- Email: [email protected]
The synthesis of connective tissue protein in smooth muscle cells
93
Biochimica et Biophysica Acta, 418 (1976) 93--103 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98479
THE SYNTHESIS OF CONNECTIVE TISSUE PROTEIN IN SMOOTH MUSCLE CELLS
B. FARIS, L.L. SALCEDO, V. COOK, L. JOHNSON, J.A. F O S T E R and C. F R A N Z B L A U Department of Biochemistry, Boston UniversitySchool of Medicine, Boston, Mass. 02118
(U.S.A.) (Received June 9th, 1975)
Summary The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C]hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [ ~4C] hydroxylysine and [ 1 4C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled e-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [ ' 4C] lysine pulsed cells with NaBH4.
Introduction The proliferation of smooth muscle cells in aortic tissue has been implicated as a major event in the pathogenesis of atherosclerosis [1,2,3]. Ross [4], Daoud et al. [5], among others, have studied the ultrastructure of these cells in culture. The ability to synthesize connective tissue protein was suggested primarily on the morphologic evidence presented by these workers. Additional studies are being undertaken in several laboratories to obtain chemical evidence as well. Indeed, Ross has presented data indicating the presence of the microfibrillarcomponent of elastic fibers [4]. That tropoelastin is present in smooth muscle cell cultures was suggested by Abraham et al. [6]. The presence of insoluble elastin and several of the lysine-derived crosslinks of collagen and elastin have not been chemically defined in the cultures. Results presented in this communication do indeed indicate the presence of insoluble collagen and elastin and yield evidence that many of the metabolic mechanisms necessary for the assembly of collagen and elastic fibers are present.
94 Material and Methods
Description of smooth muscle cell cultures Aortic medial smooth muscle cells were obtained from weanling rabbits according to the m e t h o d of Ross [4] with some modifications. Thoracic aortas were rapidly excised using sterile techniques and immediately placed in 60-mm Petri dishes with Dulbecco's Modified Eagle's Medium [7] containing 2.2 g sodium bicarbonate per 1, 10% fetal calf serum and per ml, 1000 units of penicillin, 50 pg of aureomycin, 0.25 mg fungizone and 1 ml each of minimum essential medium (MEM) non-essential amino acids (100 X) [8] and sodium pyruvate solution (100 X) per 100 ml. Under a dissecting microscope, the aortas were grossly cleaned of extraneous tissue, blood and fat and transferred to fresh medium. They were then cut open, cleaned again, placed in a fresh dish with medium and the intimal/innet medial layer pinched off and stripped according to the m e t h o d of Wolinsky and Daly [9]. These layers were cut into approx. 1 m m 3 pieces which were attached to the b o t t o m of 250-ml plastic tissue culture flasks. The flasks were allowed to stand with the cap end up to drain the pieces of most of the medium to p r o m o t e adherence. Medium as above but containing 100 units of penicillin and 100 pg of streptomycin per ml, and 3.7 g sodium bicarbonate/1 were added to cover the explants. The flasks were loosely capped and allowed to remain undisturbed for a week in a 5% CO2/95% air moist incubator at 37 ° C. The medium was changed weekly. In two to three weeks, the cells which grew out from the explants were detached with a 0.05% trypsin/0.02% EDTA solution in modified Puck's saline A solution [10], centrifuged at 200 X g for 10 min, resuspended in complete medium, c o u n t e d and seeded into new flasks. Cell culture medium was replaced twice weekly. The original attached explants were maintained to yield another batch of cells which were detached after two weeks. Routinely, 250-ml plastic tissue culture flasks were seeded with 1--1.5 • 106 cells and were passed weekly up to the fourth passage and bimonthly to the sixth using a 1 : 2 split. Experimental procedures Isolation of insoluble elastin from smooth muscle cells. Smooth muscle cells were harvested at various periods of time after the second passage. The media was removed and the resulting cell layer was homogenized in a PotterElvehjem homogenizer using a phosphate buffer (0.1 M NaH2PO4/0.15 M NaC1, pH 7.5). The homogenate was stirred overnight at 4°C and then centrifuged at 16 000 X g for 20 min. This procedure was repeated with the residue obtained. The combined supernatants were designated cell extract as stated below. The resulting cell debris from each of the time periods (2--3 flasks) was then suspended in 0.1 M NaOH and heated at 98 ° C for 45 min according to the procedure of Lansing e t a l . [11]. The resulting suspension was centrifuged at 3000 X g for 10 minutes and the residue was washed thoroughly with water, suspended in 2 ml of 6 M HC1 and h y d r o l y z e d in a sealed vial at l l 0 ° C for 20 h. The hydrolysates were placed on a Jeolco 6 AH automatic amino acid analyzer modified to analyze hydrolysates of elastin. For reference purposes,
95 insoluble elastin was prepared from the medial portion of a weanling rabbit aorta. The tissue was treated with 0.1 M NaOH as described above and analyzed for amino acid content.
Pulse label experiments (A) General description. Plastic tissue culture flasks (75 cm 2 growth area) were seeded with 1--1.5 • 106 smooth muscle cells in the second passage. The cells were maintained for different periods of time after seeding (days in culture). Before addition of the radioactive amino acid, the spent medium was aspirated off and the cell layers washed seven times with medium containing no fetal calf serum. They were then incubated for 1 h at 37°C in serum-less medium lacking lysine or proline and containing sodium ascorbate (50 pg/ml). This medium was then replaced with one containing either [14C]lysine (1.0 pCi/ml, spec. act. 312 Ci/M) or [ 14C] proline (1.0 pCi/ml, spec. act. 260 Ci/M) and sodium ascorbate as above. The pulse period lasted for 22 h. The spent radioactive medium was poured off and the flasks were drained by aspirating residual medium. 2 ml of phosphate buffer (0.1 M Nail2 PO4 / 0.15 M NaCl, pH 7.5) was added to each of the flasks which were laid on ice and the cell layers were collected with the aid of a rubber policeman and transferred to centrifuge tubes. The flasks were washed twice more with buffer solution. The cell suspension was centrifuged at 300 × g for 20 min and the cell pellets were washed with deionized water and centrifuged three more times. For those experiments with fl-aminopropionitrile fumarate the procedure is the same except the cells are incubated in medium containing/~-aminopropionitrile (15 pg/ml) during the wash and pulse periods. (B) Conversion o f [ 14 C] proline to [ 14 C] hydroxyproline. The presence of [ ~ 4C] hydroxyproline and [ ~4C] proline in the fractions described below was determined by hydrolyzing each sample in 6.0 M HC1 at l l 0 ° C for 20 h and placing the hydrolysate on a Technicon amino acid analyzer with a split-stream arrangement as described previously [12]. Also placed on the analyzer column with each hydrolysate was a standard hydroxyproline solution (not radioactive) to serve as a marker. The various fractions examined were: (1) Media: The decanted media {93--97 ml) from 12 flasks of smooth muscle cells (grown in culture either 10 or 13 days) which were pulsed for 22 h with [14C] proline was centrifuged at 16 000 X g for 20 min, and the supernatant was dialyzed versus H~ O at 4 ° C. The material remaining in the dialysis sac was recentrifuged, since a fine white precipitate appeared during dialysis. The resulting supernatant and precipitate were each lyophilized separately. (2) Intercellular material: After the spent medium was decanted from the cell layer, 2 ml of phosphate buffer was added to each flask and the cells scraped off gently with a rubber policeman. The mixture was centrifuged at 200 × g for 10 rain. The cell pellet was washed and centrifuged in the same manner t w o more times. The pooled washes were centrifuged at 16 000 × g for 20 min and the supernatant was dialyzed as described above. This dialyzed material was lyophilized. (3) Cell extract: The cell pellet from step 2 was homogenized as in the above section on "Insoluble Elastin". The combined supernatant, referred to as
96 the cell extract was dialyzed versus H 2 0 . As in the case of the media, the resulting dialyzed material was centrifuged since a precipitate was present. Again, the precipitate and supernatant were each lyophilized separately. (4) Cell debris: The residue from Step 3 was suspended in 0.1 M NaOH and placed at 98°C for 45 min. The resulting insoluble material and the supernatant after this alkali treatment were analyzed as described. (C) Incorporation of [~ 4C] lysine into connective tissue components. For this series of experiments, smooth muscle cells were grown in the presence of [~4C]lysine in place of [~4C]proline. Two separate experiments were performed. The first utilized 13 flasks of second passage cells which were 13 days old. After the pulsed media was decanted (98 ml), centrifuged at 16 000 × g for 20 min and dialyzed, the resulting precipitate was harvested and lyophilized. Approx. 0.5 mg of this lyophilized material was suspended in 5.0 ml of H2 O and 1.0 mg NaBH4 was added. Reduction was allowed to proceed for 90 min (the pH never exceeded 8.0) with occasional shaking. The pH was then adjusted to 3.0--4.0 with 50% acetic acid and the entire reaction mixture was dialyzed and lyophilized, e-Hydroxynorleucine and the reduced aldol condensation p r o d u c t of two residues of allysine (reduced aldol) were determined by hydrolyzing the NaBH4 reduced material in 2.0 M NaOH at 110°C for 20 h and employing a split-stream arrangement [ 12]. The combined cell layers from all of the flasks employed in the above experiment were washed 3 times with phosphate buffer, pH 7.5, 3 times with H2 O and then suspended in 10 ml H2 O. To this suspension, 12.5 mg NaBH4 was added and the reduction reaction was allowed to proceed as above. After dialysis, the material was divided into two equal portions, and each was treated with 0.1 M NaOH at 98°C for 45 min. The supernatant in each case was removed and one sample was h y d r o l y z e d in 6.0 M HC1 at l l 0 ° C and the other in 2.0 M NaOH at 110°C for 20 h. Separate hydrolysis in HC1 and in NaOH was also performed on the residue. The radioactive profile of each hydrolysate was determined. In a second set of experiments, 5 flasks of cells (second passage), 19 days in culture, were pulsed with [14C]lysine as above, and another 5 flasks of similar cells were pulsed with [14C]lysine in the presence of ~-aminopropionitrile (15 pg/ml). The pulsed media in each case (46--49 ml) was handled in the same fashion as in the previous experiment except that the media from the cells grown in the presence of ~-aminopropionitrile was dialyzed against H2 O containing 15 pg ~-aminopropionitrile/ml. The entire contents of each dialysis sac was lyophilized w i t h o u t centrifugation. Each sample was then suspended in 5.0 ml of H 2 0 and reduced with 2.0 mg of NaBH4. for 90 min. After dialysis, each was hydrolyzed in 2.0 M NaOH at l l 0 ° C and the radioactive elution patterns from the amino acid analyses were compared. The washed cell layers with and w i t h o u t ~-aminopropionitrile were suspended in 10 ml H2 O and reduced in the presence of 10 mg NaBH4. After dialysis and lyophilization, each was treated with NaOH according to Lansing et al. [ 11] and the insoluble material remaining was hydrolyzed in 2.0 M NaOH at 110°C. The material solubilized by the Lansing t r e a t m e n t (0.1 M NaOH at 98°C for 45 rain) was hydrolyzed separately in each case in 6.0 M HC1 and in 2.0 M NaOH.
97 To determine the total distribution of crosslinks present in the insoluble elastin, four flasks of 21 day cells were reduced with NaB3 H4 after removal of the medium. This group of cells did n o t receive any pulse label. The cell layer after reduction was treated with alkali and analyzed for radioactivity as described. For comparison purposes, the medial portion of a weanling rabbit aorta was reduced with NaB s H4, exposed to Lansing treatment and the insoluble elastin fraction was examined. The radioactive profile from the split stream amino acid analyses was determined in all cases. Results
Presence of elastin in smooth muscle cells The amino acid compositions of Lansing treated cell debris are given in Table I. The table includes the analyses from cells cultured for various days after seeding as well as that of elastin from the medial tissue of the rabbit aorta. Although no attempt to quantify cell numbers was made, approx. 5 • 106 smooth muscle cells were routinely employed if one assumes one cell-doubling occurred after seeding. To estimate the small a m o u n t of desmosines present in the insoluble elastin, two separate concentrations of the acid hydrolysate from the 20 day old cells was applied to the amino acid analyzer. The higher concentration was 15 times greater than normally used in all other samples. As can be seen from Table I, 13 day old cells yield definitive elastin amino acid analyses. It is of interest to note the difference in amino acid composition of the 9 or 10 day old cultures compared to the 13 day old alkali insoluble residue. As the cultures "age", it again becomes more difficult to remove " c o n t a m i n a n t " protein from the elastin by this alkali treatment.
Conversion of [14C]prolin e to [14C] hydroxyproline The appearance of newly synthesized collagen in these experiments was monitored b y the presence of [ 14 C] hydroxyproline. The distribution of radioactivity in proline and hydroxyproline in all fractions isolated and described above are given in Table II. In each fraction the percent of the total radioactivity represented as hydroxyproline is given as well. It can be seen that the highest percentage of labelled hydroxyproline is found in the media. Very little hydroxyproline radioactivity was found in those fractions of the cell debris (13 day) that remained insoluble after alkali treatment. This latter fraction was shown by its amino acid composition to be insoluble elastin. Studies are in progress to ascertain optimum time and conditions for soluble and insoluble collagen synthesis by these smooth muscle cells.
Lysine incorporation The alkali-insoluble residues obtained from the smooth muscle cells were examined for lysine-derived aldehydes and crosslinks. Fig. 1A shows a typical radioactive profile of a 2.0 M NaOH hydrolysate of the elastin (cell debris) derived from cells that had been pulsed with [14C]lysine and reduced with NaBH4 as described in the experimental procedures. Radioactive hydroxynorleucine and the reduced aldol c o m p o u n d are the predominant components detected. No labelled hydroxylysine can be seen, indicating little or no collagen
NC 81.2 53.0 56.0 106.7 84.9 164.1 105.7 73.9 38.8 75.6 25.5 32.5 33.5 22.2 45.4 ---
9
Days
NC 50.6 27.9 31.8 70.6 90.7 234.6 178.2 92.0 36.9 77.1 9.7 24.6 28.5 9.7 25.9 ---
10 4.8 14.9 15.8 18.7 28.3 121.8 333.2 232.0 99.1 22.9 53.5 13.7 20.7 7.7 3.5 9.2 NC NC
13 3.5 13.8 10.2 19.8 28.2 121.3 329.0 236.5 100.0 23.6 52.4 14.5 20.5 11.2 3.8 11.9 NC NC
13
* V a l u e s o b t a i n e d f r o m t w o d i f f e r e n t a m i n o a c i d a n a l y s e s (see t e x t ) .
Hyctro x y p r o l i n e Aspartic Threonine Serine Glutamic Proline Glyeine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Axginine Isodesmosine Desmosine Lysinonorleucine
A m i n o acid
NC 20.6 18.5 20.4 33.7 115.4 321.7 211.5 100.5 24.0 62.0 21.7 20.8 9.9 5.6 13.8 NC NC
19 3.8 13.3 15.1 17.3 26.2 124.6 319.1 222.9 103.9 23.1 60.1 27.1 25.0 5.9 4.6 11.7 0.5 0.4
20* NC 18.7 16.1 37.4 32.3 97.7 322.9 217.5 111.3 25.5 60.3 7.6 18.7 13.9 5.9 6.8 NC NC
23
3.7 32:2 26.7 25.8 46.0 116.0 293.7 186.4 94.2 28.3 62.5 26.3 23.8 10.3 8.7 18.5 NC NC
43
15.3 3.1 9.9 11.7 16.7 119.2 344.6 243.3 98.6 20.9 54.0 23.2 19.1 5.2 0.5 5.8 2.8 4.2 1.2
Medial tissue from rabbit aorta
V a l u e s e x p r e s s e d as r e s i d u e s / 1 0 0 0 r e s i d u e s , b u t n o t c o r r e c t e d f o r s l i g h t l o s s e s o f a m i n o a c i d s d u r i n g h y d r o l y s i s . N C = n o t c a l c u l a b l e : a m o u n t t o o s m a l l t o c a l c u l a t e .
AMINO ACID ANALYSES OF ELASTIN FROM SMOOTH MUSCLE CELLS
TABLE I
c£) 0o
99 T A B L E II IDISTRIBUTION TIONS
OF [14C] HYDROXYPROLINE
Cell f r a c t i o n
A N D [I 4 C ] P R O L I N E
CELL FRAC-
10 day
13 d a y
Hydroxyproline × I00 Proline + Hydroxyproline 10 day 13 d a y
70 000 280 000
55 000 89 000
3.2% 5.2%
13.2% 8.2%
Intercellular
28 000
26 0 0 0
0.9%
1.4%
Cell e x t r a c t 1. S u p e r n a t a n t 2. P r e c i p i t a t e
50 000
Media 1. S u p e r n a t a n t 2. P r e c i p i t a t e
Cell d e b r i s 1. S u p e r n a t a n t * 2. R e s i d u e *
Hydroxyproline (cpm)
IN V A R I O U S
28 0 0 0
1.2% 1.2%
50 000
90 000 560
2.0%
4.0% 1.6%
* Fractions from Lansing treatment.
contamination in this preparation of insoluble elastin. An unknown radioactive peak appeared in the region where valine elutes. It was not present in the ~-aminopropionitrile-treated cells described below. The reduced aldol content was calculated from the ninhydrin data obtained from the amino acid analysis. T A B L E III [ 14C] LYSINE INCORPORATION
IN V A R I O U S C E L L F R A C T I O N S
BAPN, ~-aminopropionitrlle fumarate. Cell f r a c t i o n
cpm//~M l e u c i n e (X 1 0 4) Hydroxynorleucine
Glucosylgalactosylbydroxylysine
Media 13 d a y 19 day (--BAPN) 19 day (+BAPN)
8.6 8.7 3.5
19.0 15.0 8.4
Cell d e b r i s 1. S u p e r n a t a n t * 13 day 19 day (--BAPN) 19 day (+BAPN)
2.2 2.4 0.3
2.4 1.1 0.9
2. R e s i d u e * 13 day 19 day (--BAPN) 19 d a y ( + B A P N )
2.4 3.0 0.2
----
Reduced aldol
----
1.8 2.1 0.04 4.1 3.6 0.06
Rabbit aortic medial tissue** Residue*
10.0
69.0
Cells* * Residue*
23.0
85.0
* Fractions from Lansing treatment. ** N a B 3 H 4 r e d u c e d o n l y ; n o [ 1 4 C ] l y s i n e p u l s e (see t e x t ) .
Hydroxylysine
Lysine
29.0 19.0 13.0
380.0 280,0 210.0
1.8 1.3 0.6
81.0 58.0 34.0
--
18.0
--
6.8
--
9.2
100 HNL
LYS
ALOOL
A 15
10
5
I
I
I
I
1
I
I
I
B 15
10
5
40
80
120
160
FRACTION NO. Fig. 1. R a d i o a c t i v e e i n t i o n p r o f i l e o f 2.0 M N a O H h y d r o l y s a t e s f r o m a m i n o acid a n a l y s e s of elastin f r o m cells p u l s e d w i t h [ 1 4 C ] l y s i n e a n d r e d u c e d w i t h N a B H 4. A, f r o m c o n t r o l cells; B, f r o m cells p u l s e d in p r e s e n c e o f ~3-aminopropionitrile f u m a r a t e . T h e e x p e r i m e n t a l p r o c e d u r e s f o r t h e a m i n o acid a n a l y s e s a n d d e t e r m i n a t i o n o f r a d i o a c t i v i t y h a v e b e e n o u t l i n e d in d e t a i l p r e v i o u s l y [ 1 2 ] . F o r r e f e r e n c e p u r p o s e s , v a l i n e e l u t e s at f r a c t i o n s 7 9 - - 8 2 a n d g a l a c t o s y l h y d r o x y l y s i n e w o u l d e l u t e a t f r a c t i o n s 1 1 4 - - 1 1 7 . H N L , h y d r o x y norleucine; A L D O L , reduced aldol condensation product.
The data obtained from three separate experiments indicate that 8--10 residues of reduced aldol are present per 1000 amino acid residues. Fig. 1B shows the data from Lansing-treated cell layer (cell debris) which had been subjected to fl-aminopropionitrile. This fraction revealed little radioactivity in the reduced aldol and hydroxynorleucine peaks while lysine incorporation appeared to be significant. As expected, nonradioactive reduced aldol was detected by the ninhydrin reaction from the amino acid analyzer. For comparison purposes, the radioactive profiles of the soluble fractions obtained after Lansing treatment of the cell debris are given in Fig. 2A (no /3-aminopropionitrile) and Fig. 2B (with/3-aminopropionitrile). The appearance
101 NNL
ALGOL
LY$
A
1(
o
8 t5
10
40
80
120
160
FRACTION NO.
Fig. 2. Radioactive elution profile of 2.0 M NaOH hyd~ol:fsates from amino acid analyses of alkali soluble fraction from cells pulsed w i t h [ 1 4 C ] l y s i n e and reduced w i t h NaBH 4. A, from c o n t r o l cells; B, from cells pulsed in presence of ~-aminopropionitrile fumaratc. The e x p e r i m e n t a l procedures for the amino acid analyses and d e t e r m i n a t i o n of radioactivity have been outli ne d in detail previously [ 1 2 ] . F or reference purposes, valine elutes at fractions 79--82 and p l a c t o s y l h y d r o x y l y s i n e w oul d elute at fractions 114-117. HNL, h y d r o x y n o t l e u c i n e ; GGH, glucosylgalactosylhydroxy l ys i ne ; ALDOL, reduced aldol c o n d e n s a t i o n pr oduct; HYLYS, h y d r o x y l y s i n e .
of glucosylgalactosylhydroxylysine [13] suggests the presence of solubilized collagen fragments. Of interest is the negligible amount of radioactivity found in the area where galactosylhydroxylysine [13] would elute. Examination of media from the pulse labelled cells in the presence or absence of/3-aminopropionitrile displayed e-hydroxynorleucine, glucosylgalactosyl hydroxylysine and no reduced aldol (not shown). These data, together with the presence of radioactive hydroxylysine, support the evidence that collagen is present in the media in contrast to the insoluble cell debris after Lansing treatment. ~-aminopropionitrfle-treated cells still display hydroxylysine and glucosylgalactosylhydroxylysine radioactivity (not shown).
102
Table III summarizes the total radioactivity in each of the components described. Included in the table are the radioactive data obtained from elastin prepared from rabbit aorta and reduced with NaB 3 H 4 as described. Data from NaB 3 H4 reduced 21 day cell layer is also included. Discussion Ross and his collaborators noted t h a t aortic smooth muscle cells are capable of synthesizing elastic tissue. Morphologic evidence suggested the presence of collagen, elastic fiber microfibrils, and insoluble elastin [ 14]. Chemical evidence for the soluble precursor of elastin, namely tropoelastin, was presented by Abraham et al. [6]. Isolation of tropoelastin itself however has proved somewhat elusive. Data included in this communication clearly indicate the presence of insoluble elastin in cultures of these cells. The amino acid analyses of the Lansing-treated cell layers are quite compatible with that of amorphou.s elastin isolated from the rabbit aortic tissue itself. The 9 or 10 day cultures show significant amounts of acidic amino acids suggesting the presence of glycoprotein or microfibrillar components. This observation is in keeping with the concept that such glycoprotein moieties are synthesized early in elastic tissue fibrogenesis to form a matrix for the insoluble elastin c o m p o n e n t [2]. The 20 day cultures show significant amounts of desmosine and isodesmosine when large quantities of hydrolysate were analyzed. To substantiate the presence of elastin, the [ 14 C] lysine pulse experiments indicate active synthesis of allysine and the aldol condensate of two residues of allysine. The quantity of reduced aldol is such that it suggests the presence of elastin and n o t collagen. Treatment with ~-aminopropionitrile caused almost complete inhibition of formation of allysine and its aldol condensate. Although the amino acid compositions of elastin preparations from the cell cultures are strikingly similar to t h a t found in the rabbit aorta itself, the distribution of radiolabelled e-hydroxynorleucine and reduced aldol after reduction are quite different. One must note that the ratio of these two substances from the rabbit elastin preparation is obtained from NaB 3 H4 reduction and represents the entire distribution of the two moieties in the sample. The [ 14 CI lysine pulse in the cells represent only newly synthesized elastin during a 22-h period and additional chase periods would most likely be required to approach the ratio of the e-hydroxynorleucine to reduced aldol seen in the rabbit aortic elastin itself. The NaB 3H4 reduced cell preparation reveals a ratio which resembles more closely the reduced rabbit aortic elastin. On the basis of hydroxyproline distribution, significant amounts of collagen appear in the media after a 22-h pulse experiment. This is confirmed by the appearance of labelled hydroxylysine and glucosylgalactosylhydroxylysine. It is of interest to note that little or no galactosylhydroxylysine is detected in these cells. While the distribution of the hydroxylysine-carbohydrate moieties vary from collagen to collagen, the basement membrane proteins described by Spiro [15] contain 94--97% of bound hydroxylysine in the dissaccharride form. The type or types of collagen which this cell culture is elaborating will be important to determine in future experimentation. Trelstad [16] has reported several types of collagens to be present in aortic tissue including that found in basement membrane.
103
Finally, one should mention the appearance of an additional lysinederived, borohydride-reduceable c o m p o n e n t in the elastin obtained from the [ 14 C] lysine pulse experiment. It does not appear when ~-aminopropionitrile is added, which is consistent with the fact that it is not glucosylgalactosylhydroxylysine. It may very well be an intermediate in the pathway of desmosine synthesis. These smooth muscle cells with their capacity to elaborate collagen, elastin and glycosaminoglycans, afford an ideal system to study the biosynthesis and interaction of these components under a variety of conditions. Acknowledgements The authors are indebted to Valerie Verbitski and Howard Bond for excellent technical assistance. This work was supported by National Institutes of Health Grant No. AM 07697, HL 13262, HL 15964, HD 05796. Judith Foster is an Established Investigator of the American Heart Association. References 1 Haust, M.D,, More, R.H., Bencosme, S,A. and Balis, J.U. (1965) Exp. Mol. Patho! 4, 508--524 2 Ross, R. and Glomset, J.A. (1973) Science 180, 1332--1339 3 Fisher-Dzoga, K., Chen, R. and Wissler, R.W. (1974) Advances in Experimental Medicine and Biology (Wagner, W,D. and Clarkson, T.B., eds), Vol. 43, pp. 299--311 4 Ross, R. (1971) J. Cell Biol. 50, 172--186 5 Daoud0 A.S., Fritz, K.E., Singh, J., Augustyn, J.M. and J a r m o l y c h , J. (1974) Advances in Experhnental Medicine and Biology (Wagner, W.D. and Clarkson, T.B., eds), Vol. 43, pp. 281--298 6 Abraham, P.A., Smith, D.W. and Cames, W.H. (1974) Biochem. Biophys. Res. Commun. 58, 597---604 7 Morton, H.C. ( 1 9 7 0 ) In Vitro 6, 89--108 8 Eagle, H. (1959) Science 180, 432--437 9 Wolinsky, H. and Daly, M.M. (1970) Proc. Soc. Exp. Biol. Med. 135, 364--368 10 Puck, T.T., Cieciura, S.J. and Fisher, H.W. (1957) J. Exp. Med. 106, 145--157 11 Lansing, A.I., Rosenthal, T.B., Alex, M. and D e m p s e y , E.W. (1952) Anat. Record 114, 5 5 5 - 5 7 0 12 Franzblau, C. and Lent, R.W. (1969) in Structure, F u n c t i o n and Evolution of Proteins, B r o o k h a v e n Symposium No. 21,358--377 13 Spiro, R.G. (1967) J, Biol, Chem. 242, 4 8 1 3 - 4 8 2 3 14 Ross, R. and Klebanoff, S, (1971) J. Cell Biol. 50, 159--171 15 Spiro, R.G. (1969) J. Biol. Chem. 244, 602--612 16 Treistad, R.L. (1974) Bioehem. Biophys. Res. Cornmun. 57, 717--725
Biochimica et Biophysica Acta, 418 (1976) 93--103 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98479
THE SYNTHESIS OF CONNECTIVE TISSUE PROTEIN IN SMOOTH MUSCLE CELLS
B. FARIS, L.L. SALCEDO, V. COOK, L. JOHNSON, J.A. F O S T E R and C. F R A N Z B L A U Department of Biochemistry, Boston UniversitySchool of Medicine, Boston, Mass. 02118
(U.S.A.) (Received June 9th, 1975)
Summary The synthesis of elastin by smooth muscle cells was clearly demonstrated by amino acid analyses and the presence of lysine-derived crosslinks. The values obtained were compatible with those found in amorphous elastin isolated from rabbit aortic tissue. Collagen synthesis by these same cells was monitored by the appearance of [14C]hydroxyproline when the cells were grown in the presence of [14C] proline. When the cells were pulsed with [14C] lysine, one could detect [ ~4C] hydroxylysine and [ 1 4C] glucosylgalactosylhydroxylysine. Further evidence for the synthesis of elastin and collagen was the finding of radiolabelled e-hydroxynorleucine and the reduced aldol condensate of two residues of allysine after reduction of [ ' 4C] lysine pulsed cells with NaBH4.
Introduction The proliferation of smooth muscle cells in aortic tissue has been implicated as a major event in the pathogenesis of atherosclerosis [1,2,3]. Ross [4], Daoud et al. [5], among others, have studied the ultrastructure of these cells in culture. The ability to synthesize connective tissue protein was suggested primarily on the morphologic evidence presented by these workers. Additional studies are being undertaken in several laboratories to obtain chemical evidence as well. Indeed, Ross has presented data indicating the presence of the microfibrillarcomponent of elastic fibers [4]. That tropoelastin is present in smooth muscle cell cultures was suggested by Abraham et al. [6]. The presence of insoluble elastin and several of the lysine-derived crosslinks of collagen and elastin have not been chemically defined in the cultures. Results presented in this communication do indeed indicate the presence of insoluble collagen and elastin and yield evidence that many of the metabolic mechanisms necessary for the assembly of collagen and elastic fibers are present.
94 Material and Methods
Description of smooth muscle cell cultures Aortic medial smooth muscle cells were obtained from weanling rabbits according to the m e t h o d of Ross [4] with some modifications. Thoracic aortas were rapidly excised using sterile techniques and immediately placed in 60-mm Petri dishes with Dulbecco's Modified Eagle's Medium [7] containing 2.2 g sodium bicarbonate per 1, 10% fetal calf serum and per ml, 1000 units of penicillin, 50 pg of aureomycin, 0.25 mg fungizone and 1 ml each of minimum essential medium (MEM) non-essential amino acids (100 X) [8] and sodium pyruvate solution (100 X) per 100 ml. Under a dissecting microscope, the aortas were grossly cleaned of extraneous tissue, blood and fat and transferred to fresh medium. They were then cut open, cleaned again, placed in a fresh dish with medium and the intimal/innet medial layer pinched off and stripped according to the m e t h o d of Wolinsky and Daly [9]. These layers were cut into approx. 1 m m 3 pieces which were attached to the b o t t o m of 250-ml plastic tissue culture flasks. The flasks were allowed to stand with the cap end up to drain the pieces of most of the medium to p r o m o t e adherence. Medium as above but containing 100 units of penicillin and 100 pg of streptomycin per ml, and 3.7 g sodium bicarbonate/1 were added to cover the explants. The flasks were loosely capped and allowed to remain undisturbed for a week in a 5% CO2/95% air moist incubator at 37 ° C. The medium was changed weekly. In two to three weeks, the cells which grew out from the explants were detached with a 0.05% trypsin/0.02% EDTA solution in modified Puck's saline A solution [10], centrifuged at 200 X g for 10 min, resuspended in complete medium, c o u n t e d and seeded into new flasks. Cell culture medium was replaced twice weekly. The original attached explants were maintained to yield another batch of cells which were detached after two weeks. Routinely, 250-ml plastic tissue culture flasks were seeded with 1--1.5 • 106 cells and were passed weekly up to the fourth passage and bimonthly to the sixth using a 1 : 2 split. Experimental procedures Isolation of insoluble elastin from smooth muscle cells. Smooth muscle cells were harvested at various periods of time after the second passage. The media was removed and the resulting cell layer was homogenized in a PotterElvehjem homogenizer using a phosphate buffer (0.1 M NaH2PO4/0.15 M NaC1, pH 7.5). The homogenate was stirred overnight at 4°C and then centrifuged at 16 000 X g for 20 min. This procedure was repeated with the residue obtained. The combined supernatants were designated cell extract as stated below. The resulting cell debris from each of the time periods (2--3 flasks) was then suspended in 0.1 M NaOH and heated at 98 ° C for 45 min according to the procedure of Lansing e t a l . [11]. The resulting suspension was centrifuged at 3000 X g for 10 minutes and the residue was washed thoroughly with water, suspended in 2 ml of 6 M HC1 and h y d r o l y z e d in a sealed vial at l l 0 ° C for 20 h. The hydrolysates were placed on a Jeolco 6 AH automatic amino acid analyzer modified to analyze hydrolysates of elastin. For reference purposes,
95 insoluble elastin was prepared from the medial portion of a weanling rabbit aorta. The tissue was treated with 0.1 M NaOH as described above and analyzed for amino acid content.
Pulse label experiments (A) General description. Plastic tissue culture flasks (75 cm 2 growth area) were seeded with 1--1.5 • 106 smooth muscle cells in the second passage. The cells were maintained for different periods of time after seeding (days in culture). Before addition of the radioactive amino acid, the spent medium was aspirated off and the cell layers washed seven times with medium containing no fetal calf serum. They were then incubated for 1 h at 37°C in serum-less medium lacking lysine or proline and containing sodium ascorbate (50 pg/ml). This medium was then replaced with one containing either [14C]lysine (1.0 pCi/ml, spec. act. 312 Ci/M) or [ 14C] proline (1.0 pCi/ml, spec. act. 260 Ci/M) and sodium ascorbate as above. The pulse period lasted for 22 h. The spent radioactive medium was poured off and the flasks were drained by aspirating residual medium. 2 ml of phosphate buffer (0.1 M Nail2 PO4 / 0.15 M NaCl, pH 7.5) was added to each of the flasks which were laid on ice and the cell layers were collected with the aid of a rubber policeman and transferred to centrifuge tubes. The flasks were washed twice more with buffer solution. The cell suspension was centrifuged at 300 × g for 20 min and the cell pellets were washed with deionized water and centrifuged three more times. For those experiments with fl-aminopropionitrile fumarate the procedure is the same except the cells are incubated in medium containing/~-aminopropionitrile (15 pg/ml) during the wash and pulse periods. (B) Conversion o f [ 14 C] proline to [ 14 C] hydroxyproline. The presence of [ ~ 4C] hydroxyproline and [ ~4C] proline in the fractions described below was determined by hydrolyzing each sample in 6.0 M HC1 at l l 0 ° C for 20 h and placing the hydrolysate on a Technicon amino acid analyzer with a split-stream arrangement as described previously [12]. Also placed on the analyzer column with each hydrolysate was a standard hydroxyproline solution (not radioactive) to serve as a marker. The various fractions examined were: (1) Media: The decanted media {93--97 ml) from 12 flasks of smooth muscle cells (grown in culture either 10 or 13 days) which were pulsed for 22 h with [14C] proline was centrifuged at 16 000 X g for 20 min, and the supernatant was dialyzed versus H~ O at 4 ° C. The material remaining in the dialysis sac was recentrifuged, since a fine white precipitate appeared during dialysis. The resulting supernatant and precipitate were each lyophilized separately. (2) Intercellular material: After the spent medium was decanted from the cell layer, 2 ml of phosphate buffer was added to each flask and the cells scraped off gently with a rubber policeman. The mixture was centrifuged at 200 × g for 10 rain. The cell pellet was washed and centrifuged in the same manner t w o more times. The pooled washes were centrifuged at 16 000 × g for 20 min and the supernatant was dialyzed as described above. This dialyzed material was lyophilized. (3) Cell extract: The cell pellet from step 2 was homogenized as in the above section on "Insoluble Elastin". The combined supernatant, referred to as
96 the cell extract was dialyzed versus H 2 0 . As in the case of the media, the resulting dialyzed material was centrifuged since a precipitate was present. Again, the precipitate and supernatant were each lyophilized separately. (4) Cell debris: The residue from Step 3 was suspended in 0.1 M NaOH and placed at 98°C for 45 min. The resulting insoluble material and the supernatant after this alkali treatment were analyzed as described. (C) Incorporation of [~ 4C] lysine into connective tissue components. For this series of experiments, smooth muscle cells were grown in the presence of [~4C]lysine in place of [~4C]proline. Two separate experiments were performed. The first utilized 13 flasks of second passage cells which were 13 days old. After the pulsed media was decanted (98 ml), centrifuged at 16 000 × g for 20 min and dialyzed, the resulting precipitate was harvested and lyophilized. Approx. 0.5 mg of this lyophilized material was suspended in 5.0 ml of H2 O and 1.0 mg NaBH4 was added. Reduction was allowed to proceed for 90 min (the pH never exceeded 8.0) with occasional shaking. The pH was then adjusted to 3.0--4.0 with 50% acetic acid and the entire reaction mixture was dialyzed and lyophilized, e-Hydroxynorleucine and the reduced aldol condensation p r o d u c t of two residues of allysine (reduced aldol) were determined by hydrolyzing the NaBH4 reduced material in 2.0 M NaOH at 110°C for 20 h and employing a split-stream arrangement [ 12]. The combined cell layers from all of the flasks employed in the above experiment were washed 3 times with phosphate buffer, pH 7.5, 3 times with H2 O and then suspended in 10 ml H2 O. To this suspension, 12.5 mg NaBH4 was added and the reduction reaction was allowed to proceed as above. After dialysis, the material was divided into two equal portions, and each was treated with 0.1 M NaOH at 98°C for 45 min. The supernatant in each case was removed and one sample was h y d r o l y z e d in 6.0 M HC1 at l l 0 ° C and the other in 2.0 M NaOH at 110°C for 20 h. Separate hydrolysis in HC1 and in NaOH was also performed on the residue. The radioactive profile of each hydrolysate was determined. In a second set of experiments, 5 flasks of cells (second passage), 19 days in culture, were pulsed with [14C]lysine as above, and another 5 flasks of similar cells were pulsed with [14C]lysine in the presence of ~-aminopropionitrile (15 pg/ml). The pulsed media in each case (46--49 ml) was handled in the same fashion as in the previous experiment except that the media from the cells grown in the presence of ~-aminopropionitrile was dialyzed against H2 O containing 15 pg ~-aminopropionitrile/ml. The entire contents of each dialysis sac was lyophilized w i t h o u t centrifugation. Each sample was then suspended in 5.0 ml of H 2 0 and reduced with 2.0 mg of NaBH4. for 90 min. After dialysis, each was hydrolyzed in 2.0 M NaOH at l l 0 ° C and the radioactive elution patterns from the amino acid analyses were compared. The washed cell layers with and w i t h o u t ~-aminopropionitrile were suspended in 10 ml H2 O and reduced in the presence of 10 mg NaBH4. After dialysis and lyophilization, each was treated with NaOH according to Lansing et al. [ 11] and the insoluble material remaining was hydrolyzed in 2.0 M NaOH at 110°C. The material solubilized by the Lansing t r e a t m e n t (0.1 M NaOH at 98°C for 45 rain) was hydrolyzed separately in each case in 6.0 M HC1 and in 2.0 M NaOH.
97 To determine the total distribution of crosslinks present in the insoluble elastin, four flasks of 21 day cells were reduced with NaB3 H4 after removal of the medium. This group of cells did n o t receive any pulse label. The cell layer after reduction was treated with alkali and analyzed for radioactivity as described. For comparison purposes, the medial portion of a weanling rabbit aorta was reduced with NaB s H4, exposed to Lansing treatment and the insoluble elastin fraction was examined. The radioactive profile from the split stream amino acid analyses was determined in all cases. Results
Presence of elastin in smooth muscle cells The amino acid compositions of Lansing treated cell debris are given in Table I. The table includes the analyses from cells cultured for various days after seeding as well as that of elastin from the medial tissue of the rabbit aorta. Although no attempt to quantify cell numbers was made, approx. 5 • 106 smooth muscle cells were routinely employed if one assumes one cell-doubling occurred after seeding. To estimate the small a m o u n t of desmosines present in the insoluble elastin, two separate concentrations of the acid hydrolysate from the 20 day old cells was applied to the amino acid analyzer. The higher concentration was 15 times greater than normally used in all other samples. As can be seen from Table I, 13 day old cells yield definitive elastin amino acid analyses. It is of interest to note the difference in amino acid composition of the 9 or 10 day old cultures compared to the 13 day old alkali insoluble residue. As the cultures "age", it again becomes more difficult to remove " c o n t a m i n a n t " protein from the elastin by this alkali treatment.
Conversion of [14C]prolin e to [14C] hydroxyproline The appearance of newly synthesized collagen in these experiments was monitored b y the presence of [ 14 C] hydroxyproline. The distribution of radioactivity in proline and hydroxyproline in all fractions isolated and described above are given in Table II. In each fraction the percent of the total radioactivity represented as hydroxyproline is given as well. It can be seen that the highest percentage of labelled hydroxyproline is found in the media. Very little hydroxyproline radioactivity was found in those fractions of the cell debris (13 day) that remained insoluble after alkali treatment. This latter fraction was shown by its amino acid composition to be insoluble elastin. Studies are in progress to ascertain optimum time and conditions for soluble and insoluble collagen synthesis by these smooth muscle cells.
Lysine incorporation The alkali-insoluble residues obtained from the smooth muscle cells were examined for lysine-derived aldehydes and crosslinks. Fig. 1A shows a typical radioactive profile of a 2.0 M NaOH hydrolysate of the elastin (cell debris) derived from cells that had been pulsed with [14C]lysine and reduced with NaBH4 as described in the experimental procedures. Radioactive hydroxynorleucine and the reduced aldol c o m p o u n d are the predominant components detected. No labelled hydroxylysine can be seen, indicating little or no collagen
NC 81.2 53.0 56.0 106.7 84.9 164.1 105.7 73.9 38.8 75.6 25.5 32.5 33.5 22.2 45.4 ---
9
Days
NC 50.6 27.9 31.8 70.6 90.7 234.6 178.2 92.0 36.9 77.1 9.7 24.6 28.5 9.7 25.9 ---
10 4.8 14.9 15.8 18.7 28.3 121.8 333.2 232.0 99.1 22.9 53.5 13.7 20.7 7.7 3.5 9.2 NC NC
13 3.5 13.8 10.2 19.8 28.2 121.3 329.0 236.5 100.0 23.6 52.4 14.5 20.5 11.2 3.8 11.9 NC NC
13
* V a l u e s o b t a i n e d f r o m t w o d i f f e r e n t a m i n o a c i d a n a l y s e s (see t e x t ) .
Hyctro x y p r o l i n e Aspartic Threonine Serine Glutamic Proline Glyeine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Axginine Isodesmosine Desmosine Lysinonorleucine
A m i n o acid
NC 20.6 18.5 20.4 33.7 115.4 321.7 211.5 100.5 24.0 62.0 21.7 20.8 9.9 5.6 13.8 NC NC
19 3.8 13.3 15.1 17.3 26.2 124.6 319.1 222.9 103.9 23.1 60.1 27.1 25.0 5.9 4.6 11.7 0.5 0.4
20* NC 18.7 16.1 37.4 32.3 97.7 322.9 217.5 111.3 25.5 60.3 7.6 18.7 13.9 5.9 6.8 NC NC
23
3.7 32:2 26.7 25.8 46.0 116.0 293.7 186.4 94.2 28.3 62.5 26.3 23.8 10.3 8.7 18.5 NC NC
43
15.3 3.1 9.9 11.7 16.7 119.2 344.6 243.3 98.6 20.9 54.0 23.2 19.1 5.2 0.5 5.8 2.8 4.2 1.2
Medial tissue from rabbit aorta
V a l u e s e x p r e s s e d as r e s i d u e s / 1 0 0 0 r e s i d u e s , b u t n o t c o r r e c t e d f o r s l i g h t l o s s e s o f a m i n o a c i d s d u r i n g h y d r o l y s i s . N C = n o t c a l c u l a b l e : a m o u n t t o o s m a l l t o c a l c u l a t e .
AMINO ACID ANALYSES OF ELASTIN FROM SMOOTH MUSCLE CELLS
TABLE I
c£) 0o
99 T A B L E II IDISTRIBUTION TIONS
OF [14C] HYDROXYPROLINE
Cell f r a c t i o n
A N D [I 4 C ] P R O L I N E
CELL FRAC-
10 day
13 d a y
Hydroxyproline × I00 Proline + Hydroxyproline 10 day 13 d a y
70 000 280 000
55 000 89 000
3.2% 5.2%
13.2% 8.2%
Intercellular
28 000
26 0 0 0
0.9%
1.4%
Cell e x t r a c t 1. S u p e r n a t a n t 2. P r e c i p i t a t e
50 000
Media 1. S u p e r n a t a n t 2. P r e c i p i t a t e
Cell d e b r i s 1. S u p e r n a t a n t * 2. R e s i d u e *
Hydroxyproline (cpm)
IN V A R I O U S
28 0 0 0
1.2% 1.2%
50 000
90 000 560
2.0%
4.0% 1.6%
* Fractions from Lansing treatment.
contamination in this preparation of insoluble elastin. An unknown radioactive peak appeared in the region where valine elutes. It was not present in the ~-aminopropionitrile-treated cells described below. The reduced aldol content was calculated from the ninhydrin data obtained from the amino acid analysis. T A B L E III [ 14C] LYSINE INCORPORATION
IN V A R I O U S C E L L F R A C T I O N S
BAPN, ~-aminopropionitrlle fumarate. Cell f r a c t i o n
cpm//~M l e u c i n e (X 1 0 4) Hydroxynorleucine
Glucosylgalactosylbydroxylysine
Media 13 d a y 19 day (--BAPN) 19 day (+BAPN)
8.6 8.7 3.5
19.0 15.0 8.4
Cell d e b r i s 1. S u p e r n a t a n t * 13 day 19 day (--BAPN) 19 day (+BAPN)
2.2 2.4 0.3
2.4 1.1 0.9
2. R e s i d u e * 13 day 19 day (--BAPN) 19 d a y ( + B A P N )
2.4 3.0 0.2
----
Reduced aldol
----
1.8 2.1 0.04 4.1 3.6 0.06
Rabbit aortic medial tissue** Residue*
10.0
69.0
Cells* * Residue*
23.0
85.0
* Fractions from Lansing treatment. ** N a B 3 H 4 r e d u c e d o n l y ; n o [ 1 4 C ] l y s i n e p u l s e (see t e x t ) .
Hydroxylysine
Lysine
29.0 19.0 13.0
380.0 280,0 210.0
1.8 1.3 0.6
81.0 58.0 34.0
--
18.0
--
6.8
--
9.2
100 HNL
LYS
ALOOL
A 15
10
5
I
I
I
I
1
I
I
I
B 15
10
5
40
80
120
160
FRACTION NO. Fig. 1. R a d i o a c t i v e e i n t i o n p r o f i l e o f 2.0 M N a O H h y d r o l y s a t e s f r o m a m i n o acid a n a l y s e s of elastin f r o m cells p u l s e d w i t h [ 1 4 C ] l y s i n e a n d r e d u c e d w i t h N a B H 4. A, f r o m c o n t r o l cells; B, f r o m cells p u l s e d in p r e s e n c e o f ~3-aminopropionitrile f u m a r a t e . T h e e x p e r i m e n t a l p r o c e d u r e s f o r t h e a m i n o acid a n a l y s e s a n d d e t e r m i n a t i o n o f r a d i o a c t i v i t y h a v e b e e n o u t l i n e d in d e t a i l p r e v i o u s l y [ 1 2 ] . F o r r e f e r e n c e p u r p o s e s , v a l i n e e l u t e s at f r a c t i o n s 7 9 - - 8 2 a n d g a l a c t o s y l h y d r o x y l y s i n e w o u l d e l u t e a t f r a c t i o n s 1 1 4 - - 1 1 7 . H N L , h y d r o x y norleucine; A L D O L , reduced aldol condensation product.
The data obtained from three separate experiments indicate that 8--10 residues of reduced aldol are present per 1000 amino acid residues. Fig. 1B shows the data from Lansing-treated cell layer (cell debris) which had been subjected to fl-aminopropionitrile. This fraction revealed little radioactivity in the reduced aldol and hydroxynorleucine peaks while lysine incorporation appeared to be significant. As expected, nonradioactive reduced aldol was detected by the ninhydrin reaction from the amino acid analyzer. For comparison purposes, the radioactive profiles of the soluble fractions obtained after Lansing treatment of the cell debris are given in Fig. 2A (no /3-aminopropionitrile) and Fig. 2B (with/3-aminopropionitrile). The appearance
101 NNL
ALGOL
LY$
A
1(
o
8 t5
10
40
80
120
160
FRACTION NO.
Fig. 2. Radioactive elution profile of 2.0 M NaOH hyd~ol:fsates from amino acid analyses of alkali soluble fraction from cells pulsed w i t h [ 1 4 C ] l y s i n e and reduced w i t h NaBH 4. A, from c o n t r o l cells; B, from cells pulsed in presence of ~-aminopropionitrile fumaratc. The e x p e r i m e n t a l procedures for the amino acid analyses and d e t e r m i n a t i o n of radioactivity have been outli ne d in detail previously [ 1 2 ] . F or reference purposes, valine elutes at fractions 79--82 and p l a c t o s y l h y d r o x y l y s i n e w oul d elute at fractions 114-117. HNL, h y d r o x y n o t l e u c i n e ; GGH, glucosylgalactosylhydroxy l ys i ne ; ALDOL, reduced aldol c o n d e n s a t i o n pr oduct; HYLYS, h y d r o x y l y s i n e .
of glucosylgalactosylhydroxylysine [13] suggests the presence of solubilized collagen fragments. Of interest is the negligible amount of radioactivity found in the area where galactosylhydroxylysine [13] would elute. Examination of media from the pulse labelled cells in the presence or absence of/3-aminopropionitrile displayed e-hydroxynorleucine, glucosylgalactosyl hydroxylysine and no reduced aldol (not shown). These data, together with the presence of radioactive hydroxylysine, support the evidence that collagen is present in the media in contrast to the insoluble cell debris after Lansing treatment. ~-aminopropionitrfle-treated cells still display hydroxylysine and glucosylgalactosylhydroxylysine radioactivity (not shown).
102
Table III summarizes the total radioactivity in each of the components described. Included in the table are the radioactive data obtained from elastin prepared from rabbit aorta and reduced with NaB 3 H 4 as described. Data from NaB 3 H4 reduced 21 day cell layer is also included. Discussion Ross and his collaborators noted t h a t aortic smooth muscle cells are capable of synthesizing elastic tissue. Morphologic evidence suggested the presence of collagen, elastic fiber microfibrils, and insoluble elastin [ 14]. Chemical evidence for the soluble precursor of elastin, namely tropoelastin, was presented by Abraham et al. [6]. Isolation of tropoelastin itself however has proved somewhat elusive. Data included in this communication clearly indicate the presence of insoluble elastin in cultures of these cells. The amino acid analyses of the Lansing-treated cell layers are quite compatible with that of amorphou.s elastin isolated from the rabbit aortic tissue itself. The 9 or 10 day cultures show significant amounts of acidic amino acids suggesting the presence of glycoprotein or microfibrillar components. This observation is in keeping with the concept that such glycoprotein moieties are synthesized early in elastic tissue fibrogenesis to form a matrix for the insoluble elastin c o m p o n e n t [2]. The 20 day cultures show significant amounts of desmosine and isodesmosine when large quantities of hydrolysate were analyzed. To substantiate the presence of elastin, the [ 14 C] lysine pulse experiments indicate active synthesis of allysine and the aldol condensate of two residues of allysine. The quantity of reduced aldol is such that it suggests the presence of elastin and n o t collagen. Treatment with ~-aminopropionitrile caused almost complete inhibition of formation of allysine and its aldol condensate. Although the amino acid compositions of elastin preparations from the cell cultures are strikingly similar to t h a t found in the rabbit aorta itself, the distribution of radiolabelled e-hydroxynorleucine and reduced aldol after reduction are quite different. One must note that the ratio of these two substances from the rabbit elastin preparation is obtained from NaB 3 H4 reduction and represents the entire distribution of the two moieties in the sample. The [ 14 CI lysine pulse in the cells represent only newly synthesized elastin during a 22-h period and additional chase periods would most likely be required to approach the ratio of the e-hydroxynorleucine to reduced aldol seen in the rabbit aortic elastin itself. The NaB 3H4 reduced cell preparation reveals a ratio which resembles more closely the reduced rabbit aortic elastin. On the basis of hydroxyproline distribution, significant amounts of collagen appear in the media after a 22-h pulse experiment. This is confirmed by the appearance of labelled hydroxylysine and glucosylgalactosylhydroxylysine. It is of interest to note that little or no galactosylhydroxylysine is detected in these cells. While the distribution of the hydroxylysine-carbohydrate moieties vary from collagen to collagen, the basement membrane proteins described by Spiro [15] contain 94--97% of bound hydroxylysine in the dissaccharride form. The type or types of collagen which this cell culture is elaborating will be important to determine in future experimentation. Trelstad [16] has reported several types of collagens to be present in aortic tissue including that found in basement membrane.
103
Finally, one should mention the appearance of an additional lysinederived, borohydride-reduceable c o m p o n e n t in the elastin obtained from the [ 14 C] lysine pulse experiment. It does not appear when ~-aminopropionitrile is added, which is consistent with the fact that it is not glucosylgalactosylhydroxylysine. It may very well be an intermediate in the pathway of desmosine synthesis. These smooth muscle cells with their capacity to elaborate collagen, elastin and glycosaminoglycans, afford an ideal system to study the biosynthesis and interaction of these components under a variety of conditions. Acknowledgements The authors are indebted to Valerie Verbitski and Howard Bond for excellent technical assistance. This work was supported by National Institutes of Health Grant No. AM 07697, HL 13262, HL 15964, HD 05796. Judith Foster is an Established Investigator of the American Heart Association. References 1 Haust, M.D,, More, R.H., Bencosme, S,A. and Balis, J.U. (1965) Exp. Mol. Patho! 4, 508--524 2 Ross, R. and Glomset, J.A. (1973) Science 180, 1332--1339 3 Fisher-Dzoga, K., Chen, R. and Wissler, R.W. (1974) Advances in Experimental Medicine and Biology (Wagner, W,D. and Clarkson, T.B., eds), Vol. 43, pp. 299--311 4 Ross, R. (1971) J. Cell Biol. 50, 172--186 5 Daoud0 A.S., Fritz, K.E., Singh, J., Augustyn, J.M. and J a r m o l y c h , J. (1974) Advances in Experhnental Medicine and Biology (Wagner, W.D. and Clarkson, T.B., eds), Vol. 43, pp. 281--298 6 Abraham, P.A., Smith, D.W. and Cames, W.H. (1974) Biochem. Biophys. Res. Commun. 58, 597---604 7 Morton, H.C. ( 1 9 7 0 ) In Vitro 6, 89--108 8 Eagle, H. (1959) Science 180, 432--437 9 Wolinsky, H. and Daly, M.M. (1970) Proc. Soc. Exp. Biol. Med. 135, 364--368 10 Puck, T.T., Cieciura, S.J. and Fisher, H.W. (1957) J. Exp. Med. 106, 145--157 11 Lansing, A.I., Rosenthal, T.B., Alex, M. and D e m p s e y , E.W. (1952) Anat. Record 114, 5 5 5 - 5 7 0 12 Franzblau, C. and Lent, R.W. (1969) in Structure, F u n c t i o n and Evolution of Proteins, B r o o k h a v e n Symposium No. 21,358--377 13 Spiro, R.G. (1967) J, Biol, Chem. 242, 4 8 1 3 - 4 8 2 3 14 Ross, R. and Klebanoff, S, (1971) J. Cell Biol. 50, 159--171 15 Spiro, R.G. (1969) J. Biol. Chem. 244, 602--612 16 Treistad, R.L. (1974) Bioehem. Biophys. Res. Cornmun. 57, 717--725