Expression of Small Extracellular Chondroitin/Dermatan Sulfate Proteoglycans Is Differentially Regulated in Human Endothelial Cells*

(Received for publication, October 9, 1996, and in revised form, February 10, 1997)

Lassi Nelimarkka Dagger , Varpu Kainulainen §, Elke Schönherr , Susanna Moisander Dagger , Matti Jortikka par , Mikko Lammi par , Klaus Elenius §, Markku Jalkanen § and Hannu Järveläinen Dagger **Dagger Dagger

From the Departments of Dagger  Medical Biochemistry and ** Internal Medicine, University of Turku, and § Turku Centre for Biotechnology, FIN-20520 Turku, Finland, the  Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, GER-4400 Germany, and the par  Department of Anatomy, University of Kuopio, FIN-70211 Kuopio, Finland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We have examined the expression of the small extracellular chondroitin/dermatan sulfate proteoglycans (CS/DS PGs), biglycan, decorin, and PG-100, which is the proteoglycan form of colony stimulating factor-1, in the human endothelial cell line EA.hy 926. We have also examined whether modulation of the phenotype of EA.hy 926 cells by tumor necrosis factor-alpha (TNF-alpha ) is associated with specific changes in the synthesis of these PGs. We demonstrate that EA.hy 926 cells, when they form monolayer cultures typical of macrovascular endothelial cells, express and synthesize detectable amounts of biglycan and PG-100, but not decorin. On SDS-polyacrylamide gel electrophoresis both PGs behave like proteins of the relative molecular weight of ~250,000. TNF-alpha that changed the morphology of the cells from a polygonal shape into a spindle shape and that also stimulated the detachment of the cells from culture dish, markedly decreased the net synthesis of biglycan, whereas the net synthesis of PG-100 was increased. These changes were parallel with those observed at the mRNA level of the corresponding PGs. The proportions of the different sulfated CS/DS disaccharide units of PGs were not affected by TNF-alpha . Several other growth factors/cytokines, such as interferon-gamma , fibroblast growth factors-2 (FGF-2) and -7 (FGF-7), interleukin-1beta , and transforming growth factor-beta , unlike TNF-alpha , modulated neither the morphology nor the biglycan expression of EA.hy 926 cells under the conditions used in the experiments. However, PG-100 expression was increased also in response to FGF-2 and -7 and transforming growth factor-beta . None of the above cytokines, including TNF-alpha , was able to induce decorin expression in the cells. Our results indicate that the regulatory elements controlling the expression of the small extracellular CS/DS PGs in human endothelial cells are different.


INTRODUCTION

Proteoglycans (PGs)1 are complex macromolecules that consist of a protein core and one or more glycosaminoglycan (GAG) side chains, covalently bound to the core protein (1). Nearly 30 individual PGs with various functions residing in the extracellular matrix, on the cell surface, or inside the cells, have been identified (1-4). Among the PGs of the extracellular matrix, four small chondroitin/dermatan sulfate (CS/DS) PG species, biglycan (5), decorin (6), PG-100 (7), and epiphycan (4, 8, 9) are currently known. Two of these PGs, biglycan and decorin, are highly homologous with each other in terms of their core protein structure (5), and together with epiphycan, fibromodulin, and lumican they both belong to the small leucine-rich PG gene family (4). With some exceptions biglycan and decorin differ in the number of GAG chains attached to their core proteins. Biglycan is usually substituted with two GAG chains, whereas decorin typically has only one GAG chain (10-12). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) biglycan has variously been shown to migrate at positions of protein molecular weight markers of ~200,000 or more, while decorin migrates at positions of protein molecular weight markers of 87,000-180,000 (13-17). The third small extracellular CS/DS PG species, originally isolated from the culture medium of human osteosarcoma cells and tentatively named PG-100 (7), has recently been demonstrated to be identical with the macrophage colony-stimulating factor CSF-1 (18). PG-100 is therefore a PG form of this cytokine (19, 20), and it can be called a "part-time" PG to be differentiated from biglycan, decorin, and other PGs that usually exist only in the GAG containing form (21). The fourth small CS/DS PG, epiphycan, has been shown to be closely related to osteoinductive factor (22), and the expression of this PG seems to be cartilage specific (8).

The exact functions of the small CS/DS PGs are still somewhat controversial. Decorin, the most thoroughly investigated molecule in this group, has been shown to bind to collagen fibrils and to regulate fibril formation (23-26). Decorin has also been found to interact with the cytokine transforming growth factor-beta (TGF-beta ) and to be involved in cell proliferation and differentiation (27-29). The role of biglycan in these processes is obscure, although it has been shown to bind to these same molecules (30-32). The pericellular localization of biglycan (33) as well as its ability to bind to fibronectin (34) suggest that biglycan is likely to interfere with cell adhesion (34). There is also recent evidence to suggest that biglycan is associated with cell migration (35). CSF-1 is known to control the growth and differentiation of mononuclear phagocytes (36) as well as the growth of several other cell types, including endothelial cells (37). However, PG-100 exhibits less than 1% of the biological activity of mature CSF-1 (18) and should perhaps be considered an extracellular storage form of this cytokine (19).

We have earlier shown that the principal small extracellular CS/DS PG species synthesized by bovine aortic endothelial (BAE) cells in monolayer cultures is biglycan (16). This result has been shown to be true for human umbilical vein endothelial cell monolayers as well (16). We have also shown that BAE cell monolayer cultures do not express detectable amounts of either decorin or type I collagen (16). However, when BAE cells change their phenotype and form so called sprouting cultures, the synthesis of both decorin and type I collagen is initiated (38, 39) indicating that the synthesis of decorin and type I collagen is associated with an in vitro angiogenesis phenomenon.

In this study we have examined the expression of the small extracellular CS/DS PGs in the permanent human endothelial cell line EA.hy 926 (40). Previously this cell line has been shown to possess several characteristics typical of endothelial cells (40-46). However, the production of extracellular matrix molecules, including PGs, of EA.hy 926 cells has remained unidentified. Furthermore, we have examined whether modulation of the phenotype of EA.hy 926 cells is associated with specific changes in the synthesis of the small extracellular CS/DS PGs. We demonstrate that EA.hy 926 cells express biglycan, but not decorin. We also demonstrate that these cells synthesize PG-100, the Mr of which (~250,000) is similar to that of biglycan synthesized by these same cells. In addition, the inhibitory effect of TNF-alpha on biglycan expression and its stimulatory effect on PG-100 expression by EA.hy 926 cells, associated with the altered morphology and adhesion of the cells, are shown.


EXPERIMENTAL PROCEDURES

Cell Culture

Cells of the permanent human endothelial cell line EA.hy 926 (40) were grown on plastic dishes (Nunc, Roskilde, Denmark) at 37 °C in 95% air + 5% CO2 in bicarbonate-buffered Dulbecco's modified Eagle's medium (Flow Laboratories, Irvine, United Kingdom) containing 100 IU/ml of penicillin (Oriola, Finland) and 50 µg/ml streptomycin (Oriola), and supplemented with 10% (v/v) of fetal calf serum (FCS, Life Technologies, Inc., Paisley, Scotland), and 1 × HAT (100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine; Life Technologies, Inc.). For subcultures, the cells were detached with 0.01% (w/v) trypsin (Sigma).

Human skin fibroblasts (HSFs) that were used as control cells were derived from a skin biopsy of a healthy person, and these cells were subcultured as described previously (47). The culture medium used for HSFs was the same as described above for EA.hy 926 cells with the exception that HAT was omitted.

Metabolic Labeling of the Cultures

For identification of sulfated PGs in the culture media, confluent cell cultures were labeled for 24 h with 50 µCi/ml Na2[35S]O4 (Amersham, United Kingdom) in sulfate-free Dulbecco's modified Eagle's medium supplemented with 10% (v/v) FCS, whereafter the media were collected and the detached cells were removed by low speed centrifugation (2000 rpm) for 5 min. The incorporation of [35S]sulfate into newly synthesized PGs was evaluated by the cetylpyridinium chloride (CPC) precipitation assay (48) before further analyses of the culture media (see below).

In the experiments examining the effect of TNF-alpha on the synthesis of the small extracellular CS/DS PGs, the cells were grown to confluence in the serum containing culture medium. Next, the cells were kept in the serum-free culture medium for 4 h, whereafter the cells were labeled as described above in serum-free culture medium containing TNF-alpha at various concentrations. After removing the detached cells from the culture media by low speed centrifugation (see above), the amount of [35S]sulfate-labeled PGs in the samples was determined by the CPC precipitation assay (48) before further analyses.

Cell Number Determination

Cell number calculations were performed with a hemocytometer. Before the calculations, the cells were fixed with a formaldehyde containing buffer solution (10%, v/v, of 37% formaldehyde in 0.085 M NaCl and 0.1 M Na2 SO4). Two aliquots of each cell suspension were taken for the cell counting.

Cell Viability Determination

The viability of the cells in the control and TNF-alpha treated cultures was measured using the trypan blue exclusion test as described (49).

Glycosaminoglycan Degradation

For the identification of CS/DS and heparan sulfate (HS) PGs in the culture media, equal volumes of [35S]sulfate-labeled media were ethanol-precipitated, dried, dissolved into appropriate buffers, and digested with 0.1 IU/ml of chondroitin ABC lyase from Proteus vulgaris (Seikagaku Co., Tokyo, Japan) or 6 milliunits/ml of heparitinase from Flavobacterium heparinum (Seikagaku Co.), respectively (50, 51).

Immunoprecipitation

The immunoprecipitation of biglycan and PG-100 from the culture media with polyclonal antisera against these two PGs (see below) was performed as described previously (52). Aliquots of [35S]sulfate-labeled media corresponding to 500,000 cpm as determined by the CPC precipitation assay (48) were taken for the analysis. The immunoprecipitants were run on a gradient SDS-PAGE as described (see below).

Purification of Proteoglycans by Ion Exchange Chromatography

Partial purification of PGs by ion exchange chromatography was performed by applying equal aliquots of [35S]sulfate-labeled media to a DEAE-Sephacel (Pharmacia, Uppsala, Sweden) column equilibrated with 0.2 M NaCl in the buffer containing 50 mM sodium acetate, 2.0 M urea, 1 mM phenylmethylsulfonyl fluoride, and 0.1% Triton X-100. The column was eluted with a linear gradient of 0.2-1.0 M NaCl in the same buffer as above. The amount of radioactivity in sulfated PGs in each fraction was determined by the CPC precipitation assay (48). All fractions that contained [35S]sulfate-labeled PGs were pooled before further analyses.

SDS-Polyacrylamide Gel Electrophoresis

SDS-PAGE of the samples was carried out essentially as described by Laemmli (53) on a 4-12% linear gradient gel with a 3% stacking gel (16). The positions of the radioactive bands were visualized by fluorography of dried gels previously treated with 2,5-diphenyloxazole (Fisons, UK). 14C-Methylated protein molecular weight standards (Amersham) were used to estimate the average sizes of [35S]sulfate-labeled macromolecules.

Western Blot Analysis

[35S]Sulfate-labeled medium samples or partially purified PGs from similar medium samples were lyophilized, whereafter they were digested with chondroitin ABC lyase and heparitinase to remove all GAG chains (see above). Next, SDS-PAGE was carried out (see above) under reducing conditions using a 12.5% gel, whereafter the proteins were electroblotted to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Specific bands were detected by immunostaining (18, 54) using polyclonal antisera against biglycan and PG-100 (see below).

Antisera

Biglycan antiserum was a polyclonal antiserum made in a rabbit against a synthetic peptide of human biglycan (amino acids 11-24) that was conjugated to bovine serum albumin before injections (5). This antiserum, called LF-51, was kindly provided by Dr. L. Fisher (National Institute of Dental Research, Bethesda, MD). PG-100 antiserum was a polyclonal antiserum that was raised in a rabbit against PG-100 core protein as described (7). This antiserum was kindly provided by Dr. H. Kresse (University of Münster, Münster, Germany).

Estimation of the Sulfation and the Average Chain Length of Glycosaminoglycan Chains

[35S]Sulfate-labeled medium samples from control and TNF-alpha stimulated EA.hy 926 cell cultures were digested with 0.1 IU/ml of chondroitin ABC lyase in a buffer containing 200 mM Tris, 60 mM sodium acetate, pH 8.0, and 5 µg of chondroitin sulfate A (Seikagaku Co.) as a carrier. The digestions were performed for 6 h at 37 °C. After ethanol precipitation overnight at 4 °C the supernatants and the wash solutions (70% ethanol) were combined, ethanol was removed by evaporation, and the sulfated disaccharides released by chondroitin ABC lyase treatment were separated by ion exchange chromatography (55). The average length of the GAG chains was estimated using a Sephacryl S-300 (Pharmacia) gel filtration (56, 57).

RNA Isolation and Northern Blot Analysis

Total cellular RNA was isolated from the cultures using the single-step method (58). Northern blot analyses using GeneScreen Plus membranes (DuPont NEN) were performed essentially as described previously (16). Random priming (Multiprime DNA Labeling System, Amersham, UK) was used for labeling of the cDNAs with 5'-[alpha -32P]dCTP (Amersham) to a high specific activity. For the quantitation of the hybridization signals, the membranes were probed with a rat glyceraldehyde-3-phosphate dehydrogenase cDNA (59).

cDNA Probes

The following cDNA probes were used: a biglycan cDNA (5) for the full-length human biglycan core protein; a 1338-base pair CSF-1 cDNA (18) for the PG-100 core protein; a decorin cDNA (6) for the full-length human decorin core protein; a 1.1-kilobase perlecan cDNA, called HS-1 (60); and pHCAL1U (61) for pro-alpha 1(I) collagen. These cDNA probes were kindly provided by Drs. Fisher (National Institute of Dental Research), Kresse (University of Münster, Münster, Germany), Krusius (The Finnish Red Cross, Helsinki, Finland), Iozzo (Thomas Jefferson University, Philadelphia, PA), and Vuorio (University of Turku, Turku, Finland), respectively.

Cytokines

The following cytokines were used: TNF-alpha and fibroblast growth factor-7 (FGF-7), purchased from PeproTech; fibroblast growth factor-2 (FGF-2), purchased from PeproTech or Boehringer Mannheim; interleukin-1beta (IL-1beta ), interferon-gamma (IFN-gamma ), and TGF-beta , all purchased from Boehringer Mannheim. TNF-alpha was used at concentrations of 5, 25, and 50 ng/ml. FGF-7, FGF-2, IL-1beta , IFN-gamma , and TGF-beta were used at concentrations of 50 ng/ml, 10 ng/ml, 5 IU/ml, 1000 IU/ml, and 20 ng/ml, respectively.

Statistical Analysis

Student's unpaired t test was used to describe the significance of the differences between the results of the control and cytokine-treated EA.hy 926 cell cultures.


RESULTS

Identification of Small Extracellular Chondroitin/Dermatan Sulfate Proteoglycans in Monolayer Cultures of EA.hy 926 Human Endothelial Cells

SDS-PAGE of [35S]sulfate-labeled medium sample from a confluent monolayer culture of EA.hy 926 cells demonstrated that these cells synthesize and secrete into the medium two size classes of [35S]sulfate-labeled macromolecules, one that remains on the top of the separating gel (Fig. 1, lane 1, band III), and another one with the Mr of ~250,000 (Fig. 1, lane 1, band II). Chondroitin ABC lyase and heparitinase digestions of [35S]sulfate-labeled medium samples prior to SDS-PAGE indicated that the macromolecules of band II are CS/DS PGs, while those of band III represent HS containing PGs (Fig. 1, lanes 2-4), e.g. perlecan (60), since these cells express abundant mRNA for this PG (data not shown). The composition of sulfated PGs in the culture medium of EA.hy 926 endothelial cell monolayers differed from that of sulfated PGs present in the culture medium of HSFs used as control cells. Besides containing PGs migrating in band II and band III (Fig. 1, lane 5), the culture medium of HSFs contained two additional size classes of sulfated PGs, one with the Mr of ~120,000 (Fig. 1, lane 5, band I) and another one that does not enter the separating gel (Fig. 1, lane 5, band IV). Both of these additional PG size classes were sensitive to chondroitin ABC lyase digestion (Fig. 1, lane 6) indicating that they represent CS/DS PGs. Based on the results of previous studies, the band I PGs (Mr of ~120,000) most probably represent decorin, while the PGs of band IV mainly represent versican (6, 13, 62-65).


Fig. 1. Comparison of size classes of newly synthesized, [35S]sulfate-labeled macromolecules in the culture medium of EA.hy 926 human endothelial cells and human skin fibroblasts. [35S]Sulfate-labeled medium samples from a monolayer culture of EA.hy 926 cells and HSF control culture were run under reducing conditions on a 4-12% gradient SDS-PAGE with a 3% stacking gel before and after chondroitin ABC lyase and/or heparitinase digestions. The gel was treated with 2,5-diphenyloxazole and dried, and the radioactive bands were visualized by fluorography. Lanes 1-4 represent [35S]sulfate-labeled medium samples from EA.hy 926 cell culture before GAG degrading enzyme treatments (lane 1), and after digestions with chondroitin ABC lyase (lane 2), heparitinase (lane 3), and chondroitin ABC lyase and heparitinase (lane 4). Lanes 5-8 represent [35S]sulfate-labeled medium samples from HSF culture before GAG degrading enzyme treatments (lane 5), and after digestions with chondroitin ABC lyase (lane 6), heparitinase (lane 7), and chondroitin ABC lyase and heparitinase (lane 8). The arrow indicates the upper limit of the separating gel. Roman numerals I-IV indicate the different size classes of [35S]sulfate-labeled macromolecules.
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The above results showed that EA.hy 926 cells in a monolayer culture synthesize and secrete into the medium only one size class (band II) of small CS/DS PGs. The average size of these CS/DS PG molecules is of the same range as that of HSF-derived small extracellular CS/DS PGs, previously identified as biglycan (64, 65). To clarify whether the EA.hy 926 cell derived small CS/DS PGs that migrate on SDS-PAGE in the band II also represent biglycan, immunoprecipitation of [35S]sulfate-labeled medium sample from these cells using an antiserum against biglycan (LF-51) was performed. As shown in Fig. 2 (panel A, lanes 1 and 3), the biglycan antiserum LF-51 was able to immunoprecipitate [35S]sulfate-labeled macromolecules that migrate on SDS-PAGE gel in band II. This result was true for both the EA.hy 926 endothelial cell and HSF cultures. No cross-reactivity to band I CS/DS PGs that were present only in the culture medium of HSFs and that have previously been identified as decorin (64, 65) was observed (Fig. 2, panel A, lane 3). These results confirmed that the CS/DS PGs of band II synthesized by EA.hy 926 cells contain biglycan molecules.


Fig. 2. Immunoprecipitation of biglycan and PG-100 from [35S]sulfate-labeled culture medium of EA.hy 926 cell monolayers. Antisera against biglycan (panel A) and PG-100 (panel B) were used for the immunoprecipitation of [35S]sulfate-labeled medium samples from a monolayer culture of EA.hy 926 endothelial cells. Immunoprecipitations of identical medium samples with normal rabbit serum were used as negative controls. Aliquots corresponding to 500,000 cpm as determined by CPC precipitation assay were used for immunoprecipitations. SDS-PAGE analyses identical to that described in the legend for Fig. 1 were performed. The arrows in panels A and B indicate the upper limit of the separating gel. Lanes 1 and 2 in panel A represent [35S]sulfate-labeled medium samples from EA.hy 926 cell culture after immunoprecipitation with the biglycan antiserum LF-51 (lane 1) or NRS (lane 2), and lanes 3 and 4 represent [35S]sulfate-labeled medium samples from HSF culture after immunoprecipitation with the biglycan antiserum LF-51 (lane 3) or NRS (lane 4). Lanes 1 and 2 in panel B represent [35S]sulfate-labeled medium samples from EA.hy 926 cell culture after immunoprecipitation with the antiserum against PG-100 (lane 1) or NRS (lane 2). [35S]Sulfate-labeled macromolecules representing biglycan and PG-100 are indicated by the asterisk (*).
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As not all the CS/DS PGs of band II in the culture medium of EA.hy 926 cells could be immunoprecipitated with the antiserum LF-51 (data not shown), the possibility remained that there are additional species of small CS/DS PGs migrating in the position of band II. A few years ago, evidence for the existence of a new member of the small extracellular CS/DS PGs of this size range, tentatively named PG-100, has been presented (7). Recently, this PG was shown to represent a GAG-linked form of the macrophage colony stimulating factor CSF-1 (18). The result from immunoprecipitation using an antiserum against PG-100 (66) demonstrated that EA.hy 926 cells synthesize and secrete into the medium PG-100 and that the Mr of these molecules is similar to that of biglycan synthesized by the same cells (Fig. 2, panel B, lane 1).

We also examined whether monolayer cultures of EA.hy 926 endothelial cells contain mRNA for small extracellular CS/DS PGs, biglycan, decorin, and PG-100. Northern blot analysis using a full-length cDNA probe for human biglycan core protein demonstrated that EA.hy 926 cells express mRNA that hybridizes to a biglycan cDNA probe (Fig. 3). Furthermore, mRNA for PG-100 was detected (Fig. 3). In contrast, mRNA that hybridizes to a cDNA probe for decorin core protein was not expressed in detectable amounts by monolayer cultures of EA.hy 926 cells. Human skin fibroblasts used as control cells expressed abundant mRNA for the core proteins of all three PGs (Fig. 3).


Fig. 3. The expression of mRNAs for the core proteins of the small extracellular CS/DS PGs and alpha 1(I) collagen by EA.hy 926 cell monolayer and HSF control cultures. Northern blot analysis of total cellular RNA (10 µg/lane) from a monolayer culture of EA.hy 926 cells and from a HSF culture was performed. The blot was probed with 32P-labeled cDNAs for biglycan, decorin, and PG-100 core proteins, and for pro-alpha 1(I) collagen and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as indicated. The average sizes of the mRNAs for the small CS/DS PGs and pro-alpha 1(I) collagen are shown. kb, kilobase.
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Previously, we have demonstrated that monolayer cultures of BAE cells that do not express detectable amounts of decorin also fail to express type I collagen (16). To examine whether EA.hy 926 endothelial cells are similar in this respect, expression of type I collagen by these cells was examined. Northern blot analysis of total cellular RNA from monolayer cultures of EA.hy 926 cells demonstrated that these cells do not express detectable amounts of mRNA for alpha 1(I) collagen (Fig. 3). The opposite was true for HSFs (Fig. 3).

Modulation of Small Extracellular CS/DS PG Expression in EA.hy 926 Cells by TNF-alpha

The functions of the small extracellular CS/DS PGs are still somewhat controversial. Evidence exists that these PGs, especially biglycan and decorin, interfere with the cell adhesion process (34, 67, 68). The small extracellular CS/DS PGs have also been shown to be involved in an in vitro phenomenon of angiogenesis (39). Therefore, we examined whether potentially angiogenically active cytokines modulate both the phenotype of EA.hy 926 cells and the synthesis of the small extracellular CS/DS PGs of these cells. Several cytokines, including TNF-alpha , IFN-gamma , FGF-2 and -7, IL-1beta , and TGF-beta , were tested. Of these cytokines TNF-alpha was found to be the only one that was able to modulate the phenotype of EA.hy 926 cells under the conditions used in the experiments. Specifically, this cytokine changed the morphology of EA.hy 926 cells from a polygonal shape into a spindle shape (Fig. 4). Furthermore, TNF-alpha stimulated the detachment of the cells from the culture dish which was indicated by an increase in the number of cells floating free in the culture medium (Fig. 5). As TNF-alpha is known to induce apoptosis and general cytotoxic effects on a variety of cells including endothelial cells (69, 70), we performed the trypan blue exclusion test to examine whether this was true also in the present study. The results demonstrated that almost all cells that remained attached to the culture dish were negative for trypan blue staining. This was evident for the control (96% of the cells trypan blue negative) as well as for the TNF-alpha (50 ng/ml) treated (98% of the cells trypan blue negative) cultures. In contrast, most of the cells floating free in the medium were positive for trypan blue staining, the proportion of positively stained cells being greater in the TNF-alpha (50 ng/ml) treated (80% of the cells trypan blue positive) than in the control cultures (63% of the cells trypan blue positive). These results collectively indicate that, although TNF-alpha induced some cytotoxicity on EA.hy 926 cells in this study, the changes in cell morphology and the increase in the detachment of the cells could be only partially accounted for the cytotoxic effects of TNF-alpha .


Fig. 4. The effect of TNF-alpha on the shape of EA.hy 926 cells in culture. The cells were grown to confluence in the culture medium containing 10% (v/v) FCS. Next, the cells were kept in the serum-free culture medium for 4 h, whereafter TNF-alpha at concentrations of 5, 25, and 50 ng/ml was added on the cells. The cells were grown for additional 24 h, after which phase-contrast micrographs were taken to visualize the morphology of the cells. Panel A represents the cells of the control culture (TNF-alpha not added), whereas panel B represents the cells exposed to 50 ng/ml TNF-alpha . A similar morphological change of the cells as shown in panel B was evident in the cultures exposed to 5 and 25 ng/ml TNF-alpha (data not shown).
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Fig. 5. The effect of TNF-alpha on the number of EA.hy 926 cells on plastic and in the culture medium. The cells were grown and exposed to TNF-alpha as described in the legend for Fig. 4. After an incubation time of 24 h, the number of cells on plastic and that of cells floating free in the culture medium were calculated using a hemocytometer. Each symbol represents the mean ± S.D. of duplicate cell number calculations of three separate cultures. The significance of the differences between the control and TNF-alpha treated cultures was tested using Student's unpaired t test (*, p < 0.05; **, p < 0.01).
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Proteoglycan synthesis of EA.hy 926 cells was concomitantly affected. The net incorporation of [35S]sulfate into PGs secreted into the culture medium by the cells was slightly (31-34%), but significantly increased in response to TNF-alpha stimulation (Fig. 6). The increased [35S]sulfate incorporation observed at all TNF-alpha concentrations (5, 25, and 50 ng/ml) seemed to be most prominent in band II CS/DS PGs (Fig. 7). As the degree of sulfation of the CS/DS disaccharide isomers (Table I) or the average GAG chain length (data not shown) were not altered in TNF-alpha treated cultures, we examined whether the increase in [35S]sulfate incorporation in response to TNF-alpha was due to an increase in the net synthesis of biglycan or PG-100, or both of them. Western blot analyses of chondroitin ABC lyase digested medium samples from TNF-alpha -treated EA.hy 926 cell cultures using antisera against biglycan and PG-100 demonstrated that the net synthesis of PG-100 markedly increases in response to TNF-alpha , while the net synthesis of biglycan decreases (Fig. 8). Northern blot analyses of total cellular RNA from these cultures using cDNA probes for biglycan and PG-100 core proteins demonstrated that the changes observed at the product level were parallel with those observed at the mRNA level of the corresponding PGs (Fig. 9). The other cytokines used in this study neither modulated the phenotype of EA.hy 926 cells nor influenced significantly biglycan expression of the cells under the conditions used in the experiments (Fig. 10). In contrast, the expression of PG-100 by EA.hy 926 cells was found to be increased in response to FGF-2 and -7 and TGF-beta stimulation of the cells, but not in response to IFN-gamma or IL-1beta stimulation (Fig. 10). Furthermore, as in control cultures of EA.hy 926 cells, there were no detectable amounts of mRNA for decorin core protein or alpha 1(I) collagen in any of the cytokine-stimulated cultures (data not shown).


Fig. 6. The effect of TNF-alpha on the net incorporation of [35S]sulfate into newly synthesized PGs secreted into the culture medium by EA.hy 926 cells. The cells were grown to confluence in the culture medium containing 10% (v/v) FCS, whereafter the cells were kept in the serum-free culture medium for 4 h. Next, the serum- and sulfate-free culture medium containing 50 µCi/ml [35S]sulfate and TNF-alpha at various concentrations (0, 5, 25, and 50 ng/ml) were changed. The cells were grown for an additional 24 h, after which the media were collected. The amount of [35S]sulfate-labeled PGs in the samples was determined by the CPC precipitation assay. Each symbol represents the mean ± S.D. of duplicate determinations of three separate cultures. The significance of the differences between the control and TNF-alpha treated cultures was tested using Student's unpaired t test (*, p < 0.05; **, p < 0.01).
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Fig. 7. The effect of TNF-alpha on the net incorporation of [35S]sulfate into various size classes of newly synthesized PGs secreted into the culture medium by EA.hy 926 cells. EA.hy 926 cells were cultured, treated with TNF-alpha , and labeled with [35S]sulfate as described in the legend for Fig. 6. Equal volumes of the medium from each culture condition were taken and analyzed by SDS-PAGE before and after chondroitin ABC lyase and heparitinase digestions as described in the legend for Fig. 1. Panel A represents medium samples that were not digested with GAG degrading enzymes, and panels B-D represent medium samples after digestions with chondroitin ABC lyase (panel B), heparitinase (panel C), or chondroitin ABC lyase and heparitinase (panel D).
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Table I. Effect of TNF-alpha treatment on the molar proportions of sulfated chondroitin/dermatan sulfate disaccharide isomers in the secreted proteoglycans, analyzed by CarboPac PA1 HPLC column after chondroitin ABC lyase digestion


TNF-alpha Disaccharide isomer
 Delta di-4S  Delta di-6S Disulfated Trisulfated

ng/ml
0 11.2 2.8 85.7 0.3
5 16.7 3.6 79.1 0.6
25 15.7 4.4 79.2 0.7
50 20.9 5.1 73.3 0.7


Fig. 8. Western blotting for the biglycan and PG-100 core proteins in the culture media of TNF-alpha stimulated EA.hy 926 cells and their non-stimulated counterparts. The cells were grown and stimulated with TNF-alpha as described in the legend for Fig. 6. An equal volume of the medium from each culture condition was run through a DEAE-Sephacel column to partially purify the PGs secreted into the medium by the cells. Next, the samples were dialyzed against tap water, lyophilized, and dissolved in 100 µl of double distilled water. Equal aliquots were taken for digestion with chondroitin ABC lyase and heparitinase, whereafter the samples were run on a 12.5% SDS-PAGE. The proteins were transferred to nitrocellulose filter and the blots were incubated in the presence of antisera against biglycan (panel A) and PG-100 (panel B) to determine the amounts of the core proteins in different samples. The lanes in panels A and B contain medium samples from EA.hy 926 endothelial cell control culture (0) and from cultures of EA.hy 926 cells stimulated with 5, 25, and 50 ng/ml TNF-alpha as indicated.
[View Larger Version of this Image (46K GIF file)]


Fig. 9. The effect of TNF-alpha on the mRNA level of biglycan and PG-100 in EA.hy 926 cell monolayers. The cells were grown as described in the legend for Fig. 6. Total cellular RNA was isolated and 10 µg of RNA from each culture condition was taken for Northern blot analysis. The blot was probed with 32P-labeled cDNAs for the biglycan and PG-100 core proteins, and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The average sizes of the biglycan and PG-100 core protein mRNAs are shown.
[View Larger Version of this Image (29K GIF file)]


Fig. 10. The effect of various cytokines on the mRNA level of biglycan and PG-100 in EA.hy 926 cell monolayers. The cells were grown to confluence in the culture medium containing 10% (v/v) FCS, whereafter the cells were exposed for 24 h to 1000 IU/ml of IFN-gamma , 10 ng/ml of FGF-2, 5 IU/ml of IL-1beta , 20 ng/ml of TGF-beta , and 50 ng/ml of FGF-7 under the serum-free culture medium. Total cellular RNA was isolated and 10 µg of total cellular RNA from each culture condition was taken for Northern blot analysis. The blot was probed with 32P-labeled cDNAs for the biglycan and PG-100 core proteins, and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The average sizes of the biglycan and PG-100 core protein mRNAs are shown. 1, control; 2, IFN-gamma ; 3, FGF-2; 4, IL-1beta ; 5, TGF-beta ; 6, FGF-7.
[View Larger Version of this Image (21K GIF file)]


DISCUSSION

Earlier a permanent human endothelial cell line EA.hy 926, that is capable of expressing von Willebrand factor, has been established (40). Besides von Willebrand factor, this cell line has been shown to produce several other molecules characteristic of endothelial cells, such as thrombomodulin (41, 71), prostacyclin (43), tissue-type plasminogen activator (44), type I plasminogen activator inhibitor (44), endothelin-1 (45, 46), endothelin converting enzyme (72), and vascular adhesion protein-1 (73). EA.hy 926 cells have also been shown to release an endothelial specific compound, platelet activating factor (42). However, the synthesis of the extracellular matrix molecules, including PGs, of EA.hy 926 cells has remained largely unidentified. In the present study we have examined PG expression of EA.hy 926 cells. In particular, we have focused on the expression of the small extracellular CS/DS PG species, except the cartilage specific epiphycan (4, 8), by these cells. As the small extracellular CS/DS PGs have been suggested to be involved in the regulation of endothelial cell behavior (35, 39, 67, 74), we have also examined whether modulation of the phenotype of EA.hy 926 cells is associated with specific changes in the synthesis of the small CS/DS PGs. We have demonstrated that EA.hy 926 cells, when forming monolayer cultures typical of macrovascular endothelial cells, express and synthesize detectable amounts of two small extracellular CS/DS PG species, namely biglycan and PG-100. In contrast, EA.hy 926 cells failed to express detectable amounts of decorin. The exact amounts of biglycan and PG-100 synthesized by EA.hy 926 cells were not determined. However, according to our experience (see also Figs. 8 and 9) EA.hy 926 cells normally synthesize more biglycan than PG-100 whereas the opposite is true when these cells are exposed to TNF-alpha . The apparent discrepancy between this and the result shown in Fig. 2 (see panel A, lane 1, and panel B, lane 1) is most probably due to the different precipitation capability of the two antisera used and the different exposure times of the x-ray films. In this study we have also demonstrated that the same EA.hy 926 endothelial cell monolayers that do not express decorin, fail to express type I collagen. These results are in a full agreement with those of our previous studies demonstrating that monolayer cultures of bovine aortic and human umbilical vein endothelial cells express and synthesize biglycan, but not decorin or type I collagen (16). BAE cell monolayers are also capable of producing PG-100.2 Thus, EA.hy 926 cells seem to express and synthesize a set of small extracellular CS/DS PGs typical of monolayer cultures of macrovascular endothelial cells. As the characteristic properties of human endothelial cells tend to change during culture and as the life span of human endothelial cells is highly limited, earlier studies concerning, e.g. synthesis of PGs by endothelial cells, have mainly been performed using cells of animal origin. This study has proved that EA.hy 926 cells represent an excellent in vitro human model system to be used for studies examining the factors and the regulatory mechanisms underlying the differential expression of the small extracellular CS/DS PGs. These cells are also very useful when the importance of the small extracellular CS/DS PGs in the control of endothelial cell behavior in man will be examined.

The importance of decorin in the fibrillogenesis of type I collagen (23, 25) and in the regulation of the activity of TGF-beta (28) has been demonstrated. In contrast, the functions of biglycan and PG-100 are not exactly known. Indirect evidence exists suggesting that biglycan is involved in the regulation of cell adhesion. This evidence is based, e.g. on the notion that biglycan has a pericellular localization (33), and on the fact that biglycan, like decorin (75, 76), interferes with the molecules such as fibronectin (34), important in the cell adhesion. In this study, we have shown that the morphological change of EA.hy 926 cells from a polygonal shape into a spindle shape and the increase in the number of detached cells in response to TNF-alpha treatment are associated with a significant decrease in the net synthesis of biglycan by the cells. We have also shown that several other cytokines, such as IFN-gamma , FGF-2 and -7, IL-1beta , and TGF-beta , that did not change the phenotype of EA.hy 926 cells did also not influence biglycan expression of these cells under the conditions used in the experiments. Since the above change in the cell morphology and the increase of the cell detachment could be only partially accounted for the cytotoxic effects of TNF-alpha (see "Results"), the results of this study support the finding by Bidanset et al. (34) that biglycan plays a role in cell adhesion. We are currently trying to overexpress biglycan in EA.hy 926 cells to examine whether the above changes in the morphology and adhesion of EA.hy 926 cells in response to TNF-alpha can be prevented. In regard with PG-100, the net synthesis of this part-time PG was increased in EA.hy 926 cells in response to TNF-alpha . The expression of PG-100 was also found to be increased in response to FGF-2 and -7 and TGF-beta . Thus, unlike the expression of biglycan, PG-100 expression did not show a strict correlation with the morphological change of EA.hy 926 cells. This indicates that the functional role of PG-100 for endothelial cells is different from that of biglycan. It can also be concluded that the regulatory elements controlling the expression of PG-100 and biglycan differ from each other. As regards biglycan, in a recent study with T47D cells it was shown that TNF-alpha is capable of altering biglycan promoter activity (77). The finding that the expression of PG-100 is up-regulated in response to TNF-alpha excludes the possibility that the effect of TNF-alpha on PG synthesis by endothelial cells and other cell types is merely inhibitory as demonstrated in previous studies (78-82). However, the obvious discrepancy in this study that the net incorporation of [35S]sulfate into all extracellular PGs was increased only by 31-34% in response to TNF-alpha (Fig. 6), while the net incorporation of [35S]sulfate into small extracellular CS/DS PGs was increased by severalfold (Fig. 7) suggests that the synthesis of most extracellular PG species has to decrease in response to TNF-alpha . Therefore, PG-100 may be one of the few PGs, the synthesis of which is increased in response to TNF-alpha (Fig. 8).

In the present study we have shown that EA.hy 926 cells do not express decorin or type I collagen even after having changed their phenotype from a polygonal shape into a spindle shape. Previously Iruela-Arispe et al. (38) and Järveläinen et al. (39) have demonstrated that BAE cells initiate the synthesis of both type I collagen and decorin during their morphological transition from a polygonal monolayer to a sprouting phenotype (an in vitro model for angiogenesis). Although EA.hy 926 cells of this study changed their phenotype, the cells still grew in a monolayer culture and formed no cords or tube-like structures as did the BAE cells in the studies cited above. Therefore, it is likely that decorin and type I collagen are needed in later stages of angiogenesis displayed by endothelial cells in vitro. Indeed, recently we have been able to demonstrate that EA.hy 926 cells, when they are grown on floating collagen type lattices together with rat fibroblasts, start to build up cords and after 6 days in culture human decorin can be detected by immunostaining.3

In summary, this study has shown that EA.hy 926 cells express and synthesize a set of small extracellular CS/DS PGs, characteristic of macrovascular endothelial cells of various origin. This study has also confirmed that different regulatory mechanisms are involved in the gene expression of the small extracellular CS/DS PGs. Furthermore, additional indirect evidence has been presented suggesting that biglycan plays a role in the cell adhesion process. Thus, EA.hy 926 cells represent an excellent in vitro model system to be used in studies on various aspects of PG expression in endothelial cells in man.


FOOTNOTES

*   This study was supported by the Turku University Foundation, Finnish Foundation for Cardiovascular Research, The Finnish Cultural Foundation, The Academy of Finland, and The Orion-Farmos Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger Dagger    To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Tel.: 358-2-3337444; Fax: 358-2-3337229; E-mail: hannu.jarvelainen{at}utu.fi.
1   The abbreviations used are: PG(s), proteoglycan(s); BAE, bovine aortic endothelial; CPC, cetylpyridinium chloride; CS, chondroitin sulfate; CSF-1, colony stimulating factor-1; DS, dermatan sulfate; FCS, fetal calf serum; FGF, fibroblast growth factor; GAG, glycosaminoglycan; HS, heparan sulfate; HSF, human skin fibroblast; IFN-gamma , interferon-gamma ; IL-1beta , interleukin-1beta ; PAGE, polyacrylamide gel electrophoresis; TNF-alpha , tumor necrosis factor-alpha ; TGF-beta , transforming growth factor-beta .
2   E. Schönherr, unpublished result.
3   E. Schönherr, L. Nelimarkka, and H. Järveläinen, unpublished results.

ACKNOWLEDGEMENTS

We thank Terttu Jompero, Taina Kalevo, and Liisa Peltonen for excellent technical assistance. We are also grateful to Dr. Larry Fisher for providing us with the antiserum LF-51 against biglycan and the biglycan cDNA probe, and Drs. Tom Krusius, Renato Iozzo, and Eero Vuorio for the cDNA probes for decorin, perlecan, and pro-alpha 1(I) collagen, respectively. The antibody against PG-100 and the PG-100 cDNA probe were kindly provided by Dr. Hans Kresse. We thank Dr. Thomas Hering (Case Western Reserve University, Cleveland, OH) for providing valuable comments of the manuscript.


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