Microfilament disruption in a noncycling organized tissue, the corneal endothelium, initiates mitosis.

The adult corneal endothelium represents a noncycling cell population that resides as a monolayer on its basement membrane, Descemet's membrane. Evidence is presented for the first time, showing that mitotic regulation in this organized tissue, residing on its natural basement membrane, is coupled to microfilament integrity. When mitotically quiescent rat corneal endothelia are organ cultured in medium containing serum and cytochalasin B, low levels of mitosis are initiated. Supplementing the culture medium with either insulin or IGF-2 augments this response and results in increased cell density within the tissue monolayer. Fluorescence microscopy of actin using TRITC-conjugated phalloidin revealed that cellular circumferential microfilament bundles appear unaffected by cytochalasin B treatment, whereas the cytoplasmic microfilaments appear to be completely disrupted. These results suggest the possibility that the actin cytoskeleton is involved with the regulation of cell growth in the corneal endothelium.