Effect of polyvinyl chloride plastic on the growth and physiology of human umbilical vein endothelial cells

Biomoteriok 17 (1996) 1791-1794 0 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0142-9612(95)00360-6

ELSEVIER

0142-9612/96/$15.00

Effect of polyvinyl chloride plastic on the growth and physiology of human umbilical vein endothelial cells Josef Storck, Hesham Ab Del Razek and Eberhard R. Zimrnermann University Miinster, institute of Vegetative Physiology, In this work,

human

umbilical

plates,

referred

human

umbilical

plasma

was significantly

fibronectin.

to as control endothelial

Cells

than control

from

collagen

A. After

higher

grown

role in their

inhibitor, Received

growth

Endothelial von Willebrand 22 August

cells

cells,

the PAI-

polyvinyl

factor,

1995; accepted

plates

coated inhibitor

release

grown

0

1996 Elsevier

chloride,

tissue

substance

plasminogen

1 (PAI-I),

more

coated

plasminogen

PAI-

or

activator was

with gelatin

and

on PVC was significantly

on the fibronectin-coated

and coat used to culture

Science

of

A and heparin

with the same

plates

cell culture

(PVC). Growth

collagen

of HUVEC cultured

of cells

polystyrene

plastics

as much tissue

on the control

that the type of plastic

and function.

on common chloride

with gelatin,

activator

those

with the exception

It is concluded

cultured

up to four times

to plasminogen

stimulation,

than that of control

definite

cells were

as on soft polyvinyl on PVC coated

on PVC than from

endotoxin

no difference.

(HUVEC)

on PVC produced

With reference

cells

showed

Keywords:

cells

as well

less than that on the control

cultured

cells.

released

vein endothelial plates,

Robert Koch StraRe 27a, D-48149 Monster, Germany

PVC that

HUVEC

play a

Limited. activator,

plasminogen

activator

endotoxin 20 November

1995

MATERIALS

Local activation of plasmin at the site of perturbed endothelial cells is still the key for the process of degradation of the deposited fibrin in viva. Tissueand urokinase-type plasminogen activator are the serine proteases which are released mainly from cleave the circulating endothelial cells to plasminogen in the blood to plasmin. Plasminogen activator inhibitor 1 (PAI-I), which is also released from endothelial cells, is the physiologcal inhibitor of plasminogen activator (tPA). The major tissue function of PAI- is probably to prevent the removal of a fibrin clot’. Bacterial endotoxin was observed to increase the release of PAI- in vivo and in cultured Endotoxin is also capable of endothelial cells’. changes in procoagulant vascular initiating An augmented release of von endothelium. Willebrand factor (vWf) antigen from endothelial cells induced by endotoxin compared with that of the control was also observed3. The variable circumstances under which the endothelial cells may be cultured in vitro gives the opportunity for the change in type of plastic or coating used to culture human umbilical endothelial cells (HUVEC) to affect the behaviour of these cells, and our attention was directed to study the effect of these variants on the release of tPA, PAI- and vWf from the endothelial cells.

AND METHODS

Materials The cell culture reagents, medium RPM1 1640, phosphate buffered saline (PBS) with Ca2+ and Mg’+, PBS without Ca2+ and Mg*+, collagenase type CLS III solution, trypsin/EDTA penicillin/ (154 Umg-‘), amphotericin B and L-glutamine, streptomycin, collagen A were all supplied by Biochrom KG (Seromed, Berlin, Germany). Fetal calf serum and human fibronectin were purchased horn Boehringer Mannheim, Germany. Cell culture plates 10, 40 and 100mm in diameter were from NUNC GmbH, Wiesbaden, Germany. The soft polyvinyl chloride plastics (PVC) used were kindly provided by Hills AG, Germany. Heparin plasma was collected using a lOm1 syringe monovette blood collection system containing lithium heparin (15 I.E. heparin per ml blood) from Sarstedt, Niimbrecht, Germany. Gelatin and lipopolysaccharide from Escherichia coli serotype 026:B6 containing 10,OOOU endotoxin per mg were obtained from Sigma Chemical Co., USA. Crystal violet used to stain cells was obtained from Aldrich Chemical Company, Inc., Milwauke, USA. The Imunolyse tPA ELISA and PAI- ELISA used in this study were supplied by Biopool, AB Umea, Sweden, while Asserachrom vWf ELISA came from Boehringer Mannheim.

Correspondence to Dr J. Storck. 1791

Biomaterials

1996,

Vol. 17 No. 18

HUVEC growth

1792

and physiology

on PVC: J. Storck

et al.

RESULTS

Cell culture Human umbilical cords were used to isolate and culture the endothelial cells of their veins as described by Jaffe et a1.4. Z&well plates, with a surface area of 1.9cm2 per well, were used. To ensure disinfection of the plastics, the Z&well plates of PVC were constructed one day in advance, and immersed in 70% ethanol for 30 min and then removed and left under the flow bank overnight to allow the evaporation of alcohol. On the day of the experiment, the Z&well plates of PVC, together with those of polystyrene plates (Nunc, Germany), referred to as control plates, were coated with 0.4% gelatin (400~1 per well), 0.5 mgml-’ of collagen A, heparin plasmaPBS 2:3 (v/v) or with 25 pgml-’ of human fibronectin. For the preparation of heparin plasma, blood was collected from normal volunteers, who had taken no medicaments during the previous two weeks. The blood was centrifuged at 3,000rpm for 20min to separate plasma. To coat plates with human fibronectin, plates were incubated at room temperature for 1 h, while gelatin, collagen A, and heparin plasma were incubated on plates for 30min at 37X, followed by additional 30min at room temperature. In the case of the gelatin, collagen A and human fibronectin, the coating solution was aspirated directly before plating the cells. However, heparin plasma was aspirated after the first 30min, leaving a thin film to dry during the following 30min before inoculation of the cells. Confluent endothelial cells of the first or second passage were then dispersed by trypsin/EDTA and Cells centrifuged. were resuspended in culture medium and counted. The medium was further diluted with culture medium to a final concentration of 20,000 cells per ml, and 1 ml was inoculated to each well of the various 24-well plates. One day later, cells were refed with 1 ml per well of culture medium.

Growth of HUVEC on PVC HUVEC were grown on PVC coated with different substances and stained. The growth of HUVEC showed variation according to the coat as illustrated in Figure Z. In the case of the control plates, the growth of cells on gelatin and fibronectin was reaching values between found to be maximal, and 58 078cm-“, and 51 648cmm2 and 36 328 cm-’ 58 693 cm-‘, respectively, without any significant difference between each other. Cell growth on collagen A and heparin plasma was nearly equal, but significantly less (= P < 0.05) than that on other coats. The growth of HUVEC on fibronectin-coated PVC was nearly the same as that on the control plates. However, with gelatin coating, usually used in cell culture, and collagen A coating, the growth of cells on PVC was about 50% less than that on control plates. With heparin plasma-coated plates, the cell growth on PVC was minimal (< 1OOOcells per cm”).

60000

--

50000

I

-

I

1 control

1

Stimulation of cells After 4d inoculation of endothelial cells on the culture plates, cells were washed with 1 ml per well of PBS with Ca” and Mg2+, and incubated with culture medium containing 0, 0.1, 1 and 10~gml~’ of lipopolysaccharide, with endotoxin activity of After 24 h of incubation, medium was lounge’. collected, centrifuged at 3,000rpm and stored at -20°C until its contents of tPA, PAIand vWf antigens were measured by ELISAs. After collection of samples, plates were incubated with 1 ml per well of normal saline for 2 min, followed by 500~1 of 0.2% crystal violet (w/v) in 2% ethanol in water (v/ v) for 10min. Plates were then washed twice with tap water, and left to dry. Stained cells were counted using a microscope video camera connected to a computer by a video grabber card. Automatic cell counting was performed using the AUTOSCOPE (Digithurst, USA) evaluation software. The results are expressed as mean j, standard deviation (SD). All significant data in this work were demonstrated using the paired t-test and the significant level was set to p < 0.05. Biomaterials

1996.

Vol. 17 No. 18

gelatin

collagen

plasma

fibronectin

Figure 1 HUVEC growth on PVC. Endothelial cells were inoculated to control plates or PVC plates coated with gelatin, collagen A, heparin plasma (plasma) or human fibronectin, and left to grow for 4d. The supernatant was collected and cells were counted. Results are expressed as mean f SD of five experiments. *p < 0.05 for cells grown on PVC versus control cells grown on polystyrene. + p < 0.05 for cells grown on gelatin coating versus cells grown on collagen, plasma, or fibronectin coating.

HUVEC

growth

500 T

and physiology

on PVC: J. Storck et al.

_*

=

tPA

=

PAI-1

=

VWf

1793

1 D

0

500 -

;

400

.-

si

$ 400 C 5 E 300 -

h g

%

300

g

8 % s

100

8 i

200

z

200

0 1

a

control 300

250

I m m

gelatin collagen A fibronectin

100 200

150

0 gelatin

collagen

A

100

fibronectin

Figure 2 Effect of coating on the basic release of tPA, PAI1, and vWf from HUVEC. After 4d of growth, cells were incubated with culture medium for 24 h and the amounts of tPA, PAI-1, and vWf in the supernatant were determined. The influence of PVC on the production of enzymes is expressed as a percentage of the control plate. Results are expressed as mean &SD of five experiments. lp < 0.05 for cells grown on PVC versus control cells grown on polystyrene.

tPA, PAI- and vWf release from HUVEC grown on PVC coated with different substances Cells grown on PVC released more tPA than those grown on control plates. The difference in tPA release was at a maximum with the gelatin-coated plastics, and at a minimum with the fibronectin-coated plastics. While the amount of PAL1 released from the gelatinand collagen A-coated PVC was more than that released from the control plates coated with the same chemical, PAI- released from the fibronectin-coated PVC was nearly the same (94.6% f 7.6%) as from the control plates. The relative amount of vWf released from cells grown on PVC was less (about 70%) than that released from the control plates coated with the same chemical (Figure 2).

Influence of endotoxin on the tPA, PA&l, and vWf release from HUVEC Exposure of endothelial cells to endotoxin markedly increased the release of PAI-1. Cells stimulated with 0.1-10 pg ml-’ endotoxin showed a dose-dependent increase of the PAI- release in comparison with that of the quiescent cells (data not shown). Figure 3a

50

0

b

PVC

control

Figure 3 Influence of PVC on the response of endothelial cells to endotoxin. Gelatin, collagen A, or human fibronectin were used to coat. After 24 h of stimulation with endotoxin (10 pg ml-‘), the amounts of PAI(a) and vWf (b) released from the cells were calculated as a percentage of that released from non-stimulated cells. Results are expressed as mean+ SD of five experiments. ‘p < 0.05 for cells grown on PVC versus the corresponding control cells grown on polystyrene. + p < 0.05 for endotoxin stimulated cells versus the corresponding non-stimulated cells.

shows the effect of the type of plastic on PAI- release from HUVEC stimulated with 10 pg ml-' of endotoxin as a percentage of that released from non-stimulated cells, where the cells grown on PVC released more PAI- than that observed with cells grown on control plates. In the case of PVC, cells grown on gelatin or collagen A were more sensitive to endotoxin than those cultured on appropriately coated control plates. PVC However, cells grown on fibronectin-coated showed an increase of PAI- release relative to that of the non-stimulated cells which was not significant when compared with the corresponding PAI- increase from cells grown on the control plates. Although the amount of tPA released from the HLJVEC grown on different plastics was significantly different, incubation of these cells with different doses of endotoxin resulted in a non-significant variation in Biomaterials

1996. Vol. 17 No. 18

1794

their tPA release to that of the unperturbed cells (data not shown). The release of vWf from endotoxin stimulated cells increased nongrown on control plates was significantly in comparison with the non-stimulated cells. However, HUVEC grown on gelatin-coated PVC showed a significant response for the endotoxin induced vWf release. Cells grown on collagen A- and fibronectin-coated PVC showed an even higher vWf response to endotoxin than those grown on gelatincoated PVC (Figure 3b).

DISCUSSION The difference in the rate of growth of HUVEC according to the type of coating or plastic observed in this study reflects the role of the matrix in the in vitro culture of these cells. However, the equal growth of HUVEC on different plastics coated with human fibronectin, a normal constituent of the subendothelial matrix in vivo, indicates the suitability of this coating in the in vitro culture and study of endothelial cells in order to approach their in vivo physiological functions. The difference of cell number based on different coatings, in spite of using the same plastic, may be explained by a different adherence of the various chemicals to PVC. Our study demonstrates that the type of plastic not only affects the rate of growth of cells but also the functional behaviour of these cells. The type of plastic coating also plays a role in the behaviour of these cells. While cells grown on fibronectin showed no difference in their release of the observed proteins, a variation was found with the other types of coating. HUVEC cultured on PVC plastics showed more fibrinolytic activity than those cultured on the control plate, which is represented by the release of more tPA without an associated equal increase in the PAIrelease. Again, the response of the cells to endotoxin was different according to the culture conditions. Perturbation of endothelial cells grown on PVC by endotoxin resulted mostly in a reinforced increase of the PAIrelease than the cells of the control plates. The marked significant increase of PAI-1, but not tPA, observed in our study indicates that the accumulation of endotoxin

Biomaterials 1996.Vol. 17 No. 18

HUVEC growth

and physiology

on PVC: J. Storck

et al.

in vivo during infection or inflammation does not favour the fibrinolytic activity of plasmin. In fact, this larger amount of PAI-1, released under this condition, will bind rapidly to the released tPA, preventing activation of the circulating plasminogen, and, in turn, helping to stabilize the formed fibrin clot. Concerning vWf, the mild change in its release after incubation of HUVEC with endotoxin for 24 h is in agreement with other investigators3. However, they reported a greater variation in vWf when it was measured after a shorter time of 6 h incubation of the stimulant. It could be postulated that a rapid release of vWf may lead to exhaustion of the storage with the consequence of releasing a smaller amount of the protein in the next few hours leading to an end release of vWf nearly equal to that released from the unperturbed cells. This non-significant change in vWf after 24 h of incubation of endothelial cells with endotoxin may reflect the short-lived effect of endotoxin on this adhesive protein, which minimizes its role during chronic bacterial infections, or even the next few days of acute bacterial infections. Comparing the variable effects of endotoxin on the release of the different enzymes from HUVEC grown on different coats and plastics, it was concluded that the type of plastic and coat not only influences the basic release of the enzyme but also the change in this release in response to stimuli.

REFERENCES Zoellner H, Wojta J, Gallicchio M, et al. Cytokinine regulation of the synthesis of plasminogen activator inhibitor-2 by human vascular endothelial cells. Comparison with plasminogen activator inhibitor-l. Thromb Haemost 1993; 69: 135-140. Emeis JJ. Regulation of the acute release of tissue-type plasminogen activator from the endothelium by coagulation activation products. Arm NY Acad Sci 1992; 667:249-258. Schorer AE, Moldow CF, Rick ME. Interleukin 1 or endotoxin increases the release of von Willebrand factor from human endothelial cells. Br 1 Haematol 1987; 67:193-197. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973; 52:2645-2756.