Proc. Nati. Acad. Sci. USA Vol. 73, No. 7, pp. 2361-2364, July 1976
Biochemistry
Characterization of the collagen synthesized by endothelial cells in culture (basement membrane collagen/biosynthesis/calf aorta)
BARBARA V. HOWARD,* EDWARD J. MACARAK, DIANE GUNSON, AND NICHOLAS A. KEFALIDESt Departments of Biochemistry and Medicine, University of Pennsylvania and the Philadelphia General Hospital, Philadelphia, Pa. 19104
Communicated by Robert E. Forster, May 10, 1976
ABSTRACT [14CJProline and ["4Clysine were incorporated into collagen by cultures of endothelial cells derived from calf aortae. The isomer 3-hydroxy["4Cproline accounted for 10% of the total hydroxy[14Clproline in the collagen isolated from the medium. Approximately 81% of the hydroxy"4C~lysine isolated from the medium was glycosylated, and 91% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosylgalactose. Gel filtration chromatography or acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the initially synthesized peptide chain of [14Cjcollagen had a molecular weight of about 135,000; after pepsin digestion this was converted to 115,000. The ratio of hydroxy[14Clproline to total ['4Clproline X 100 in the pepsin-resistant fraction was 59. Wen examined by immunofluorescence microscopy, the endothelial cultures stained positively with antiserum to (Type IV) collagen from basement membrane of bovine anterior lens capsule. The data indicate that cultured endothelial cells derived from calf aortae synthesize collagen that resembles that of basement membrane collagen.
The establishment of cultures of endothelial cells in vitro provides an opportunity to study the metabolic functions of these cells (1, 2). It has been proposed (3) that endothelial cells are the source of the basement membrane that invariably underlies these cells. The nature of the collagen component of vascular basement membrane has been characterized in capillaries of the glomerulus and the choroid plexus (4, 5). However, biosynthetic studies of collagen of vascular basement membrane are scanty (6-11), and there are no reports of studies of this collagen from large vessel endothelium. This report presents a partial characterization of the collagen synthesized by primary cultures of aortic endothelial cells. The results indicate that cultured endothelial cells synthesize collagen that is similar to basement membrane (Type IV) collagen synthesized by other cell culture systems in vitro. MATERIALS AND METHODS Cell Culture. For the isolation and culture of endothelial cells from calf aortae the methods of jaffe et al. (1) and Gimbrone et al. (2) were adapted with modifications. Calf aortae were obtained from the slaughter house within 1 hr after the animals' death. Thoracic aortae, approximately 15-20 cm long, were removed from the animals aseptically and immediately placed in sterile chilled Dulbecco's phosphate-buffered saline supplemented with glucose (5 mg/liter), amphotericin B (2.5 ,gg/ml), penicillin (50 units/ml), and streptomycin (50 ,g/ml). Six to eight vessels were collected and subsequently processed for culture within 2 hr. To isolate endothelial cells, we washed Abbreviation: NaDodSO4, sodium dodecyl sulfate. * Present address. Department of Biochemistry, Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, Pa. 19129. t To whom correspondence should be addressed at Clinical Research Center, Philadelphia General Hospital, 700 Civic Center Boulevard,
aortae in Dulbecco's phosphate-buffered saline and defatted them by dissection. The paired intercostal vessels were tied and the lower end of the vessel was clamped with an intestinal clamp. The vessel was then filled with 10-15 ml of collagenase (1 mg/ml; Type II, Worthington) in Medium 199, and the upper end was clamped with a second intestinal clamp. The vessels were incubated at room temperature for 45 min and the contents were collected. This collagenase effluent usually contained few cells and was discarded. The vessels were then washed four times with 10 ml of growth medium, which consisted of Medium 199 as modified by Lewis et al. (12) supplemented with 20% fetal bovine serum, gentamicin (50 ,g/ml), and amphotericin B (2.5 sug/ml). During each wash, the vessel was clamped and the medium within the vessel was agitated gently by inverting the vessel four times. The pooled effluents containing the freed endothelial cells were distributed evenly among 75-cm2 glass or plastic petri dishes (10 ml per dish), and the cells were incubated at 370 in a humidified atmosphere of 5% CO2 in air. The medium was changed in all cultures every 3 days. For subculture, cells were treated briefly (1-2 min) with a calcium- and magnesium-free buffered saline containing trypsin (0.25%) and EDTA (0.05%). Released cells were seeded in new petri plates and the medium was changed 4-6 hr after the trypsin-EDTA treatment. Cultures established in this manner from calf aortae have
been identified as endothelial by light and electron microscopic criteria, and the growth of the cells has been characterized. These data are described elsewheret. Endothelial cultures from human umbilical veins were derived by standard methods (1, 2), and human diploid fibroblast cultures were established from foreskins as described (13). Collagen Synthesis. Monolayers of primary cultures of endothelial cells (approximately 7 days after isolation, three to five cell generations in vitro) were incubated in a buffered balanced salt solution (14) supplemented with fetal bovine serum (10%), glucose (5 mg/ml), BME vitamins and amino acids (Gibco), glutamine (1 mM), ascorbic acid (50 ug/ml), 13-aminopropionitrile (50 jg/ml), and either [U-14C]proline (2 gCi/ml; 200 mCi/mmol) or [U-14C]lysine (1IuCi/ml; 342 mCi/mmol). After 48 hr the incubation medium and cell fractions were separated and calf skin collagen was added as carrier. Incorporation of total 14C into nondialyzable protein was determined after extensive dialysis against 0.1% sodium dodecyl sulfate in 0.1% sodium phosphate, pH 7.4 (NaDodSO4/PJ). 4-Hydroxy[14C]proline was quantitated in this material by the method of Juva and Prockop (15). For gel chromatography and gel electrophoresis, the newly synthesized collagen in the medium was precipitated with ammonium sulfate (176 mg/ml). This ret E. Macarak, B. V. Howard, and N. A. mitted.
Philadelphia, Pa. 19104.
2361
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2362
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Proc. Natl. Acad. Sci. USA 73 (1976)
Table 1. Incorporation of [ 14C] proline into total protein and synthesis of hydroxy l4 C ] proline by endothelial cells in culture (average of four determinations)
Table 2. Hydroxy[ 'I4 C ]proline isomers in collagen synthesized by endothelial cells in culture
(3-Hydroxyproline/ (B) Hydroxy
(A) Total
'"C
['4C]proline
(B/A)
x
536,000 1,270,000
27,700 27,100
5.2 2.1
collagen (Fig.
2) Fibroblasts Medium
59*
153,000
764,000
20
Monolayer cultures were incubated for 48 hr in an incubation medium containing [14C]proline (2 MCi/ml) as described in Materials and Methods. Medium was removed and NaDodSO4 added to a 0.1% final concentration. The cell layer was harvested by scraping in NaDodSO4/P1. After samples were boiled for 3 min, they were extensively dialyzed against NaDodSO4/Pi, and total 14C radioactivity, as well as hydroxy[14C]proline were determined as described in Materials and Methods. Pepsin-digested collagen was obtained as described in Fig. 2. * For pepsin-digested material, the number represents the ratio of hydroxy[14C]proline to total [14C]proline in fractions 38-41 of Fig. 2.
sulted in a considerable enrichment (6- to 9-fold) of the [14C]collagen in the preparation. Characterization of Synthesized Collagen. The material precipitated by ammonium sulfate was prepared for chromatography or electrophoresis by reduction with 5% 2-mercaptoethanol in 1% NaDodSO4/Pi for 3 hr at 400, or by reduction with 50 mM dithioerythritol, 1 M urea, 0.5 M Tris-HCl at pH 8.2 for 15 min at room temperature followed by alkylation with 120 mM iodoacetamide. Gel filtration chromatography was performed on a calibrated column of 6% agarose (Bio-Gel A-5 M) eluted with NaDodSO4/Pj as described (7). 4-Hydroxy[14C]proline was quantitated according to the method of Juva and Prockop (15). NaDodSO4-polyacrylamide gel electrophoresis was accomplished according to the procedure of Goldberg et al. (16), using a polymerizing solution of 7% acrylamide monomer, 0.137% N,N'-diallyltartardiamide, 0.1% NaDodSO4, and 0.5 M urea. Migration was compared to and ,B components of carrier calf skin collagen. The gels were fractionated into 1-mm slices with a Mickle Gel Slicer (Brinkman), and slices were solubilized in 2% periodic acid before their radioactivity was determined (17). The content of 3- and 4-hydroxy[14C]proline was determined in material precipitated by ammonium sulfate or fractions of chromatograms as described (6). Hydroxy[14C]lysine and glycosides were quantitated after alkaline hydrolysis on the amino acid analyzer by the method of Askenasi and Kefalides (18). For pepsin digestion, the ammonium sulfate precipitate was dialyzed against 0.05 M acetic acid and treated with pepsin (100 ,gg/ml) at 150 for 6 hr. The reaction was stopped by dialyzing against 15% KCl in 0.01 M sodium phosphate, pH 9, and the material was prepared for chromatography as described above. Immunologic Studies. Basement membrane collagen was extracted from anterior lens capsules of calf eyes by limited pepsin digestion for 24 hr at 15°, and the collagen was precipitated three times with 15% KCl, 0.02 M Na2HPO4 (5). The a
Bovine aortic Primary Passage 7 Human umbilical Human fibroblast
10.2 ± 1.4 (5)* 13.0 9.3 1.6
100 (%)
(dpm/mg cell protein) Endothelial Medium Cells Pepsin-digested medium ['I 4C]-
Sample
total hydroxyproline) x 100
Primary cultures of aortic or umbilical endothelial cells were incubated for 48 hr with [14C]proline as described in Materials and Methods. Cultures from aortae were also labeled in a similar manner after seven subcultivations. Medium was precipitated with ammonium sulfate, and the proportions of 3-hydroxyproline and 4-hydroxyproline were determined with an amino acid analyzer (6). * No. of determinations.
collagen wasdissolved in 0.1 M acetic acid at a concentration of 5 mg/ml, dialyzed against physiological saline, and mixed with an equal volume of Freund's complete adjuvant. Four New Zealand white rabbits were immunized with this method, using a previously described immunization schedule (19), and bled 1 week after the fifth injection. All sera were stored at -70°. Coverslip cultures of endothelial cells were washed in phosphate-buffered saline and air-dried. Fifty microliters of antiserum or preimmunization serum were placed on each coverslip; they were incubated in a moist chamber for 1 hr at room temperature. Cellular localization of antibody was detected by the indirect immunofluorescence reaction using fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin (Cappel Laboratories, Downingtown, Pa.) RESULTS
Collagen synthesis was demonstrated in the endothelial cell cultures by the synthesis of hydroxy['4C]proline in both cellular and extracellular protein fractions (Table 1). The amount of collagen synthesized by these cells was very low as compared to fibroblast cultures, and the excreted collagen comprised a small proportion of the total extracellular protein. The rate of collagen synthesis per mg of cell protein in the endothelial cultures was of the same order of magnitude as that observed in freshly isolated endothelial cell preparations. Table 2 shows data for the isomers of hydroxy[14C]proline present in material after ammonium sulfate precipitation or gel filtration. The collagen synthesized by primary cultures of these cells contained 10.2% of the total hydroxy[14C]proline as the 3 isomer. In addition, comparable amounts of the 3-hydroxy['4C]proline were also found in cultures after seven passages in vtro and in endothelial cultures derived from umbilical veins. Control values from fibroblast cultures averaged 1.6%. Determination of glycosylated derivatives of hydroxy[14C]lysine in the cell and medium fractions indicated that 91% of the derivatives were in the form of the disaccharide, glucosylgalactose (Table 3). The percent glycosylation of hydroxy['4C]lysine averaged 81% in the medium fraction. Values for percent glycosylation of hydroxy[14C]lysine in the cell fraction were lower, averaging 72% (Table 3). Both these values were significantly higher than those obtained in fibroblast cultures, although the skin fibroblast data differed from values reported for Type I collagen synthesized by tendon fibroblasts in vitro (20).
Biochemistry: Howard et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
22363
Table 3. Glycosylation of hydroxy [ 4 C ] lysine by endothelial cells in culture
Disaccharide % Glycosylation (Di- + monosaccharide) V0 Endothelial Medium Cell fraction Fibroblast Cell + medium
Vt
4x
91 ± 0.62 (7)* 91 (2)
81 ± 1.4 (3) 72 (2)
0
81 ± 3.3
46 ± 4.7 (5)
V
z
I.-
(5)
Monolayers of primary aortic endothelial cells or skin fibroblasts incubated for 48 hr with [14C]lysine as described in Materials and Methods. Medium and cell layer were separated and hydrolyzed separately. Hydroxy[14C]lysine and glycosides were quantitated after alkaline hydrolysis on an amino acid analyzer (18). Monosaccharide refers to galactosyl-hydroxy[14C]lysine, and disaccharide is glucosyl-galactosyl-hydroxy['4C]lysine. * No. of determinations. were
4
U-
E
01 020
Upon NaDodSO4-acrylamide gel electrophoresis, after reduction, the majority of the ['4C]protein migrated as a species with a molecular weight of about 135,000 (Fig. 1). When large amounts of material were applied, a significant proportion of the 14C remained at the origin. On gel filtration, a significant proportion of the hydroxy[14C]proline eluted with an apparent molecular weight of approximately 135,000. However, greater than 50% of the hydroxy['4C]proline was found in a peak close to the exclusion volume. The percent hydroxylation of proline in the larger molecular weight fraction averaged 14%. After pepsin digestion and gel filtration (Fig. 2), the [14C]collagen synthesized by the endothelial cell cultures had an apparent molecular weight of 115,000. Determination of the total hydroxy['4C]proline in the peak tubes of this fraction indicated 59% hydroxylation of the ['4C]proline. When endothelial cell cultures were incubated with antiserum to basement membrane collagen of anterior lens capsule and counterstained with fluorescein goat anti-rabbit gamma^1 ^2
P11PI2
2
'CO
0
0U.
E
0
40
20
DISTANCE FROM
40
ORIGIN
60
(mm) FIG. 1. NaDodSO4-polyacrylamide gel electrophoresis of [14C]protein from endothelial medium after 48-hr pulse with [14C]proline. Material was precipitated with ammonium sulfate and reduced in 1% NaDodSO4, 1% mercaptoethanol as described in Materials and Methods. Samples contained approximately 20,000 cpm in 100 Ml. Gels were stained before slicing to determine migration of
carrier collagen components.
40
60
FRACTION NUMBER FIG. 2. Gel filtration chromatography on NaDodSO4-agarose A5m of ["4C]protein from endothelial medium. Cells were incubated for 48 hr in medium containing [14C]proline, and material in medium was precipitated with ammonium sulfate as described in Materials and Methods. Material was dialyzed against 0.05 M acetic acid and treated with pepsin at 15°. The reaction was stopped by dialyzing against 15% KCl, and the precipitated material was treated with NaDodSO4-5% mercaptoethanol and dialyzed against the column buffer. Percent hydroxylation of [14C]proline was determined in fraction 38-41, as indicated by the brackets (see Table 1).
globulin, a yellow-green fluorescence disseminated throughout the entire cytoplasm. Cells incubated with normal serum showed no fluorescence (Fig. 3). By use of l25-Ilabeled Types I, II, III, and IV bovine collagen in a radioimmunoassay it has been shown that the above antisera are quite specific, having high titers (over 1:100,000) to Type IV collagen but showing no crossreaction with Types I, II, or III (25). DISCUSSION The data indicate that cultures of endothelial cells derived from calf aorta synthesize collagen, and the chemical characteristics resemble basement membrane (Type IV) collagen. Further evidence that the endothelial cells isolated from calf aorta synthesize a basement membrane-like collagen is derived from the immunological studies, which show that antiserum prepared against collagen of calf anterior lens capsule (Type IV) localizes in the cytoplasm of these cells as demonstrated by the indirect immunofluorescent technique. It is of interest to compare the properties of the material synthesized by the endothelial cells to those of basement membrane collagen made in other systems in vitro. These include endodermal cells of the parietal yolk sac (21-23), chick lens (6-8), glomeruli (11), and Descemet's membrane (24). The amount of 3-hydroxy[14C]proline in the collagen synthesized by the endothelial cells is similar to that of the other basement membrane systems. Less than 2% of this isomer is found in Types I, II, or III. Moreover, the pepsin-resistant fraction, which eluted with apparent molecular weight of 115,000, had 59% of its ['4C]proline as hydroxy['4C]proline. This is similar to values obtained for basement membrane isolated from mature tissues (4). The percent glycosylation of hydroxy[14C]lysine and the proportion of the hydroxy[14C]lysine-linked glycoside, glucosylgalactose, are similar to those observed in the other
basement-membrane-synthesizing systems.
22364
Biochemistry: Howard et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
of the collagen with a serum protein, or a specific affinity among macromolecules secreted by these cells. In conclusion, the synthesis of collagen in endothelial cell cultures has been demonstrated and the product partially characterized. Data so far indicate the product is similar to a basement membrane collagen. Since few collagens other than Type I from cell culture systems have been extensively studied, it remains to be determined whether the unique characteristics of the endothelial cell collagen can be attributed to the culture conditions in vitro or whether there are differences in basement membrane collagen from different sources. The authors wish to thank Dr. Ronald Minor for assistance in the isolation of endothelium, Dr. Bradley Arbogast for data on hydroxy[14C]lysine and its glycosides, and Mrs. Margaret Lu and Miss Kathy Spause for excellent technical assistance. This study was supported by NIH Grants AM-14526, AM-14805, and HL-16058. 1. Jaffe, E A., Nachinan, R. L, Becker, C. G. & Minick, C. R. (1973) J. Clin. Invest. 52,2745-2756. 2. Gimbrone, M. A., Cotran, R. S. & Folkman, J. (1974) J. Cell Biol. 60, 673-684. 3. Palade, G. E. & Bruns, A. R. (1964) in Small Blood Vessel Inaolvement in Diabetes Mellitus, eds. Siperstein, M. D., Colwell, A. R., & Meyer, K. (American Institute of Biological Sciences, Publications) p. 39. 4. Kefalides, N. A. (1973) Int. Rev. Conn. Tiss. Res. 6,63-104. 5. Kefalides, N. A. & Denduchis, B. (1969) Biochemistry 8, 4613-4621. 6. Grant, M. E., Kefalides, N. A. & Prockop, D. J. (1972) J. Biol. Chem. 247, 3539-3544. 7. Grant, M. E., Kefalides, N. A. & Prockop, D. J. (1972) J. Biod.
Chem. 247,3545-3551.
8. Grant, M. E., Schofield, J. D., Kefalides, N. A. & Prockop, D. J. (1973) J. Biol. Chem. 248, 7432-7437. 9. Killen, P. D., Quadracci, L. J. & Stricker, G. E. (1974) Fed. Proc. 33, 617, abstr. 10. Krisco, L. & Walker, W. G. (1974) Proc. Soc. Exp. Biol. Med. 146, 942-947. 11. Grant, M. E., Harwood, R. & Williams, I. F. (1975) Eur. J. Biochem. 54, 531-540. 12. Lewis, L. J., Hoak, J. C., Maca, R. D. & Fry, G. L. (1973) Scietce 181,453-458. 13. Howard, B. V., Howard, W. J., deLaLlera, M. & Kefalides, N. FIG. 3. (a) Immunofluorescence reaction between rabbit antiserum to collagen of bovine lens capsule and bovine aortic endothelial cells. Note the strong cytoplasmic fluorescence. (b) Control reaction using preimmunization rabbit serum. Note limited autofluorescence over the nucleus only. In other in vitro systems known to synthesize basement
membrane collagen, the initially synthesized polypeptide chain (procollagen) has an apparent molecular weight ranging between 140,000 and 155,000 (7, 11, 24). The difference in molecular weight between the initial polypeptide chain synthesized by calf aorta endothelium and other cell systems may in part be due to experimental variability and in part to variability among basement membrane collagens synthesized by different cell types. It should also be noted that the proteins synthesized by the endothelial cells of calf aorta appear to aggregate when large quantities are applied to gel filtration columns or to acrylamide gels. This could represent nonspecific association
A. (1976) Atherosclerosis 23,521-534. 14. Cristofalo, V. J. & Kritchevsky, D. (1966) J. Cell. Physiol. 67, 125-132. 15. Juva, K. & Prockop, D. J. (1966) Anal. Biochem. 15,77-83. 16. Goldberg, B., Epstein, E. H. & Stein, C. J. (1972) Proc. Natd. Acad. Sci. USA 69,3655-3659. 17. Anker, H. S. (1970) FEBS Lett. 7,293. 18. Askenasi, R. S. & Kefalides, N. A. (1972) Anal. Biochem. 47, 67-72. 19. Kefalides, N. A. (1972) Connect. Tissue Res. 1, 3. 20. Dehm, P. & Prockop, D. J. (1973) Eur. J. Biochem. 35,159-166. 21. Clark, C. C., Tomichek, E. A., Koszalka, T. R., Brent, R. L. & Kefalides, N. A. (1974) Proc. Int. Congr. Biochem. 9th, 423A. 22. Clark, C. C., Tomichek, E. A., Koszalka, T. R., Minor, R. R. & Kefalides, N. A. (1975) J. Biol. Chem. 250,5259-5267.
.23. Minor, R. R., Clark, C. C., Koszalka, T. R., Brent, R. L. & Kefalides, N. A. (1973) J. Cell Biol. 59,228, abstr. 24. Kefalides, H. A., Cameron, J. D., Tomichek, E. A. & Yanoff, M. (1976) j. Blol. Chem., 251,730-733. 25. Gunson, D., Kefalides, N. A. (1976) Immunology, in press.
Biochemistry
Characterization of the collagen synthesized by endothelial cells in culture (basement membrane collagen/biosynthesis/calf aorta)
BARBARA V. HOWARD,* EDWARD J. MACARAK, DIANE GUNSON, AND NICHOLAS A. KEFALIDESt Departments of Biochemistry and Medicine, University of Pennsylvania and the Philadelphia General Hospital, Philadelphia, Pa. 19104
Communicated by Robert E. Forster, May 10, 1976
ABSTRACT [14CJProline and ["4Clysine were incorporated into collagen by cultures of endothelial cells derived from calf aortae. The isomer 3-hydroxy["4Cproline accounted for 10% of the total hydroxy[14Clproline in the collagen isolated from the medium. Approximately 81% of the hydroxy"4C~lysine isolated from the medium was glycosylated, and 91% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosylgalactose. Gel filtration chromatography or acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the initially synthesized peptide chain of [14Cjcollagen had a molecular weight of about 135,000; after pepsin digestion this was converted to 115,000. The ratio of hydroxy[14Clproline to total ['4Clproline X 100 in the pepsin-resistant fraction was 59. Wen examined by immunofluorescence microscopy, the endothelial cultures stained positively with antiserum to (Type IV) collagen from basement membrane of bovine anterior lens capsule. The data indicate that cultured endothelial cells derived from calf aortae synthesize collagen that resembles that of basement membrane collagen.
The establishment of cultures of endothelial cells in vitro provides an opportunity to study the metabolic functions of these cells (1, 2). It has been proposed (3) that endothelial cells are the source of the basement membrane that invariably underlies these cells. The nature of the collagen component of vascular basement membrane has been characterized in capillaries of the glomerulus and the choroid plexus (4, 5). However, biosynthetic studies of collagen of vascular basement membrane are scanty (6-11), and there are no reports of studies of this collagen from large vessel endothelium. This report presents a partial characterization of the collagen synthesized by primary cultures of aortic endothelial cells. The results indicate that cultured endothelial cells synthesize collagen that is similar to basement membrane (Type IV) collagen synthesized by other cell culture systems in vitro. MATERIALS AND METHODS Cell Culture. For the isolation and culture of endothelial cells from calf aortae the methods of jaffe et al. (1) and Gimbrone et al. (2) were adapted with modifications. Calf aortae were obtained from the slaughter house within 1 hr after the animals' death. Thoracic aortae, approximately 15-20 cm long, were removed from the animals aseptically and immediately placed in sterile chilled Dulbecco's phosphate-buffered saline supplemented with glucose (5 mg/liter), amphotericin B (2.5 ,gg/ml), penicillin (50 units/ml), and streptomycin (50 ,g/ml). Six to eight vessels were collected and subsequently processed for culture within 2 hr. To isolate endothelial cells, we washed Abbreviation: NaDodSO4, sodium dodecyl sulfate. * Present address. Department of Biochemistry, Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, Pa. 19129. t To whom correspondence should be addressed at Clinical Research Center, Philadelphia General Hospital, 700 Civic Center Boulevard,
aortae in Dulbecco's phosphate-buffered saline and defatted them by dissection. The paired intercostal vessels were tied and the lower end of the vessel was clamped with an intestinal clamp. The vessel was then filled with 10-15 ml of collagenase (1 mg/ml; Type II, Worthington) in Medium 199, and the upper end was clamped with a second intestinal clamp. The vessels were incubated at room temperature for 45 min and the contents were collected. This collagenase effluent usually contained few cells and was discarded. The vessels were then washed four times with 10 ml of growth medium, which consisted of Medium 199 as modified by Lewis et al. (12) supplemented with 20% fetal bovine serum, gentamicin (50 ,g/ml), and amphotericin B (2.5 sug/ml). During each wash, the vessel was clamped and the medium within the vessel was agitated gently by inverting the vessel four times. The pooled effluents containing the freed endothelial cells were distributed evenly among 75-cm2 glass or plastic petri dishes (10 ml per dish), and the cells were incubated at 370 in a humidified atmosphere of 5% CO2 in air. The medium was changed in all cultures every 3 days. For subculture, cells were treated briefly (1-2 min) with a calcium- and magnesium-free buffered saline containing trypsin (0.25%) and EDTA (0.05%). Released cells were seeded in new petri plates and the medium was changed 4-6 hr after the trypsin-EDTA treatment. Cultures established in this manner from calf aortae have
been identified as endothelial by light and electron microscopic criteria, and the growth of the cells has been characterized. These data are described elsewheret. Endothelial cultures from human umbilical veins were derived by standard methods (1, 2), and human diploid fibroblast cultures were established from foreskins as described (13). Collagen Synthesis. Monolayers of primary cultures of endothelial cells (approximately 7 days after isolation, three to five cell generations in vitro) were incubated in a buffered balanced salt solution (14) supplemented with fetal bovine serum (10%), glucose (5 mg/ml), BME vitamins and amino acids (Gibco), glutamine (1 mM), ascorbic acid (50 ug/ml), 13-aminopropionitrile (50 jg/ml), and either [U-14C]proline (2 gCi/ml; 200 mCi/mmol) or [U-14C]lysine (1IuCi/ml; 342 mCi/mmol). After 48 hr the incubation medium and cell fractions were separated and calf skin collagen was added as carrier. Incorporation of total 14C into nondialyzable protein was determined after extensive dialysis against 0.1% sodium dodecyl sulfate in 0.1% sodium phosphate, pH 7.4 (NaDodSO4/PJ). 4-Hydroxy[14C]proline was quantitated in this material by the method of Juva and Prockop (15). For gel chromatography and gel electrophoresis, the newly synthesized collagen in the medium was precipitated with ammonium sulfate (176 mg/ml). This ret E. Macarak, B. V. Howard, and N. A. mitted.
Philadelphia, Pa. 19104.
2361
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2362
Biochemistry: Howard et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 1. Incorporation of [ 14C] proline into total protein and synthesis of hydroxy l4 C ] proline by endothelial cells in culture (average of four determinations)
Table 2. Hydroxy[ 'I4 C ]proline isomers in collagen synthesized by endothelial cells in culture
(3-Hydroxyproline/ (B) Hydroxy
(A) Total
'"C
['4C]proline
(B/A)
x
536,000 1,270,000
27,700 27,100
5.2 2.1
collagen (Fig.
2) Fibroblasts Medium
59*
153,000
764,000
20
Monolayer cultures were incubated for 48 hr in an incubation medium containing [14C]proline (2 MCi/ml) as described in Materials and Methods. Medium was removed and NaDodSO4 added to a 0.1% final concentration. The cell layer was harvested by scraping in NaDodSO4/P1. After samples were boiled for 3 min, they were extensively dialyzed against NaDodSO4/Pi, and total 14C radioactivity, as well as hydroxy[14C]proline were determined as described in Materials and Methods. Pepsin-digested collagen was obtained as described in Fig. 2. * For pepsin-digested material, the number represents the ratio of hydroxy[14C]proline to total [14C]proline in fractions 38-41 of Fig. 2.
sulted in a considerable enrichment (6- to 9-fold) of the [14C]collagen in the preparation. Characterization of Synthesized Collagen. The material precipitated by ammonium sulfate was prepared for chromatography or electrophoresis by reduction with 5% 2-mercaptoethanol in 1% NaDodSO4/Pi for 3 hr at 400, or by reduction with 50 mM dithioerythritol, 1 M urea, 0.5 M Tris-HCl at pH 8.2 for 15 min at room temperature followed by alkylation with 120 mM iodoacetamide. Gel filtration chromatography was performed on a calibrated column of 6% agarose (Bio-Gel A-5 M) eluted with NaDodSO4/Pj as described (7). 4-Hydroxy[14C]proline was quantitated according to the method of Juva and Prockop (15). NaDodSO4-polyacrylamide gel electrophoresis was accomplished according to the procedure of Goldberg et al. (16), using a polymerizing solution of 7% acrylamide monomer, 0.137% N,N'-diallyltartardiamide, 0.1% NaDodSO4, and 0.5 M urea. Migration was compared to and ,B components of carrier calf skin collagen. The gels were fractionated into 1-mm slices with a Mickle Gel Slicer (Brinkman), and slices were solubilized in 2% periodic acid before their radioactivity was determined (17). The content of 3- and 4-hydroxy[14C]proline was determined in material precipitated by ammonium sulfate or fractions of chromatograms as described (6). Hydroxy[14C]lysine and glycosides were quantitated after alkaline hydrolysis on the amino acid analyzer by the method of Askenasi and Kefalides (18). For pepsin digestion, the ammonium sulfate precipitate was dialyzed against 0.05 M acetic acid and treated with pepsin (100 ,gg/ml) at 150 for 6 hr. The reaction was stopped by dialyzing against 15% KCl in 0.01 M sodium phosphate, pH 9, and the material was prepared for chromatography as described above. Immunologic Studies. Basement membrane collagen was extracted from anterior lens capsules of calf eyes by limited pepsin digestion for 24 hr at 15°, and the collagen was precipitated three times with 15% KCl, 0.02 M Na2HPO4 (5). The a
Bovine aortic Primary Passage 7 Human umbilical Human fibroblast
10.2 ± 1.4 (5)* 13.0 9.3 1.6
100 (%)
(dpm/mg cell protein) Endothelial Medium Cells Pepsin-digested medium ['I 4C]-
Sample
total hydroxyproline) x 100
Primary cultures of aortic or umbilical endothelial cells were incubated for 48 hr with [14C]proline as described in Materials and Methods. Cultures from aortae were also labeled in a similar manner after seven subcultivations. Medium was precipitated with ammonium sulfate, and the proportions of 3-hydroxyproline and 4-hydroxyproline were determined with an amino acid analyzer (6). * No. of determinations.
collagen wasdissolved in 0.1 M acetic acid at a concentration of 5 mg/ml, dialyzed against physiological saline, and mixed with an equal volume of Freund's complete adjuvant. Four New Zealand white rabbits were immunized with this method, using a previously described immunization schedule (19), and bled 1 week after the fifth injection. All sera were stored at -70°. Coverslip cultures of endothelial cells were washed in phosphate-buffered saline and air-dried. Fifty microliters of antiserum or preimmunization serum were placed on each coverslip; they were incubated in a moist chamber for 1 hr at room temperature. Cellular localization of antibody was detected by the indirect immunofluorescence reaction using fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin (Cappel Laboratories, Downingtown, Pa.) RESULTS
Collagen synthesis was demonstrated in the endothelial cell cultures by the synthesis of hydroxy['4C]proline in both cellular and extracellular protein fractions (Table 1). The amount of collagen synthesized by these cells was very low as compared to fibroblast cultures, and the excreted collagen comprised a small proportion of the total extracellular protein. The rate of collagen synthesis per mg of cell protein in the endothelial cultures was of the same order of magnitude as that observed in freshly isolated endothelial cell preparations. Table 2 shows data for the isomers of hydroxy[14C]proline present in material after ammonium sulfate precipitation or gel filtration. The collagen synthesized by primary cultures of these cells contained 10.2% of the total hydroxy[14C]proline as the 3 isomer. In addition, comparable amounts of the 3-hydroxy['4C]proline were also found in cultures after seven passages in vtro and in endothelial cultures derived from umbilical veins. Control values from fibroblast cultures averaged 1.6%. Determination of glycosylated derivatives of hydroxy[14C]lysine in the cell and medium fractions indicated that 91% of the derivatives were in the form of the disaccharide, glucosylgalactose (Table 3). The percent glycosylation of hydroxy['4C]lysine averaged 81% in the medium fraction. Values for percent glycosylation of hydroxy[14C]lysine in the cell fraction were lower, averaging 72% (Table 3). Both these values were significantly higher than those obtained in fibroblast cultures, although the skin fibroblast data differed from values reported for Type I collagen synthesized by tendon fibroblasts in vitro (20).
Biochemistry: Howard et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
22363
Table 3. Glycosylation of hydroxy [ 4 C ] lysine by endothelial cells in culture
Disaccharide % Glycosylation (Di- + monosaccharide) V0 Endothelial Medium Cell fraction Fibroblast Cell + medium
Vt
4x
91 ± 0.62 (7)* 91 (2)
81 ± 1.4 (3) 72 (2)
0
81 ± 3.3
46 ± 4.7 (5)
V
z
I.-
(5)
Monolayers of primary aortic endothelial cells or skin fibroblasts incubated for 48 hr with [14C]lysine as described in Materials and Methods. Medium and cell layer were separated and hydrolyzed separately. Hydroxy[14C]lysine and glycosides were quantitated after alkaline hydrolysis on an amino acid analyzer (18). Monosaccharide refers to galactosyl-hydroxy[14C]lysine, and disaccharide is glucosyl-galactosyl-hydroxy['4C]lysine. * No. of determinations. were
4
U-
E
01 020
Upon NaDodSO4-acrylamide gel electrophoresis, after reduction, the majority of the ['4C]protein migrated as a species with a molecular weight of about 135,000 (Fig. 1). When large amounts of material were applied, a significant proportion of the 14C remained at the origin. On gel filtration, a significant proportion of the hydroxy[14C]proline eluted with an apparent molecular weight of approximately 135,000. However, greater than 50% of the hydroxy['4C]proline was found in a peak close to the exclusion volume. The percent hydroxylation of proline in the larger molecular weight fraction averaged 14%. After pepsin digestion and gel filtration (Fig. 2), the [14C]collagen synthesized by the endothelial cell cultures had an apparent molecular weight of 115,000. Determination of the total hydroxy['4C]proline in the peak tubes of this fraction indicated 59% hydroxylation of the ['4C]proline. When endothelial cell cultures were incubated with antiserum to basement membrane collagen of anterior lens capsule and counterstained with fluorescein goat anti-rabbit gamma^1 ^2
P11PI2
2
'CO
0
0U.
E
0
40
20
DISTANCE FROM
40
ORIGIN
60
(mm) FIG. 1. NaDodSO4-polyacrylamide gel electrophoresis of [14C]protein from endothelial medium after 48-hr pulse with [14C]proline. Material was precipitated with ammonium sulfate and reduced in 1% NaDodSO4, 1% mercaptoethanol as described in Materials and Methods. Samples contained approximately 20,000 cpm in 100 Ml. Gels were stained before slicing to determine migration of
carrier collagen components.
40
60
FRACTION NUMBER FIG. 2. Gel filtration chromatography on NaDodSO4-agarose A5m of ["4C]protein from endothelial medium. Cells were incubated for 48 hr in medium containing [14C]proline, and material in medium was precipitated with ammonium sulfate as described in Materials and Methods. Material was dialyzed against 0.05 M acetic acid and treated with pepsin at 15°. The reaction was stopped by dialyzing against 15% KCl, and the precipitated material was treated with NaDodSO4-5% mercaptoethanol and dialyzed against the column buffer. Percent hydroxylation of [14C]proline was determined in fraction 38-41, as indicated by the brackets (see Table 1).
globulin, a yellow-green fluorescence disseminated throughout the entire cytoplasm. Cells incubated with normal serum showed no fluorescence (Fig. 3). By use of l25-Ilabeled Types I, II, III, and IV bovine collagen in a radioimmunoassay it has been shown that the above antisera are quite specific, having high titers (over 1:100,000) to Type IV collagen but showing no crossreaction with Types I, II, or III (25). DISCUSSION The data indicate that cultures of endothelial cells derived from calf aorta synthesize collagen, and the chemical characteristics resemble basement membrane (Type IV) collagen. Further evidence that the endothelial cells isolated from calf aorta synthesize a basement membrane-like collagen is derived from the immunological studies, which show that antiserum prepared against collagen of calf anterior lens capsule (Type IV) localizes in the cytoplasm of these cells as demonstrated by the indirect immunofluorescent technique. It is of interest to compare the properties of the material synthesized by the endothelial cells to those of basement membrane collagen made in other systems in vitro. These include endodermal cells of the parietal yolk sac (21-23), chick lens (6-8), glomeruli (11), and Descemet's membrane (24). The amount of 3-hydroxy[14C]proline in the collagen synthesized by the endothelial cells is similar to that of the other basement membrane systems. Less than 2% of this isomer is found in Types I, II, or III. Moreover, the pepsin-resistant fraction, which eluted with apparent molecular weight of 115,000, had 59% of its ['4C]proline as hydroxy['4C]proline. This is similar to values obtained for basement membrane isolated from mature tissues (4). The percent glycosylation of hydroxy[14C]lysine and the proportion of the hydroxy[14C]lysine-linked glycoside, glucosylgalactose, are similar to those observed in the other
basement-membrane-synthesizing systems.
22364
Biochemistry: Howard et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
of the collagen with a serum protein, or a specific affinity among macromolecules secreted by these cells. In conclusion, the synthesis of collagen in endothelial cell cultures has been demonstrated and the product partially characterized. Data so far indicate the product is similar to a basement membrane collagen. Since few collagens other than Type I from cell culture systems have been extensively studied, it remains to be determined whether the unique characteristics of the endothelial cell collagen can be attributed to the culture conditions in vitro or whether there are differences in basement membrane collagen from different sources. The authors wish to thank Dr. Ronald Minor for assistance in the isolation of endothelium, Dr. Bradley Arbogast for data on hydroxy[14C]lysine and its glycosides, and Mrs. Margaret Lu and Miss Kathy Spause for excellent technical assistance. This study was supported by NIH Grants AM-14526, AM-14805, and HL-16058. 1. Jaffe, E A., Nachinan, R. L, Becker, C. G. & Minick, C. R. (1973) J. Clin. Invest. 52,2745-2756. 2. Gimbrone, M. A., Cotran, R. S. & Folkman, J. (1974) J. Cell Biol. 60, 673-684. 3. Palade, G. E. & Bruns, A. R. (1964) in Small Blood Vessel Inaolvement in Diabetes Mellitus, eds. Siperstein, M. D., Colwell, A. R., & Meyer, K. (American Institute of Biological Sciences, Publications) p. 39. 4. Kefalides, N. A. (1973) Int. Rev. Conn. Tiss. Res. 6,63-104. 5. Kefalides, N. A. & Denduchis, B. (1969) Biochemistry 8, 4613-4621. 6. Grant, M. E., Kefalides, N. A. & Prockop, D. J. (1972) J. Biol. Chem. 247, 3539-3544. 7. Grant, M. E., Kefalides, N. A. & Prockop, D. J. (1972) J. Biod.
Chem. 247,3545-3551.
8. Grant, M. E., Schofield, J. D., Kefalides, N. A. & Prockop, D. J. (1973) J. Biol. Chem. 248, 7432-7437. 9. Killen, P. D., Quadracci, L. J. & Stricker, G. E. (1974) Fed. Proc. 33, 617, abstr. 10. Krisco, L. & Walker, W. G. (1974) Proc. Soc. Exp. Biol. Med. 146, 942-947. 11. Grant, M. E., Harwood, R. & Williams, I. F. (1975) Eur. J. Biochem. 54, 531-540. 12. Lewis, L. J., Hoak, J. C., Maca, R. D. & Fry, G. L. (1973) Scietce 181,453-458. 13. Howard, B. V., Howard, W. J., deLaLlera, M. & Kefalides, N. FIG. 3. (a) Immunofluorescence reaction between rabbit antiserum to collagen of bovine lens capsule and bovine aortic endothelial cells. Note the strong cytoplasmic fluorescence. (b) Control reaction using preimmunization rabbit serum. Note limited autofluorescence over the nucleus only. In other in vitro systems known to synthesize basement
membrane collagen, the initially synthesized polypeptide chain (procollagen) has an apparent molecular weight ranging between 140,000 and 155,000 (7, 11, 24). The difference in molecular weight between the initial polypeptide chain synthesized by calf aorta endothelium and other cell systems may in part be due to experimental variability and in part to variability among basement membrane collagens synthesized by different cell types. It should also be noted that the proteins synthesized by the endothelial cells of calf aorta appear to aggregate when large quantities are applied to gel filtration columns or to acrylamide gels. This could represent nonspecific association
A. (1976) Atherosclerosis 23,521-534. 14. Cristofalo, V. J. & Kritchevsky, D. (1966) J. Cell. Physiol. 67, 125-132. 15. Juva, K. & Prockop, D. J. (1966) Anal. Biochem. 15,77-83. 16. Goldberg, B., Epstein, E. H. & Stein, C. J. (1972) Proc. Natd. Acad. Sci. USA 69,3655-3659. 17. Anker, H. S. (1970) FEBS Lett. 7,293. 18. Askenasi, R. S. & Kefalides, N. A. (1972) Anal. Biochem. 47, 67-72. 19. Kefalides, N. A. (1972) Connect. Tissue Res. 1, 3. 20. Dehm, P. & Prockop, D. J. (1973) Eur. J. Biochem. 35,159-166. 21. Clark, C. C., Tomichek, E. A., Koszalka, T. R., Brent, R. L. & Kefalides, N. A. (1974) Proc. Int. Congr. Biochem. 9th, 423A. 22. Clark, C. C., Tomichek, E. A., Koszalka, T. R., Minor, R. R. & Kefalides, N. A. (1975) J. Biol. Chem. 250,5259-5267.
.23. Minor, R. R., Clark, C. C., Koszalka, T. R., Brent, R. L. & Kefalides, N. A. (1973) J. Cell Biol. 59,228, abstr. 24. Kefalides, H. A., Cameron, J. D., Tomichek, E. A. & Yanoff, M. (1976) j. Blol. Chem., 251,730-733. 25. Gunson, D., Kefalides, N. A. (1976) Immunology, in press.
