21 (I 975) 259-272 (‘1Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

.4rhuws&rosis,

THE

PORCINE

D. N. SLATER Division

AND

ENDOTHELIAL

CELL

IN TISSUE

259

CULTURE

J. M. SLOAN*

of Pathology,

University

qf‘ Shefield,

ShcTjfield (Great

Buitaitl

J

(Received September 9th, 1974) (Accepted November l9th, 1974)

SUMMARY

Endothelial cells from porcine aorta and inferior vena cava have been harvested, using trypsin, EDTA or collagenase, and grown in tissue culture. Growth-behaviour. cytology, scanning and electronmicroscopy findings are reported. It is hoped this technique will prove useful in the investigation of atherosclerosis.

Key words : Atherosclerosis Pig - Pinocytotic

- Endothelial

cell - Microjlaments

- Multitubular

that

bodies -

vesicles - Tissue culture

INTRODUC’i-IOh

Despite the importance about

its metabolism

of endothelium

and the changes

as emphasised

by Oliver’, little is known

that occur in atherosclerosis.

An operational

difficulty is that the endothelial cell is intimately attached to other vascular structural components. There have been few tissue culture studies using pure cell lines of endothelial cells. Fryer et a/.2, extending the original work of Maruyama3, obtained cultures of human umbilical arterial and venous endothelium after removing the cells from the aorta using a technique with trypsin. However, cellular replication was not good and the ultrastructure was not studied. Buonassis? isolated rabbit aortic endothelium with collagenase; he achieved good cellular replication and showed that the cells had the capacity to synthesise and excrete sulphated mucopolysaccharides. Jaffe et al.” and Gimbrone et al.6 have recently obtained replicating human venous endothelium using the collagenase technique. This communication reports that replicating endo-

* Present address: Ireland.

Institute

of Pathology,

Queen’s University

of Belfast, Belfast, Northern

I>. N.

260 thelial

cells can be obtained

using trypsin,

MATERIALS

EDTA

from

both

porcine

arterial

SLATER,

and venous

J. M. SLOAN

endothelium,

or collagenase.

AND METHODS

All materials

were supplied

by Biocult

Laboratories

unless

otherwise

stated.

Harvesting endotfzelial cells Five to ten cm segments of aorta, inferior vena cava or pulmonary veins were obtained within 3 min of slaughter from young male pigs (20-30 weeks old). The segments were washed in Hanks’ balanced salt solution (BSS) and transported in Hanks’ BSS at 4°C. All minor vessels were ligated and the ends of the segments were clamped trypsin

with bull-dog

w/v in Hanks’

clips. The sacs were inflated with 5-10 ml of 0.25 x,

BSS, 1 in 5000 EDTA

in Dulbecco’s

calcium

and magnesium

free phosphate buffered saline (Flow Laboratories) or phosphate buffered saline with calcium and magnesium containing 1 mg/ml collagenase (Sigma Type III: Fraction “A”). After incubation at 37°C one bull-dog clip was removed and the contents transferred

into a sterile 30 ml universal

container

(Sterilin

Ltd.). The sac was cut

open in its longitudinal axis and the luminal surface gently scraped with a stainless steel scalpel blade (Swann-Morton No. 22). The blade must be inclined at an angle of approximately 60” to the intimal surface. The endothelial cells were washed off the blade with a jet of culture medium (see below) and the samples centrifuged at 1000 rpm for 5 min. The supernatants resuspended

were discarded

in 2 ml of culture

finally added to 4 ml aliquots

medium; of culture

and the white pellets of endothelial cells I ml suspension of endothelial cells were

medium.

For scanning

electron

microscopy

the cells were grown in 5 cm glass petri dishes containing 13 mm diameter cover slips (Chance Ltd.). For electron microscopy they were grown in 30 ml tissue culture flasks (Falcon Plastics). Culture media At first we used Medium

199 with Earle’s

BSS containing

2.00 g/l NaHCOs

(Flow Laboratories) gassed in 5 “/oCOa/air or Medium 199 with Hanks’ BSScontaining 0.35 g/l NaHCOs (Flow Laboratories). In later experiments we used Medium 199 with Hanks’ BSS containing Hepes buffer. 100 ml were all supplemented by 20 ml of foetal bovine serum, 0.5 ml of 200 mM glutamine and 100 units of penicillin and 100 pg of streptomycin/ml. Subculture and quantitation Subculture was achieved using 0.15% trypsin w/v Hanks’ BSS. Single cell suspensions were obtained by ultrasonification for 5 set in an ultrasonic bath (Kerry K5200). Cells were counted in a haemocytometer and viability was assessed with I “/;; trypan blue.

PORCINE

tNDOTHELlAL

CELL IN I’ISSUE CULTURL

261

Microscopy

Cells were studied For light microscopy I5 min in methanol.

during

growth

using a Leitz Diavert

inverted

microscope.

cells were either fixed for 30 min in IO”,, formal-saline or for For scanning electron microscopy the cells were fixed for I ht

in 3 % phosphate-buffered

glutaraldehyde,

washed in phosphate-buffered

distilled water and finally slowly dried in air. The coverslips were mounted stubs with DAG915 conductivity paint and coated with gold-palladium. viewed on a Cambridge S4 Stereoscan at IO kV. Cells were prepared for transmission electron ley’s techniquei.

Cells were fixed in 37; phosphate

microscopy buffered

saline, glass on scanning They were

by modifying

glutaraldehyde

Brink-

and post-

fixed in I :‘:, osmium tetroxide for 30 min. They were stained with 2 y,, aqueous uranyl acetate for 30 min and dehydrated in ethanol. Subsequently they were cleared with upgraded strengths of hydroxypropylmethacrylate to Epon. The Epon was peeled off the flasks and selected areas bored out. Sections were cut on a Porter-Blum MT2B ultramicrotome and viewed on an AEI Corinth SO0 electron microscope at 60 kV. RESULTS

Light microscopy and electron microscopy showed that the endothelial ccli lining of the aortic and venous sacs no longer remains after harvesting. There appear5 to have been minimal

intimal

disruption

with the internal

elastic

lamina

remaining

intact. Incubation

for 20 min appeared

cells using 0.25 ‘/” trypsin, the incubation venous

EDTA

satisfactory

for harvesting

(I in 5000) or collagenase

times had to be approximately

doubled

aortic

(I mg/ml).

to obtain

similar

endothelial However. numbers

of

cells. Cell viability

of both aortic and venous cells after incubation was approximatelc 95 ‘:,, and effluent cell numbers were between 2 and 5 Y IO”. Cellular attachment varied between 20 and 60’%, of the initial inocula; this proportion depended more on the number of cells in an aggregate rather than on the number of cells per flask. Aggregates over 20 cells appeared to establish themselves less readily. Cellular attachment was present in 3-6 hr and confluent monolayers of 2-3 IO” cells per flask were achieved in 6-9 days. To achieve confluent monolayers the initial inoculum had to be greater than 5-7 x 10” cells per flask; with sm2ller numbers individual cell colonies were obtained. With the exception of the initial incubation time no significant differences were found between aortic and venous cells. Although all experiments involved primary cultures, subcultures at the end of every week were possible and one cell-line has been maintained for 3 months.

D.

262

Fig.

I,

A confluent

endothelial

Fig. 2. Three-day

cells appear

culture

cells. HE,

of h-day arterial

showing

its monolayer

J. M. SLOAN

nature

with gian

40.

venous endothelitm

indistinct

endothelium

N. SLATER,

and perinuclear

showing

nuclei in various

halos can be seen. HE,

stages of mitosis. The edges

” 100.

of the

PORCINE

ENDOTHELIAL

CELL

IN TISSUE

The cells had a uniform

‘6.3

C‘ULTURL

epithelioid

polygonal

appearance,

occasionally

being

slightly elongated. Measurements in the longitudinal axis varied between 40 and 60~. Occasionally giant cells were present, being approximately 150,u in diameter, but the nuclei were of a similar size (Fig. I). Mitotic especially proached.

figures were numerous

in young colonies

around the periphery. Mitotic figures became less as confluency was apThe nuclei contained one to three nucleoli (Figs. 2 and 3). Although edges

were indistinct,

staining

with silver nitrate

showed a patchy distribution

of “cement”

lines; thisevidentlydependedon howclosethecontact was betweencells. Phasecontrast microscopy showed a granular cytoplasm that, because of greater cytoplasmic depth, was more marked in the perinuclear region. In some cells there was a less granular perinuclear

area which appeared

ence between

as a semicircular

arterial

and venous

electron

microscope

endothelium

halo. There was no apparent in both young

differ-

(3 day) and confluent

cultures.

Fig. 3. Scanning The nucleoli

appear

(S.E.M.)

as white dots in darkened

appearance

of 3-day colonies

areas representing

of arterial

the nuclei. S.E.M..

endothelium

180.

D. N. SLA’TER,

264

J. M. SLOA N

Fig. 4. Numerous pits are seen on the surface of the cell representing pinocytotic vesicles. S.E.M., 2200.

hj

electron microscopic The outstanding feature was the presence of numerous pits in the cell surface, which were interpreted as possibly representing pinocytotic vesciles (Fig. 4). Most varied in size between 100 nm and 2500 nm. The number of vesicles of this size on the Scunnillg

upper surface varied between I50 and 250 per cell. They were more numerous in the perinuclear region and occasionally giant vesicles were present up to I x I04 nm. However, these were rare in normal endothelial cell cultures and when present were limited to not more than two per cell. The nuclei assumed a flattened hollow appearance with nucleoli standing out prominently. At high magnification occasional gaps were evident between adjacent cell membranes (Fig. 5). No surface spikes were seen comparable to those seen in transmission electron microscopy. The surface appearance of arterial and venous endothelium appeared similar at all ages. Transmission

electron microscopy

Each cell is banded

by a cell membrane

which has the usual

unit membrane

PORCINE

EkDOTHtLIAL

Fig. 5. Small layer.

S.E.M.,

CELI.

gaps are apparent

IN TISSUE

between

CULTURL

adjacent

arterial

265

endothelial

cells of a &day

confluent

mono-

7200.

(Fig. 6). Microvilli

were seen projecting

from the upper cell surface:

1000 nm in height and 100 nm in diameter (Fig. 7). The intercellular junctions had three main configurations.

these were 300--

The cells “butted”

onto each other, slightly overlapped or formed a “mortice” type ofjunction. Although cell membranes became closely apposed, they did not fuse. However, the cytoplasm was always

dense

near the regions

of apposition

(Fig. 8). Areas of cytoplasm

also

occasionally seemed dense where the cell membrane happened fortuitously to rest on the base of the flask. The pinocytotic vesicles (Fig. 7) were either smooth or “coated”x. The smooth vesicles measured from 70 nm to 150 nm and sometimes two or three appeared partially fused. Their membrane was of the same thickness as the cell membrane, from which they seem to be derived by evagination. They were distributed reasonably uniformly on both cell surfaces in the cytoplasm. The coated vesicles were seen less frequently; they measured between 80 nm and 120 nm. The coating appeared to possess radiating projections about I5 nm in length and 15 nm apart.

266

D. N. SLATER,

Fig. 6. Transmission cell sectioned

electron

vertically.

Fig. 7. An arterial and a microvillus.

microscope

Its flattened

6-day endothelial T.E.M..

50.000.

nature

appearance is apparent.

cell showing

(T.E.M.)

of a 3-day-old

T.E.M..

pinocytotic

J. M. SLOAN

arterial

endothelial

/’ 5000.

vesicles, myofilaments,

a microtubule

PORCINE

ENDOTHELIAL

Fig. 8. Cells from bowing

cytoplasmic

CELL

confluent density.

h-day

IN TISSUE

venous

CULTURF

endothelium

267

showing

As seen here. a microvillus

close apposition

commonly

occurs

and increased

in this position.

neighT.E.M..

50.000.

Long and short fragments

of microtubules

(Fig. 7) were present

measuring

25

nm in diameter. No attachment to cellular structures was seen except to centrioles during mitosis. Cytoplasmic filaments (Fig. 7) were present varying in diameter between 4 and 7 nm. The fine filaments tended to form aggregates, especially near the cell membrane, but again no direct attachment to the cell membrane was seen. The thicker filaments tended to form apparently haphazard arrangements within the cytoplasm. The Golgi apparatus contained the classical stacked smooth-surfaced cislernae and vesicles. Varying amounts of free ribosomes and rough-surfaced endoplasmic reticulum were present, but smooth-surfaced endoplasmic reticulum was less frequent. The mitochondria were of two types. Some were of the classical type with linear cristae, while others were of the tubular-vesicular type that is associated with steroidproducing cells. The latter type were up to 4,~ in length and of varying shape including branched and horse-shoe forms (Fig. 9). one to two Microtubular dense bodies (Fig. 10) were also seen, approximately

268

D. N. SLATER. J. M. SLOAN

Fig. 9. Tubular-vesicular

mitochondria

in a 3-day-old

venous

endothelial

cell. T.E.M.,

,’ 50,000.

per ultra-thin section. Usually these were round structures, derived from unit membrane, of diameter 100-700 nm and with a dense granular cytoplasm containing up to 200 microtubules of diameter 15-20 nm. Some had an irregular outline and others a more elongated appearance. Rarely a second type of microtubular body possessed a double

membrane

with microtubules

of diameter

25 nm.

Osmiophilic droplets (Fig. I I) within the cytoplasm. were limited to one or two per cell. Free electron-dense granules were consistent with small amounts of glycogen. No significant differences were noted between arterial and venous endothelium at 7 days. However, in younger cells the cytoplasmic free ribosomes were more numerous than the attached ribosomes on the rough endoplasmic reticulum (Fig. 9). DISCUSSION

After overcoming the technical difficulties of separating a cell line, believed to be endothelial in origin, the next problem was that of finding a satisfactory marker for

PORCINE

Fig.

ENDOTHELIAL

10. A 6-day-old

appear

to be related

CELL

IN TISSUE

venous endothelial to the formation

CULTURE

cell showing

of a pinocytotic

269

a multibubular vesicle. T.E.M.,

dense body and the microvilli j

50,000.

the identification of endothelial cells. Although unit membrane multitubular dense bodies are usually accredited to Weibel and Palades, similar bodies were probably first described by Pease and Paule lo. To our knowledge they have not been described in fibroblasts and smooth muscle cells - the major source of cellular contamination in endothelial cell cultures - but they have been described in hamster plateletsll and human capillary

pericytests.

However,

they provide

a useful ultrastructural

marker

for

vascular endothelium. Burri and Weiberts considered they were concerned with blood coagulation but their origin and function remain an enigma. Imai and Thomasl” consider that, in swine, the endothelial cells in the proliferative atheromatous lesions develop an increased number of multibubular dense bodies. Although ultrastructural differences have been reported between human and swine arterial and venous endothelium, after 4 days no major morphological difference was apparent between cultured arterial and venous endothelium. These results seem to suggest that ultrastructural differences tend to disappear when the cells are exposed to a common environment. However, functional differences - such as the time required to harvest the

D. N. SLATER, J. M. SLOAN

270

Fig. 11. A 6-day arterial endothelial cell showing an osmophilic droplet within the cytoplasm. T.E.M., s 125,000.

cells and differences terial endothelium

in fibrinolytic adapted

activity l5 - persist for at least 7-10 days. Why arto high pressure is easier to detach than the more “ad-

hesive” venous endothelium is a problem that we are investigating and which could be relevant to the vascular distribution and pathogenesis of atherosclerosis. The exact function of pinocytotic vesicles in metabolic transport and permeability remains speculative. Moreover, many scanning electron micrographs of endothelium show in our opinion artefacts such as deposited fibrin on the cell surface. Buck16 illustrates vesicles in dog aorta using the palladium-shadowed carbon-replica technique, that are similar to those in our cells. Maruffo and Portman found increased pinocytotic vesicles in experimental coronary atherosclerosis in monkeys, whereas Imai and ThomasI reported no change in porcine cerebral atheroma. The three types of intercellular junctions observed are similar to those found by Schwartz and Benditt in the ratl*. However, the gap apparatus - although it can be of several types’s - is limited to a narrowing of the space between cells or increased

271

PORCINE ENDOTHELIAL CELL IN TISSUE CULTURE

density

of the neighbouring

cytoplasm

were not seen. The absence of microvilli suit to explain but a similar inconsistency The presence

of microtubules

or both.

Areas of fusion

(“tight

on the scanning electron microscope is diffihas been reported by Albarracin and Bain20.

and microfilaments

has been widely reported

endothelium; Becker and Murphy d1 showed that the filaments contain and this laid a firm foundation for the view that endothelial cells contract. We think that an extension investigation the technique

of these studies could provide

and understanding of atherosclerosis to the human vasculature.

We have already labelled

studied

the incorporation

acetate, glucose, palmitic

into mucopolysaccharides

into lipids

the incorporation

and studied

the production

for the

we have extended

of [32P]PO4 and I%-

acid, cholesterol

and chol-

of [35S]S04 and [l%]glucose of proactivator

All these aspects

of endothelial

concentration of insulin and oestrogens. Similarly, significant metahave been found in endothelial cells isolated from patients with atherosclerotic peripheral vascular disease and in rabbits fed atherowill be subsequently

are significantly

and activator.

and by increasing bolic alterations diabetes mellitus,

genie diets. All these findings

cell metabolism

in

actomycin

new avenues

and recently

acid, oleic acid, linoleic

esterol esters. We have also studied

junctions”)

altered

by anoxia

reported.

ACKNOWL.EDGMENTS

The authors are grateful to Mr. Cox and the staff of the Sheffield Corporation Abattoir for providing porcine tissue; to Miss S. Geary for assistance with tissue culture: electron

to Mr. T. Durrant, Miss E. A. Parry and Miss V. Graham for assistance with microscopy; to Mrs. M. Row for photographic assistance and Mrs. J.

Jacques

for preparing

the manuscript.

REFERENCES I OLIVER, M.. Causes and control of atherosclerosis and ischaemic heart disease, Mdicitw, 17 (1973) 1056. 2 FRYER, D. G., BIRNBAUM, G. AND LUTTRELL, C. N.. Human endothelium in cell culture, J. Atheroscler. Res., 6 (I 966) 15 I . 3 MARUYAMA, Y., Cited by D. G. FRYER et al. (Ref. 2). Z. Zelif~rsch., 60 (1963) 69. 4 BUONASSISI, V., Sulphated mucopolysaccharide synthesis and secretion in endothelial cell cultures, Exptl. Cell. Rex, 76 (1973) 363. 5 JAFFE, E. A., NACHMAN, R. L., BECKER, C. G., AND MINICK, R. D., Culture of human endothelial cells derived from human umbilical cord veins, Circulation, 46 (1972) II. 259. 6 GIMBRONE. M. A. J., COTRAN, S. AND FOLKMAN. J., Human vascular endothelial cells and culture. J. Cell Biol., 60 (I 974) 673. 7 BRINKLEY, B. R., MURPHY, P. AND RICHARDSON, L. C., Procedure for embedding in situ selected cell cultures itz vitro, J. Cell Biol., 35 (1967) 279. 8 SUN, C. N. AND GHIDONI, J. J., The ultrastructure of aortic endothelial cells of normal dogs, Cytologia, 38 (1973) 667. 9 WEIBEL, E. R. AND PALADE, G. E., New cytoplasmic components in arterial endothelium, J. Cell Biol.. 23 (1964) 101. IO PEASE, D. C., AND PAULE, W. J., The electron microscopy of elastic arteries in the thoracic aorta of rat, J. Ultrastruct. Res.. 3 (1960) 469

D. N. SLATER, I. M. SLOAN

272

I1 HAYDON,G. B., AND TAYLOR,D., Microtubules in hamster platelets, J. Cell Biol., 26 (1965) 673. 12 ZELICKSON,A. S., A tubular structure in the endothelial cells and pericytes of human endothelium, J. invest. Derm., 46 (1966) 167. 13 BURRY, P. H. AND WEIBER,E. R., Beeinflussung einer spezifischen cytoplasmatischen Organelle und Endothelzellen durch Adrenalin, 2. Zelfforsch. mikrosk. Anat., 88 (1968) 426. 14 IMAI, H. AND THOMAS,W. A., Cerebral arteriosclerosis in swine, Expt. Mol. Path., 8 (1968) 330. 15 SLATER, D. N., Unpublished observations. 16 BUCK, R. C., The fine structure of endothelium and large arteries, J. Biophys. Biochem. Cytol.,

4 (1958) 187. 17 MARUFFO,C. A. AND PORTMAN,0. W., Nutritional control of coronary artery atherosclerosis in the squirrel monkey, J. Atheroscler. Res., 8 (1968) 237. 18 SCHWARTZ,S. M., AND BENDITT,E. P., Structure and permeability of rat thoracic intima, Am. J. Path., 66 (1972) 241. 19 PARRY,E. W. AND ABRANOVICH,D. R., The ultrastructure of human umbilical vessel endothelium from early pregnancy to full term, J. Anat. (Lo&.), 111 (1972) 29. 20 ALBARRACIN,N. S. J. AND BAIN, B., Plasmalemmal configurations assumed by lymphocytes in vitro, Am. J. Clin. Puth., 60 (1973) 628. 21 BECKER,C. G. AND MURPHY, G. E., Demonstration

thelium, intima, the atherosclerotic Am. J. Path., 52 (1968) 22A.

of actinomycin in cells of heart valve, endoplaque and endocardial and myocardial Aschoff bodies,

The porcine endothelial cell in tissue culture.

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