James Gailit

Wound repair in the context of extracellular matrix.

Wound repair requires a continually evolving network of interactions among cells, cytokines and the extracellular matrix. Cell-surface integrins provide a mechanical connection between matrix components and the cytoskeleton, and integrins can transduce an astonishing variety of signals along pathways that may intercept the pathways triggered by cytokine receptors.

Fibrinogen Is a Ligand for Integrin α5β1 on Endothelial Cells

Previous studies have shown that fibrinogen can associate with endothelial cells via an Arg-Gly-Asp (RGD) recognition specificity. In the present study, we have characterized the specificity of fibrinogen binding to endothelial cells under different cation conditions. Fibrinogen binding to suspended endothelial cells was selectively supported by Mn2+ and was suppressed by Ca2+. The Mn2+-supported interaction was completely inhibited by RGD peptides but not by αvβ3 blocking monoclonal antibodies. In contrast, the interaction was completely blocked by two α5β1 monoclonal antibodies. This interaction was not mediated by fibronectin bound to the integrin; could be demonstrated with purified α5β1; and also was observed with a second α5β1-bearing cell type, platelets. The binding of fibrinogen to α5β1 on endothelial cells in the presence of Mn2+ was time-dependent, specific, saturable, and of high affinity (Kd = 65 nM). By employing anti-peptide monoclonal antibodies, the carboxyl-terminal RGD sequence at Aα 572-574 was implicated in fibrinogen recognition by α5β1. Two circumstances were identified in which α5β1 interacted with fibrinogen in the presence of Ca2+: when the receptor was activated with monoclonal antibody (8A2) or when the fibrinogen was presented as an immobilized substratum. These results identify fibrinogen as a ligand for α5β1 on endothelial and other cells, an interaction which may have broad biological implications.

Platelet‐derived growth factor and inflammatory cytokines have differential effects on the expression of integrins α1β1 and α5β1 by human dermal fibroblasts in vitro

Dermal fibroblasts are essential for the repair of cutaneous wounds. Fibroblasts presumably use cell surface receptors of the integrin family during migration into a wound from the adjacent uninjured tissue and for the subsequent matrix repairs. We have investigated the possible roles of platelet‐derived growth factor and inflammatory cytokines in the regulation of integrin expression on wound fibroblasts using a porcine cutaneous wound model and cultured human cells. Tissue specimens collected from 4‐day pig wounds were stained with antibodies specific for the α1 and α5 integrin subunits. Staining for α1 was markedly decreased on fibroblasts adjacent to the wound and in the granulation tissue, while staining for α5 was clearly enhanced in both locations. Normal adult human dermal fibroblasts in culture express the integrins α1β1, a collagen receptor, and α5β1, a fibronectin receptor. Quantitative flow cytometry was used to measure cell surface integrin expression after treatment with platelet‐derived growth factor (PDGF)‐AA, PDGF‐AB, or PDGF‐BB. Each isoform of PDGF produced a significant decrease in the level of α1 present on the cell surface and an increase in the level of α5. Furthermore, PDGF‐BB produced a corresponding decrease in α1 mRNA and an increase in α5 mRNA. In contrast, treatment with three inflammatory cytokines, IL‐1β, TNF‐α, and IFN‐γ, produced clear increases in the levels of α1 and α5 present on the cell surface. Our observations suggest that the differential effects of PDGF and inflammatory cytokines may be part of the mechanism regulating the expression of α1 and α5 integrins by dermal fibroblasts during wound repair.

Osteoclast integrin alphaVbeta3 is present in the clear zone and contributes to cellular polarization.

Abstract.Osteoclasts are primary bone-resorbing cells with highly polarized cytoplasmic structures, such as ruffled borders and clear zones. In the present study, we have examined the subcellular localization and function of the αVβ3 integrin in primary rat osteoclasts and mouse osteoclast-like multinucleated cells (OCLs) formed in vitro. At the ultrastructural level, the specific immunoreactivity of both αV and β3 subunits is localized not only along the plasma membranes of osteoclast ruffled borders and basolateral membranes but also in clear zones. Addition of GRGDS (Gly−Arg−Gly−Asp−Ser) peptide to the culture medium during a pit formation assay reduces resorbed areas on dentine slices in a dose-dependent manner. Treatment with the GRGDS peptide also dose-dependently inhibits the formation of a ringed structure of F-actin (an ”actin ring”) in OCLs on dentine slices. Electron-microscopic analysis has revealed that OCLs treated with GRGDS peptide at 1 mM can adhere to dentine slices with unusually broad or poorly defined clear zones but do not form the ruffled burder structure. These results suggest that integrin αV and β3 subunits localized in the ruffled border/clear zone complex of osteoclasts are essential for the functional osteoclast-to-bone matrix interaction and the structural polarization of osteoclasts.

Integrins in Wound Repair

The integrin family of cell adhesion receptors consists of over 20 members, which mediate cell surface interactions with extracellular matrix or (in some cases) with other cells (Akiyama et al., 1990b; Albelda and Buck, 1990; Clark, 1990; Hemler, 1990; Hogg, 1991; Ruoslahti, 1991; Shattil and Brugge, 1991; Yamada, 1991; Damsky and Werb, 1992; Ginsberg et al., 1992; Hynes, 1992; Akiyama and Yamada, 1993; Gailit and Clark, 1993; Glukhova and Thiery, 1993; Gumbiner, 1993; Juliano and Haskill, 1993; Sastry and Horwitz, 1993; Sonnenberg, 1993; Tuckwell et al., 1993; Zetter, 1993; Springer, 1994). Each integrin is a heterodimer, consisting of one a and one β subunit in a noncovalent complex. As summarized in Fig. 1, only certain combinations of integrins are observed: major groupings include integrins of the β1 subfamily and integrins containing the αv subunit. Changes in either the α or the β subunit of integrin heterodimers alter their specificity for ligands, as summarized in Table I.

Echistatin, a disintegrin, inhibits sperm-oolemmal adhesion but not oocyte penetration**Funded in part by Research Foundation Grant No. 9384, State University of New York at Stony Brook, Stony Brook, New York.

OBJECTIVE To study the effects of echistatin, a disintegrin known to block the binding of fibronectin (FN) and vitronectin to their respective integrin receptors, alpha 5 beta 1 and alpha v beta 3, on the adhesion of human spermatozoa to the oolemma of zona-free hamster eggs and their subsequent penetration. DESIGN Motile capacitated human spermatozoa and zona-free hamster eggs were coincubated in the presence of echistatin or in its absence and observed at short serial intervals. Whole mounts of these eggs, washed out of sperm suspension and stained with acridine orange, were scored for numbers of oolemmal adherent and penetrating sperm. SETTING University Hospital laboratories. PATIENTS Known fertile semen donors. MAIN OUTCOME MEASURES Numbers of spermatozoa adherent to the oolemma and those penetrating the oocyte. RESULTS Sperm adherence to the oolemma was reduced significantly at micromolar concentrations of echistatin, in a concentration-dependent manner. In contrast, echistatin did not inhibit the penetration of oocytes by sperm that had become adherent to the oolemma despite the presence of echistatin. CONCLUSION We propose that two processes occur in the binding of sperm to the oolemma, one that is echistatin sensitive and possibly involving the integrin receptors that recognize FN and vitronectin, and a second process, resistant to echistatin, that leads to gamete membrane fusion.