Frederick Grinnell

Cellular Adhesiveness and Extracellular Substrata

Publisher Summary This chapter discusses the cellular adhesiveness and extracellular substrata. Cell adhesiveness is a fundamental cell property. It plays a role in developmental processes such as cell migration during embryogenesis and morphogenesis in response to particular extracellular matrices; it plays a role in homeostatic processes such as tissue and organ stability, thrombosis, inflammation, and wound healing; and it plays a role in the pathology of various disease states, for instance, in the invasive and metastatic behavior of malignant cells, in disorders of platelet function, and in disorders of leukocyte function. Adhesiveness is common to many different cell types under a variety of conditions, and the underlying mechanisms are analogous for active cell-substratum adhesion occurring in vitro to artificial and to model physiological substrata and in vivo to fibrinogen or fibrin deposits, to the basement membrane, and to other acellular components of the connective tissue.

Fibroblast biology in three-dimensional collagen matrices

Research on fibroblast biology in three-dimensional collagen matrices offers new opportunities to understand the reciprocal and adaptive interactions that occur between cells and surrounding matrix in a tissue-like environment. Such interactions are integral to the regulation of connective tissue morphogenesis and dynamics that characterizes tissue homeostasis and wound repair. During fibroblast-collagen matrix remodeling, mechanical signals from the remodeled matrix feed back to modulate cell behavior in an iterative process. As mechanical loading (tension) within the matrix increases, the mechanisms used by cells to remodel the matrix change. Fibroblasts in matrices that are under tension or relaxed respond differently to growth factor stimulation, and switching between mechanically loaded and unloaded conditions influences whether cells acquire proliferative/biosynthetic active or quiescent/resting phenotypes.

Fibroblast–collagen-matrix contraction: growth-factor signalling and mechanical loading

Fibroblast-collagen-matrix contraction provides a unique way to study reciprocal geometric and mechanical interactions between fibroblasts and extracellular matrix. Such interactions are difficult to appreciate or examine in routine cell culture because the culture surface is usually fixed in place. Forces exerted on collagen fibrils by cells cause isometric tension to develop in the cells if the collagen resists deformation; by contrast, the cells remain mechanically unloaded in the absence of matrix resistance. Recent evidence suggests that the state of cellular mechanical loading determines the mechanism that cells use to regulate contraction.

Fibroblast adhesion to fibrinogen and fibrin substrata: Requirement for cold-insoluble globulin (plasma fibronectin)

We carried out experiments to determine conditions for fibroblast adhesion to fibrinogen and fibrin substrata. Baby hamster kidney (BHK) cells did not attach to substrata composed of purified fibrinogen or fibrin. When cold-insoluble globulin (CIG) (plasma fibronectin) was bound to fibrinogen or fibrin substrata, adhesion of BHK sells was observed and the extent of adhesion was dependent upon the CIG conecntration. Binding of CIG to fibrinogen or fibrin substrata in the presence of Factor XIII (factor) under covalent crosslinking conditions resulted in a marked increased in the ability of the substrata to support cell adhesion. Control experiments indicated that CIG formed the sites on the fibrinogen and fibrin substrata to which the cells were attaching. In addition, the effect of factor XIII was shown to require covalent crossliking of CIG to the fibrinogen or fibrin, which involved a glutamine residue on the CIG molecule and could be prevented by prior crosslinking of CIG with putrescine or with itself. The enhanced ability of Factor XIII-crosslinked CIG substrata to support cell adhesion could not be accounted for by the absolute amount of CIG bound to the substrata. We present in this paper the possibility that the orientation of CIG on the substrata is the critical factor.

Studies on the biocompatibility of materials: fibroblast reorganization of substratum-bound fibronectin on surfaces varying in wettability.

The ability of human fibroblasts to remove and reorganize fibronectin (FN) bound on material surfaces was studied as a novel feature of material surface biocompatibility. Other traditional parameters of biocompatibility analyzed included cell spreading, clustering of fibronectin receptors into focal adhesions, development of stress fibers, and cell growth. Five different materials with surface wettability ranging from hydrophilic (underwater contact angle 25 degrees) to hydrophobic (underwater contact angle 111 degrees) were used, i.e., clean glass (GLASS), aminopropylsilane (APS), octadecylsilane (ODS), polylactate (PL), and silicone (SI). When cells were cultured on these materials in serum-containing medium, formation of FN receptor-rich focal adhesions and actin stress fibers were more evident on the hydrophilic surfaces (GLASS and APS) compared to the hydrophobic ones (PL, ODS, and SI). Cell growth showed a similar pattern, that is, increased cell proliferation with increasing material surface wettability. Preadsorption of FN on the material surfaces increased subsequent cell spreading and cytoskeletal reorganization on hydrophobic surfaces except SI. Removal and reorganization of FN from the material surfaces into extracellular matrixlike structures occurred on GLASS but not on less wettable surfaces, suggesting that this removal/reorganization process may be more sensitive to changes in surface wettability than other parameters of biocompatibility.

Initial adhesion of human fibroblasts in serum-free medium: Possible role of secreted fibronectin

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.

Cell Motility and Mechanics in Three-Dimensional Collagen Matrices

Fibrous connective tissues provide mechanical support and frameworks for other tissues of the body and play an integral role in normal tissue physiology and pathology. Three-dimensional collagen matrices exhibit mechanical and structural features that resemble fibrous connective tissue and have become an important model system to study cell behavior in a tissue-like environment. This review focuses on motile and mechanical interactions between cells—especially fibroblasts—and collagen matrices. We describe several matrix contraction models, the interactions between fibroblasts and collagen fibrils at global and subcellular levels, unique features of mechanical feedback between cells and the matrix, and the impact of the cell-matrix tension state on cell morphology and mechanical behavior. We develop a conceptual framework to explain the balance between cell migration and collagen translocation including the concept of promigratory and procontractile growth factor environments. Finally, we review the significance of these concepts for the physiology of wound repair.

Stress relaxation of contracted collagen gels: Disruption of actin filament bundles, release of cell surface fibronectin, and down-regulation of DNA and protein synthesis☆

Relaxation of stressed collagen gels provides a model system uniquely suited to studying the regulation of cell morphology and biosynthetic function by tissue organization. Stress relaxation results in rapid, synchronous changes in cell morphology without enzymatic or other drug treatments, and makes possible an analysis of the initial cellular events associated with changes in tissue organization. During the first hour after stress relaxation, we observed transient hypercontraction of collagen gels and loss of collagen fibril organization as stress in the system dissipated. Morphological changes in the fibroblasts included retraction of pseudopodia, collapse of cytoplasmic actin filament bundles, and loss of cell surface fibronectin. Accompanying these morphological changes, we observed marked decreases in DNA and protein synthesis, especially of fibronectin and type I procollagens. These results show that changes in tissue organization can exert rapid and profound effects on the morphology and biosynthetic function of cells within the tissue.

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.

Differences in the Regulation of Fibroblast Contraction of Floating Versus Stressed Collagen Matrices

To learn more about the regulation of contraction of collagen matrices by fibroblasts, we compared the ability of lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) to stimulate contraction of floating and stressed collagen matrices. In floating collagen matrices, PDGF and LPA stimulated contraction with similar kinetics, but appeared to utilize complementary signaling pathways since contraction obtained by the combination of growth factors exceeded that observed with saturating concentrations of either alone. The PDGF-simulated pathway was selectively inhibited by the protein kinase inhibitor KT5926. In stressed collagen matrices, PDGF and LPA stimulated contraction with different kinetics, with LPA acting rapidly and PDGF acting only after an ∼1-h lag period. Pertussis toxin, known to block signaling through the Gi class of heterotrimeric G-proteins, inhibited LPA-stimulated contraction of floating but not stressed matrices, suggesting that LPA-stimulated contraction depends on receptors coupled to different G-proteins in floating and stressed matrices. On the other hand, the Rho inhibitor C3 exotransferase blocked contraction of both floating and stressed collagen matrices. These results suggest the possibility that distinct signaling mechanisms regulate contraction of floating and stressed collagen matrices.