Mechanochemical Switching between Growth and Differentiation during Fibroblast Growth Factor-stimulated Angiogenesis In Vitro : Role of Extracellular Matrix

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tensiondependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (>500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (<100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm 2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM. ENTRAL question in developmental biology concerns how groups of interacting cells and molecules give rise to three-dimensional tissues that exhibit specialized form as well as function. We are interested in the process by which endothelial cell growth and capillary tube formation are controlled during angiogenesis. Capillary development is an excellent system for study of histodifferentiation because cloned endothelial cells retain the ability to form branching tubular networks; i.e., to undergo "angiogenesis in vitro" (Folkman and Haudenschild, 1980). Morphogenesis of the embryonic vasculature involves two modes of vessel formation: (a) accumulation of endothelial cells into networks composed of loosely associated cellular cords that eventually form into tubes; or (b) neovascularization by sprouting from these early vessel rudiments (Coffin and Poole, 1988). In vitro angiogenesis systems best model the former. In vivo studies clearly demonstrate that neovascularization can be initiated by soluble endothelial mitogens such as basic fibroblast growth factor (FGF; Shing et al., 1985; Esch et al., 1985). However, the regulatory signals that determine whether capillary endothelial cells will grow, branch, differentiate, or involute in response to FGF appear to be provided by the local tissue microenvironment. For example, during initiation of the first capillary branches, one endothelial cell grows in response to mitogenic stimulation while its neighbors, only microns away, do not (Auprunk and Folkman, 1977). Furthermore, during later stages of neovascularization, rapidly growing capillary sprouts appear juxtaposed to regressing capillaries as well as differentiating tubes that © The Rockefeller University Press, 0021-9525/89/07/317/14 $2.00 The Journal of Cell Biology, Volume 109, July 1989 317-330 317 on July 9, 2017 jcb.rress.org D ow nladed fom have become quiescent (Clark and Clark, 1938). FGF similarly retains its multifunctionality in vitro: FGF both stimulates endothelial cell growth (Shing et al., 1985; Esch et al., 1985) and promotes formation of differentiated capillary tubes (Montesano et al., 1986). The local regulatory signals that modulate FGF action and control capillary development may be conveyed by extracetlular matrix (ECM) ~ molecules. Localized alterations of ECM composition and integrity parallel changes of vascular form during capillary initiation, elongation, differentiation, and involution (Ausprunk and Folkman, 1977; Folkman, 1982; Sariola et al., 1984; Ingber et al., 1986; Form et al., 1986). Purified matrix components also modulate the effects of angiogenic factors on endothelial cell growth (Schor et al., 1979; Ingber et al., 1987) and capillary differentiation in vitro (Maciag et al., 1982; Madri and Williams, 1983; Schor et al., 1983; Montesano et al., 1986). Yet, little is known about the mechanism by which insoluble ECM molecules transmit regulatory information to endothelial cells. Matrix proteins alter cell behavior as a result of specific binding interactions with distinct types of cell surface ECMreceptors, such as the integrin family of receptors (Hynes, 1987; Ruoslahti and Pierschbacher, 1987). However, ECM molecules cannot transmit growth and pattern-regulating signals based solely upon occupancy of cell surface ECMreceptors since the biological effects of matrix components vary greatly depending upon their structural configuration. For example, a variety of cells proliferate on rigid, collagencoated dishes (Wicha et al., 1979; Madri and Williams, 1983; Ben Ze'ev et al., 1988), but differentiate when cultured on or within malleable collagen gels (Emerman and Pitelka, 1977; Schor et al., 1983; Montesano et al., 1983; Ben Ze'ev et al., 1988). The differentiation-inducing effects of collagen gels and complex ECM substrata (e.g., laminin gels, matrigel) also can be varied by altering their mechanical integrity (Lee et al., 1984; Li et al., 1987). Cell shape is determined through the action of tensile forces that are generated within the intracellular cytoskeleton and resisted by ECM attachment points (Harris et al., 1980; Ingber and Jamieson, 1985). Thus, one of the major effects of altering ECM structural integrity is induction of cell shape changes; rigid dishes support cell extension whereas malleable substrata promote rounding (Emerman and Pitelka, 1977; Ingber and Jamieson, 1985). Endothelial cells also take on different forms on rigid dishes depending upon the type of ECM molecules used for cell attachment (Ingber et al., 1987). ECM molecules may modulate cell growth and differentiation in response to soluble factors based upon their ability to alter cell shape. Anchorage-dependent cells, such as endothelial cells, proliferate more rapidly in serum-containing medium as they become more flattened and cease growing as they take on increasingly rounded forms (Folkman and Moscona, 1978; Gospodarowicz et al., 1978). Similarly, the growth-promoting effects of different ECM molecules increase in parallel with their relative ability to support capillary cell extension in serum-free medium supplemented with FGF (Ingber et al., 1987). Matrix-dependent changes of cell shape that inhibit growth may also promote differentiation. For example, hepatocytes and mammary epithelial cells 1. Abbreviations used in this paper: ECM, extracellular matrix; FGF, basic fibroblast growth factor. cease growing and increase their expression of differentiation-specific genes when cultured on substrata that promote cell rounding (Lee et al., 1984; Li et al., 1987; Ben Ze'ev et al., 1988). The differentiated phenotype of chondrocytes (Glowacki et al., 1983), adipocyte precursors (Spiegelman and Ginty, 1983), and pheochromocytoma cells (Bethea and Kozak, 1984) can be similarly altered by modulating substrate adhesivity and controlling cell form. In this article, we explore the possibility that the ability of FGF to stimulate endothelial cell growth in one microenvironment and promote capillary differentiation in another depends on the mechanical context in which it acts. We focus on the role of ECM and tension-dependent changes of endothelial cell shape during regulation of FGF-stimulated angiogenesis in vitro. Materials and Methods In Vitro Culture Systems for Study of Angiogenesis Capillary endothelial cells were isolated from bovine adrenal cortex or human foreskin, cloned, and passaged as previously described (Folkman et al., 1979). Capillary endothelial cells from both species produce tubular netv~rks of similar size and shape when cultured under similar conditions (Folkman and Haudenschild, 1980). In our time-lapse cinematographic studies, spontaneous formation of capillary tubes was promoted by refceding human capillary cells every other day with DME (Gibco Laboratories, Grand Island, NY) supplemented with 15% human serum, endothelial cell growth supplement (5,4 mg/ml; Collaborative Research Incorporated, Bedford, MA), and tumor cell-conditioned medium (Folkman et al., 1979) mixed 1:1 with conditioned medium obtained from confluent cultures of bovine aortic endothelial cells. Tubes formed within '~1 mo after plating on gelatinized dishes. In our other spontaneous angiogenesis models, bovine capillary endothelial cells were cultured in complete medium comprised of DME supplemented with 10% calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 5 ~l/ml retinal extract (Gitlin, 1981). Similar results were also obtained using tumor-conditioned medium (Folkman et al., 1979) in place of retinal extract as a source of endothelial mitogens. In one set of experiments, cell Cultures were refed every 3 d until the cell monolayers spontaneously detached from the gelatinized surfaces of 6 well culture plates (Costar, Cambridge, MA). We