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

Abstract

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-Descemet's 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(-8) M colchicine or 2.5 X 10(-3) M 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. However, when injured endothelia were cultured in the presence of colchicine, no reaction product was observed at the cell-DM interface, although it could be detected intracellularly within RER. Incubating the tissues in the presence of puromycin abolished all extracellular and intracellular staining. These results indicate that during wound repair, corneal endothelial cells produce fibronectin and deposit it upon Descemet's membrane by a mechanism that may be mediated by microtubules.

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

“…Findings presented here demonstrate the reverse effect, that is, alteration of free tubulin subunits with colchicine, so that they become ineffective in microtubular dynamics, initiates the upregulation of tubulin synthesis. Since microtubules play pivitol roles for cell movement in the corneal endothelium during wound repair [Sabet and Gordon, 1989], and in other systems as well [Liao et al, 1995], there is a critical need to maintain a viable subunits pool. Colchicine-tubulin complexes are known to bind microtubules at either end in vitro, reducing their growth rate [Vandecandelaere et al, 1994].…”

“…Similarly, in the corneal endothelium, MTOC translocation may be coupled not only with directed migration but also to the polarized secretion of extracellular matrix protein. Disrupting microtubule integrity with colchicine prevents not only the deposition of fibronectin, but results in the restriction of cell movement [Sabet and Gordon, 1989;Gordon and Staley, 1990]. In this present study, we have investigated cytoskeletal changes in the organ cultured corneal endothelium during wound repair.…”

Differential adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells in primary culture.

“…To ascertain whether modulation of the expression of fibronectin (FN) during migration (15,16) in parallel. IF specimens were observed on a microscope equipped with epifluorescence illumination (Axiophot Zeiss) and selected areas were analyzed on a confocal laser system (MRC 600; Bio-Rad Laboratories, Richmond, CA) connected to the Axiophot microscope.…”

Endoglin Regulates the Activation and Quiescence of Endothelium by Participating in Canonical and Non-Canonical TGF-β Signaling Pathways

“…We next determined the production of various ECM proteins in Eng+/+ and Eng+/2 EC. Tenascin-C, fibronectin, osteopontin, collagen IV, thrombospondin-1 (TSP-1), and TSP-2 are the major ECM components secreted by EC and play important roles in cell migration, wound repair, and inflammation (Lund et al, 2009;Sabet and Gordon, 1989;Tucker, 1991). Fig.…”