Spatiotemporal Segregation of Endothelial Cell Integrin and Nonintegrin Extracellular MatiN-binding Proteins during Adhesion Events

Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin/~1, /33, and associated t~ subunits as well as a/31 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrixbinding specificities of each of these integrin subclasses. By 1 h after plating, organization of/~1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas/~3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrixbinding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence,/~1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69-e.g. , antibodies to LB69 as well as YIGSR-NH2 pept ide-do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs. NOOTHELIAL cells are generally nonproliferative stationary polar cells that create a highly metabolic nonthrombogenic luminal surface within blood vessels. In intimate contact with the basal aspect of the endothelium is a complex basement membrane comprised of a variety of glycoproteins and proteoglycans (Madri et al., 1980a). In response to an injury, such as balloon catheter denudation, endothelial cells migrate over and proliferate on this matrix to reconstitute a nonthrombogenic vascular lining (Haudenschild and Schwartz, 1979). The matrix, itself, is a dynamic surface whose composition changes in response to synthetic and degradatory properties of endothelial, smooth muscle, and inflammatory cells (Pratt et al., 1985). In vitro, extracellular matrix components such as fibronectin (Fn),~ laminin (Ln), interstitial collagens, and basement 1. Abbreviations used in this paper: BAEC, bovine aortic endothelial cell; Fb, fibrinogen; Fn, fibronectin; HIFCS, heat-inactivated FCS; HUVEC, membrane collagens can all modulate the shape and behavior of bovine aortic endothelial cells (BAECs) (Madri et al., 1988). BAEC-substratum interaction may differ from many other cell systems since workers have long recognized that chelators such as EDTA are insufficient to detach BAECs from tissue culture vessels, and even routine tissue culture requires a combination of trypsin and EDTA (Pratt et al., 1984). Nevertheless, the cellular binding proteins for extracellular matrix proteins that investigators have documented in other systems may also be potential mediators of BAEC interaction with matrix elements (Rao et al., 1983; Van Mourik et al., 1985; Ruoslahti and Pierschbacher, 1987). In earlier studies (Yannariello-Brown et al., 1988), this laboratory has demonstrated the presence of a previously described human umbilical vein endothelial cell; Ln, laminin; Sulfo-MBS, maleimidobenzoyl-sul fosuccinimido ester. © The Rockefeller University Press, 0021-9525/90/03/789/13 $2.00 The Journal of Cell Biology, Volume 110, March 1990 789-801 789 on A uust 6, 2017 jcb.rress.org D ow nladed fom 69-kD Ln-binding protein (Rao et al., 1983; Malinoff and Wicha, 1983; Wewer et al., 1987; Yow et al., 1988) on the BAEC surface. This protein, referred to as LB69, exhibits an organization modulated by extracellular Ln, can be radioactively labeled at the cell surface, and binds to Ln immobilized on Sepharose (Yannariello-Brown et al., 1988). Some investigators have suggested that LB69 may bind Ln via a B1 chain tyrosine-isoleucine-glycine-serine-arginine (YIGSR) sequence (Graf et al., 1987a,b). However, the observation that LB69 is not the only species purified by such Ln affinity chromatography (Yannariello-Brown et al., 1988) suggested the existence of other Ln-binding proteins. Furthermore, the relative specificity of LB69 for Ln implied the existence of different binding proteins for other matrix components such as Fn and fibrinogen (Fb). The "integrin" class of proteins are good candidates for such binding proteins (Hynes, 1987). Integrins have been shown to bind largely in a divalent cation-dependent manner to extracellular matrix proteins such as Fn, Fb, and Ln via an arginine-glycine-aspartate (RGD) ligand sequence (Ruoslahti and Pierschbacher, 1986). This sequence appears in a presumed/3 turn of Fn in the sequence GRGDSP (Pierschbacher and Ruoslahti, 1984) and in the Ln A chain as well (Sasaki et al., 1988). Fb also contains such an RGD sequence (Plow et al., 1985). Previously described integrin receptors (Buck et al., 1986; Tamkun et al., 1986; Argraves et al., 1987; Fitzgerald et al., 1987a,b; Hynes, 1987) are comprised of two disulfide-linked subunits, an ot and smaller /$, and the molecular weights of several bands purified from BAECs by Ln affinity chromatography in addition to LB69 (Yannariello-Brown et al., 1988) are not inconsistent with those of integrin subunits. Moreover, others have documented that two classes of integrins, referred to as/~1 and 83 for their class of/~ subunits (Hynes, 1987), are present in human umbilical vein endothelium (Plow et al., 1986; Newman et al., 1986; Charo et al., 1987; Cheresh, 1987; Fitzgerald et al., 1987b; Dejana et al., 1988a,b) and in rat liver endothelial cells (Johansson et al., 1987) and they have noted their involvement in cell attachment to extracellular matrix. Fitzgerald et al. (1985) and Hayman et al. (1985) have identified/~3 integrins in bovine aortic endothelium. In this communication, we have used RGD peptides and specific antisera to integrins to demonstrate that BAECs do express/$1 and/33 class integrin proteins and that they participate in BAEC attachment to Ln, Fn, and Fb. Furthermore, despite sharing a common ligand sequence (RGD) these integrins possess certain binding specificities for their extracellular matrix ligands which can in turn modulate integrin organization. We also confirm that nonintegrin-dependent mechanisms contribute to BAEC adhesion to extracellular matrix and we show that integrin and nonintegrin BAEC proteins exhibit a spatiotemporal segregation during attachment, spreading, and migration on Ln. Materials and Methods