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Culture of rat and pig aortic endothelial cells Differences in their isolation, growth rate and glycosaminoglycan synthesis
19
Atherosclerosis, 38 (1981) 19-26 @Elsevier/North-Holland Scientific Publishers, Ltd.
CULTURE
OF RAT AND PIG AORTIC ENDOTHELIAL
Differences
in their Isolation,
Growth
CELLS
Rate and Glycosaminoglycan
Synthesis
M.J. MERRILEES and LESLEY SCOTT Department of Anatomy, (New Zealand)
School of Medicine,
University of Auckland,
Auckland
(Received 16 April, 1980) (Revised, received 5 June, 1980) (Accepted 19 June, 1980)
Summary Aortic endothelial cells of the rat, an animal not normally predisposed to atherosclerosis, were found to resist removal from the vessel way by collagenase digestion, whereas endothelial cells of the pig, which is susceptible to atherosclerosis, were readily removed by the same treatment. Under the same tissue culture conditions, rat endothelial cells, obtained from explants, had a faster rate of growth and could be passaged many more times than pig endothelial cells. Furthermore, in culture, rat endothelial cells produced large amounts of glycosaminoglycans (GAG), mostly (> 80%) hyaluronic acid, while pig endothelial Cells produced more moderate amounts which were mostly (>70%) sulphated. While it is not known whether these differences accurately reflect in vivo characteristics of rat and pig endothelium, it is suggested that species variability in the strength of attachment of endothelium to the underlying intima, in the rate of regeneration after damage, and in possible contribution to the GAG composition of the intimal matrix, may be correlated with susceptibility to atherosclerosis. .Key words:
Aortic
endothelial
cells -
Culture -
Glycosaminoglycans
-
Growth rate -
Pig - Rat
This work was supported by a grant from the Medical Research Council of New Zealand. Abbreviations: CS = chondroitin sulphate: DS = dermatan sulphate; GAG = glycosamino&can; hyaluronic acid; HS = heparan sulphate.
HA =
20
Introduction Endothelial damage, caused by such factors as hemodynamic stress, hypertension, hypoxia, vaso-active substances and certain lipids, and followed by platelet-stimulated myointimal thickening, is considered to be one of the major factors in the initiation of the atherosclerosis lesion [l]. It would be expected therefore that resistance to endothelial damage or denudation might confer some protection against lesion formation. We have found that aortic endothelial cells of the rat, an animal not normally predisposed to atherosclerosis [2], resist removal by collagenase digestion, whereas endothelial cells of the pig, which is susceptible to atherosclerosis [3], are readily removed by the same treatment. In addition, we have noted that under the same tissue culture conditions endothelial cells from the rat and the pig show marked differences in their survival, growth rate, and synthesis of glycosaminoglycans (GAGS). Methods and Materials Epdothelial cell isolation and culture Pig endothelial cells were obtained from 5-6 cm segments of thoracic aorta taken from pigs weighing 30-40 kg. One end of each vessel segment plus the intercostal twigs were ligatured with suture thread; the lumen was filled with Ml99, containing 0.025% collagenase type IV (Sigma); the other end was ligatured, and then the preparation was incubated at 37°C for 15 min. The collagenase solution was next recovered and the lumen rinsed with Ml99 containing 10% tryptose phosphate broth, 100 units/ml of penicillin and 100 I.cg/ml of streptomycin (hereafter referred to as SM199) and supplemented with 10% fetal calf serum (FCS). Endothelial cells and endothelial-cell sheets from the initial digest and the rinse were pooled, seeded at an estimated density of 0.75 X 10’ cells per 25 sq. cm Falcon flask, and cultured in SM199 containing 10% FCS. Two-centimeter segments of rat aorta, taken from approximately 300 g rats, were treated by the same procedure as for the pig aortas, except that the intercostal twigs were not ligatured. During the incubation the aortas were orientated with the twigs facing upwards to prevent leakage. In addition to the 0.025% collagenase treatment, rat aortas were also incubated with 0.05% and 0.1% collagenase and with 0.05% collagenase plus 0.05% trypsin and 0.01 M EDTA. Small pieces (0.5 mm X 0.5 mm) of rat aorta, stripped of adventitia, were cultured in SM199 plus 10% fetal calf serum in 25 sq. cm flasks. These explants produced outgrowths of endothelial cells within 5 days and the rapid growth of these cells ensured their dominance over smooth muscle cells which first appeared at the margin of the explants at about 14 days. On day 16 or 17 these primary explant cultures were subcultured and, by the second subculture, only endothelial cells had passaged. Although the morphological characteristics of both the rat and pig cells in culture were typical of endothelial cells, electron microscopy was undertaken to confirm that they were endothelial and not smooth muscle cells or fibroblasts.
21
All cultures were maintained at 37°C in an atmosphere of 95% air and 5% COZ. In selected cultures GAG synthesis was monitored by the incorporation of [ 3H] acetate into the individual GAGS. Synthesis was measured over a 24-h period beginning with the addition of 5 ml of fresh medium containing 200 E.tCiof [ 3H]acetate and 10% (or in some instances 2%) FCS. Glycosaminoglycan analysis Glycosaminoglycans synthesised and released into the growth medium or retained in the cell layer were measured by the method of Merrilees et al. [ 41, with a modification for the determination of incorporation of [ 3H]acetate into the various GAGS. This method has been detailed previously [ 51. Briefly, growth medium or cell layer was digested with protease, protein extracted under high salt and acid conditions and GAGS precipitated with ethanol. The precipitate was taken up in a small volume of water, dried and redissolved in a known volume of water and electrophoresed on cellulose acetate membranes using a Beckman Microzone system. The membranes were stained with Alcian Blue and GAG bands corresponding to hyaluronic acid, dermatan sulphate and chondroitin sulphates 4 and .6 were identified using co-electrophoretic standards and enzymatic digestion. Heparan sulphate, for which no standard was available, was identified on the basis of its known position in relation to the other GAGS. Each band containing an individual GAG was cut out of the membrane and solubilised in Dioxan before adding scintillation fluid for counting. Results Pig endothelial cells and endothelial-cell sheets, obtained by collagenase digestion and seeded at approximately 0.75 X lo5 cells/25 sq. cm, attached to the plastic substrate within a few hours and were confluent by 7-10 days. Cells were then lifted with 0.05% trypsin-0.01 M EDTA and subcultured at a seeding density of approximately 1.5 X 10’ cells per flask, or approximately half the confluent density. This subculture and the next (S2), seeded at the same density, were confluent within 1 week. By the third subculture, however, the typical pavement pattern of endothelial cells showed signs of being lost. A number of the cells became enlarged and flattened with a loss of close contact with their neighbours. The typical pattern was usually completely lost by the 4th subculture and the surviving cells were generally stellate and scattered. Cultures were terminated at this stage. Segments of rat aorta, treated by exactly the same procedure as for the pig aortas, did not yield endothelial cells. Incubation with increased concentrations of 0.05% and 0.1% collagenase and with 0.05% collagenase plus 0.05% trypsin and 0.01 M EDTA also failed to remove the endothelial cells, as did extended incubation of up to 30 min. That the cells remained attached to the subendothelial layer throughout these procedures was confirmed by microscopic examination of segments of opened aorta fixed in glutaraldehyde and stained with toluidine blue or hematoxglin. Rat endothelial cells were obtained, however, by explant culture. In subculture these cells, seeded at 0.5 X lo5 cells/25 sq. cm, grew rapidly and reached confluency (3.0 X 10’ cells/25 sq. cm) after 5 days. Throughout successive sub-
22 TABLE 1 COMPARISON OF THE GROWTH AND SURVIVAL OF RAT CELLS UNDER THE SAME CULTURE CONDITIONS (see text)
Rat Pig
AND PIG AORTIC
ENDOTHELIAL
Seeding density per 25 so. cm
Days to reach confluency
Confluent density at 2nd subculture
Maximum no. of subcultures before degeneration
0.5 x lo5 1.5 x lo5
5 I
3.0 x lo5 3.0 x lo5
>12 3-4
cultures the cells maintained a typical pavement pattern and, in one case, cells were passaged 13 times before being deliberately terminated. The differences between rat and pig endothelial cells in culture are summarised in Table 1. Both pig and rat endothelial cells in culture synthesised and released into the growth medium the GAGS hyaluronic acid (HA), dermatan sulphate (DS) and chondroitin sulphates 4 and 6 (CS), and on the basis of correct electrophoretic position, heparan sulphate (HS). Differences were found, however, between the 2 species, both in the amounts of individual GAG synthesised, expressed on a TABLE 2 AMOUNT AND PERCENTAGE OF LABELLED (r3HlACETATE) GLYCOSAMINOGLYCANS SYNTHESISED AND RELEASED INTO THE GROWTH MEDIUM (OVER A 24-h PERIOD) BY CULTURES OF RAT AND PIG AORTIC ENDOTHELIAL CELLS, PIG AORTIC SMOOTH MUSCLE CELLS, AND CHICK EMBRYO FIBROBLASTS Each determination is a mean from two flasks. Cell type and species
Subcul-
cpm/106
%
cells
% GAG
Serum
HA
ture
HS
DS
CS
Total
HA
HS
DS
CS
1 2 1 2
10 I 8 -
2 1 3
5 4 8 11
12 19 12
19 27 26
52 44 40
Endothelial Rat 1
10 10 10 2
5170 8232 5725 8474
115 207 73 178
683 101 523 -
404 388 120 383
6912 9528 6441 9035
83 81 89 94
SZ S4 S5 S6
2 2 2 2
8943 5653 1393 1612
205 19 98 321
497 291 643 982
470 326 429 472
10113 6289 8563 9387
88 90 86 81
Sl
10 10
392 1143 117
656 1682 315
1808 2696 561
3405 6158 1421
11 10 22
s2 s3 s6 Sl
Rat 2
Endotheiial Pig1
s2 s2 Pig 2 Pig 3
Aortic
smooth
Pig
2
519 631 314
Sl
10
152
82
511
492
1303
12
6
44
38
Sl
2
411
167
411
412
1461
28
11
28
32
S4
2
892
112
512
811
2393
37
7
21
35
Sl
0.2
320
31
123
975
1455
22
3
8
61
muscle
Fibroblast Chick embryo
-
HA = hyaluronic acid, HS = heparan sulphate, DS = dermatan sulphate. and CS = chondroitin sulphate.
23
cpm/106 cell basis, and in the proportion
of these GAGS in total GAGS in the growth medium (Table 2). The most marked difference was the very high level of synthesis of HA by the rat endothelial cells. These cells produced on average 15 times as much HA as the pig cells and this GAG accounted for over 80% of the total GAG in the growth medium. Furthermore, this high level of synthesis was maintained in successive subcultures. Two features suggested that the level of HA synthesis by the rat cells was high rather than that the synthesis by the pig cells was low. Firstly, a comparison with cultured pig aortic smooth muscle cells and with chick embryo fibroblasts (Table 2) - cells known to be active in the secretion of GAGS - indicated that HA synthesis by the rat cells was exceptionally high. Secondly, it was noticeable that fresh growth medium on the rat cell cultures became quite viscous within a few days, a feature consistent with high levels of HA. Differences in the amount of the sulphated GAGS synthesised by the 2 cell types were less marked, and intraspecies variability prevented a meaningful comparison. The proportion of sulphated GAGS, especially DS and CS, in total GAGS, however, did show a marked difference. Whereas the rat sulphated GAGS accounted for less than 20% of the total, pig sulphated GAGS accounted for more than 70%. While there are not enough data to compare statistically the effects of 10% and 2% serum levels (Table 2) or GAG synthesis, it would appear that changes in the serum level did not affect the high level of HA synthesis by rat endotheha1 cells, or alter the relative proportion of GAGS in either species. The amount of GAG synthesised and retained in the cell layers of both the rat and pig was only 5% of the total GAG synthesised and the proportion of the individual GAGS did not differ significantly from the proportions in the growth medium. Discussion These results demonstrate that: (1) rat aortic endothelial cells resist removal from the vessel wall by collagenase digestion whereas pig aortic endothelial cells do not; (2) under the same conditions of tissue culture, rat endothelial cells have a much faster growth rate and can be maintained for many more subcultures than pig endothelial cells, and (3) rat endothelial cells in culture synthesise and secrete large amounts of GAGS into the growth medium, mostly hyaluronic acid, while pig endothelial cells synthesise only moderate amounts of GAG, and these are mostly sulphated. While these findings pertain to particular culture conditions they, nevertheless, suggest that endothelial cells among different species may behave differently in vivo, and in a way that might account for their known resistance or susceptibility to atherosclerosis. The rat is normally resistant to lesions [2] and the difficulty in removing endothelial cells by collagenase digestion and other treatments would suggest that in vivo it is also resistant to damage. However, should damage occur and cell division and regeneration take place at a rapid rate, as occurs in tissue culture, then denuded areas would be quickly covered and the normal morphology maintained. It has been established that rapid
24
endothelial regeneration occurs in rat aortas damaged by balloon catheters [ 71 or air drying [2], and that myointimal thickenings, which develop as a result of the denudation, regress following endothelial regeneration [ 21. In contrast to the rat, the pig is susceptible to atherosclerosis and has been frequently used as a model for human atherosclerosis studies [8]. If the ease of removal of endothelial cells by collagenase digestion is a reflection of in vivo characteristics, then it may account, at least in part, for this predisposition to lesion formation. The slower rate of growth of the pig endothelial cells and the difficulty in passaging the cells beyond 4 subcultures is more difficult to interpret since the culture conditions may not have been optimal. The cells in the first 2 subcultures, however, appeared to be in good health according to the criteria of cell attachment: the formation of a typical pavement pattern at confluency, and stable cell size (confluent densities the same as for the rat endothelial cells in culture). Comparison with the rat is based on data from these healthy cultures only. Pig endothelial cells, however, have been apparently successfully maintained in culture for longer periods than were achieved in this study, although it is notable that either earlier subcultures (1 through 6) [9] or primaries only [lo] were used for experiments. However, variations in the age of the animals and in culture conditions make comparisons with our system of doubtful value. The reason for the difference in susceptibility of the two types of endothelial cells to collagenase removal is unknown but it might be related to differences in intimal morphology. In the pig, as with most other large mammals, the intima is relatively broad and the subendothelial space contains a network of collagen and elastic fibres arranged more loosely towards the endothelial surface [ 81. In the rat, however, there is a very much reduced subendothelial space and in places the endothelial cells have been reported to be attached directly to the irregular inner surface of the internal elastic lamina [ll]. It has been suggested that this serves to anchor the endothelium [12]. A further difference between the two species is apparent from various published micrographs. While the subendothelial surface of pig endothelial cells are generally smooth [ 8,131, rat endothelial cells frequently have an irregular subendothelial surface with cytoplasmic projections, or in some cases small hooklets, that extend into the narrow subendothelial space [ 8,11,14-161. These hooklets are especially prominent where subendothelial vacuolation occurs in response to a high fat diet [14]. More recently, Buck [17] has demonstrated that the cytoplasmic projections in the subendothelial space are in fact longitudinal folds, or rugae, that run parallel to the long axis of the vessel and project to within a few nanometers of the internal elastic lamina’or into fenestrations in the lamina. It is again postulated that this arrangement serves to attach more firmly the endothelial cells to the vessel wall. These marked differences in intimal morphology may then account for the differences in susceptibility of rat and pig endotheliurn to collagenase digestion. The synthesis and secretion of GAGS by rat and pig endothelial cells in culture is not unexpected since both bovine [ 181 and rabbit [ 191 endothelial cells in culture synthesise GAGS, although only the sulphated GAGS have been measured and thus a full comparison with our results is not possible. The significance of GAG synthesis by aortic endothelial cells, however, is unknown and,
25
before any conjecture can be made about the role of GAGS, it will be necessary to establish whether endothelial cells in vivo secrete GAGS of the same type and amount as in culture, and whether the secretion is towards the lumen or towards the underlying matrix or both. It is of interest that in a preliminary autoradiographic investigation of rat aorta in organ culture (unpublished), where the integrity of the arterial wall is maintained and approximates to the in vivo state, we have found that - as in tissue culture - endothelium synthesises high levels of HA in comparison with the cells of the media and adventitia. The results in this present study indicate that endothelial cells should at least be considered as possible contributors to the extracellular components of the arterial wall, and that this contribution may show species variability. Variation in the synthesis of HA by the endothelial cells in culture is of particular interest since this GAG plays an important role in the organisation of tissues during morphogenesis [ 201. It is notable that HA differs from the sulphated GAGS in that it does not bind lipoprotein [21]. Also while it usually forms only a small percentage of the total GAG in the intact arterial wall [22], and relatively less in lesions [23], the intimal concentration is generally higher than that in the media [ 221. References 1 Ross. R.. Glomset. J. and Hsrker, L., Response to injury and atherogenesis, Amer. J. Path., 86 (1977) 675. 2 Fishman. J.A., Ryan, G.B. and Kamovsky, M.J., Endothelial regeneration in the rat carotid artery and the significance of endothelisl denudation in the pathogenesis of myointimal thickening, Lab. Invest., 32 (1975) 339. 3 French, J.E.. Atherosclerosis in relation to the structure and function of the arterial intima. with special reference to the endothelium. Int. Rev. EXP. Path., 5 (1966) 253. 4 Merrilees, M.J.. Merrilees. M.A.. Bimbaum. P.S., Scott, P.J. and Flint, M.H.F., The effect of centrlfugal force on glycosaminoglycan production by aortic smooth muscle cells in culture, Atherosclerosis, 27 (1977) 259. 5 Gillard, G.C., Bimbaum, P.. Reilly, H.C., Merrilees, M.J. and Flint, M.H.. The effect of charged synthetic polymers on proteoglycan synthesis and sequestration in chick embryo fibroblast cultures, Biochim. Biophys. Acta, 584 (1979) 520. 6 Breen, M.. Weinstein, H.G., Andersen. M. and Veiss. A., Microanalysis and characterisation of acidic glycosaminoglycans in human tissues, Anal. Biochem.. 35 (1970) 146. 7 Schwartz, S.M., Haudenschild, C.C. end Eddy, E.M.. Endothelial regeneration, Part 1 (Quantitative analysis of initial stages of endothelial regeneration ln rat aortic intima), Lab. Invest., 38 (1978) 568. 8 French, J.E.. Jennings. M.A. and Florey. H.W., Morphological studies on atherosclerosis in swine, Ann. N.Y. Acad. Sci, 127 (1965) 780. 9 Pearson, J.D., Carleton, J.S.. Hutchinks, A. and Gordon, J.L., Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture. Biochem. J., 170 (1978) 265. 10 Slater. D.N. and Sloan, J.M., The porcine endothelial cellin tissue cuhure. Atherosclerosis, 21 (1975) 259. 11 Pease. D.C. and Paule, W.J., Electron microscopy of elastic arteries - The thomcic aorta of the rat, J. Ultrastr. Res., 3 (1960) 469. 12 Keech, M.K., Electron microscope study of the normal rat aorta. J. Cell. BioL. 7 (1960) 533. 13 Gutstein. W.H.. Farrell, G.A. and Armellini, C., Blood flow disturbance and endothelial cell injury in preatherosclerotic swine, Lab. Invest., 29 (1973) 134. 14 Still, W.J.S. and O’Neal, R.M., Electron microscopic study of experimental atherosclerosis in the rat, Amer. J. Path., 40 (1962) 21. 15 Huttner. I., More, R.H. and Rona, G.. Fine structural evidence of specific mechanism for increased endothelial permeability in experimental hypertension, Amer. J. Path., 61 (1970) 395. 16 Huttner, I., Boutet. M. and More, R.H.. Studies on protein passage through arterial endothelium, Part 2 (Regional differences in permeability to fine structural protein tracers in arterial endothelium of normotensive rat), Lab. Invest.. 28 (1973) 678.
26 17 Buck, R.C., The longitudinal orientation of structures in the subendotheliai space of rat aorta, Amer. J. Anat., 156 (1979) 1. 18 Gamse, G.. Fromme. H.S. and Kresse. H., Metabolism of sulphated glycosaminogiycans in cultured endothelial cells and smooth muscle cells from bovine aorta. Biochim. Biophys. Acta. 544 (1978) 514. 19 Buonassi, V., Suiphated mucopolysaccharide synthesis and secretion in endotheliai cell cultures, EXP. CelL Res.. 76 (1973) 363. 20 Toole, B.P., Morphogenetic role of glycosaminoglycans (acid mucopolysaccharides) in brain and other tissues. In: S.H. Barondes (Ed.), Neuronal Recognition, Plenum Press, New York, London, 1976, p. 276. 21 Iverius, P.H., Possible role of glycosaminoglycans in the genesis of atherosclerosis. In: Atherogeneais Initiating Factors (Ciba Foundation Symposium, No. 12). Elsevier/North-Holland, Amsterdam, 1973. p. 185. 22 Massaro. T.A. and Glatz. C.E.. Distribution of glycosaminoglycans in consecutive layers of the rabbit aorta. Artery, 5 (1979) 1. 23 Dalferes, Jr., E.R., Ruiz. H., Kumar, V., Radhakrishnamurthy, B. and Berenson, G.S.. Acid mucopolysaccharides of fatty streaks in young human male aortas, Atherosclerosis. 13 (1971) 121.
Atherosclerosis, 38 (1981) 19-26 @Elsevier/North-Holland Scientific Publishers, Ltd.
CULTURE
OF RAT AND PIG AORTIC ENDOTHELIAL
Differences
in their Isolation,
Growth
CELLS
Rate and Glycosaminoglycan
Synthesis
M.J. MERRILEES and LESLEY SCOTT Department of Anatomy, (New Zealand)
School of Medicine,
University of Auckland,
Auckland
(Received 16 April, 1980) (Revised, received 5 June, 1980) (Accepted 19 June, 1980)
Summary Aortic endothelial cells of the rat, an animal not normally predisposed to atherosclerosis, were found to resist removal from the vessel way by collagenase digestion, whereas endothelial cells of the pig, which is susceptible to atherosclerosis, were readily removed by the same treatment. Under the same tissue culture conditions, rat endothelial cells, obtained from explants, had a faster rate of growth and could be passaged many more times than pig endothelial cells. Furthermore, in culture, rat endothelial cells produced large amounts of glycosaminoglycans (GAG), mostly (> 80%) hyaluronic acid, while pig endothelial Cells produced more moderate amounts which were mostly (>70%) sulphated. While it is not known whether these differences accurately reflect in vivo characteristics of rat and pig endothelium, it is suggested that species variability in the strength of attachment of endothelium to the underlying intima, in the rate of regeneration after damage, and in possible contribution to the GAG composition of the intimal matrix, may be correlated with susceptibility to atherosclerosis. .Key words:
Aortic
endothelial
cells -
Culture -
Glycosaminoglycans
-
Growth rate -
Pig - Rat
This work was supported by a grant from the Medical Research Council of New Zealand. Abbreviations: CS = chondroitin sulphate: DS = dermatan sulphate; GAG = glycosamino&can; hyaluronic acid; HS = heparan sulphate.
HA =
20
Introduction Endothelial damage, caused by such factors as hemodynamic stress, hypertension, hypoxia, vaso-active substances and certain lipids, and followed by platelet-stimulated myointimal thickening, is considered to be one of the major factors in the initiation of the atherosclerosis lesion [l]. It would be expected therefore that resistance to endothelial damage or denudation might confer some protection against lesion formation. We have found that aortic endothelial cells of the rat, an animal not normally predisposed to atherosclerosis [2], resist removal by collagenase digestion, whereas endothelial cells of the pig, which is susceptible to atherosclerosis [3], are readily removed by the same treatment. In addition, we have noted that under the same tissue culture conditions endothelial cells from the rat and the pig show marked differences in their survival, growth rate, and synthesis of glycosaminoglycans (GAGS). Methods and Materials Epdothelial cell isolation and culture Pig endothelial cells were obtained from 5-6 cm segments of thoracic aorta taken from pigs weighing 30-40 kg. One end of each vessel segment plus the intercostal twigs were ligatured with suture thread; the lumen was filled with Ml99, containing 0.025% collagenase type IV (Sigma); the other end was ligatured, and then the preparation was incubated at 37°C for 15 min. The collagenase solution was next recovered and the lumen rinsed with Ml99 containing 10% tryptose phosphate broth, 100 units/ml of penicillin and 100 I.cg/ml of streptomycin (hereafter referred to as SM199) and supplemented with 10% fetal calf serum (FCS). Endothelial cells and endothelial-cell sheets from the initial digest and the rinse were pooled, seeded at an estimated density of 0.75 X 10’ cells per 25 sq. cm Falcon flask, and cultured in SM199 containing 10% FCS. Two-centimeter segments of rat aorta, taken from approximately 300 g rats, were treated by the same procedure as for the pig aortas, except that the intercostal twigs were not ligatured. During the incubation the aortas were orientated with the twigs facing upwards to prevent leakage. In addition to the 0.025% collagenase treatment, rat aortas were also incubated with 0.05% and 0.1% collagenase and with 0.05% collagenase plus 0.05% trypsin and 0.01 M EDTA. Small pieces (0.5 mm X 0.5 mm) of rat aorta, stripped of adventitia, were cultured in SM199 plus 10% fetal calf serum in 25 sq. cm flasks. These explants produced outgrowths of endothelial cells within 5 days and the rapid growth of these cells ensured their dominance over smooth muscle cells which first appeared at the margin of the explants at about 14 days. On day 16 or 17 these primary explant cultures were subcultured and, by the second subculture, only endothelial cells had passaged. Although the morphological characteristics of both the rat and pig cells in culture were typical of endothelial cells, electron microscopy was undertaken to confirm that they were endothelial and not smooth muscle cells or fibroblasts.
21
All cultures were maintained at 37°C in an atmosphere of 95% air and 5% COZ. In selected cultures GAG synthesis was monitored by the incorporation of [ 3H] acetate into the individual GAGS. Synthesis was measured over a 24-h period beginning with the addition of 5 ml of fresh medium containing 200 E.tCiof [ 3H]acetate and 10% (or in some instances 2%) FCS. Glycosaminoglycan analysis Glycosaminoglycans synthesised and released into the growth medium or retained in the cell layer were measured by the method of Merrilees et al. [ 41, with a modification for the determination of incorporation of [ 3H]acetate into the various GAGS. This method has been detailed previously [ 51. Briefly, growth medium or cell layer was digested with protease, protein extracted under high salt and acid conditions and GAGS precipitated with ethanol. The precipitate was taken up in a small volume of water, dried and redissolved in a known volume of water and electrophoresed on cellulose acetate membranes using a Beckman Microzone system. The membranes were stained with Alcian Blue and GAG bands corresponding to hyaluronic acid, dermatan sulphate and chondroitin sulphates 4 and .6 were identified using co-electrophoretic standards and enzymatic digestion. Heparan sulphate, for which no standard was available, was identified on the basis of its known position in relation to the other GAGS. Each band containing an individual GAG was cut out of the membrane and solubilised in Dioxan before adding scintillation fluid for counting. Results Pig endothelial cells and endothelial-cell sheets, obtained by collagenase digestion and seeded at approximately 0.75 X lo5 cells/25 sq. cm, attached to the plastic substrate within a few hours and were confluent by 7-10 days. Cells were then lifted with 0.05% trypsin-0.01 M EDTA and subcultured at a seeding density of approximately 1.5 X 10’ cells per flask, or approximately half the confluent density. This subculture and the next (S2), seeded at the same density, were confluent within 1 week. By the third subculture, however, the typical pavement pattern of endothelial cells showed signs of being lost. A number of the cells became enlarged and flattened with a loss of close contact with their neighbours. The typical pattern was usually completely lost by the 4th subculture and the surviving cells were generally stellate and scattered. Cultures were terminated at this stage. Segments of rat aorta, treated by exactly the same procedure as for the pig aortas, did not yield endothelial cells. Incubation with increased concentrations of 0.05% and 0.1% collagenase and with 0.05% collagenase plus 0.05% trypsin and 0.01 M EDTA also failed to remove the endothelial cells, as did extended incubation of up to 30 min. That the cells remained attached to the subendothelial layer throughout these procedures was confirmed by microscopic examination of segments of opened aorta fixed in glutaraldehyde and stained with toluidine blue or hematoxglin. Rat endothelial cells were obtained, however, by explant culture. In subculture these cells, seeded at 0.5 X lo5 cells/25 sq. cm, grew rapidly and reached confluency (3.0 X 10’ cells/25 sq. cm) after 5 days. Throughout successive sub-
22 TABLE 1 COMPARISON OF THE GROWTH AND SURVIVAL OF RAT CELLS UNDER THE SAME CULTURE CONDITIONS (see text)
Rat Pig
AND PIG AORTIC
ENDOTHELIAL
Seeding density per 25 so. cm
Days to reach confluency
Confluent density at 2nd subculture
Maximum no. of subcultures before degeneration
0.5 x lo5 1.5 x lo5
5 I
3.0 x lo5 3.0 x lo5
>12 3-4
cultures the cells maintained a typical pavement pattern and, in one case, cells were passaged 13 times before being deliberately terminated. The differences between rat and pig endothelial cells in culture are summarised in Table 1. Both pig and rat endothelial cells in culture synthesised and released into the growth medium the GAGS hyaluronic acid (HA), dermatan sulphate (DS) and chondroitin sulphates 4 and 6 (CS), and on the basis of correct electrophoretic position, heparan sulphate (HS). Differences were found, however, between the 2 species, both in the amounts of individual GAG synthesised, expressed on a TABLE 2 AMOUNT AND PERCENTAGE OF LABELLED (r3HlACETATE) GLYCOSAMINOGLYCANS SYNTHESISED AND RELEASED INTO THE GROWTH MEDIUM (OVER A 24-h PERIOD) BY CULTURES OF RAT AND PIG AORTIC ENDOTHELIAL CELLS, PIG AORTIC SMOOTH MUSCLE CELLS, AND CHICK EMBRYO FIBROBLASTS Each determination is a mean from two flasks. Cell type and species
Subcul-
cpm/106
%
cells
% GAG
Serum
HA
ture
HS
DS
CS
Total
HA
HS
DS
CS
1 2 1 2
10 I 8 -
2 1 3
5 4 8 11
12 19 12
19 27 26
52 44 40
Endothelial Rat 1
10 10 10 2
5170 8232 5725 8474
115 207 73 178
683 101 523 -
404 388 120 383
6912 9528 6441 9035
83 81 89 94
SZ S4 S5 S6
2 2 2 2
8943 5653 1393 1612
205 19 98 321
497 291 643 982
470 326 429 472
10113 6289 8563 9387
88 90 86 81
Sl
10 10
392 1143 117
656 1682 315
1808 2696 561
3405 6158 1421
11 10 22
s2 s3 s6 Sl
Rat 2
Endotheiial Pig1
s2 s2 Pig 2 Pig 3
Aortic
smooth
Pig
2
519 631 314
Sl
10
152
82
511
492
1303
12
6
44
38
Sl
2
411
167
411
412
1461
28
11
28
32
S4
2
892
112
512
811
2393
37
7
21
35
Sl
0.2
320
31
123
975
1455
22
3
8
61
muscle
Fibroblast Chick embryo
-
HA = hyaluronic acid, HS = heparan sulphate, DS = dermatan sulphate. and CS = chondroitin sulphate.
23
cpm/106 cell basis, and in the proportion
of these GAGS in total GAGS in the growth medium (Table 2). The most marked difference was the very high level of synthesis of HA by the rat endothelial cells. These cells produced on average 15 times as much HA as the pig cells and this GAG accounted for over 80% of the total GAG in the growth medium. Furthermore, this high level of synthesis was maintained in successive subcultures. Two features suggested that the level of HA synthesis by the rat cells was high rather than that the synthesis by the pig cells was low. Firstly, a comparison with cultured pig aortic smooth muscle cells and with chick embryo fibroblasts (Table 2) - cells known to be active in the secretion of GAGS - indicated that HA synthesis by the rat cells was exceptionally high. Secondly, it was noticeable that fresh growth medium on the rat cell cultures became quite viscous within a few days, a feature consistent with high levels of HA. Differences in the amount of the sulphated GAGS synthesised by the 2 cell types were less marked, and intraspecies variability prevented a meaningful comparison. The proportion of sulphated GAGS, especially DS and CS, in total GAGS, however, did show a marked difference. Whereas the rat sulphated GAGS accounted for less than 20% of the total, pig sulphated GAGS accounted for more than 70%. While there are not enough data to compare statistically the effects of 10% and 2% serum levels (Table 2) or GAG synthesis, it would appear that changes in the serum level did not affect the high level of HA synthesis by rat endotheha1 cells, or alter the relative proportion of GAGS in either species. The amount of GAG synthesised and retained in the cell layers of both the rat and pig was only 5% of the total GAG synthesised and the proportion of the individual GAGS did not differ significantly from the proportions in the growth medium. Discussion These results demonstrate that: (1) rat aortic endothelial cells resist removal from the vessel wall by collagenase digestion whereas pig aortic endothelial cells do not; (2) under the same conditions of tissue culture, rat endothelial cells have a much faster growth rate and can be maintained for many more subcultures than pig endothelial cells, and (3) rat endothelial cells in culture synthesise and secrete large amounts of GAGS into the growth medium, mostly hyaluronic acid, while pig endothelial cells synthesise only moderate amounts of GAG, and these are mostly sulphated. While these findings pertain to particular culture conditions they, nevertheless, suggest that endothelial cells among different species may behave differently in vivo, and in a way that might account for their known resistance or susceptibility to atherosclerosis. The rat is normally resistant to lesions [2] and the difficulty in removing endothelial cells by collagenase digestion and other treatments would suggest that in vivo it is also resistant to damage. However, should damage occur and cell division and regeneration take place at a rapid rate, as occurs in tissue culture, then denuded areas would be quickly covered and the normal morphology maintained. It has been established that rapid
24
endothelial regeneration occurs in rat aortas damaged by balloon catheters [ 71 or air drying [2], and that myointimal thickenings, which develop as a result of the denudation, regress following endothelial regeneration [ 21. In contrast to the rat, the pig is susceptible to atherosclerosis and has been frequently used as a model for human atherosclerosis studies [8]. If the ease of removal of endothelial cells by collagenase digestion is a reflection of in vivo characteristics, then it may account, at least in part, for this predisposition to lesion formation. The slower rate of growth of the pig endothelial cells and the difficulty in passaging the cells beyond 4 subcultures is more difficult to interpret since the culture conditions may not have been optimal. The cells in the first 2 subcultures, however, appeared to be in good health according to the criteria of cell attachment: the formation of a typical pavement pattern at confluency, and stable cell size (confluent densities the same as for the rat endothelial cells in culture). Comparison with the rat is based on data from these healthy cultures only. Pig endothelial cells, however, have been apparently successfully maintained in culture for longer periods than were achieved in this study, although it is notable that either earlier subcultures (1 through 6) [9] or primaries only [lo] were used for experiments. However, variations in the age of the animals and in culture conditions make comparisons with our system of doubtful value. The reason for the difference in susceptibility of the two types of endothelial cells to collagenase removal is unknown but it might be related to differences in intimal morphology. In the pig, as with most other large mammals, the intima is relatively broad and the subendothelial space contains a network of collagen and elastic fibres arranged more loosely towards the endothelial surface [ 81. In the rat, however, there is a very much reduced subendothelial space and in places the endothelial cells have been reported to be attached directly to the irregular inner surface of the internal elastic lamina [ll]. It has been suggested that this serves to anchor the endothelium [12]. A further difference between the two species is apparent from various published micrographs. While the subendothelial surface of pig endothelial cells are generally smooth [ 8,131, rat endothelial cells frequently have an irregular subendothelial surface with cytoplasmic projections, or in some cases small hooklets, that extend into the narrow subendothelial space [ 8,11,14-161. These hooklets are especially prominent where subendothelial vacuolation occurs in response to a high fat diet [14]. More recently, Buck [17] has demonstrated that the cytoplasmic projections in the subendothelial space are in fact longitudinal folds, or rugae, that run parallel to the long axis of the vessel and project to within a few nanometers of the internal elastic lamina’or into fenestrations in the lamina. It is again postulated that this arrangement serves to attach more firmly the endothelial cells to the vessel wall. These marked differences in intimal morphology may then account for the differences in susceptibility of rat and pig endotheliurn to collagenase digestion. The synthesis and secretion of GAGS by rat and pig endothelial cells in culture is not unexpected since both bovine [ 181 and rabbit [ 191 endothelial cells in culture synthesise GAGS, although only the sulphated GAGS have been measured and thus a full comparison with our results is not possible. The significance of GAG synthesis by aortic endothelial cells, however, is unknown and,
25
before any conjecture can be made about the role of GAGS, it will be necessary to establish whether endothelial cells in vivo secrete GAGS of the same type and amount as in culture, and whether the secretion is towards the lumen or towards the underlying matrix or both. It is of interest that in a preliminary autoradiographic investigation of rat aorta in organ culture (unpublished), where the integrity of the arterial wall is maintained and approximates to the in vivo state, we have found that - as in tissue culture - endothelium synthesises high levels of HA in comparison with the cells of the media and adventitia. The results in this present study indicate that endothelial cells should at least be considered as possible contributors to the extracellular components of the arterial wall, and that this contribution may show species variability. Variation in the synthesis of HA by the endothelial cells in culture is of particular interest since this GAG plays an important role in the organisation of tissues during morphogenesis [ 201. It is notable that HA differs from the sulphated GAGS in that it does not bind lipoprotein [21]. Also while it usually forms only a small percentage of the total GAG in the intact arterial wall [22], and relatively less in lesions [23], the intimal concentration is generally higher than that in the media [ 221. References 1 Ross. R.. Glomset. J. and Hsrker, L., Response to injury and atherogenesis, Amer. J. Path., 86 (1977) 675. 2 Fishman. J.A., Ryan, G.B. and Kamovsky, M.J., Endothelial regeneration in the rat carotid artery and the significance of endothelisl denudation in the pathogenesis of myointimal thickening, Lab. Invest., 32 (1975) 339. 3 French, J.E.. Atherosclerosis in relation to the structure and function of the arterial intima. with special reference to the endothelium. Int. Rev. EXP. Path., 5 (1966) 253. 4 Merrilees, M.J.. Merrilees. M.A.. Bimbaum. P.S., Scott, P.J. and Flint, M.H.F., The effect of centrlfugal force on glycosaminoglycan production by aortic smooth muscle cells in culture, Atherosclerosis, 27 (1977) 259. 5 Gillard, G.C., Bimbaum, P.. Reilly, H.C., Merrilees, M.J. and Flint, M.H.. The effect of charged synthetic polymers on proteoglycan synthesis and sequestration in chick embryo fibroblast cultures, Biochim. Biophys. Acta, 584 (1979) 520. 6 Breen, M.. Weinstein, H.G., Andersen. M. and Veiss. A., Microanalysis and characterisation of acidic glycosaminoglycans in human tissues, Anal. Biochem.. 35 (1970) 146. 7 Schwartz, S.M., Haudenschild, C.C. end Eddy, E.M.. Endothelial regeneration, Part 1 (Quantitative analysis of initial stages of endothelial regeneration ln rat aortic intima), Lab. Invest., 38 (1978) 568. 8 French, J.E.. Jennings. M.A. and Florey. H.W., Morphological studies on atherosclerosis in swine, Ann. N.Y. Acad. Sci, 127 (1965) 780. 9 Pearson, J.D., Carleton, J.S.. Hutchinks, A. and Gordon, J.L., Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture. Biochem. J., 170 (1978) 265. 10 Slater. D.N. and Sloan, J.M., The porcine endothelial cellin tissue cuhure. Atherosclerosis, 21 (1975) 259. 11 Pease. D.C. and Paule, W.J., Electron microscopy of elastic arteries - The thomcic aorta of the rat, J. Ultrastr. Res., 3 (1960) 469. 12 Keech, M.K., Electron microscope study of the normal rat aorta. J. Cell. BioL. 7 (1960) 533. 13 Gutstein. W.H.. Farrell, G.A. and Armellini, C., Blood flow disturbance and endothelial cell injury in preatherosclerotic swine, Lab. Invest., 29 (1973) 134. 14 Still, W.J.S. and O’Neal, R.M., Electron microscopic study of experimental atherosclerosis in the rat, Amer. J. Path., 40 (1962) 21. 15 Huttner. I., More, R.H. and Rona, G.. Fine structural evidence of specific mechanism for increased endothelial permeability in experimental hypertension, Amer. J. Path., 61 (1970) 395. 16 Huttner, I., Boutet. M. and More, R.H.. Studies on protein passage through arterial endothelium, Part 2 (Regional differences in permeability to fine structural protein tracers in arterial endothelium of normotensive rat), Lab. Invest.. 28 (1973) 678.
26 17 Buck, R.C., The longitudinal orientation of structures in the subendotheliai space of rat aorta, Amer. J. Anat., 156 (1979) 1. 18 Gamse, G.. Fromme. H.S. and Kresse. H., Metabolism of sulphated glycosaminogiycans in cultured endothelial cells and smooth muscle cells from bovine aorta. Biochim. Biophys. Acta. 544 (1978) 514. 19 Buonassi, V., Suiphated mucopolysaccharide synthesis and secretion in endotheliai cell cultures, EXP. CelL Res.. 76 (1973) 363. 20 Toole, B.P., Morphogenetic role of glycosaminoglycans (acid mucopolysaccharides) in brain and other tissues. In: S.H. Barondes (Ed.), Neuronal Recognition, Plenum Press, New York, London, 1976, p. 276. 21 Iverius, P.H., Possible role of glycosaminoglycans in the genesis of atherosclerosis. In: Atherogeneais Initiating Factors (Ciba Foundation Symposium, No. 12). Elsevier/North-Holland, Amsterdam, 1973. p. 185. 22 Massaro. T.A. and Glatz. C.E.. Distribution of glycosaminoglycans in consecutive layers of the rabbit aorta. Artery, 5 (1979) 1. 23 Dalferes, Jr., E.R., Ruiz. H., Kumar, V., Radhakrishnamurthy, B. and Berenson, G.S.. Acid mucopolysaccharides of fatty streaks in young human male aortas, Atherosclerosis. 13 (1971) 121.