Endothelial cell culture on fibrillar collagen: model to study platelet adhesion and liposome targeting to intercellular collagen matrix.

Abstract

Human umbilical endothelial cells (ECs) were grown on fibrillar type I collagen in 16.4-mm multiwell tissue culture plates. Human platelets were added to the wells, and platelet adhesion to collagen was examined by scanning electron microscopy and radioisotopic technique in the absence of ECs and in preconfluent and confluent EC cultures. Single adherent platelets of different shapes as well as small aggregates were seen on collagen surface. Human plasma fibronectin added to the system stimulated platelet adhesion and their spreading on collagen. ECs had no effect on the percentage of platelets adherent to collagencoated gaps in preconfluent culture but decreased the number of spread platelets. It is demonstrated that collagen-coated gaps can bind ' C-labeled liposome.antibody and "'C-labeled liposome-fibronectin conjugates. ECs grown on fibrillar collagen are suggested as useful models for screening of antiplatelet drugs and for the study of drug targeting tothe areas of vascular injury for prevention of thrombosis..At present, injury of the endothelial lining of the vessel wall is viewed as one of the key mechanisms of thrombogenesis and, possibly, ofatherogenesis (for review, see refs. 1-3). The injury or loss of endothelial cells (ECs) results in the loss of athrombogenic properties by the vessel wall and this leads to adhesion of blood platelets to the injured surface. It is thought that the recognition of injury occurs through a specific interaction of platelets with subendothelium, of which several constituents, particularly collagen, have high affinity toward platelets (1, 2).To study the mechanism of platelet adhesion to the injured vessel wall, everted vessel segments or surfaces coated with fibrillar collagen have been used (2, 4-6). In the work reported here, an experimental model is described which includes partial reconstitution of the luminal surface of "normal" and "injured" vessel wall. This surface is imitated by a bilayer structure of confluent or preconfluent EC cultures grown on fibrillar collagen. The model was used for: (i) quantitative studies ofplatelet adhesion to collagen-coated gaps in preconfluent EC cultures and to the surface of ECs; (ii) examination of effects produced by plasma fibronectin and ECs on the adhesion and spreading ofplatelets on fibrillar collagen; and (iii) attempts to develop an approach for specific recognition ofcollagen-coated gaps in preconfluent EC culture by "4C-labeled liposome-antibody to type I collagen andd "4C-labeled liposome-fibronectin conjugates. MATERIALS AND METHODSIsolation of Fibronectin, Collagen, and Antibodies to Collagen. Human plasma fibronectin was isolated by affinity chromatography on gelatin-Sepharose (7) 5603The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The influence of biomaterials on endothelial cell thrombogenicity

“…These studies suggested overall that EC exhibit a relatively non-thrombogenic phenotype on a number of common polymer substrates with or without ECM coatings. Limited platelet adhesion and activation (serotonin release and spreading) occurs on non-stimulated HUVEC cultured on TCPS [175][176][177], fibrillar collagen [178], crosslinked albumin heparin gels with [179] and without heparin [180], thermanox coverslips [181] and fibronectin coated polyethylene [182]. Similar trends were also observed for BAEC cultured on TCPS [183,184], and for sheep pulmonary artery EC cultured on gelatinized polycarbonate or TCPS [185].…”

“…In a different kind of assay measuring fibrin deposition from blood plasma, canine aortic EC on both TCPS and microporous polymer (fluropore filter) with and without a fibronectin coating showed reduced fibrin deposition, that was dependent on the proliferative state of the cells [191]. Moreover in pre-confluent EC cultures platelet adhesion has been observed not just in regions of the exposed underlying matrix but also on the non-confluent EC themselves [176,178,179,183,185] suggesting that the EC are thrombogenic at non-confluent densities.…”

Binding of antibodies in liposomes to extracellular matrix antigens

“…Lipo- [8], it might be possible to direct liposomes to specific matrices by using antibodies to tissue-specific matrix components. In previous studies, we showed that liposomes prepared with antibodies to type I collagen and fibronectin bind to the matrix deposited by endothelial cells in culture [5]. Here we have utilized antibodies to laminin and to fibronectin in the preparation of liposomes and have found that the antibodies, when incorporated into liposomes, retain their specificity for these matrix components.…”

A new, rapid and reproducible method to obtain high quality endothelium in vitro

“…Isolated HUVECs can be propagated using different types of endothelial-specific media, supplements and coatings for the culture vessels (Albelda et al 1989; Chazov et al 1981; Clark et al 1986; Gimbrone et al 1974; Jaffe et al 1973b; Lewis et al 1973; Maciag et al 1981). Confluent HUVECs can differentiate into a monolayer of tightly packed cells, the so-called cobblestone phenotype, that resembles endothelium morphology in vivo (Smeets et al 1992).…”