Detection of surface-adsorbed (lipo)proteins by means of a two-step enzyme-immunoassay: a study on the Vroman effect

In view of reports on the involvement of high-molecular-weight (HMW) kininogen and high-density lipoprotein (HDL) in the Vroman effect, we studied the adsorption of fibrinogen, HMW kininogen, HDL and several other proteins from pooled human plasma and congenitally HMW kininogen-deficient plasma onto glass and low-density polyethylene, both as a function of the plasma concentration and the contact time. Mixtures of purified (lipo)proteins were also included in the study. Protein adsorption was determined by means of a two-step enzyme-immunoassay. Our results support the hypothesis that HMW kininogen is involved in the displacement of fibrinogen, which is almost instantly adsorbed from normal plasma onto glass. On hydrophobic polymers like polyethylene, the low amounts of adsorbed fibrinogen and HMW kininogen from plasma and concentrated plasma solutions may be due to a preferential adsorption of HDL

Interaction of cultured human endothelial cells with polymeric surfaces of different wettabilities

The in vitro interaction of human endothelial cells (HEC) and polymers with different wettabilities in culture medium containing serum was investigated. Optimal adhesion of HEC generally occurred onto moderately wettable polymers. Within a series of cellulose type of polymers the cell adhesion increased with increasing contact angle of the polymer surfaces. Proliferation of HEC occurred when adhesion was followed by progressive flattening of the cells.\ud \ud Our results suggest that moderately wettable polymers exhibit a serum and/or cellular protein adsorption pattern that is favourable for growth of HEC

Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated polytetrafluoroethylene

From in vitro experiments it is known that human endothelial cells show poor adhesion to hydrophobic polymers. The hydrophobicity of vascular prostheses manufactured from Teflon® or Dacron® may be the reason why endothelialization of these grafts does not occur after implantation in humans. We modified films of polytetrafluoroethylene (Teflon®) by nitrogen plasma and oxygen plasma treatments to make the surfaces more hydrophilic. Depending on the plasma exposure time, modified polytetrafluoroethylene surfaces showed water-contact angles of 15–58°, versus 96° for unmodified polytetrafluoroethylene. ESCA measurements revealed incorporation of both nitrogen- and oxygen-containing groups into the polytetrafluoroethylene surfaces, dependent on the plasma composition and exposure time. The thickness of the modified surface layer was ~1 nm. The adhesion of cultured human endothelial cells from 20% human serum-containing culture medium to modified polytetrafluoroethylene surfaces with contact angles of 20–45° led to the formation of a monolayer of cells, which was similar to the one formed on tissue culture polystyrene, the reference surface. This was not the case when endothelial cells were seeded upon unmodified polytetrafluoroethylene. Surface-modified expanded polytetrafluoroethylene prosthesis material (GORE TEX® soft tissue) also showed adhesion of endothelial cells comparable to cell adhesion to the reference surface. The amounts of serum proteins, including fibronectin, adsorbed from serumcontaining medium to modified polytetrafluoroethylene surfaces were larger than those adsorbed to unmodified polytetrafluoroethylene. Moreover, the modified surfaces probably allow the exchange of adsorbed serum proteins with cellular fibronectin

The influence of protein adsorption on interactions of cultured human endothelial cells with polymers

A systematic study of the effects of polymer surface properties on the interaction with human endothelial cells (HEC) may lead to the development of small-diameter vascular grafts. HEC, suspended in culture medium containing 20% serum adhered and spread onto moderately wettable polymers such as TCPS (tissue culture polystyrene). Reduced or no adhesion of HEC was observed upon the hydrophobic polymers PETP (polyethyleneterephthalate, Dacron) and FEP (fluoroethylenepropylene copolymer, Teflon). Polymers precoated with the proteins albumin (Alb), high density lipoprotein (HDL), and immunoglobulin G (IgG) inhibited the adhesion of HEC, whereas fibronectin (Fn) coátings promoted cell adhesion. Endothelialization of PETP and FEP only occurred after precoating of these materials with Fn. The adsorption of Fn, Alb, HDL, and IgG from solutions of different serum concentrations onto TCPS, PETP, and FEP was related to the adhesion of HEC. Serum Fn only adsorbed onto TCPS, with the maximum at 0.1% serum concentration. Maximal cell adhesion onto TCPS was also observed after pretreatment with a solution containing 0.1% serum. The cell adhesion inhibiting proteins Alb and HDL preferentially adsorbed at higher serum concentrations. Desorption of these proteins and exchange for, e. g., cellular Fn may result in cell spreading and proliferation of HEC upon TCPS

Deposition of endothelial fibronectin on polymeric surfaces

Cellular fibronectin is deposited on tissue culture polystyrene during the adhesion and spreading of cultured human endothelial cells (HEC). Following the seeding of HEC upon this polymer, larger amounts of fibronectin are deposited as both cell density and incubation time increase. Our results indicate that the ability to deposit cellular fibronectin onto a polymeric surface is a condition for the spreading and proliferation of HEC

Adsorption of fibronectin derived from serum and from human endothelial cells onto tissue culture polystyrene

Human endothelial cells (HEC) suspended in a culture medium containing 20% human serum (CMS) adhere and spread on(to) moderately wettable polymers, such as tissue culture polystyrene (TCPS). We have previously shown that serum derived-fibronectin, which is a cell adhesion promoting protein, has a high affinity for TCPS, but that the amount of fibronectin which adsorbed from CMS was relatively small. In this study we investigated whether fibronectin derived from HEC contributes to the adhesion and spreading of the cells on(to) TCPS. Therefore, HEC were seeded in the presence of fibronectin-depleted CMS. The amount of fibronectin detected on TCPS increased with both cell seeding density and incubation time. Although initial HEC adhesion is delayed on TCPS which had been precoated with albumin (Alb), high density lipoprotein (HDL) or immunoglobulin G(IgG), maximal numbers of adhering and spreading HEC were found on these surfaces 6 h after seeding of HEC. Fibronectin was detected on these surfaces, but an exchange of preadsorbed Alb, HDL, or IgG for fibronectin could not be demonstrated. We conclude that HEC deposit fibronectin onto TCPS, irrespective of the presence of a preadsorbed layer of proteins which delay cell adhesion

Dependence of endothelial cell growth on substrate-bound fibronectin

A better understanding of the mechanism of adhesion, spreading and proliferation of human endothelial cells (HEC) on polymeric surfaces may lead to the development of vascular prostheses which allow the formation of an endothelial lining on the luminal surface. In the present investigation the interaction of HEC with polyethylene precoated with monoclonal antibodies directed against HEC membrane antigens and against extracellular matrix compounds was studied. F(ab¿)2 fragments of a monoclonal antibody, directed against an endothelial cell membrane antigen, and F(ab')2 fragments of a monoclonal antibody, directed against cellular fibronectin, were also included in this study. Preadsorption of these antibodies and F(ab')2 fragments, including mixtures of antibodies and mixtures of F(ab')2 fragments, resulted in cell adhesion and spreading as well as moderate cell proliferation (or no proliferation) for several days. However, a good proliferation of HEC was only observed on polyethylene precoated with fibronectin or CLB-HEC-FN-140 (directed against fibronectin). These results strongly suggest that fibronectin, bound to a solid substrate, provides a biochemical signal necessary for the proliferation of HEC. The initial proliferation of HEC on other preadsorbed antibodies or F(ab')2 fragments may be explained by the fact that suspended HEC, used for cell seeding, still possess cell membrane-bound fibronectin