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[35S]Proteoglycan metabolism of arterial smooth muscle cells cultured from normotensive and hypertensive rats
299
Atherosclerosis, 45 (1982) 299-310 Elsevier Scientific Publishers Ireland, Ltd.
[ 35SlProteoglycan Metabolism of Arterial Smooth Muscle Cells Cultured from Normotensive and Hypertensive Rats A. Schmidt, J. Griinwald Institute of Atherosclerosis
and E. Buddecke
Research and Institute of Physiological Chemistry, Miinster (F. R. G.)
University of Miinster,
(Received 26 February, 1982) (Revised, received 17 May, 1982) (Accepted 22 June, 1982)
Summary Arterial smooth muscle cells cultured from normotensive and hypertensive rats incorporated [ ‘%]sulfate into the extracellular and pericellular sulfated proteoglycans and endocytose extracellular [ %]proteoglycans at a significantly higher rate in the phase of logarithmic growth than did nondividing cells. 35S incorporation into proteoglycans was positively correlated with [ 3H]thymidine incorporation into the cellular TCA-precipitable material. The rates of [ 35S]proteoglycan synthesis and endocytosis per cell per day were higher in smooth muscle cells from hypertensive than from normotensive animals, the observed differences being related to a higher average protein content of smooth muscle cells cultured from hypertensive rats as compared with cells of normotensive animals. Gel filtration under dissociative conditions separated the [ ‘%]proteoglycans into high and low molecular weight fractions (A, B) differing in glycosaminoglycan composition and their ability to be endocytosed by smooth muscle cells. The relative proportion of the high molecular weight proteoglycan fraction A decreased continuously from sparse to confluent cell cultures. Key words: Cultured arterial smooth muscle cells - Hypertension
- Proteoglycan
metabolism
Introduction
Arterial smooth muscle cells in culture retain many of the characteristics associated with their behaviour in vivo. The synthesis of the arterial wall connective tissue 0021-9150/82/0000-0000/$02.75
0 1982 Elsevier Scientific Publishers Ireland, Ltd
300
matrix constituents, including collagen [1,2] elastic fiber proteins [2-41 and proteoglycans [5], under cell culture conditions has been described. The characteristic distribution pattern of arterial tissue glycosaminoglycans has been shown to be expressed by cultured bovine [6] and pig [7] arterial smooth muscle cells. Moreover, the response of arterial tissue to injury or experimental atherogenesis is detectable in organ and cell culture., Thus, an enhanced in vivo and in vitro incorporation of [35S]sulfate into the glycosaminoglycans has been reported for the arterial tissue of rats under the influence of various risk factors [8,9]. Furthermore, rabbit smooth muscle cells were observed to incorporate more [14C]glucosamine into the acidic glycosaminoglycans when cultured from cholesterol-induced arteriosclerotic lesions of the aorta than when derived from the aorta of control animals [lo]. Proliferation of arterial smooth muscle cells is considered to be an early event in the development of arteriosclerotic lesions [ 1 l- 131. Exposure to arteriosclerotic risk factors such as hypertension and diabetes mellitus promotes the proliferation of arterial smooth muscle cells in vivo [ 141 as well as in culture [ 151, but information about the proteoglycan metabolism of proliferating smooth muscle cells is as yet limited. The present study compares the proteoglycan metabolism of smooth muscle cells cultured from the aorta of hypertensive and normotensive rats. Material and Methods Materials
Chondroitin AC lyase, chondroitin ABC lyase and the disaccharide standards (3-O-A4-glucuronosyl-N-acetylgalactosamine 4-sulfate and -6-sulfate) were purchased from Miles Seravac/Lausanne. Sodium [ 35S]sulfate (carrier free) was obtained from the Radiochemical Centre, Amersham-Buchler (Braunschweig). All chemicals were of analytical grade or the best grade available and were purchased from BoehringerMannheim GmbH (Mannheim), Merck (Darmstadt) and Serva (Heidelberg). Analytical
methodr
Glucuronic acid was determined as described by Bitter and Muir [ 161,cell protein by the method of Lowry et al. [17] with bovine serum albumin as standard. Cells were counted in a Coulter counter (Coulter Electronics, Krefeld) with the following settings: attenuation 707, aperture 64, threshold 50.5. Radioactivity was measured in a liquid scintillation spectrometer (Packard, Model1 B 2450) with Instagel (Zinsser, Frankfurt). Quench corrections were made by internal standards. Cell culture
Arterial smooth muscle cells were cultured from the thoracic aorta of male Wistar rats (200-250g) according to Ross et al. [18]. Normotensive and hypertensive rats were weight- and age-matched; renal hypertension was produced by the method of Page [19]. After 4-6 days the arterial pressure, as measured in the tail artery, increased from 80-90 mm Hg to about 200 mm Hg. Biopsies from the thoracic aorta of the animals were taken for cell culturing after the hypertension had lasted about 2 weeks. The vascular changes observed in hypertension of the rat (myocyte hyper-
301
trophy, medial thickening and fibrosis) were proportional in degree to the duration of hypertension and differed in frequency and intensity [20]. Second passage cell cultures were used in all experiments. Cell population growth curves were obtained by incubation of the cells with an initial density of 2000 cells/cm’ (5 X 104/5 ml) in Dulbecco’s minimal essential medium with 10% fetal calf serum. The cell count for each of the plates harvested (25 cm’ Falcon flasks) was estimated by counting the cells that were suspended in a known volume of 0.25% trypsin solution. Cultures used for studies of [ 35S]proteoglycan and [ 35S]glycosaminoglycan synthesis at different cell densities or in relation to [3H]thymidine incorporation were grown in a medium containing 5 PCi [35S]sulfate, or [ 35S]sulfate in combination with 1 PCi [3H]thymidine/ml for 24 h prior to harvesting. To assure that all replicate cultures in a particular experiment were grown at the same concentration of [ 35S]sulfate and [3H]thymidine, the same batch of culture medium was used prior to and during the labelling period. All experiments were performed under strictly identical cell culture conditions. It was ensured that the pH value of the culture medium (pH 7.35) remained constant during the period of 35S labelling or endocytosis of proteoglycans. For protein determination the cell layers were washed 5 times with Hank’s solution. After adding 2 ml of 0.1% trypsin solution, cells were kept at 37°C for 10 min, then dispersed in the flask by vigorous shaking and transferred into conical centrifugation tubes. The cell suspension was centrifuged for 10 min at 800 X g, washed twice with 0.15 M NaCl and the cell pellet was dissolved in 1 ml 10% NaOH. Aliquots were used for protein determination (see above). Incorporation of [ I’*S]sulfate into proteoglycans and glycosaminoglycans Determination of extracellular and surface associated [ 35S]proteoglycans [ 35S]glycosaminoglycans was carried out as previously described [6].
and
Incorporation of [ ‘Hlthymidine
Cells were harvested by trypsinization, washed twice in Dulbecco’s medium to remove extraneous protein followed by repeated washes in 5% trichloroacetic acid to remove precursor radioactivity and acid-soluble material. The pellet was dissolved in tissue solubilizer (Packard, Frankfurt) for scintillation counting. Preparation of extracellular [ .“S] proteoglycans
Six confluent cultures were incubated in the presence of 5 ml of serum-free medium (Waymouth modified according to ref. 21) and 5 PCi [35S]sulfate/ml for 5 days. The medium (30 ml) was then concentrated to 0.5-1.0 ml by ultrafiltration, dialyzed against 0.1 M ammonium sulfate and 0.15 M NaCl, for 24 h each and chromatographed on Sepharose 2B CL (115 X 1 cm) at 4°C using 0.15 M NaCl as eluent. Aliquots of the [35S]proteoglycan fractions A and B were analyzed for chondroitin 4,6-sulfate, dermatan sulfate and heparan sulfate after subsequent degradation with alkali (0.15 M NaOH, 4 h, 37’C) and chondroitinase AC and ABC according to Saito (221. The [35S]sulfate-containing proteoglycan fraction B (see
302
Fig. 5) was pooled, concentrated for endocytosis experiments. Determination
of [ 35S] proteoglycan
to a small volume by ultrafiltration
and then used
endocytosis
The rate of endocytosis was measured at nonsaturating [35S]proteoglycan concentrations. Subconfluent and confluent cultures (25 cm2 Falcon flasks) were incubated for 24 h after plating with 2.0 ml medium (pH 7.4) containing 30 X lo3 cpm [ 35S]proteoglycans. Adsorbed, endocytozed and degraded [ 35S]proteoglycans were determined after a 16-h incubation period precisely as described previously [6]. The rate of endocytosis is expressed as a percentage of the added 35S radioactivity per mg protein. Gel filtration of [ 35S] proteoglycans 10 ml of medium from cell cultures at different stages of growth were reduced to a volume of 0.5 ml by ultrafiltration. The concentrated medium was exhaustively dialyzed against 0.10 M ammonium sulfate in the presence of a protease-inhibiting cocktail [23], adjusted to a final concentration of 4M guanidinium chloride by addition of 8 M guanidinium chloride buffered with 20 mM Tris, pH 7.0. 0.8 ml was applied to a Sepharose 4 B CL column (0.9 X 100 cm), equilibrated and eluted with 4 M buffered guanidinium chloride. 0.8~ml fractions were collected and monitored for radioactivity (see Fig. 5).
I
I
I
I
I
2
4
6
8
Time (days) Fig. 1. Cell population
growth curve for rat smooth muscle cells cultured from the aorta of normotensive (0) and hypertensive (A) rats. Second passage cells were incubated in 5 ml, 25 cm2 Falcon flasks with an initial cell density of 2X 10’ cells/cm2 and counted at the specified intermediate time points. Means and standard deviations of 7 experiments done in duplicate.
303
Results Cell population growth curue and incorporation of [“‘S]sulfate proteoglycans
into extracellular
Growth curves of smooth muscle cells cultured from normotensive and hypertensive rats after seeding 1 X lo5 cells per flask are shown in Fig. 1. No differences in the plating efficiency between the cells from normotensive or hypertensive rats were observed. Cell population doubling time during the logarithmic growth phase was 26-30 h for the cells from normotensive rats and 18-20 h for the cells from hypertensive rats. Following confluence, arterial smooth muscle cell growth slows but does not stop. The clear differences between the cells from normotensive and hypertensive animals are in accordance with the previous observation of a higher proliferation rate of smooth muscle cells cultured from hypertensive rats as compared with cells from normotensive rats [ 151. When the amount of [%]sulfate which was incorporated into the proteoglycans secreted from the cells into the growth medium over a period of 24 h was determined on the 5th, 7th and 11th day after plating, an increased amount of incorporated 35S
Cell density(x 10m6 cells/flask)
Fig. 2. Incorporation of [35S]sulfate into extracellular proteoglycans at different cell densities. Cell suspensions with an initial density of 2-4X IO3 cells/cmr were cultured and harvested between the 3rd and 11th day after plating. 24 h prior to harvesting the cultures were refed with 5 ml of medium containing 25 gCi [3sS]sulfate. Differences between cells from normotensive (0) and hypertensive (A) rats disappear when radioactivity is related to the cell protein content.
304
radioactivity per flask up to the 7th day was noted. This was expected from the increased number of cells. However, on day 11 a lower amount of incorporated 35S radioactivity per flask was found despite a further increase in cell number. Cell proliferation and proteoglycan synthesis The rate of [35S]proteoglycan synthesis declines in both cell lines as the cells progress from low to high densities (Fig. 2). The slopes reflecting the change in the rate of [35S]proteoglycan synthesis as cell density increases appear to be similar for cells from normotensive and hypertensive rats, but decline when the cells reach confluence at densities of 0.8- 1.0 X IO6 cells/flask. At all densities the net rate of [ 35S]proteoglycan synthesis per cell is higher for the cells from hypertensive than for those from normotensive animals. However, as is evident from Fig. 3, this result is due to a higher protein content in the cells of hypertensive as compared to normotensive rats (see Discussion). Regardless of this difference the protein concentration per cell decreases in both cell lines as cell density increases. When seen in relation to the protein content, progressively decreasing but roughly equal net rates of [35S]proteoglycan synthesis are calculated for cells from normotensive and hypertensive rats. The results shown in Figs. 2 and 3 reveal that both normotensive and hypertensive cells in the exponential phase of growth (Fig. 1) secrete more [3SS]proteoglycans into the extracellular medium than do confluent cells.
Cell density (x
10m6 cells/flask)
Fig. 3. Cell protein concentration at different cell densities. Cell culture conditions as in Fig. 2. Cultures from normotensive (0) and hypertensive (A) rats were made in triplicate for cell counting and protein determination (means of 2 values).
305
20 t 0
I 20
I 40
I 60 3H
1
I 80
l&3
ll0
-3 cpm x 10 /mg cell protein
Fig. 4. Correlation between [ 3H]thymidine incorporation into trichloroacetic acid-precipitable material of arterial smooth muscle cells and [ “S]sulfate incorporation into extracellular proteoglycans. Cell cultures at various stages of growth were incubated in the presence of 1 pCi [3H]thymidine and 5 pCi [ 35S]sulfate/ml medium for 24 h prior to harvesting (see Methods). Correlation coefficient 0.75, slope of regression line 0.24 (P ~0.05).
Further evidence of a correlation between cell division and proteoglycan synthesis was obtained from double labelling experiments with [3H]thymidine and [ 35S]sulfate. A plot of “S radioactivity incorporated into the external sulfated proteoglycans versus the 3H radioactivity incorporated into the cellular TCA-precipitable material reveals (Fig. 4) a correlation coefficient of 0.75 and a slope of the regression line of 0.24. This indicates a proportional increase in [35S]proteoglycan synthesis with increasing [ 3H]thymidine incorporation. No differences between cells derived from normotensive and hypertensive animals could be ascertained.
TABLE
1
RELATIVE CONCENTRATIONS OF INDIVIDUAL FROM SECRETIONS OF CONFLUENT ARTERIAL
[3sS]GLYCOSAMINOGLYCANS ISOLATED SMOOTH MUSCLE CELL CULTURES
Glycosaminoglycan compositions of the proteoglycan fractions A and B were determined after enzymatic degradation with chondroitinase AB and ABC. Values are means of 5 experiments and expressed as cpm/lOO cpm of total sulfated glycosaminoglycans. Cell type
Normotensive
Hypertensive
PG fraction
Total glycosaminoglycans
(cpm/lOO
cpm)
c4s
C6S
DS
HS
A B
81 46
6 7
10 40
3
A B
81 43
7 7
10 43
2 7
1
306
10 8 6 4 2
Fraction Number Fig. 5. Elution profile of intracellular [ 35S]proteoglycans at different cell densities. The medium of sparse (5th day), subconfluent (7th day) and confluent (1 Ith day) cultures was concentrated, dialyzed exhaustively against 0.10 M ammonium sulfate, adjusted to 4 M guanidinium chloride and chromatographed on Sepharose 4B CL (see Methods). 15000 cpm were applied to the columns.
Proteoglycan pattern and endocytosis The macromolecular state of extracellular [ ‘%]proteoglycans
produced by arterial smooth muscle cells was judged from gel filtration experiments. In order to exclude interactions of proteoglycans with each other or with other extracellular constituents, the gel chromatography was performed with buffered 4 M guanidinium chloride as eluent (Fig. 5). The extracellular proteoglycans resolved into species of two molecular weights (A, B) differing in hydrodynamic size, a higher molecular weight fraction being excluded from the gel, and a lower molecular weight fraction having a K,,
307
TABLE
2
INFLUENCE OF CELL DENSITY ON UPTAKE AND DEGRADATION GLYCANS (FRACTION B) BY ARTERIAL SMOOTH MUSCLE CELLS NORMOTENSIVE AND HYPERTENSIVE RATS
OF [3sS]PROTEOCULTURED FROM
Cells from the first subculture were suspended with 0.25% trypsin solution and diluted with fresh medium to the specified density. Values are means of two experiments done in triplicate. Cells were preincubated for 24 h, then for 16 h in the endocytotic experiments, with 2 nmole [%]proteoglycan disaccharide units/ml medium (30X IO3 cpm/ml). For calculation of proteoglycan molarity, the equivalence of 1 pmole of proteoglycan-bound disaccharide to 1 mg proteoglycan (protein content about 40%) was assumed. Proteoglycan disaccharide units were calculated from glucuronic acid determination. Cell type
Cell density (cellX IO’/25
cm*)
Uptake (W of added [ 35S]proteoglycans/ mg cell protein)
Degradation (W of endocytosed proteoglycans) -
Normotensive
1.23 2.46 4.92 9.84
19.2 14.8 12.4 10.2
97 96 98 95
Hypertensive
1.65 3.30 6.60 13.20
18.7 13.9 10.7 9.4
98 98 94 97
value of 0.36. The ratio of 35S radioactivity incorporated into fractions A and B depended on culture conditions. With increasing cell density fraction B became the predominant proteoglycan, the ratio of A/B decreasing continuously from 0.3 in subconfluent cultures (5th day) to 0.06 in confluent cultures (11 th day). Fractions A and B differed in their relative glycosaminoglycan composition (Table l), the chondroitin 4+ulfate/dermatan sulfate ratio of fraction A being significantly higher than that of fraction B. The pericellular [35S]proteoglycans were obtained by extraction with 4 M guanidinium chloride after removing the medium from the washed cell layer. The coextracted intracellular 35S radioactivities accounted for only 3-5% of the total radioactivity and were not considered. The elution profile of pericellular proteoglycans (data not shown) resembled that of the extracellular proteoglycans, with the exception that only lo-20% of the 35S radioactivity incorporated into total proteoglycans was detected in the pericellular proteoglycan fraction, and that the proportion of heparan sulfate was higher in the pericellular proteoglycans in relation to the extracellular proteoglycans. The [ 35S]proteoglycan fraction B is internalized by smooth muscle cells, while no endocytotic uptake of the proteoglycan fraction A was observed. Table 2 provides evidence that the [35S]proteoglycan fraction B is internalized at rates which decline as the cell density increases. More than 95% of [3SS]proteoglycans endocytosed within the incubation period are degraded intracellularly as judged by the release of inorganic [35S]sulfate into the culture medium. There are no differences between
cells cultured from normotensive and hypertensive animals with respect to the rate of endocytosis as related to the cell protein content. I
Discussion
The results here provide evidence for metabolic differences (a) between rapidly proliferating and nonproliferating arterial smooth muscle cell cultures, and (b) between cultured smooth muscle cells derived from normotensive and hypertensive rats. The [ 35S]proteoglycan metabolism of arterial smooth muscle cells in the logarithmic phase of growth differs from that of non-dividing cells in a higher rate of [ 35S]proteoglycan synthesis, by differences in the glycosaminoglycan composition of extracellular proteoglycans and an enhanced rate of endocytotic uptake of [ 35S]proteoglycans. The positive correlation between cell proliferation and proteoglycan synthesis is confirmed by [3H]thymidine/[35S]sulfate labelling experiments. A more complex effect of cell density on the net synthesis of chondroitin sulfate has been described for human skin fibroblasts [24] and mouse 3T3 cells [25]. The finding that cells at low density endocytose proteoglycans at a higher rate than confluent cells (Table2) suggests higher receptor activity of cells in the exponential phase of growth. An analogous result has been obtained for the receptor-mediated uptake of LDL which was higher in non-confluent cultures of human skin fibroblasts [26,27], rat aortic smooth muscle cells [28], and bovine [29] and rat [30] endothelial cells than in contact-inhibited, confluent cultures. Likewise, the receptor-independent (fluid) endocytosis of [U-‘4C]sucrose by arterial smooth muscle cells cultured from monkey thoracic aorta was observed to be inversely proportional to cell density [31]. The proteoglycans produced by smooth muscle cells and secreted into the culture medium differ in size and glycosaminoglycan content (Table 1). Under both dissociative and non-dissociative conditions two proteoglycan fractions with different hydrodynamic sizes could be resolved by gel filtration (Fig. 5). This confirms earlier studies by Kresse et al. [6], CSster [32] and Carlstedt [33], who isolated and characterized proteoglycans of varying size from the medium of human skin fibroblasts. The additional information provided by the present work concerns the observation that the high molecular weight proteoglycan fraction (A of Fig. 5) is not endocytosed by the parent cells and decreases in relation to the low molecular weight proteoglycan fractions as the cultured cells progress from low to high cell densities. With the exception of a higher cell protein content of cells derived from hypertensive animals, no significant differences between the cell lines from hypertensive and normotensive animals could be detected. The higher cell protein content of arterial smooth muscle cells derived from hypertensive animals was explained by the observation that arterial smooth muscle cells contain a subpopulation of large, partly polynucleated cells. The percentage of these cells is significantly higher in cells from hypertensive (26%) as compared to control animals (13%) [34]. In summary, our results suggest that the enhanced in vivo incorporation of [35S]sulfate into the sulfated glycosaminoglycans of the arterial wall of hypertensive
animals [14] is closely related to the higher number of proliferating cells in the arterial tissue as compared to normotensive animals. Thus, the rate of [3SS]proteoglycan synthesis depends rather on the ratio of dividing to nondividing cells than on metabolic diversities between smooth muscle cells cultured from normotensive and hypertensive animals. No evidence for a higher metabolic activity of cells from hypertensive animals could be detected in a comparison of cells in the same growth phase, with the metabolic parameters stated, in relation to the cell protein content. Our investigation emphasizes the importance of cell proliferation as an early event in the development of hypertension-induced metabolic alterations of arterial tissue. Acknowledgements
The authors are indebted skillful technical assistance.
to Ms. H. Stockmann
and Ms. M. Ahler for their
References 1 Ross, R. and Klebanoff, S.J., The smooth muscle cell, Part 1 (In viva synthesis of connective tissue proteins), J. Cell Biol., 50 (1971) 159. 2 Ross, R., The arterial smooth muscle cell. In: R.W. Wissler and J.C. Geer (Eds.), The Pathogenesis of Atherosclerosis, Williams and Wilkins Co., Baltimore, MD, 1972, p. 147. 3 Narayanan, S., Sandberg, L.B., Ross, R. and Layman, D., The smooth muscle cell, Part 3 (Elastin synthesis in arterial smooth muscle cell culture), J. Cell Biol., 68 (1976) 411. 4 Smith, D.W., Abraham, P.A. and Cames, W.H., Studies of the biosynthesis of a soluble precursor of elastin. In: L.B. Sandberg, W.R. Gray and C. Franzblau (Eds.), Elastin and Elastic Tissue, Plenum Press, New York, NY, 1977, p. 385. 5 Wight, T.N. and Ross, R., Proteoglycans in primate arteries, Part 2 (Synthesis and secretion of glycosaminoglycans by arterial smooth muscle cells in culture, J. Cell Biol., 67 (1975) 675. 6 Kresse, H., Figura, K., Buddecke, E. and Fromme, H.G., Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells, Part 1 (Characterization of different pools of sulfated glycosaminoglycans), Hoppe-Seylefs Z. Physiol. Chem., 356 (1975) 929. 7 Deudon, E., Breton, M., Benou, E. and Picard, J., Metabolism of glycosaminoglycans in cultured smooth muscle cells from pig aorta, Biochimie, 62 (1 l- 12) (1980) 8 11(Extr. of Vol. 62). 8 Hauss, W.H., The role of the mesenchymal cells in arteriosclerosis, Front. Matrix Biol., 2 (1976) 89. 9 Kresse, H., Filipovic, I., Wessels, F., Wessels, G. and Buddecke, E., Selektive Zunahme der Syntheserate von Dermatansulfat und Heparansulfat des Arteriengewebes bei genetischem bzw. experimentellem Hypertonus der Ratte, Z. Klin. Chem., 9 (1971) 21. 10 Pietila, K., Yla-Herttuala, S., Jaakkola, 0. and Nikkari, T., Metabolism of glycosaminoglycans and lipids in smooth muscle cells from atherosclerotic rabbit aortas in culture, Atherosclerosis, 37 (1980) 449. 11 Titus, J.L. and Weilbaecher, D.G., Smooth muscle cells in atherosclerosis. In: A.M. Gotto. Jr., L.C. Smith and B. Allen (Eds.), Atherosclerosis V, Springer-Verlag, Heidelberg, 1980. 126. 12 Ross, R., Glomset, J. and Harker, L., The response to injury and atherogenesis - The role of endothelium and smooth muscle. In: R. Paoletti and A.M. Gotto, Jr., (Eds.), Atherosclerosis Review 3. Raven Press, New York, NY, 1978, p. 69. 13 Hauss, W.H., Mey, J. and Schulte, H., Effect of risk factors and antirheumatic drugs on the proliferation of aortic wall cells, Atherosclerosis, 34 (1979) 119. 14 Hauss, W.H., Role of arterial wall cells in sclerogenesis, Ann. N.Y. Acad. Sci., 275 (1976) 286. 15 Mey, J., Grtinwald, J. and Hauss, W.H., Growth rate differences between arterial smooth muscle cells cultivated from rat impaired by short- or long-term hypertension respectively, Artery, 8 (1980) 348.
310 16 Bitter, T. and Muir, H., A modified uranic acid carbazole reaction, Anal. B&hem., 4 (1962) 330. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (195 1) 265. 18 Ross, R. and Glomset, J., Atherosclerosis and the arterial smooth muscle cell, Science, 180 (1973) 1332. 19 Page, J.H. and Graef, J., Hypertension following experimental perinephritis induced by cellophane, Arch. Path., 28 (1939) 613. 20 Hadjilsky, P., Morphogenese de l’arteriopathie central et p&ipherique du rat spontanement hypertendu, Paroi arterielle (Arterial Wall), 6 (1980) 119. 21 Fratantoni, J.C., Hall, C.W. and Neufeld, E.F., The defect in Hurler and Hunter syndromes, Part 2 (Deficiency of specific factors involved in mucopolysaccharide degradation), Proc. Nat. Acad. Sci. (USA), 64 (1969) 360. 22 Saito, H., Yamagata, T. and Suzuki, S., Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates, J. Biol. Chem., 243 (1968) 1536. 23 Oegema, Jr., T.R., Hascall, V.C. and Dziewiatkowski, D.D., Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma, J. Biol. Chem., 250 (1975) 6151. 24 Hopwood, J.J. and Dorfman, A., Glycosaminoglycan synthesis by cultured human skin fibroblasts after transformation with Simian virus 40, J. Biol. Chem., 252 (1977) 4777. 25 Cohn, R.H., Cassiman, J.J. and Bernfield, M.R., Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan, J. Cell Biol., 71 ( 1976) 280. 26 Goldstein, J.L. and Brown, M.S., Binding and degradation of low density lipoproteins by cultured human fibroblasts, J. Biol. Chem., 249 (1974) 5153. 27 Kruth, H.S., Avigan, J., Gamble, W. and Vaughan, M., Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts, J. Cell Biol., 83 (1979) 588. 28 Stein, 0. and Stein, Y., Surface binding and interiorixation of homologous and heterologous serum lipoproteins by rat aortic smooth muscle cells in culture, B&him. Biophys. Acta, 398 (1975) 377. 29 Coetzee, G.A., Stein, 0. and Stein, Y., Uptake and degradation of low density lipoproteins (LDL) by confluent, contact-inhibited bovine and human endothelial cells exposed to physiological concentrations of LDL, Atherosclerosis, 33 (1979) 425. 30 Vlodavsky, I., Fielding, P.E., Fielding, C.J. and Gospodarowicz, D., Role of contact inhibition in the regulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells, Proc. Nat. Acad. Sci. (USA), 75 (1978) 356. 31 Davies, P.F. and Ross, R., Growth-mediated, density-dependent inhibition of endocytosis in cultured arterial smooth muscle cells, Exp. Cell Res., 129 (1980) 329. 32 Caster, L., Carlstcdt, I. and Malmstrom, A., Isolation of ‘%- and ‘H-labelled proteoglycans from cultures of human embryonic skin fibroblasts, B&hem. J., 183 (1979) 669. 33 Carlstedt, I., Caster, L. and Malmstrom, A., Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture, B&hem. J., 197 (198 1) 2 17. 34 Grtinwald, J., Robenek, H., Mey, J. and Hauss, W.H., In vivo and in vitro cellular changes in experimental hypertension - Electronmicroscopic and morphometric studies of aortic smooth muscle cells, Exp. Mol. Path., 36 (1982) 164.
Atherosclerosis, 45 (1982) 299-310 Elsevier Scientific Publishers Ireland, Ltd.
[ 35SlProteoglycan Metabolism of Arterial Smooth Muscle Cells Cultured from Normotensive and Hypertensive Rats A. Schmidt, J. Griinwald Institute of Atherosclerosis
and E. Buddecke
Research and Institute of Physiological Chemistry, Miinster (F. R. G.)
University of Miinster,
(Received 26 February, 1982) (Revised, received 17 May, 1982) (Accepted 22 June, 1982)
Summary Arterial smooth muscle cells cultured from normotensive and hypertensive rats incorporated [ ‘%]sulfate into the extracellular and pericellular sulfated proteoglycans and endocytose extracellular [ %]proteoglycans at a significantly higher rate in the phase of logarithmic growth than did nondividing cells. 35S incorporation into proteoglycans was positively correlated with [ 3H]thymidine incorporation into the cellular TCA-precipitable material. The rates of [ 35S]proteoglycan synthesis and endocytosis per cell per day were higher in smooth muscle cells from hypertensive than from normotensive animals, the observed differences being related to a higher average protein content of smooth muscle cells cultured from hypertensive rats as compared with cells of normotensive animals. Gel filtration under dissociative conditions separated the [ ‘%]proteoglycans into high and low molecular weight fractions (A, B) differing in glycosaminoglycan composition and their ability to be endocytosed by smooth muscle cells. The relative proportion of the high molecular weight proteoglycan fraction A decreased continuously from sparse to confluent cell cultures. Key words: Cultured arterial smooth muscle cells - Hypertension
- Proteoglycan
metabolism
Introduction
Arterial smooth muscle cells in culture retain many of the characteristics associated with their behaviour in vivo. The synthesis of the arterial wall connective tissue 0021-9150/82/0000-0000/$02.75
0 1982 Elsevier Scientific Publishers Ireland, Ltd
300
matrix constituents, including collagen [1,2] elastic fiber proteins [2-41 and proteoglycans [5], under cell culture conditions has been described. The characteristic distribution pattern of arterial tissue glycosaminoglycans has been shown to be expressed by cultured bovine [6] and pig [7] arterial smooth muscle cells. Moreover, the response of arterial tissue to injury or experimental atherogenesis is detectable in organ and cell culture., Thus, an enhanced in vivo and in vitro incorporation of [35S]sulfate into the glycosaminoglycans has been reported for the arterial tissue of rats under the influence of various risk factors [8,9]. Furthermore, rabbit smooth muscle cells were observed to incorporate more [14C]glucosamine into the acidic glycosaminoglycans when cultured from cholesterol-induced arteriosclerotic lesions of the aorta than when derived from the aorta of control animals [lo]. Proliferation of arterial smooth muscle cells is considered to be an early event in the development of arteriosclerotic lesions [ 1 l- 131. Exposure to arteriosclerotic risk factors such as hypertension and diabetes mellitus promotes the proliferation of arterial smooth muscle cells in vivo [ 141 as well as in culture [ 151, but information about the proteoglycan metabolism of proliferating smooth muscle cells is as yet limited. The present study compares the proteoglycan metabolism of smooth muscle cells cultured from the aorta of hypertensive and normotensive rats. Material and Methods Materials
Chondroitin AC lyase, chondroitin ABC lyase and the disaccharide standards (3-O-A4-glucuronosyl-N-acetylgalactosamine 4-sulfate and -6-sulfate) were purchased from Miles Seravac/Lausanne. Sodium [ 35S]sulfate (carrier free) was obtained from the Radiochemical Centre, Amersham-Buchler (Braunschweig). All chemicals were of analytical grade or the best grade available and were purchased from BoehringerMannheim GmbH (Mannheim), Merck (Darmstadt) and Serva (Heidelberg). Analytical
methodr
Glucuronic acid was determined as described by Bitter and Muir [ 161,cell protein by the method of Lowry et al. [17] with bovine serum albumin as standard. Cells were counted in a Coulter counter (Coulter Electronics, Krefeld) with the following settings: attenuation 707, aperture 64, threshold 50.5. Radioactivity was measured in a liquid scintillation spectrometer (Packard, Model1 B 2450) with Instagel (Zinsser, Frankfurt). Quench corrections were made by internal standards. Cell culture
Arterial smooth muscle cells were cultured from the thoracic aorta of male Wistar rats (200-250g) according to Ross et al. [18]. Normotensive and hypertensive rats were weight- and age-matched; renal hypertension was produced by the method of Page [19]. After 4-6 days the arterial pressure, as measured in the tail artery, increased from 80-90 mm Hg to about 200 mm Hg. Biopsies from the thoracic aorta of the animals were taken for cell culturing after the hypertension had lasted about 2 weeks. The vascular changes observed in hypertension of the rat (myocyte hyper-
301
trophy, medial thickening and fibrosis) were proportional in degree to the duration of hypertension and differed in frequency and intensity [20]. Second passage cell cultures were used in all experiments. Cell population growth curves were obtained by incubation of the cells with an initial density of 2000 cells/cm’ (5 X 104/5 ml) in Dulbecco’s minimal essential medium with 10% fetal calf serum. The cell count for each of the plates harvested (25 cm’ Falcon flasks) was estimated by counting the cells that were suspended in a known volume of 0.25% trypsin solution. Cultures used for studies of [ 35S]proteoglycan and [ 35S]glycosaminoglycan synthesis at different cell densities or in relation to [3H]thymidine incorporation were grown in a medium containing 5 PCi [35S]sulfate, or [ 35S]sulfate in combination with 1 PCi [3H]thymidine/ml for 24 h prior to harvesting. To assure that all replicate cultures in a particular experiment were grown at the same concentration of [ 35S]sulfate and [3H]thymidine, the same batch of culture medium was used prior to and during the labelling period. All experiments were performed under strictly identical cell culture conditions. It was ensured that the pH value of the culture medium (pH 7.35) remained constant during the period of 35S labelling or endocytosis of proteoglycans. For protein determination the cell layers were washed 5 times with Hank’s solution. After adding 2 ml of 0.1% trypsin solution, cells were kept at 37°C for 10 min, then dispersed in the flask by vigorous shaking and transferred into conical centrifugation tubes. The cell suspension was centrifuged for 10 min at 800 X g, washed twice with 0.15 M NaCl and the cell pellet was dissolved in 1 ml 10% NaOH. Aliquots were used for protein determination (see above). Incorporation of [ I’*S]sulfate into proteoglycans and glycosaminoglycans Determination of extracellular and surface associated [ 35S]proteoglycans [ 35S]glycosaminoglycans was carried out as previously described [6].
and
Incorporation of [ ‘Hlthymidine
Cells were harvested by trypsinization, washed twice in Dulbecco’s medium to remove extraneous protein followed by repeated washes in 5% trichloroacetic acid to remove precursor radioactivity and acid-soluble material. The pellet was dissolved in tissue solubilizer (Packard, Frankfurt) for scintillation counting. Preparation of extracellular [ .“S] proteoglycans
Six confluent cultures were incubated in the presence of 5 ml of serum-free medium (Waymouth modified according to ref. 21) and 5 PCi [35S]sulfate/ml for 5 days. The medium (30 ml) was then concentrated to 0.5-1.0 ml by ultrafiltration, dialyzed against 0.1 M ammonium sulfate and 0.15 M NaCl, for 24 h each and chromatographed on Sepharose 2B CL (115 X 1 cm) at 4°C using 0.15 M NaCl as eluent. Aliquots of the [35S]proteoglycan fractions A and B were analyzed for chondroitin 4,6-sulfate, dermatan sulfate and heparan sulfate after subsequent degradation with alkali (0.15 M NaOH, 4 h, 37’C) and chondroitinase AC and ABC according to Saito (221. The [35S]sulfate-containing proteoglycan fraction B (see
302
Fig. 5) was pooled, concentrated for endocytosis experiments. Determination
of [ 35S] proteoglycan
to a small volume by ultrafiltration
and then used
endocytosis
The rate of endocytosis was measured at nonsaturating [35S]proteoglycan concentrations. Subconfluent and confluent cultures (25 cm2 Falcon flasks) were incubated for 24 h after plating with 2.0 ml medium (pH 7.4) containing 30 X lo3 cpm [ 35S]proteoglycans. Adsorbed, endocytozed and degraded [ 35S]proteoglycans were determined after a 16-h incubation period precisely as described previously [6]. The rate of endocytosis is expressed as a percentage of the added 35S radioactivity per mg protein. Gel filtration of [ 35S] proteoglycans 10 ml of medium from cell cultures at different stages of growth were reduced to a volume of 0.5 ml by ultrafiltration. The concentrated medium was exhaustively dialyzed against 0.10 M ammonium sulfate in the presence of a protease-inhibiting cocktail [23], adjusted to a final concentration of 4M guanidinium chloride by addition of 8 M guanidinium chloride buffered with 20 mM Tris, pH 7.0. 0.8 ml was applied to a Sepharose 4 B CL column (0.9 X 100 cm), equilibrated and eluted with 4 M buffered guanidinium chloride. 0.8~ml fractions were collected and monitored for radioactivity (see Fig. 5).
I
I
I
I
I
2
4
6
8
Time (days) Fig. 1. Cell population
growth curve for rat smooth muscle cells cultured from the aorta of normotensive (0) and hypertensive (A) rats. Second passage cells were incubated in 5 ml, 25 cm2 Falcon flasks with an initial cell density of 2X 10’ cells/cm2 and counted at the specified intermediate time points. Means and standard deviations of 7 experiments done in duplicate.
303
Results Cell population growth curue and incorporation of [“‘S]sulfate proteoglycans
into extracellular
Growth curves of smooth muscle cells cultured from normotensive and hypertensive rats after seeding 1 X lo5 cells per flask are shown in Fig. 1. No differences in the plating efficiency between the cells from normotensive or hypertensive rats were observed. Cell population doubling time during the logarithmic growth phase was 26-30 h for the cells from normotensive rats and 18-20 h for the cells from hypertensive rats. Following confluence, arterial smooth muscle cell growth slows but does not stop. The clear differences between the cells from normotensive and hypertensive animals are in accordance with the previous observation of a higher proliferation rate of smooth muscle cells cultured from hypertensive rats as compared with cells from normotensive rats [ 151. When the amount of [%]sulfate which was incorporated into the proteoglycans secreted from the cells into the growth medium over a period of 24 h was determined on the 5th, 7th and 11th day after plating, an increased amount of incorporated 35S
Cell density(x 10m6 cells/flask)
Fig. 2. Incorporation of [35S]sulfate into extracellular proteoglycans at different cell densities. Cell suspensions with an initial density of 2-4X IO3 cells/cmr were cultured and harvested between the 3rd and 11th day after plating. 24 h prior to harvesting the cultures were refed with 5 ml of medium containing 25 gCi [3sS]sulfate. Differences between cells from normotensive (0) and hypertensive (A) rats disappear when radioactivity is related to the cell protein content.
304
radioactivity per flask up to the 7th day was noted. This was expected from the increased number of cells. However, on day 11 a lower amount of incorporated 35S radioactivity per flask was found despite a further increase in cell number. Cell proliferation and proteoglycan synthesis The rate of [35S]proteoglycan synthesis declines in both cell lines as the cells progress from low to high densities (Fig. 2). The slopes reflecting the change in the rate of [35S]proteoglycan synthesis as cell density increases appear to be similar for cells from normotensive and hypertensive rats, but decline when the cells reach confluence at densities of 0.8- 1.0 X IO6 cells/flask. At all densities the net rate of [ 35S]proteoglycan synthesis per cell is higher for the cells from hypertensive than for those from normotensive animals. However, as is evident from Fig. 3, this result is due to a higher protein content in the cells of hypertensive as compared to normotensive rats (see Discussion). Regardless of this difference the protein concentration per cell decreases in both cell lines as cell density increases. When seen in relation to the protein content, progressively decreasing but roughly equal net rates of [35S]proteoglycan synthesis are calculated for cells from normotensive and hypertensive rats. The results shown in Figs. 2 and 3 reveal that both normotensive and hypertensive cells in the exponential phase of growth (Fig. 1) secrete more [3SS]proteoglycans into the extracellular medium than do confluent cells.
Cell density (x
10m6 cells/flask)
Fig. 3. Cell protein concentration at different cell densities. Cell culture conditions as in Fig. 2. Cultures from normotensive (0) and hypertensive (A) rats were made in triplicate for cell counting and protein determination (means of 2 values).
305
20 t 0
I 20
I 40
I 60 3H
1
I 80
l&3
ll0
-3 cpm x 10 /mg cell protein
Fig. 4. Correlation between [ 3H]thymidine incorporation into trichloroacetic acid-precipitable material of arterial smooth muscle cells and [ “S]sulfate incorporation into extracellular proteoglycans. Cell cultures at various stages of growth were incubated in the presence of 1 pCi [3H]thymidine and 5 pCi [ 35S]sulfate/ml medium for 24 h prior to harvesting (see Methods). Correlation coefficient 0.75, slope of regression line 0.24 (P ~0.05).
Further evidence of a correlation between cell division and proteoglycan synthesis was obtained from double labelling experiments with [3H]thymidine and [ 35S]sulfate. A plot of “S radioactivity incorporated into the external sulfated proteoglycans versus the 3H radioactivity incorporated into the cellular TCA-precipitable material reveals (Fig. 4) a correlation coefficient of 0.75 and a slope of the regression line of 0.24. This indicates a proportional increase in [35S]proteoglycan synthesis with increasing [ 3H]thymidine incorporation. No differences between cells derived from normotensive and hypertensive animals could be ascertained.
TABLE
1
RELATIVE CONCENTRATIONS OF INDIVIDUAL FROM SECRETIONS OF CONFLUENT ARTERIAL
[3sS]GLYCOSAMINOGLYCANS ISOLATED SMOOTH MUSCLE CELL CULTURES
Glycosaminoglycan compositions of the proteoglycan fractions A and B were determined after enzymatic degradation with chondroitinase AB and ABC. Values are means of 5 experiments and expressed as cpm/lOO cpm of total sulfated glycosaminoglycans. Cell type
Normotensive
Hypertensive
PG fraction
Total glycosaminoglycans
(cpm/lOO
cpm)
c4s
C6S
DS
HS
A B
81 46
6 7
10 40
3
A B
81 43
7 7
10 43
2 7
1
306
10 8 6 4 2
Fraction Number Fig. 5. Elution profile of intracellular [ 35S]proteoglycans at different cell densities. The medium of sparse (5th day), subconfluent (7th day) and confluent (1 Ith day) cultures was concentrated, dialyzed exhaustively against 0.10 M ammonium sulfate, adjusted to 4 M guanidinium chloride and chromatographed on Sepharose 4B CL (see Methods). 15000 cpm were applied to the columns.
Proteoglycan pattern and endocytosis The macromolecular state of extracellular [ ‘%]proteoglycans
produced by arterial smooth muscle cells was judged from gel filtration experiments. In order to exclude interactions of proteoglycans with each other or with other extracellular constituents, the gel chromatography was performed with buffered 4 M guanidinium chloride as eluent (Fig. 5). The extracellular proteoglycans resolved into species of two molecular weights (A, B) differing in hydrodynamic size, a higher molecular weight fraction being excluded from the gel, and a lower molecular weight fraction having a K,,
307
TABLE
2
INFLUENCE OF CELL DENSITY ON UPTAKE AND DEGRADATION GLYCANS (FRACTION B) BY ARTERIAL SMOOTH MUSCLE CELLS NORMOTENSIVE AND HYPERTENSIVE RATS
OF [3sS]PROTEOCULTURED FROM
Cells from the first subculture were suspended with 0.25% trypsin solution and diluted with fresh medium to the specified density. Values are means of two experiments done in triplicate. Cells were preincubated for 24 h, then for 16 h in the endocytotic experiments, with 2 nmole [%]proteoglycan disaccharide units/ml medium (30X IO3 cpm/ml). For calculation of proteoglycan molarity, the equivalence of 1 pmole of proteoglycan-bound disaccharide to 1 mg proteoglycan (protein content about 40%) was assumed. Proteoglycan disaccharide units were calculated from glucuronic acid determination. Cell type
Cell density (cellX IO’/25
cm*)
Uptake (W of added [ 35S]proteoglycans/ mg cell protein)
Degradation (W of endocytosed proteoglycans) -
Normotensive
1.23 2.46 4.92 9.84
19.2 14.8 12.4 10.2
97 96 98 95
Hypertensive
1.65 3.30 6.60 13.20
18.7 13.9 10.7 9.4
98 98 94 97
value of 0.36. The ratio of 35S radioactivity incorporated into fractions A and B depended on culture conditions. With increasing cell density fraction B became the predominant proteoglycan, the ratio of A/B decreasing continuously from 0.3 in subconfluent cultures (5th day) to 0.06 in confluent cultures (11 th day). Fractions A and B differed in their relative glycosaminoglycan composition (Table l), the chondroitin 4+ulfate/dermatan sulfate ratio of fraction A being significantly higher than that of fraction B. The pericellular [35S]proteoglycans were obtained by extraction with 4 M guanidinium chloride after removing the medium from the washed cell layer. The coextracted intracellular 35S radioactivities accounted for only 3-5% of the total radioactivity and were not considered. The elution profile of pericellular proteoglycans (data not shown) resembled that of the extracellular proteoglycans, with the exception that only lo-20% of the 35S radioactivity incorporated into total proteoglycans was detected in the pericellular proteoglycan fraction, and that the proportion of heparan sulfate was higher in the pericellular proteoglycans in relation to the extracellular proteoglycans. The [ 35S]proteoglycan fraction B is internalized by smooth muscle cells, while no endocytotic uptake of the proteoglycan fraction A was observed. Table 2 provides evidence that the [35S]proteoglycan fraction B is internalized at rates which decline as the cell density increases. More than 95% of [3SS]proteoglycans endocytosed within the incubation period are degraded intracellularly as judged by the release of inorganic [35S]sulfate into the culture medium. There are no differences between
cells cultured from normotensive and hypertensive animals with respect to the rate of endocytosis as related to the cell protein content. I
Discussion
The results here provide evidence for metabolic differences (a) between rapidly proliferating and nonproliferating arterial smooth muscle cell cultures, and (b) between cultured smooth muscle cells derived from normotensive and hypertensive rats. The [ 35S]proteoglycan metabolism of arterial smooth muscle cells in the logarithmic phase of growth differs from that of non-dividing cells in a higher rate of [ 35S]proteoglycan synthesis, by differences in the glycosaminoglycan composition of extracellular proteoglycans and an enhanced rate of endocytotic uptake of [ 35S]proteoglycans. The positive correlation between cell proliferation and proteoglycan synthesis is confirmed by [3H]thymidine/[35S]sulfate labelling experiments. A more complex effect of cell density on the net synthesis of chondroitin sulfate has been described for human skin fibroblasts [24] and mouse 3T3 cells [25]. The finding that cells at low density endocytose proteoglycans at a higher rate than confluent cells (Table2) suggests higher receptor activity of cells in the exponential phase of growth. An analogous result has been obtained for the receptor-mediated uptake of LDL which was higher in non-confluent cultures of human skin fibroblasts [26,27], rat aortic smooth muscle cells [28], and bovine [29] and rat [30] endothelial cells than in contact-inhibited, confluent cultures. Likewise, the receptor-independent (fluid) endocytosis of [U-‘4C]sucrose by arterial smooth muscle cells cultured from monkey thoracic aorta was observed to be inversely proportional to cell density [31]. The proteoglycans produced by smooth muscle cells and secreted into the culture medium differ in size and glycosaminoglycan content (Table 1). Under both dissociative and non-dissociative conditions two proteoglycan fractions with different hydrodynamic sizes could be resolved by gel filtration (Fig. 5). This confirms earlier studies by Kresse et al. [6], CSster [32] and Carlstedt [33], who isolated and characterized proteoglycans of varying size from the medium of human skin fibroblasts. The additional information provided by the present work concerns the observation that the high molecular weight proteoglycan fraction (A of Fig. 5) is not endocytosed by the parent cells and decreases in relation to the low molecular weight proteoglycan fractions as the cultured cells progress from low to high cell densities. With the exception of a higher cell protein content of cells derived from hypertensive animals, no significant differences between the cell lines from hypertensive and normotensive animals could be detected. The higher cell protein content of arterial smooth muscle cells derived from hypertensive animals was explained by the observation that arterial smooth muscle cells contain a subpopulation of large, partly polynucleated cells. The percentage of these cells is significantly higher in cells from hypertensive (26%) as compared to control animals (13%) [34]. In summary, our results suggest that the enhanced in vivo incorporation of [35S]sulfate into the sulfated glycosaminoglycans of the arterial wall of hypertensive
animals [14] is closely related to the higher number of proliferating cells in the arterial tissue as compared to normotensive animals. Thus, the rate of [3SS]proteoglycan synthesis depends rather on the ratio of dividing to nondividing cells than on metabolic diversities between smooth muscle cells cultured from normotensive and hypertensive animals. No evidence for a higher metabolic activity of cells from hypertensive animals could be detected in a comparison of cells in the same growth phase, with the metabolic parameters stated, in relation to the cell protein content. Our investigation emphasizes the importance of cell proliferation as an early event in the development of hypertension-induced metabolic alterations of arterial tissue. Acknowledgements
The authors are indebted skillful technical assistance.
to Ms. H. Stockmann
and Ms. M. Ahler for their
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310 16 Bitter, T. and Muir, H., A modified uranic acid carbazole reaction, Anal. B&hem., 4 (1962) 330. 17 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (195 1) 265. 18 Ross, R. and Glomset, J., Atherosclerosis and the arterial smooth muscle cell, Science, 180 (1973) 1332. 19 Page, J.H. and Graef, J., Hypertension following experimental perinephritis induced by cellophane, Arch. Path., 28 (1939) 613. 20 Hadjilsky, P., Morphogenese de l’arteriopathie central et p&ipherique du rat spontanement hypertendu, Paroi arterielle (Arterial Wall), 6 (1980) 119. 21 Fratantoni, J.C., Hall, C.W. and Neufeld, E.F., The defect in Hurler and Hunter syndromes, Part 2 (Deficiency of specific factors involved in mucopolysaccharide degradation), Proc. Nat. Acad. Sci. (USA), 64 (1969) 360. 22 Saito, H., Yamagata, T. and Suzuki, S., Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates, J. Biol. Chem., 243 (1968) 1536. 23 Oegema, Jr., T.R., Hascall, V.C. and Dziewiatkowski, D.D., Isolation and characterization of proteoglycans from the swarm rat chondrosarcoma, J. Biol. Chem., 250 (1975) 6151. 24 Hopwood, J.J. and Dorfman, A., Glycosaminoglycan synthesis by cultured human skin fibroblasts after transformation with Simian virus 40, J. Biol. Chem., 252 (1977) 4777. 25 Cohn, R.H., Cassiman, J.J. and Bernfield, M.R., Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan, J. Cell Biol., 71 ( 1976) 280. 26 Goldstein, J.L. and Brown, M.S., Binding and degradation of low density lipoproteins by cultured human fibroblasts, J. Biol. Chem., 249 (1974) 5153. 27 Kruth, H.S., Avigan, J., Gamble, W. and Vaughan, M., Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts, J. Cell Biol., 83 (1979) 588. 28 Stein, 0. and Stein, Y., Surface binding and interiorixation of homologous and heterologous serum lipoproteins by rat aortic smooth muscle cells in culture, B&him. Biophys. Acta, 398 (1975) 377. 29 Coetzee, G.A., Stein, 0. and Stein, Y., Uptake and degradation of low density lipoproteins (LDL) by confluent, contact-inhibited bovine and human endothelial cells exposed to physiological concentrations of LDL, Atherosclerosis, 33 (1979) 425. 30 Vlodavsky, I., Fielding, P.E., Fielding, C.J. and Gospodarowicz, D., Role of contact inhibition in the regulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells, Proc. Nat. Acad. Sci. (USA), 75 (1978) 356. 31 Davies, P.F. and Ross, R., Growth-mediated, density-dependent inhibition of endocytosis in cultured arterial smooth muscle cells, Exp. Cell Res., 129 (1980) 329. 32 Caster, L., Carlstcdt, I. and Malmstrom, A., Isolation of ‘%- and ‘H-labelled proteoglycans from cultures of human embryonic skin fibroblasts, B&hem. J., 183 (1979) 669. 33 Carlstedt, I., Caster, L. and Malmstrom, A., Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture, B&hem. J., 197 (198 1) 2 17. 34 Grtinwald, J., Robenek, H., Mey, J. and Hauss, W.H., In vivo and in vitro cellular changes in experimental hypertension - Electronmicroscopic and morphometric studies of aortic smooth muscle cells, Exp. Mol. Path., 36 (1982) 164.