Cells and Materials Volume 6 Number 1 Numbers 1-3 Article 20 1996 The Effect of Vitronectin and Other Extracellular Matrix Molecules on Endothelial Expansion and Plasminogen Activation P. Anne Underwood CSIRO Molecular Science Penny A. Bean CSIRO Molecular Science Follow this and additional works at: https://digitalcommons.usu.edu/cellsandmaterials Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Underwood, P. Anne and Bean, Penny A. (1996) "The Effect of Vitronectin and Other Extracellular Matrix Molecules on Endothelial Expansion and Plasminogen Activation," Cells and Materials: Vol. 6 : No. 1 , Article 20. Available at: https://digitalcommons.usu.edu/cellsandmaterials/vol6/iss1/20 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Cells and Materials by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Cells and Materials Vol. 6, No. 1-3, 1996 (Pages 193-207) 1051-6794/96$5.00 + . 25 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA THE EFFECT OF VITRONECTIN AND OTHER EXTRACELLULAR MATRIX MOLECULES ON E~'DOTHELIAL EXPANSION AND PLASMINOGEN ACTIVATION P. Anne Underwood• and Penny A. Bean Cooperative Research Centre for Cardiac Technology and CSIRO Division of Biomolecular Engineering, Sydney Laboratory, N. Ryde, NSW, Australia (Received for publication August 17, 1996, and in revised form November 11, 1996) Abstract Introduction Endothelial recovery following procedures used to The endothelium forms an important barrier to con­ alleviate blood vessel occlusion is modulated by the local tact of the plasma and blood cells with underlying tissue, extracellular matrix upon which it has to migrate and as well as presenting a non-thrombogenic surface to proliferate. This extracellular material is derived from circulating blood. Procedures used to alleviate blood vessel wall cells, and plasma proteins which bind to the vessel occlusion such as balloon angioplasty, end­ exposed surfaces. We have demonstrated that arterectomy, laser ablation and vascular grafting result vitronectin adsorbs efficiently to tissue culture in varying degrees of endothelial damage and denuda­ polystyrene in competition with other plasma proteins, tion. Such denuded areas are prone to the development which suggests that it may adsorb to biomaterial surfaces of intimal thickening involving migration and proli­ in vivo. We have compared the adhesion, migration and feration of vascular smooth muscle cells, which proliferation of human umbilical artery endothelial cells frequently results in restenosis (Casscells, 1992). More on surface-coated vitronectin, with other extracellular recently stents have been introduced as a mechanism of matrix molecules encountered in this environment, maintaining lumen size against the forces of elastic namely fibronectin, laminin and collagen types I and IV. recoil and narrowing due to vessel remodeling. Endothelial proliferation was significantly reduced on the Although these devices have reduced the incidence of vitronectin surface. This was correlated with an restenosis somewhat (Goldberg et al., 1995), they increase in the ratio of plasminogen inhibitor-! to actually increase vascular smooth muscle cell hyperplasia urokinase in the cell/matrix layer. Laminin coated (Rogers and Edelman, 1995). The rate and extent of surfaces limited increases in endothelial culture area, due endothelial recovery is thought to be a controlling factor to poorer cell spreading on this surface. Such of the magnitude of the smooth muscle cell response combination of cellular responses to vitronectin and (Schwartz et al., 1980; Reidy et al., 1982; Clowes et laminin would discourage endothelial recovery, and al., 1983; Doomekamp et al. 1996). Recovering encourage smooth muscle hyperplasia in vivo. These endothelium has to attach, migrate and proliferate over considerations are important in the design of biomaterial the damaged surface and is likely to be profoundly surfaces to optimise endothelial recovery. affected by the nature of the local extracellular matrix (ECM) with which it makes contact (Madri et al., 1989; Key Words: Endothelium, extracellular matrix, Madri and Marx, 1992). The source of this ECM is vitronectin, expansion assay, adhesion, migration, damaged basement membrane and underlying connective plasminogen activator. tissue, material secreted by vascular smooth muscle cells, and plasma protein molecules such as vitronectin and fibronectin, which readily bind to ECM components of exposed connective tissue (Tomasini and Mosher, .. Address for correspondence 1991; Preissner and Potzsch, 1995; Yamada, 1991). P. Anne Underwood Vitronectin also binds competitively to certain CSIRO Molecular Science biomaterial surfaces in the presence of other plasma Sydney Laboratory, P.O. Box 184, proteins (Bale et al. 1989; Horbett, 1994). .t ... North Ryde, NSW 2113 Australia The effects of the abundant plasma protein Telephone Number: +61-2-9490-5131 vitronectin upon endot~eliiil cell, behavi9.ur ·have been FAX Number: +61-2-9480-5005 little studied to date. This contrasts 'with the e~te~sive E.mail: [email protected] in vitro studies on th~ effects ' of a number of .ECM. 193 P.A. UndeJWood and P.A. Bean molecules (such as fibronectin, laminin and collagens) normal wound healing and to optimise design of upon endothelial cell adhesion, spreading, migration and biomaterial surfaces. proliferation (reviewed by Madri et al., 1992; Herman, 1987; Ingber, 1991). When tissue culture polystyrene is Materials and Methods exposed to growth media containing serum, vitronectin adsorbs to the surface in significant concentrations while Materials fibronectin does not (Steele et al., 1993). We have The following ECM molecules were used to coat previously shown that bovine endothelial cells are surfaces. Bovine fibronectin (FN), rat type I collagen dependent on this adsorbed vitronectin for attachment to (col I), mouse Engelbreth HolmSwarm sarcoma (EHS) and growth on the culture surface (UndeJWood and type IV collagen (col IV), EHS laminin (LM) and Bennett, 1989). In contrast, it is well known that most gelatin type B solution were obtained from Sigma (St. human endothelial cells will not grow well on tissue Louis, MO, USA). Bovine vitronectin (VN) was culture polystyrene unless it is precoated with fibronectin purified from bovine serum using a monoclonal antibody or gelatin. By inference these cells are therefore affinity column as previously described (UndeJWood and refractory to a vitronectin coated surface, unlike the Bennett, 1989). Chicken FN was purified from fresh bovine endothelial cells. Vitronectin has been localised plasma by affinity chromatography on gelatin Sepharose in atherosclerotic lesions (Nicolescu et al., 1987, 1989; (Pharmacia, Uppsala, Sweden) as described (Ruoslahti Guettier et al., 1989) and binds avidly to biomaterial et al., 1982). All ECM molecules were stored in surfaces and to several ECM components (Horbett, aliquots at -70°C. All other chemicals were of 1994; Tomasini and Mosher, 1991). The biological analytical grade. ECM molecules were tested for purity consequences of this adsorption for endothelial cell by a combination of SDS electrophoresis with protein recoverage are currently unknown. At sites of staining and Western blotting and sensitive ELISA angioplasty, stent placement, or vascular graft assays using monoclonal antibodies (mAbs) or polyclonal anastomosis, resulting endothelial denudation, platelet antisera which displayed cross-species reactivity. No activation and local thrombus formation are likely to cross contamination of ECM molecules was found activate plasma vitronectin (Tomasini and Mosher, 1991; except for a trace of col IV present in the col I Preissner et al., 1993), with resultant localised binding preparation. All solutions used were prepared using to exposed ECM and biomaterial surfaces. This would pyrogen free water (Baxter Healthcare Pty Ltd, Old present a potentially unfavourable surface for endothelial Toongabbie, NSW, Australia) and small aliquots were recovery. filtered through sterile Zetapore 0.2 J.tm pore nylon The purpose of the studies reported here was to membranes (Cuno Pacific Pty Ltd, Blacktown, NSW, investigate the effects of surface coated vitronectin upon Australia). All reusable glassware was treated with E­ human arterial endothelial cell recovery, in a tissue Toxa Clean (Sigma) to remove pyrogens, and following culture model of wound repair. Fibronectin, laminin, several washes in tap and distilled water, was rinsed in collagen types I and IV, which the cells would also be pyrogen free water before sterilization by autoclaving at likely to encounter in the wounded vessel environment, l20°C for 1 h followed by dry heat at 170°C for 3 h. were included for comparison. For these studies All handling of materials was done using surgical fibronectin and vitronectin were specifically removed gloves. Tissue culture polystyrene (TCPS) and non­ from serum used in the culture media, to avoid tissue culture treated polystyrene (PS) were from Nunc confounding effects. Adsorption of the plasma proteins (Roskilde, Denmark). Flexible polyvinyl (PV) ELISA vitronectin and fibronectin to tissue culture polystyrene plates were from Dynatec (Alexandria, VA, USA). was determined in competition with other proteins, and Cell culture stability of the surface-coated proteins over time