Proc. Nati. Acad. Sci. USAVol. 73, No. 7, pp. 2361-2364, July 1976Biochemistry

Characterization of the collagen synthesized by endothelial cells inculture

(basement membrane collagen/biosynthesis/calf aorta)

BARBARA V. HOWARD,* EDWARD J. MACARAK, DIANE GUNSON, AND NICHOLAS A. KEFALIDEStDepartments 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 incorporatedinto collagen by cultures of endothelial cells derived from calfaortae. The isomer 3-hydroxy["4Cproline accounted for 10%of the total hydroxy[14Clproline in the collagen isolated fromthe medium. Approximately 81% of the hydroxy"4C~lysineisolated from the medium was glycosylated, and 91% of theglycosylated hydroxy[14C]lysine was in the form of the disac-charide glucosylgalactose. Gel filtration chromatography oracrylamide gel electrophoresis in the presence of sodium do-decyl sulfate indicated that the initially synthesized peptidechain of [14Cjcollagen had a molecular weight of about 135,000;after pepsin digestion this was converted to 115,000. The ratioof hydroxy[14Clproline to total ['4Clproline X 100 in the pep-sin-resistant fraction was 59.Wen examined by immunofluo-rescence microscopy, the endothelial cultures stained positivelywith antiserum to (Type IV) collagen from basement membraneof bovine anterior lens capsule. The data indicate that culturedendothelial cells derived from calf aortae synthesize collagenthat resembles that of basement membrane collagen.

The establishment of cultures of endothelial cells in vitro pro-vides an opportunity to study the metabolic functions of thesecells (1, 2). It has been proposed (3) that endothelial cells are thesource of the basement membrane that invariably underliesthese cells. The nature of the collagen component of vascularbasement membrane has been characterized in capillaries ofthe glomerulus and the choroid plexus (4, 5). However, bio-synthetic studies of collagen of vascular basement membraneare scanty (6-11), and there are no reports of studies of thiscollagen from large vessel endothelium. This report presentsa partial characterization of the collagen synthesized by pri-mary cultures of aortic endothelial cells. The results indicatethat cultured endothelial cells synthesize collagen that is similarto basement membrane (Type IV) collagen synthesized by othercell culture systems in vitro.

MATERIALS AND METHODSCell Culture. For the isolation and culture of endothelial cells

from calf aortae the methods of jaffe et al. (1) and Gimbroneet al. (2) were adapted with modifications. Calf aortae wereobtained from the slaughter house within 1 hr after the animals'death. Thoracic aortae, approximately 15-20 cm long, wereremoved from the animals aseptically and immediately placedin sterile chilled Dulbecco's phosphate-buffered saline sup-plemented 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 processedfor culture within 2 hr. To isolate endothelial cells, we washed

Abbreviation: NaDodSO4, sodium dodecyl sulfate.* Present address. Department of Biochemistry, Medical College ofPennsylvania, 3300 Henry Avenue, Philadelphia, Pa. 19129.

t To whom correspondence should be addressed at Clinical ResearchCenter, Philadelphia General Hospital, 700 Civic Center Boulevard,Philadelphia, Pa. 19104.

aortae in Dulbecco's phosphate-buffered saline and defattedthem by dissection. The paired intercostal vessels were tied andthe lower end of the vessel was clamped with an intestinalclamp. The vessel was then filled with 10-15 ml of collagenase(1 mg/ml; Type II, Worthington) in Medium 199, and theupper end was clamped with a second intestinal clamp. Thevessels were incubated at room temperature for 45 min and thecontents were collected. This collagenase effluent usuallycontained few cells and was discarded. The vessels were thenwashed four times with 10 ml of growth medium, which con-sisted of Medium 199 as modified by Lewis et al. (12) supple-mented with 20% fetal bovine serum, gentamicin (50 ,g/ml),and amphotericin B (2.5 sug/ml). During each wash, the vesselwas clamped and the medium within the vessel was agitatedgently by inverting the vessel four times. The pooled effluentscontaining the freed endothelial cells were distributed evenlyamong 75-cm2 glass or plastic petri dishes (10 ml per dish), andthe cells were incubated at 370 in a humidified atmosphere of5% CO2 in air. The medium was changed in all cultures every3 days. For subculture, cells were treated briefly (1-2 min) witha calcium- and magnesium-free buffered saline containingtrypsin (0.25%) and EDTA (0.05%). Released cells were seededin new petri plates and the medium was changed 4-6 hr afterthe trypsin-EDTA treatment.

Cultures established in this manner from calf aortae havebeen identified as endothelial by light and electron microscopiccriteria, and the growth of the cells has been characterized.These data are described elsewheret.

Endothelial cultures from human umbilical veins were de-rived by standard methods (1, 2), and human diploid fibroblastcultures were established from foreskins as described (13).

Collagen Synthesis. Monolayers of primary cultures of en-dothelial cells (approximately 7 days after isolation, three to fivecell generations in vitro) were incubated in a buffered balancedsalt 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-aminopro-pionitrile (50 jg/ml), and either [U-14C]proline (2 gCi/ml; 200mCi/mmol) or [U-14C]lysine (1IuCi/ml; 342 mCi/mmol). After48 hr the incubation medium and cell fractions were separatedand calf skin collagen was added as carrier. Incorporation oftotal 14C into nondialyzable protein was determined after ex-tensive 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 Juvaand Prockop (15). For gel chromatography and gel electro-phoresis, the newly synthesized collagen in the medium wasprecipitated with ammonium sulfate (176 mg/ml). This re-

t E. Macarak, B. V. Howard, and N. A. Kefalides, manuscript sub-mitted.

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Table 1. Incorporation of [ 14C] proline into total proteinand synthesis of hydroxy l4 C ] proline by endothelial cells

in culture (average of four determinations)

(B)(A) Hydroxy (B/A) x

Total '"C ['4C]proline 100 (%)

(dpm/mg cell protein)EndothelialMedium 536,000 27,700 5.2Cells 1,270,000 27,100 2.1Pepsin-digestedmedium ['I 4C]-collagen (Fig.

2) 59*FibroblastsMedium 764,000 153,000 20

Monolayer cultures were incubated for 48 hr in an incubationmedium containing [14C]proline (2 MCi/ml) as described in Ma-terials and Methods. Medium was removed and NaDodSO4 addedto a 0.1% final concentration. The cell layer was harvested byscraping in NaDodSO4/P1. After samples were boiled for 3 min,they were extensively dialyzed against NaDodSO4/Pi, and total14C radioactivity, as well as hydroxy[14C]proline were determinedas described in Materials and Methods. Pepsin-digested collagenwas obtained as described in Fig. 2.* For pepsin-digested material, the number represents the ratio ofhydroxy[14C]proline to total [14C]proline in fractions 38-41 ofFig. 2.

sulted in a considerable enrichment (6- to 9-fold) of the [14C]-collagen in the preparation.

Characterization of Synthesized Collagen. The materialprecipitated by ammonium sulfate was prepared for chroma-tography or electrophoresis by reduction with 5% 2-mercap-toethanol in 1% NaDodSO4/Pi for 3 hr at 400, or by reductionwith 50 mM dithioerythritol, 1 M urea, 0.5 M Tris-HCl at pH8.2 for 15 min at room temperature followed by alkylation with120 mM iodoacetamide. Gel filtration chromatography was

performed on a calibrated column of 6% agarose (Bio-Gel A-5M) eluted with NaDodSO4/Pj as described (7). 4-Hy-droxy[14C]proline was quantitated according to the method ofJuva and Prockop (15).

NaDodSO4-polyacrylamide gel electrophoresis was ac-

complished 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 0.5M urea. Migration was compared to a and ,B components ofcarrier calf skin collagen. The gels were fractionated into 1-mmslices with a Mickle Gel Slicer (Brinkman), and slices were

solubilized in 2% periodic acid before their radioactivity wasdetermined (17).The content of 3- and 4-hydroxy[14C]proline was determined

in material precipitated by ammonium sulfate or fractions ofchromatograms as described (6). Hydroxy[14C]lysine and gly-cosides were quantitated after alkaline hydrolysis on the aminoacid analyzer by the method of Askenasi and Kefalides (18).

For pepsin digestion, the ammonium sulfate precipitate wasdialyzed against 0.05M acetic acid and treated with pepsin (100,gg/ml) at 150 for 6 hr. The reaction was stopped by dialyzingagainst 15% KCl in 0.01 M sodium phosphate, pH 9, and thematerial was prepared for chromatography as described above.Immunologic Studies. Basement membrane collagen was

extracted from anterior lens capsules of calf eyes by limitedpepsin digestion for 24 hr at 15°, and the collagen was precip-itated three times with 15% KCl, 0.02 M Na2HPO4 (5). The

Table 2. Hydroxy[ 'I4 C ]proline isomers in collagensynthesized by endothelial cells in culture

(3-Hydroxyproline/total hydroxyproline)

Sample x 100

Bovine aorticPrimary 10.2 ± 1.4 (5)*Passage 7 13.0

Human umbilical 9.3Human fibroblast 1.6

Primary cultures of aortic or umbilical endothelial cells wereincubated for 48 hr with [14C]proline as described in Materials andMethods. Cultures from aortae were also labeled in a similarmanner after seven subcultivations. Medium was precipitatedwith ammonium sulfate, and the proportions of 3-hydroxyprolineand 4-hydroxyproline were determined with an amino acidanalyzer (6).* No. of determinations.

collagen wasdissolved in 0.1 M acetic acid at a concentration of5 mg/ml, dialyzed against physiological saline, and mixed withan equal volume of Freund's complete adjuvant. Four NewZealand white rabbits were immunized with this method, usinga previously described immunization schedule (19), and bled1 week after the fifth injection. All sera were stored at -70°.

Coverslip cultures of endothelial cells were washed inphosphate-buffered saline and air-dried. Fifty microliters ofantiserum or preimmunization serum were placed on eachcoverslip; they were incubated in a moist chamber for 1 hr atroom temperature. Cellular localization of antibody was de-tected by the indirect immunofluorescence reaction usingfluorescein isothiocyanate-labeled goat anti-rabbit immuno-globulin (Cappel Laboratories, Downingtown, Pa.)

RESULTS

Collagen synthesis was demonstrated in the endothelial cellcultures by the synthesis of hydroxy['4C]proline in both cellularand extracellular protein fractions (Table 1). The amount ofcollagen synthesized by these cells was very low as comparedto fibroblast cultures, and the excreted collagen comprised asmall proportion of the total extracellular protein. The rate ofcollagen synthesis per mg of cell protein in the endothelialcultures was of the same order of magnitude as that observedin freshly isolated endothelial cell preparations.

Table 2 shows data for the isomers of hydroxy[14C]prolinepresent in material after ammonium sulfate precipitation orgel filtration. The collagen synthesized by primary cultures ofthese cells contained 10.2% of the total hydroxy[14C]proline asthe 3 isomer. In addition, comparable amounts of the 3-hy-droxy['4C]proline were also found in cultures after seven pas-sages in vtro and in endothelial cultures derived from umbilicalveins. 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% ofthe derivatives were in the form of the disaccharide, glucosyl-galactose (Table 3). The percent glycosylation of hy-droxy['4C]lysine averaged 81% in the medium fraction. Valuesfor percent glycosylation of hydroxy[14C]lysine in the cellfraction were lower, averaging 72% (Table 3). Both these valueswere significantly higher than those obtained in fibroblastcultures, although the skin fibroblast data differed from valuesreported for Type I collagen synthesized by tendon fibroblastsin vitro (20).

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Table 3. Glycosylation of hydroxy [ 4C ] lysine byendothelial cells in culture

Disaccharide % Glycosyla-(Di- + monosaccharide) tion

EndothelialMedium 91 ± 0.62 (7)* 81 ± 1.4 (3)Cell fraction 91 (2) 72 (2)

FibroblastCell + medium 81 ± 3.3 (5) 46 ± 4.7 (5)

Monolayers of primary aortic endothelial cells or skin fibroblastswere incubated for 48 hr with [14C]lysine as described in Materialsand Methods. Medium and cell layer were separated and hydro-lyzed separately. Hydroxy[14C]lysine and glycosides were quanti-tated after alkaline hydrolysis on an amino acid analyzer (18).Monosaccharide refers to galactosyl-hydroxy[14C]lysine, anddisaccharide is glucosyl-galactosyl-hydroxy['4C]lysine.* No. of determinations.

Upon NaDodSO4-acrylamide gel electrophoresis, after re-duction, the majority of the ['4C]protein migrated as a specieswith a molecular weight of about 135,000 (Fig. 1). When largeamounts of material were applied, a significant proportion ofthe 14C remained at the origin. On gel filtration, a significantproportion of the hydroxy[14C]proline eluted with an apparentmolecular weight of approximately 135,000. However, greaterthan 50% of the hydroxy['4C]proline was found in a peak closeto the exclusion volume. The percent hydroxylation of prolinein the larger molecular weight fraction averaged 14%. Afterpepsin digestion and gel filtration (Fig. 2), the [14C]collagensynthesized by the endothelial cell cultures had an apparentmolecular weight of 115,000. Determination of the total hy-droxy['4C]proline in the peak tubes of this fraction indicated59% hydroxylation of the ['4C]proline.When endothelial cell cultures were incubated with antise-

rum to basement membrane collagen of anterior lens capsuleand counterstained with fluorescein goat anti-rabbit gamma-

P11PI2 ^1 ^2

2

'CO

0

0-U.

E

0 20 40 60

DISTANCE FROM ORIGIN (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 re-

duced in 1% NaDodSO4, 1% mercaptoethanol as described in Mate-rials and Methods. Samples contained approximately 20,000 cpm in100 Ml. Gels were stained before slicing to determine migration ofcarrier collagen components.

4

V0 40 Vt

xz0I.-V 4

U-E

01

020 40 60FRACTION NUMBER

FIG. 2. Gel filtration chromatography on NaDodSO4-agaroseA5m of ["4C]protein from endothelial medium. Cells were incubatedfor 48 hr in medium containing [14C]proline, and material in mediumwas precipitated with ammonium sulfate as described in Materialsand Methods. Material was dialyzed against 0.05 M acetic acid andtreated with pepsin at 15°. The reaction was stopped by dialyzingagainst 15% KCl, and the precipitated material was treated withNaDodSO4-5% mercaptoethanol and dialyzed against the columnbuffer. Percent hydroxylation of [14C]proline was determined infraction 38-41, as indicated by the brackets (see Table 1).

globulin, a yellow-green fluorescence disseminated throughoutthe entire cytoplasm. Cells incubated with normal serumshowed no fluorescence (Fig. 3). By use of l25-Ilabeled TypesI, II, III, and IV bovine collagen in a radioimmunoassay it hasbeen shown that the above antisera are quite specific, havinghigh titers (over 1:100,000) to Type IV collagen but showingno crossreaction with Types I, II, or III (25).

DISCUSSIONThe data indicate that cultures of endothelial cells derived fromcalf aorta synthesize collagen, and the chemical characteristicsresemble basement membrane (Type IV) collagen. Furtherevidence that the endothelial cells isolated from calf aortasynthesize a basement membrane-like collagen is derived fromthe immunological studies, which show that antiserum preparedagainst collagen of calf anterior lens capsule (Type IV) localizesin the cytoplasm of these cells as demonstrated by the indirectimmunofluorescent technique.

It is of interest to compare the properties of the materialsynthesized by the endothelial cells to those of basementmembrane collagen made in other systems in vitro. These in-clude endodermal cells of the parietal yolk sac (21-23), chicklens (6-8), glomeruli (11), and Descemet's membrane (24). Theamount of 3-hydroxy[14C]proline in the collagen synthesizedby the endothelial cells is similar to that of the other basementmembrane systems. Less than 2% of this isomer is found inTypes I, II, or III. Moreover, the pepsin-resistant fraction, whicheluted with apparent molecular weight of 115,000, had 59%of its ['4C]proline as hydroxy['4C]proline. This is similar tovalues obtained for basement membrane isolated from maturetissues (4). The percent glycosylation of hydroxy[14C]lysine andthe proportion of the hydroxy[14C]lysine-linked glycoside,glucosylgalactose, are similar to those observed in the otherbasement-membrane-synthesizing systems.

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22364 Biochemistry: Howard et al.

FIG. 3. (a) Immunofluorescence reaction between rabbit anti-serum to collagen of bovine lens capsule and bovine aortic endothelialcells. Note the strong cytoplasmic fluorescence. (b) Control reactionusing preimmunization rabbit serum. Note limited autofluorescenceover the nucleus only.

In other in vitro systems known to synthesize basementmembrane collagen, the initially synthesized polypeptide chain(procollagen) has an apparent molecular weight ranging be-tween 140,000 and 155,000 (7, 11, 24). The difference in mo-lecular weight between the initial polypeptide chain synthe-sized by calf aorta endothelium and other cell systems may inpart be due to experimental variability and in part to variabilityamong basement membrane collagens synthesized by differentcell types. It should also be noted that the proteins synthesizedby the endothelial cells of calf aorta appear to aggregate whenlarge quantities are applied to gel filtration columns or toacrylamide gels. This could represent nonspecific association

of the collagen with a serum protein, or a specific affinityamong macromolecules secreted by these cells.

In conclusion, the synthesis of collagen in endothelial cellcultures has been demonstrated and the product partiallycharacterized. Data so far indicate the product is similar to abasement membrane collagen. Since few collagens other thanType I from cell culture systems have been extensively studied,it remains to be determined whether the unique characteristicsof the endothelial cell collagen can be attributed to the cultureconditions in vitro or whether there are differences in basementmembrane 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 hy-droxy[14C]lysine and its glycosides, and Mrs. Margaret Lu and MissKathy Spause for excellent technical assistance. This study was sup-ported by NIH Grants AM-14526, AM-14805, and HL-16058.

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