Sheldon R. Gordon

Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement

The distribution of the extracellular matrix (ECM) protein, fibronectin (FN), has been examined ultrastructurally in noninjured and injured rat corneal endothelium in vivo and in vitro by immunoperoxidase cytochemistry. In noninjured endothelia, FN was observed within the rough endoplasmic reticulum (RER) cisternae but not along the cell‐Descemets membrane (DM) interface. Twenty‐four and 48 h after a circular freeze injury, immunoperoxidase reaction product was detected at the cell‐DM interface as well as within cytoplasmic vesicles and intercellular spaces. By 1 and 2 wk post‐injury, a line of reaction product could still be demonstrated at the cell‐DM interface and evidence for newly deposited basement membrane material was observed in this region. In order to understand whether fibronectin deposition during wound repair was dependent on cytoskeletal influences, organ culture experiments were performed in which the media was supplemented with either 10−8M colchicine or 2.5 × 10−3M cytochalasin B. Without inhibitors, injured corneas cultured for 24 h had FN deposition at the cell‐DM interface similar to the in vivo results. Corneas cultured in the presence of cytochalasin B also showed FN deposition at the cell‐DM interface.

Demonstration of microfibrils in Bruch's membrane of the eye.

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruchs membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruchs membrane may depend on both the elastin and microfibrillar components.

Inhibition of cytoskeletal reorganization stimulates actin and tubulin syntheses during injury-induced cell migration in the corneal endothelium

A single layer of squamous epithelial cells termed the “endothelium” resides upon its natural basement membrane (Descemets membrane) along the posterior surface of the vertebrate cornea. A well‐defined circular freeze injury to the center of the tissue exposes the underlying basement membrane and results in the directed migration of surrounding cells into the wound center. This cellular translocation is characterized by the reorganization of the actin and tubulin cytoskeletons. During migration, circumferential microfilament bundles are replaced by prominent stress fibers while microtubules, observed as delicate lattices in non‐injured cells, become organized into distinct web‐like patterns. To determine whether this cytoskeletal reorganization requires actin or tubulin synthesis, injured rabbit endothelia were organ cultured for various times and metabolically labeled with 35S‐methionine/cystine (250 μCi/ml) for the final 6 h of each experiment. Analysis of actin and tubulin immunoprecipitates indicated no significant increases in 35S incorporation occurred during the course of wound repair when compared to isotope incorporation in noninjured tissues. However, when cytoskeletal reorganization was hampered, either by pre‐treating tissues with 7 μM phalloidin to stabilize their circumferential microfilament bundles, or culturing in the presence of 10−8M colchicine to dissociate microtubules, 35S incorporation increased significantly into both actin and tubulin immunoprecipitates at 48 h post‐injury. Furthermore, in both cases, exposure to actinomycin D substantially suppressed isotope incorporation. These results indicate that cytoskeletal rearrangement of microfilaments and microtubules during wound repair, in corneal endothelial cells migrating along their natural basement membrane, utilizes existing actin and tubulin subunits for filament reorganization. Disrupting this disassembly/reassembly process prevents cytoskeletal restructuring and leads to the subsequent initiation of actin and tubulin syntheses, as a result of increased transcriptional activity. J. Cell. Biochem. 67:409–421, 1997.

Actin, myosin, and laminin localization in retinal vessels of the rat

SummaryActin, myosin, and laminin have been localized in retinal vessels of normal rats by fluorescence microscopy. Actin was localized with the fluorescent F-actin binding toxin nitrobenzoxadiazole phallacidin (NBD-Ph). Indirect immunofluorescence was used to localize myosin and laminin. In addition, laminin localization was also performed with the Protein A-horseradish peroxidase (PA-HRP) method. NBD-Ph staining gave strong fluorescence in both retinal capillaries and larger vessels. Anti-myosin fluorescence could also be observed in trypsin digests of the retinal vasculature. Strong fluorescence of PA-HRP reaction product could be detected in the walls of vessels exposed to antilaminin antibody. Actin distribution in vessels of the RCS rat with inherited retinal degeneration (retinal dystrophic RCS rat) was also studied. After exposure to NBD-Ph, all capillaries showed fluorescence. However, it was more intense in many of the capillaries in the outer retina, which also appeared morphologically abnormal. Electron microscopy of retinal capillaries fixed in 2.5% glutaraldehyde containing 8% tannic acid revealed numerous micro filaments in the pericyte cytoplasm amd some in the basal portion of endothelial cells. In pericytes, these microfilaments are in close association with the endothelial side of the cell. Tangential sections through this region indicate that these filaments may be anchored to the membrane at this site.

Surface-associated vesicles in retinal arterioles and venules.

SummaryThe surface-associated vesicles in retinal arterioles and venules were studied after fixation in glutaraldehyde-tannic acid or after intravitreal injection of peroxidase or lactoperoxidase. The vesicles were concentrated along the abluminal (basal) surface of the endothelial cells and along the plasma membranes of smooth muscle cells in arterioles and of pericytes in post-capillary venules. They were rarely encountered in the deeper regions of these cells. In perpendicular sections through the cell surface the majority of vesicles were in continuity with the plasma membrane whereas in tangential sections, they appeared to lie “free” in the cytoplasm. All such vesicles were labeled after exposure to tannic acid or to the heme-proteins. Peroxidase-reaction product was never seen in the lumen of the vessels. These observations suggest that the surface vesicles in endothelial cells, smooth muscle cells and pericytes are invaginations of the plasma membrane and are thus not involved in the transcytosis or endocytosis of proteins. The vesicles in the latter two cell types may be involved in some aspect of contractility rather than pinocytosis.

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

Abstract 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.