W.G. van Aken

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. 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 cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge

The adhesion of human endothelial cells (HEC) onto a series of well-characterized methacrylate polymer surfaces with varying wettabilities and surface charges was studied either in serum-containing (CMS) or in serum-free (CM) culture medium. HEC adhesion in CMS onto (co)polymers of hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) was found to be optimal on the moderately wettable copolymer (mol ratio 25 HEMA/75 MMA). Positively-charged copolymers of HEMA or MMA with trimethylaminoethyl methacrylate-HCl salt (TMAEMA-Cl), both with mol ratios of 85/15 and a negatively-charged copolymer of MMA with methacrylic acid (MAA), mol ratio 85/15, showed high numbers of adhering HEC. In CM, HEC adhered onto the three charged copolymers mentioned above, but neither onto the copolymer of HEMA and MAA (mol ratio 85/15) nor onto the HEMA/MMA co- and homopolymers. Complete cell spreading in CM was only observed on the positively-charged copolymers.

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 degrees, versus 96 degrees 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 approximately 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 degrees 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 serum-containing 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.

Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells

Abstract To determine if human vascular endothelium influences the growth of smooth muscle cells (SMC), we have investigated the effect of media conditioned by cultured human vascular endothelial cells on smooth muscle cell proliferation. Cell growth was measured by [ 3 H]thymidine incorporation in endothelial and smooth muscle cells. The conditioned medium of confluent endothelial cells contains factors that inhibit the growth of actively dividing endothelial and smooth muscle cells. Media conditioned by proliferating endothelial cells or confluent smooth muscle cells were inactive. The generation of the inhibitory activity was time-dependent, not correlated with cell lysis and was demonstrated in both serum-free and serum-containing medium. Protein synthesis was involved in the generation of the inhibitory activity. Upon gel filtration, the inhibitory activity was associated with a substance having a molecular weight (MW) of >10 5 . Treatment with trypsin or alpha chymotrypsin did not abolish the activity. Furthermore, the activity remained stable after incubation for 30 min at 56 °C, whereas only 50% of the activity remained present after heating for 2 min at 100 °C. It is speculated that this inhibitory activity plays an important role in the regulation of smooth muscle cell proliferation.

Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1

We examined the influence of prostaglandins on the initiation of proliferation of growth-arrested human adult aortic and fetal smooth muscle cells. Prostaglandins of the E series (25 nM) exerted a significant (p less than or equal to 0.05) inhibitory effect on DNA synthesis. Inhibition was observed when PGE1 was added in the G1 phase of the cell cycle. PGE1 had no effect when added once DNA synthesis had started. Thus prostaglandins of the E series may inhibit the responsiveness of smooth muscle cells to the mitogenic action of critical growth factors, such as PGDF. This inhibitory response is cell-cycle dependent. Once smooth muscle cells have entered S phase, PGE1 is no longer effective. Our data also suggest that cAMP is involved in the PGE1-induced growth inhibition, since concomitant with PGE1 addition, cAMP levels rose rapidly; addition of the cAMP analogue db-cAMP resulted in a cell-cycle-dependent inhibition pattern comparable to that observed with PGE1.

Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.

To study the biology of the endothelium under conditions that mimic the architecture of the vessel wall, endothelial cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cultures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter.

Deposition of cellular fibronectin and desorption of human serum albumin during adhesion and spreading of human endothelial cells on polymers

More insight into the mechanism of adhesion of human endothelial cells (HEC) on to polymeric surfaces may lead to the development of improved small-diameter vascular grafts. HEC suspended in 20% human serum-containing culture medium adhere and spread well on moderately water-wettable polymers such as tissue culture polystyrene (TCPS). Earlier it was demonstrated that during adhesion and spreading of HEC on TCPS, cellular fibronectin is deposited on to this surface. It was postulated that fibronectin deposition is accompanied by desorption of adsorbed serum proteins, e.g. human serum albumin (HSA). The amounts of adsorbed (cellular) fibronectin and HSA on TCPS surfaces pretreated for 1 h with solutions of human serum (ranging from 0.01%–20%), were determined after incubation of these surfaces for 6 h with HEC in culture medium and after incubation with culture medium without cells. Protein adsorption was determined by means of a two-step enzyme-immunoassay (EIA). HEC adhesion and spreading on TCPS resulted in a significant deposition of fibronectin irrespective of the serum concentration in the solution used for the pretreatment of TCPS. The deposition of cellular fibronectin on to TCPS, pretreated with human serum, was accompanied by displacement of adsorbed HSA. Desorption of HSA from TCPS was only detectable with the EIA at serum concentrations ranging from 0.01%–1%. Using131-l-labelled HSA as tracer protein; it could, however, be demonstrated that HSA was also displaced from TCPS, pretreated with solutions of higher serum concentrations. Pretreatment of the hydrophobic vascular graft material PET (poly(ethylene terephthalate); Dacron) and of FEP (fluoroethylenepropylene copolymer; a Teflon-like polymer) with a solution containing 20% human serum resulted in a reduced adhesion of HEC compared to uncoated surfaces. We suggest that this may be caused by a poor displacement of adsorbed serum proteins from these hydrophobic surfaces by cellular fibronectin. This may explain why HEC normally fail to adhere on to prosthetic surfaces.

Adhesion and spreading of cultured endothelial cells on modified and unmodified poly(ethylene terephthalate): a morphological study

The in vitro adhesion and spreading of human endothelial cells (HEC) on hydrophobic poly(ethylene terephthalate) (PETP) and moderately wettable tissue culture poly(ethylene terephthalate) (TCPETP) were studied with light microscopy and electron microscopy. Numbers of HEC adhering on TCPETP were always higher than those found on PETP. When cells were seeded in the presence of serum, extensive cell spreading on both PETP and TCPETP was observed after the first 30 min. Thereafter, spread cells appeared to withdraw from the PETP surface, resulting in irregularly shaped cells. Complete cell spreading occurred on TCPETP. Complete cell spreading also occurred on PETP and TCPETP when HEC had first been seeded from phosphate buffer solution and serum was supplied after 30 min. Furthermore, HEC spread on both PETP and TCPETP when the surfaces were precoated with protein(s), which promotes cell adhesion. However, when plasma was used for the coating, spread cells did not proliferate in a monolayer pattern. This study shows that TCPETP is, in general, a better surface for adhesion and proliferation of HEC than is PETP, suggesting that vascular prostheses with a TCPETP-like surface will perform better in vivo than prostheses made of PETP.

Platelet deposition in a capillary perfusion model: quantitative and morphological aspects

The capillary perfusion model according to Cazenave and co-workers was characterized by investigating the effects of protein precoating, perfusion time and shear rate on platelet deposition using 111Indium labelling of human platelets and scanning electron microscopy (SEM). Compared with uncoated polyethylene, platelet deposition was increased after precoating with purified human von Willebrand factor, fibrinogen or fibronectin, and decreased by preadsorbed immunoglobulin G, albumin or whole plasma. Platelet aggregates were observed on immunoglobulin G-coated polyethylene, whereas all other surfaces showed single adherent platelets. Complete platelet spreading was only observed after precoating with fibronectin. The quantitative data concerning platelet deposition were evaluated by using the convective-diffusion theory. Our results indicate the applicability of this perfusion model for the in vitro testing of biomaterials.

Arachidonate metabolism in cultured human vascular endothelial cells: Evidence for two prostaglandin synthetic pathways sensitive to acetylsalicylic acid

The effect of acetylsalicylic acid on endothelial prostaglandin synthesis was measured in the presence of exogenous and endogenous substrates. In both types of measurement, a rate of inhibition was found similar to that observed for acetylsalicylic acid inhibition of cyclooxygenase activity in platelets. After withdrawal of acetylsalicylic acid, a rapid restoration of cyclooxygenase activity was observed when exogenous [1-14C]arachidonate was used as a substrate (50% of initial activity at 5 h). However, when endogenous substrate, released after phospholipase activation induced by thrombin treatment of the cells, was used to test cyclooxygenase activity, only partial restoration of enzymic activity was observed (30% after 48 h). Phospholipase activity, measured by the release of free fatty acids, was not inhibited by acetylsalicylic acid. Measurement of turnover times by incubating the cells with cycloheximide revealed a short turnover time for the enzymic activity tested with exogenous [1-14C]arachidonate (2.3 h) and a relatively long turnover time for the cyclooxygenase activity tested with endogenous substrate released after thrombin treatment of the cells (54 h). These results suggest that at least two pools of cycloxygenase are involved in endothelial prostaglandin synthesis.