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The effect of heparin on fibronectin and thrombospondin synthesis and mRNA levels in cultured human endothelial cells
EXPERIMENTALCELLRESEARCH
186,X-46
(1990)
The Effect of Heparin on Fibronectin and 16 d mRNA Levels in Cultured Human Endothelia BERNADETTE Connective
LYONS-GIORDANO,
Tissue
JANE M. BRINKER,
AND NICHOLAS
A. ~E~~~~~E~'
Research Institute and Departments of Medicine, and Biociaemistry University of Pennsylvania, Philadelphia, Pennsylvania 19104
and Biophysics,
potential importance of heparin in the regulation of new vessel formation. Bn vitro heparin potentiates human umbilical vein EC chemotactie an endothelial cell growth factor (ECGF), a member sf the fibroblast growth factor family of mitogens 12, 31. The growth and migration stimulator-y a6tiVity of heparin is neutralized by protamine, an arginine-rich basic protein which is known to bind avidly to heparin [2, 41. In vivo local administration of protamine inhibits neovascularization induced by inflammatory agents or by immune reactions. Goadministration of heparin reverses the effect of protamine [5]. The mechanism of action of heparin in the regulation of EC migration and growth has not been elucidated. Studies investigating the interaction of heparin with EC have shown that it is specifically bound to and internalized by these cells [6, 71. While these data suggest that the mode of heparin regulation of C behavior may be directly through plasma membrane rece s, the effect of heparin on human umbilical vein EC avior in the absence of ECGF provides evidence for an indirect aetion of heparin. In the absence of ECGF and at low serum concentrations, heparin inhibits the growth of these cells [8]. Likewise, it lacks chemotactic akptivity for endothelium in the absence of ECGF [3]. These data demonstrate the importance of recognizing the dinate nature of the re lation of EC beh genie factors. Previous reports have demonstra the importance of extracellular matrix components he regulation of a variety of cellular activities ~~cIndi~~ growth [9]. In culture, EC synthesize an extraeellular matrix which contains proteogly6ans [lo, IlIp collagens [X2, 131, and other glycoproteins including fibr~necti~ (FIT) [14-163, laminin [ 171)and thrombospondin (TS dition, it has recently been reported t fibroblast growth factor which they matrix [ZO]. In this report, we provid onstrate that heparin, in the present a specific decrease in FN synthesis by EC compared to ECGF-treated control cells. This decrease is accompanied by a coordinate decrease in EC for FN. Heparin does not change these cells although it does reduce their TSP mRNA lev-
Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 pg/ ml) and endotbelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No ehange in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of beparin on EC FN synthesis was independent of .whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cellls. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-tranSlatiOnally regulated. 0 1990 Academic~ress, IIIC.
INTRQDUGTION Vascular endothelial cells (EC)2 are in a quiescent growth state in the healthy blood vessel. In response to vessel wall injury, these cells undergo proliferation in the sequence of events which lead to angiogenesis. Elucidation of the mechanism(s) by which EC! growth is stimulated in vitro may provide insight into the physiology of vascular pathology. Since the initial observations that mast cells containing heparin accumulate at tumor sites prior to neovascularization [ 11, numerous studies have underscored the I To whom reprint requests should be addressed at: Connective Tissue Research Institute, 3624 Market St., Philadelphia, PA 19104. ’ Abbreviations used: CCM, complete culture medium; EC, endothelial cell; ECGF, endothelial cell growth factor; FN, fibronectin; SSC, standard saline citrate; TSP, thrombospondin.
$3.00
39 All
Copyright 0 1990 rights of reproductmr,
0014~4827/90 by Academic Press, Inc. in any form reserved.
40
LYONS-GIORDANO,
BRINKER,
els. The effect of heparin on EC FN synthesis shows a high correlation to its enhancement of growth with respect to concentration dependency. These data suggest a potential role for heparin in the remodeling of the extracellular matrix concomitant with neovascularization. MATERIALS
AND
METHODS
Cell culture. Human umbilical vein EC were obtained as described previously [21]. Primary and subcultured EC cultures were grown in complete culture medium (CCM): Modified Ml99 growth medium containing 16.5% FCS, 2.0 mM glutamine, 15 mM Hepes, 10 @g/ml gentamycin, 0.4 fig/ml amphotericin B, and 30 pg/ml ECGF (Meloy Laboratories, Inc., Springfield, VA) in the absence and presence of heparin from porcine intestinal mucosa (Sigma H3125, St. Louis, MO). EC were maintained at 37°C in a humidified 5% CO, atmosphere. Analyses of matrix protein biosynthesis and steady-state mRNA levels were conducted using cells passaged one to three times. For the 22-h metabolic labeling studies, culMetabolic labeling. tures were incubated in medium containing either radiolabeled proline or radiolabeled methionine. In studies using [14C]proline labeling, subconfluent cultures were washed twice with PBS and incubated at 37°C with 4 &i/ml [14C]proline (250 mCi/mmol; Amersham Corp., Arlington Heights, IL) in MEM supplemented with 0.1% glucose, 1 mM glutamine, 50 pg/ml ascorbic acid, 50 pg/ml &aminoproprionitrile fumarate, 15 mM Hepes, and in the absence and presence of various concentrations of heparin. For pulse-labeling studies of the effect of heparin on FN and TSP biosynthesis, cultures were incubated for 2 h at 37°C with 40 pCi/ml [a5S]methionine (tran.s-35S-labeled; 1090 Ci/ mmol; ICN Radiochemicals, Irvine, CA) in methionine-free DMEM supplemented as described for [14C]proline labeling. For all analyses, medium and cell-matrix fractions were collected separately. Prior to analysis of radiolabeled proteins in the medium, protease inhibitors, N-ethylmaleimide (1 m&f) and PMSF (1 mM), were added to the medium. After removal of the medium, the cell monolayers were washed three times with cold (4°C) PBS. The cell-matrix fraction was then extracted with 62.5 mM Tris-HCl (pH 6.8) containing 4 M urea and 2% SDS. Cell debris present in the medium was sedimented by centrifugation at 500g for 20 min at 4°C. The medium fractions were dialyzed exhaustively against 0.5 M acetic acid at 4°C and then lyophilized prior to analysis by SDS-PAGE. SDS-PAGE. The media, matrix, and cell-matrix proteins were analyzed by SDS-PAGE using 3% stacking and 5% resolving gels [22]. Prior to analysis the lyophilized medium samples were dissolved in 62.5 mM Tris-HCl (pH 6.8) containing 4 M urea, 2% SDS, 10% glycerol, 0.0025% bromphenol blue, and 5% 2-mercaptoethanol. The cellmatrix samples were reduced prior to electrophoresis by addition of 5% 2-mercaptoethanol. The gels were stained with 0.25% Coomassie brilliant blue in 20% TCA and destained in 7% acetic acid-15% methanol mixture. The gels were prepared for fluorography using En3Hance (New England Nuclear, Boston, MA). The gels were dried and exposed to Kodak XAR-5 film at -70°C. For quantitation of FN and TSP in the medium, cultures ELISA. were incubated in MEM supplemented as described for metabolic labeling. After the medium was collected and centrifuged, it was dialyzed against PBS containing 0.05% Tween. FN and TSP levels in the medium were quantitated by indirect ELISA as described by Rennard [23]. Human FN standard was purchased from Biomedical Technologies, Inc. (Cambridge, MA). Rabbit antibody against human FN was purchased from Calbiochem-Behring (San Diego, CA). TSP standard and rabbit anti-human TSP antibody were a gift from Dr. George Tuszynski, Lankenau Hospital (Philadelphia, PA). Goat anti-rabbit IgG conjugated to alkaline phosphatase was purchased from Sigma (A8025, St. Louis, MO) as was p-nitrophenyl phosphate (Sigma 104-O). Radiolabeled FN and TSP were immunoImmunoprecipitation. precipitated from the combined medium (which had been dialyzed
AND
KEFALIDES
against water) and cell-matrix fractions recovered from metabolically labeled EC grown in the absence and presence of 90 pg/ml heparin. For immunoprecipitation, samples (1 X lOa cpm) in 400 ~1 of 23.4 mM Tris-HCl (pH 6.8) containing 1.5 M urea and 0.75% SDS were mixed with an equal volume of immunoprecipitation buffer: PBS (pH 7.4) containing 0.5% Tween 20,0.1% BSA, and 0.02% sodium azide. Rabbit antibody (30 ~1) specific for human FN or for human TSP was added to the samples. To assess nonspecific binding, samples were also treated with normal rabbit serum. Following an overnight incubation at 4”C, antibody-antigen complexes were precipitated using 70 ~1 of swelled protein A-Sepharose. The precipitate was washed twice with immunoprecipitation buffer and then reconstituted for analysis by SDS-PAGE. Cell proliferation assay. During the first passage of EC, approximately 7.0 X lo4 cells were seeded into a 9.5-cm2 well in CCM. Cells to be treated with heparin were allowed to attach after seeding, prior to addition of the reagent (approximately 3 h postseeding), so that potential variations in attachment efficiency would not affect the calculated proliferation. The cells were incubated for 3 days at 37°C in a humidified atmosphere of 5% CO, in air and then trypsinized and counted using a Coulter counter. The trypsinized cultures were examined by microscopy to ensure complete removal of the cells and lack of lysis by the process. The net growth was calculated by subtracting the product of the number of cells seeded and the attachment efficiency from the number of cells counted at the termination of the experiment. The percentage stimulation of proliferation was determined: Percentage
stimulation = 100 x
The attachment CCM was found
of proliferation net growth
in heparin - net growth net growth in control
efficiency of EC plated onto experimentally to be 95%.
a plastic
in control
substrate
in
RNA isolation. For isolation of total RNA, 50 pg/ml ascorbic acid and 50 pg/ml @-aminoproprionitrile fumerate were added to EC cultured in CCM in the absence or presence of heparin concentrations ranging from 0.09 to 90 fig/ml 24 h prior to harvesting. At the time of harvest, the cell layer was removed by trypsinization and the cells were washed with PBS. The cells were then lyzed, homogenized with a glass/Teflon homogenizer, and incubated in 10 mM Tris-HCl (pH 7.5), 5 mMEDTA, and 0.5% SDS buffer containing 125 pg/ml proteinase K (Boehringer-Mannheim Biochemicals, Indianapolis, IN) for 90 min at 42°C. Total cellular RNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1), selectively precipitated with 3 M sodium acetate, 5 mM EDTA, pH 7.0, and then washed with ethanol as adapted from the method described by Rowe et al. [24]. RNA levels were quantitated by spectrophotometry at 260 nm. Approximately 12 pg of total RNA was recovered per 106EC, independent of the presence of heparin. RNA blotting and hybridization. Total cellular RNA (0.16-0.64 pg/ slot) was applied to nitrocellulose using the Minifold II slot-blot apparatus and the procedure of Schleicher and Schuell, Inc. (Keene, NH) [25]. The nitrocellulose filters were baked for 2 h at 78°C under vacuum. For hybridization analysis, cDNA probes were nick translated [26] with [a-32P]dCTP and [cu-32P]dTTP (3000 Ci/mmol; Amersham, Arlington Heights, IL) without excision from the plasmid to a specific activity of 5-8 X 10’ cpm/pg. The filters were prehybridized overnight and then hybridized with the denatured labeled probes for 22 h at 42°C in a solution containing 50% formamide, 5X standard saline citrate (SSC; 0.15 MNaCI, 0.015 Mtrisodium citrate), 1 mMEDTA, 2X Denhardt’s solution [27], 100 Fg/ml salmon sperm DNA, and 0.1% SDS. The filters were then washed with a final stringency of 0.5X SSC containing 0.1% SDS at 65°C. The filters were exposed to Kodak XAR-5 film with a DuPont Cronex Lightening Plus intensifying screen at -70°C for various lengths of time. Radioactivity hybridizing to RNA on the filters was quantitated by scanning densitometry of the autoradiograms, with the band intensities being within the linear response
HEPARIN
EFFECT
TABLE
ON
FIBRONECTIN
1
Heparin Effects on EC Medium FN and TSP Levels
Substrate
Heparin concentration (M/mu
FN (pg/106 cells)
TSP (fig/lo6 cells)
Gelatin
0 30 180
4.63 + 0.40 (4) 1.93 i 0.26 (4) 2.35 i 0.11 (4)
39.3 2 0.75 (2) 40.1 + 2.70 (2) 37.7 k 2.50 (3)
Plastic
0 30 180
9.32 i 0.29 (2) 4.36 i 0.07 (2) 4.51 t 0.10 (2)
55.7 Ifr 5.70 (2) 59.2 t 0.10 (2) 71.0 2 3.60 (2)
Note. EC were grown on gelatin or plastic substrates in the absence or presence of heparin. For determination of FN and TSP levels in the medium, cells were incubated in serum-free medium for 22 h. At the termination of incubation, the medium was removed and processed for competitive ELISA analysis as described under Materials and Methods. The cells were trypsinized and counted. The heparin concentrations shown were present both during the growth of the cells and in the serum-free incubation. The numbers in parentheses indicate the number of experiments. The data represent the mean + standard deviation.
range of the film. The levels of radioactivity were linear with respect to the quantity of total RNA appiied to the filters. CDNA probes. The probes used included the cDNA clone pFHl containing a 2.1-kb insert encoding about 1300 bp of the coding region plus the complete 3’ untranslated region of human FN mRNA, which was obtained from Dr. Francisco Baralle, University of Oxford [28]. The hybridization probe for TSP was a cDNA clone with a 1.9-kb insert which specifies the entire heparin-binding domain and part of the type V collagen binding domain and was a gift from Dr. Vishva Dixit, University of Michigan [29]. Human fibroblast actin and tubulin cDNA clones were provided by Dr. Larry Kedes, Stanford University. The 1.8-kb actin insert includes sequences from the coding region up to amino acid 145 plus the complete 3’ untranslated region of the message. It hybridizes to both the y- and P-actin mRNAs [30]. The fl-tubulin cDNA clone pHFPT-1, with its 2.1-kb insert, is virtually a full-length clone specific for fi-tubulin mRNAs. This clone has the same sequences as the P-tubulin cDNA clone (D/3-1) described by Hall et al. [31].
RESULTS FN and TSP levels in the medium from heparintreated and control EC grown on plastic were analyzed by ELISA. As shown in Table I, EC cultivated in the presence of 90 or 180 pg/ml heparin exhibited a greater than 50% decrease in FN levels, while no change in TSP levels was evident. To eliminate the possibility that the heparin effect reflected heparin interference in the ELISA, FN levels were compared in control culture medium supplemented with heparin (90 pg/ml) and in unsupplemented medium. The presence of heparin did not interfere with the ELISA quantitation since FN levels were similar under both conditions. Both ELISA analyses and metabolic labeling studies demonstrated that heparin decreased FN levels (data not shown). To assess whether the heparin effect on FN levels was modulated by the substrate upon which the cells were
AND
THROMBOSPONDIN
SVNTHESIS
41
cultivated, EC were seeded in culture flasks coated with 1% gelatin in PBS and grown in CC in either the absence or the presence of heparin. Tine levels of TSP in the medium recovered after ~~c~bati~~ serum for 22 h were determined using ELISA. The data are summarized in Table 1. Like be~a~~~-t,reated EC grown on plastic, heparin-treated EC grown cm gelatin exhibited a greater than 50% ease in FN levels, whereas the TSP levels were una cted. The substrate upon which the cells were cultivated did not modulate the capacity of heparin to decrease levels of FN. Ail subsequent analyses were conducted using EC grown on plastic. Studies of the Effect of Heparin Biosynthesis o,fFN
on EC
The kinetics of FN synthesis and been extensively studied using cultured chick embryo fibroblasts 1321. FN has a half-life of 36 h on the surface of these cells. A significant loss of cell surface FN was found to occur by release of the protein into the medium. Once released into the medium, FN accumulates over time since it is not graded by proteolysis and very little (~10%) of the m m FN becomes reassociated with the cell surface. Given these findings, the els of FN in the medium of heparin-treated EC observed following a 22-h incubation is most likely a consequence of lowered biosynthesis of the protein rather ‘than an increased rate of degradation. To verify this assu EC cultures grown in the absence or presence o ml heparin were pulse-labeled for 2 h with [“‘§I nine. Medium and cell-matrix fractions were combined for analysis by SDS-PA . As shown in Fig. 1; the level of radiolabeled FN was reased in the medium-cellmatrix fraction from EC treated with hepa ning densitometry, it was determined that ment resulted in a 50% reduction in r contrast, the level of radiolabeled TS by heparin treatment. ~mmuno~reci~~tai~i~~ using antibodies specific for FN corroborated th’e apparent decrease in total radiolabeled FN in ~~~ari~-treated EC (Fig. ZA). Likewise, immu~opreci~itat~or~ using antibodies specific for TSP showed no decrease in radiolabeled TSP in the combined medium-cell,rix as a conse.quence of heparin treatment (Fig. f, Thus, using pulse-labeling conditions to minimize t effects of heparin on the rates of degradation of FN an TSP, the levels of radiolabeled FN were still decreased i heparin-treated EC cultures, wbereas ~a~~l~beled TSP levels were not. These a further substantiate the premise that heparin dec ses EC synthesis of FN but not that of TSP. ECGF Modulation
oy’ Heparin
Effect
The preceding studies of the effect of heparin synthesis were conducted using cells culture
on FN in the
42
LYONS-GIORDANO,
BRINKER.
AND
KEFALIDES
and ECGF was dependent upon the presence of ECGF in the culture medium (Fig. 3). The difficulty in maintaining EC in culture medium without exogenous ECGF as well as the fact that EC synthesize ECGF [20] pose a problem in assessingthe effects of heparin, independent of ECGF, on EC biosynthetic phenotype. FN -
TSP
Concentration Dependence of the Heparin Effect on FN Synthesis EC synthesis of FN in response to different concentrations of heparin was quantitated using ELISA. EC were incubated for 12 h in serum-free medium in the presence of heparin concentrations ranging from 0.09 to 90 pg/ml. FN levels per lo6 cells in the heparin-treated cultures were normalized as percentage inhibition relative to the levels in untreated cultures. The percentage inhibition of FN synthesis observed with each heparin concentration was plotted as a function of heparin concentration as shown in Fig. 4. By interpolation, a half-maximal response would be expected with 2.5 pg/ml heparin. Maximal heparin-induced reduction of FN levels was observed with a heparin concentration of 90 pg/ml. No increase in suppression was evident in cultures treated with 180 pg/ml heparin as shown in Table 1. No effect on TSP levels was detected by ELISA over the-same range of heparin concentrations (data not shown).
-
Correlation of the Heparin Inhibitory Effect on FN Synthesis and the Heparin Stimulatory Effect on EC Proliferation
HEPARIN FIG. 1. Heparin synthesis of FN and EC was compared by [%]methionine. For and heparin-treated contains 10,000 cpm.
-
+
effects on EC biosynthesis of FN and TSP. The TSP by control and heparin-treated (90 pg/ml) pulse-labeling the cultures for 2 h with 40 &X/ml analysis, total radiolabeledproteins from control cultures were resolved by SDS-PAGE. Each lane
presence of 30 pg/ml ECGF. Heparin is known to modulate the activity of ECGF. Specifically, heparin enhances the mitogenic efficacy of this peptide [2], potentiates its chemotactic activity [31, and increases the affinity of the peptide for its receptor [33]. To assesswhether the effect of heparin on FN synthesis was modulated by ECGF, cells grown in CCM lacking ECGF were treated with 90 pg/ml heparin in the absence and presence of 30 pgjml ECGF for 24 h. Control cells were grown in medium containing ECGF but lacking heparin. After 24 h, the cells were labeled with [14C]proline for 22 h. Radiolabeled proteins recovered in the medium and cell-matrix fractions were resolved by SDS-PAGE and visualized by fluorography. Heparin and/or ECGF did not affect cellmatrix levels of FN (data not shown). The decrease in FN synthesis by cells cultured in the presence of heparin
The concentration dependence of heparin-induced EC proliferation is presented in Fig. 4. The percentage stimulation of proliferation was calculated as described under Materials and Methods. The concentration of heparin expected to yield half-maximal stimulation of proliferation was 2.5 pg/ml. This was the same concentration as that required for half-maximal inhibition of FN synthesis. The correlation coefficient for heparin-induced stimulation of EC proliferation and inhibition of FN synthesis was determined to be -0.94 by correlation analysis. Effects of Heparin on FN and TSP mRNA Levels The rates of synthesis of extracellular matrix components may be regulated through changes in the steadystate level of their mRNAs, effects on the translatability of these mRNAs, or alterations in the rate of intracellular degradation of the newly synthesized proteins. To elucidate the mechanism by which heparin reduced EC synthesis of FN, the effect of heparin on FN mRNA levels was assessed.Total RNA was prepared from subconfluent EC grown in CCM in the absence or presence of concentrations of heparin ranging from 0.09 to 90 pg/ml. For analysis, equivalent amounts of RNA from heparintreated and control EC were applied to nitrocellulose us-
IIEPARIN
EFFECT
ON
FIBRONECTIN
AND
THR~~~~SPQND~N
SYNTHESIS
43
FN
TSP
HEPARIN FIG. 2.
-
+
Immunoprecipitation of newly synthesized or TSP in the medium-cell-matrix fraction from control nine was immunoprecipitated using specific antibodies. respectively, using antibody to FN or TSP. Lanes 3 and using preimmune serum. The amount of radiolabeled FN and by scintillation counting of the immunoprecipitates.
-
+
HEPARlN
-
FN (A) and TSP (B) from control and heparin-treated EC cultures. Radiolabeled FN and heparin-treated cultures following a 2-h pulse-labeling with 40 &i/ml [“%]methioLanes 1 and 2 contain immunoprecipitates from control and heparin-treated cultures, 4 contain immunoprecipitates from control and heparin-treated cultures, respectively, or TSP immunoprecipitated was assessed by scanning densitometry of the fluorogram
ing a slot-blot apparatus and hybridized with a 32P-labeled cDNA probe specific for FN. Concomitantly, hybridizations using radiolabeled cDNA probes for TSP, actin, and tubulin messageswere performed to assessthe specificity of the effects of heparin. Typical autoradiograms of slot-blotted RNA from EC grown in the absence or presence of increasing concentrations of heparin when hybridized with cDNA probes specific for FN or TSP are shown in Figs. 5A and 5B, respectively. Heparin caused a concentration-dependent decrease in FN and TSP messagelevels (Fig. 6). Maximal inhibition of both FN and TSP mRNA levels was observed with EC treated with 9.0 pg/ml heparin. Maximal heparin-induced suppression of FN mRNA was approximately 85% whereas that of TSP mRNA was 40%. By interpolation, the concentrations of heparin expected to yield halfmaximal suppression of FN and TSP mRNA levels were
the same (2.6 pg/ml). This concentration approximated that requisite for half-maximal inh tion of EC FN synthesis by heparin (Fig. 4). Hepari ffected FN protein and mRNA levels in a coordinate manner. fn contrast, heparin suppression of TSP mRNA Ieve accompanied by decreased TSP synthesis i res (Figs. 1 and 2B). The effects of heparin on SP mRNA levels were not a consequence of a nonspecific suppression of RNA since approximately equal a of total RNA/cell were recovered fr beparin and control cultures. Also, actm a -tubuhn levels were not affected by treatment of EC with the same range of heparin concentrations to assay the concentration dependence of heparin s ession of FN and TSP mRNA levels. In addition, the specificity of the hybridization reaction was verified by the lac ization of EC RNA to the cDNA clone
44
LYONS-G10
)RDANO.
BRI :NKER,
Medium
FN
TSP
AND
KEFALIDES
substrate, including adhesion, cytoskeletal organization, migration, proliferation, and differentiation [36]. Enhanced migration and proliferation of the endothelium are fundamental events in the process of angiogenesis [37]. Despite the current view that FN and other extracellular matrix components play a central role in the regulation of these events, limited data are available concerning the effects of angiogenic factors on EC synthesis of extracellular matrix components. The data presented here provide the first evidence that heparin, an angiogenic factor [5], inhibits EC FN synthesis. EC synthesis of TSP, an extracellular matrix component believed to be important in the regulation of smooth muscle cell growth [38] and in the attachment of a variety of cells, including cells of mesenchymal origin [39], is not affected by heparin treatment. The inhibition of FN synthesis by heparin is independent of the substrate upon which the cells are cultivated but is modulated by the presence of exogenous ECGF in the culture medium. A requirement for ECGF has previously been reported for enhancement of EC migration and proliferation by heparin [2, 31. The possibility that the growth regulatory effects of heparin in the presence of ECGF
-
-
120
250
200 .-5 5 $ z 150
+
ECGF
+
-
E 60
Heparin
-
+
procu2(1) collagen mRNA [34] since EC do not synthesize collagen type I. Moreover, a Northern blot of the same RNA was included in each hybridization reaction to further ensure the specificity. Finally, hybridization probe
PA,
a genomic
clone
E 100
+
FIG. 3. ECGF modulation of heparin effect. EC grown in CCM in the absence and presence of heparin (90 rig/ml) or grown in CCM lacking ECGF but supplemented with heparin (90 pg/ml) were labeled for 22 h with [14C]proline. Cells grown in the presence of heparin were labeled in the presence of heparin. ECGF was not present during the labeling of the cultures. Medium proteins visualized by SDS-PAGE and fluorography are shown. Each lane contains 10,000 cpm.
to the
encod&?
g
most
of the
human 28s rDNA gene [35], confirmed that equal amounts of RNA from control and heparin-treated cultures were bound to the nitrocellulose filters. DISCUSSION
Fibronectin has been implicated in the regulation of activities involving the interaction of the cell with its
;
40 50
20 .o 1
I
1”“‘1
.I
1
Heparin
r “““I
“““I 10
100
r *-
0 1,000
(vg/ml)
Concentration dependence of heparin effects on EC FN synthesis and proliferation. EC cultivated in the absence or presence of heparin concentrations ranging from 0.09 to 90 pg/ml were incubated for 12 h in serum-free medium supplemented with the same concentration of heparin used to culture the cells. The FN level in the medium was then quantitated by ELISA and the cells were counted. Values for FN levels in control and treated cultures were compared after normalization by the cell number in the respective cultures. To assay the concentration dependence of heparin-augmented proliferation, cells were cultured in the presence of heparin concentrations ranging from 0.09 to 90 pg/ml for 3 days and then trypsinized and counted. Data are presented as a percentage of control cell proliferation (+) and FN (U) synthesis.
HEPARIN
5
%
EFFECT
b
ON
FIBRONECTIN
C
AND
THROMBOSPONDIN
45
SYNTHESIS
affected by heparin treat centration response ana nate, concentration-depe and TSP messages. The of the FN steady-state m than that of the TSP mRNA level. The s FN mRNA levels by heparin correlated tion of FN synthesis with respect to the ~o~~e~tratio~ dependence of the phenomena. that heparin decreases EC expres 1 part by decreasing the amount of for translation. This decrease ma e a consequence of reduced transcription and/or increased degradation of FN mRNA. These results do not preclude the possibility that heparin lowers FN synthesis by reducing the eEiciency of translation of FN effect of heparin on steady-state message levels for FN and TSP suggests that heparin may control the activity of cellular elements which regulate their RNA levels in a concerted manner. At present little is known about the gene expression Previous work in our Iaborat demonstrated that heparin increases human vascular smooth muscle cell synthesis of increased synthesis is reflected TSP mRNA [44]. The failure of expression, alth h it reduces TS points to the pos lity that the rate of EC! synthesis of
120
FIG. 5. Heparin effects on FN and TSP mRNA levels in EC. Total cellular RNA from cells grown in the absence (row 1) or presence ofO.09 (row Z), 0.9 (row 3), 9 (row 4), and 90 (row 5) pg/ml of heparin was applied to nitrocellulose filters using a slot-blot apparatus. The RNA was hybridized with either the 32P-labeled cDNA probe for (A) FN or (B) TSP (see Materials and Methods). The autoradiograms are representative of hybridizations from which the data in Fig. 6 were obtained. RNA/slot: (a) 0.16, (b) 0.32, and (c) 0.64 pg.
are related to the biosynthetic modulatory effects of the combined factors is further suggested by the high correlation between the concentration dependence of the two phenomena. Other studies provide precedents for translational as well as transcriptional regulation of FN gene expression [40-421. Previous work from our laboratory has demonstrated that heparin increases human vascular smooth muscle cell synthesis of FN 1431 and that this increase is independent of changes in steady-state FN mRNA levels [44]. To elucidate the mechanism by which heparin suppresses EC synthesis of FN, the steady-state levels of FN mRNA in control and heparin-treated cells were determined. For comparison, the levels of TSP transcripts were likewise assessed since EC TSP synthesis was not
100 I
100
Heparin
(q$mE)
FIG. 6. Quantitation of the concentration dependence of beparin suppression of FN and TSP mRNA levels in EC. The mRNA levels of FN (m), TSP (@), actin (Cl), and P-tubulin (0) in EC grown in the presence of increasing concentrations of heparin were determined by scanning densitometry of autoradiograms following hybridization of total cellular RNA to the specific 32P-labeled cDNA probes. The percentage of each message relative to that in control cells is plotted as a function of heparin concentration present in the culture medium.
46
LYONS-GIORDANO,
BRINKER,
AND
KEFALIDES
TSP is regulated at the translational or post-translational level. The data presented herein demonstrate that heparin, in the presence of ECGF, induces changes in EC secretory phenotype concomitantly with its stimulation of EC growth in uitro. In uivo the combined effects of ECGF and heparin on EC growth and matrix protein biosynthesis may be particularly important in the repair of the vessel wall following injury or in events leading to neovascularization. Local increases in the concentrations of heparin or heparin-like molecules may increase the availability of matrix-associated ECGF to EC near the damaged area and facilitate the restoration of vessel wall integrity.
17.
Gospodarowicz, D., Greenburg, G., Foidart, (1981) J. Cell. Physiol. 107,171-183.
18.
McPherson, J., Sage, H., and Bornstein, P. (1981) J. Biol. Chem. 256,11330-11336. Mosher, D. E., Doyle, M. J., and Jaffe, E. A. (1982) J. Cell Biol. 93,343-348. Vlodavsky, J., Folkman, J., Sullivan, R., Fridman, R., Ishai-Michaeli, R., Sasse, J., and Klagsburn, M. (1987) Proc. N&l. Acud. Sci. USA 84,2292-2296. Gimbrone, M. A., Jr., Cotran, R. S., and Folkman, J. (1974) J. Cell Biol. 60,673-684. Laemmli, U. K. (1970) Nature (London) 227,680-685.
The expert technical assistance of Helen Conaway is gratefully acknowledged. This work was supported in part by NIH Grants AR20553, HL-29492, and HL-07502 and an award from the W. W. Smith Charitable Trust.
25.
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Olden, K., and Yamada, K. M. (1977) Cell 11,957-969. Schreiber, A. B., Kenney, J., Friesel, R., Mehlman, T., and iag, T. (1985) Proc. Natl. Acad. Sci. USA 82,6138-6142. Myers, J. C., Chu, M.-L., Faro, S. H., Clark, W. J., Prockop, and Ramirez, F. (1981) Proc. N&l. Acad. Sci. USA 78, 3520. Erickson, J. M., Rushford, C. L., Dorney, D. J., Wilson, and Schmickel, R. D. (1981) Gene 16,1-g.
23,
MacD. J., 3516G. N.,
Hynes, R. O., and Yamada, K. (1982) J. Cell Biol. 95,369-377. Folkman, J. (1983) Ann. N. Y. Acad. Sci. 401,212-227. Majack, R. A., Cook, S. C., and Bornstein, P. (1986) Proc. Natl. Acad. Sci. USA 83,9050-9053. Tuszynski, G. P., Rothman, V., Murphy, A., Siegler, K., Smith, L., Smith, S., Karczewski, J., and Knudsen, K. A. (1987) Science 236,1570-1573. Fagan, J. B., Sobel, M. E., Yamada, K. M., de Crombrugghe, B., and Pastan, I. (1981) J. Biol. Chem. 256,520-525. Tyagi, J. S., Hirano, H., Merlino, G. T., and Pastan, I. (1983) J. Biol. Chem. 258,5787-5793. Adams, S. L., Pacifici, M., Boettiger, D., and Pallante, K. M. (1987) J. Cell Biol. 105,483-488. Lyons-Giordano, B., Conaway, H., and Kefalides, N. A. (1987) Biochem. Biophys. Res. Commun. 148,1264-1269. Lyons-Giordano, B., Brinker, J. M., and Kefalides, N. A. (1989) Biochem. Biophys. Res. Commun. 162,1100-1104.
186,X-46
(1990)
The Effect of Heparin on Fibronectin and 16 d mRNA Levels in Cultured Human Endothelia BERNADETTE Connective
LYONS-GIORDANO,
Tissue
JANE M. BRINKER,
AND NICHOLAS
A. ~E~~~~~E~'
Research Institute and Departments of Medicine, and Biociaemistry University of Pennsylvania, Philadelphia, Pennsylvania 19104
and Biophysics,
potential importance of heparin in the regulation of new vessel formation. Bn vitro heparin potentiates human umbilical vein EC chemotactie an endothelial cell growth factor (ECGF), a member sf the fibroblast growth factor family of mitogens 12, 31. The growth and migration stimulator-y a6tiVity of heparin is neutralized by protamine, an arginine-rich basic protein which is known to bind avidly to heparin [2, 41. In vivo local administration of protamine inhibits neovascularization induced by inflammatory agents or by immune reactions. Goadministration of heparin reverses the effect of protamine [5]. The mechanism of action of heparin in the regulation of EC migration and growth has not been elucidated. Studies investigating the interaction of heparin with EC have shown that it is specifically bound to and internalized by these cells [6, 71. While these data suggest that the mode of heparin regulation of C behavior may be directly through plasma membrane rece s, the effect of heparin on human umbilical vein EC avior in the absence of ECGF provides evidence for an indirect aetion of heparin. In the absence of ECGF and at low serum concentrations, heparin inhibits the growth of these cells [8]. Likewise, it lacks chemotactic akptivity for endothelium in the absence of ECGF [3]. These data demonstrate the importance of recognizing the dinate nature of the re lation of EC beh genie factors. Previous reports have demonstra the importance of extracellular matrix components he regulation of a variety of cellular activities ~~cIndi~~ growth [9]. In culture, EC synthesize an extraeellular matrix which contains proteogly6ans [lo, IlIp collagens [X2, 131, and other glycoproteins including fibr~necti~ (FIT) [14-163, laminin [ 171)and thrombospondin (TS dition, it has recently been reported t fibroblast growth factor which they matrix [ZO]. In this report, we provid onstrate that heparin, in the present a specific decrease in FN synthesis by EC compared to ECGF-treated control cells. This decrease is accompanied by a coordinate decrease in EC for FN. Heparin does not change these cells although it does reduce their TSP mRNA lev-
Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 pg/ ml) and endotbelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No ehange in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of beparin on EC FN synthesis was independent of .whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cellls. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-tranSlatiOnally regulated. 0 1990 Academic~ress, IIIC.
INTRQDUGTION Vascular endothelial cells (EC)2 are in a quiescent growth state in the healthy blood vessel. In response to vessel wall injury, these cells undergo proliferation in the sequence of events which lead to angiogenesis. Elucidation of the mechanism(s) by which EC! growth is stimulated in vitro may provide insight into the physiology of vascular pathology. Since the initial observations that mast cells containing heparin accumulate at tumor sites prior to neovascularization [ 11, numerous studies have underscored the I To whom reprint requests should be addressed at: Connective Tissue Research Institute, 3624 Market St., Philadelphia, PA 19104. ’ Abbreviations used: CCM, complete culture medium; EC, endothelial cell; ECGF, endothelial cell growth factor; FN, fibronectin; SSC, standard saline citrate; TSP, thrombospondin.
$3.00
39 All
Copyright 0 1990 rights of reproductmr,
0014~4827/90 by Academic Press, Inc. in any form reserved.
40
LYONS-GIORDANO,
BRINKER,
els. The effect of heparin on EC FN synthesis shows a high correlation to its enhancement of growth with respect to concentration dependency. These data suggest a potential role for heparin in the remodeling of the extracellular matrix concomitant with neovascularization. MATERIALS
AND
METHODS
Cell culture. Human umbilical vein EC were obtained as described previously [21]. Primary and subcultured EC cultures were grown in complete culture medium (CCM): Modified Ml99 growth medium containing 16.5% FCS, 2.0 mM glutamine, 15 mM Hepes, 10 @g/ml gentamycin, 0.4 fig/ml amphotericin B, and 30 pg/ml ECGF (Meloy Laboratories, Inc., Springfield, VA) in the absence and presence of heparin from porcine intestinal mucosa (Sigma H3125, St. Louis, MO). EC were maintained at 37°C in a humidified 5% CO, atmosphere. Analyses of matrix protein biosynthesis and steady-state mRNA levels were conducted using cells passaged one to three times. For the 22-h metabolic labeling studies, culMetabolic labeling. tures were incubated in medium containing either radiolabeled proline or radiolabeled methionine. In studies using [14C]proline labeling, subconfluent cultures were washed twice with PBS and incubated at 37°C with 4 &i/ml [14C]proline (250 mCi/mmol; Amersham Corp., Arlington Heights, IL) in MEM supplemented with 0.1% glucose, 1 mM glutamine, 50 pg/ml ascorbic acid, 50 pg/ml &aminoproprionitrile fumarate, 15 mM Hepes, and in the absence and presence of various concentrations of heparin. For pulse-labeling studies of the effect of heparin on FN and TSP biosynthesis, cultures were incubated for 2 h at 37°C with 40 pCi/ml [a5S]methionine (tran.s-35S-labeled; 1090 Ci/ mmol; ICN Radiochemicals, Irvine, CA) in methionine-free DMEM supplemented as described for [14C]proline labeling. For all analyses, medium and cell-matrix fractions were collected separately. Prior to analysis of radiolabeled proteins in the medium, protease inhibitors, N-ethylmaleimide (1 m&f) and PMSF (1 mM), were added to the medium. After removal of the medium, the cell monolayers were washed three times with cold (4°C) PBS. The cell-matrix fraction was then extracted with 62.5 mM Tris-HCl (pH 6.8) containing 4 M urea and 2% SDS. Cell debris present in the medium was sedimented by centrifugation at 500g for 20 min at 4°C. The medium fractions were dialyzed exhaustively against 0.5 M acetic acid at 4°C and then lyophilized prior to analysis by SDS-PAGE. SDS-PAGE. The media, matrix, and cell-matrix proteins were analyzed by SDS-PAGE using 3% stacking and 5% resolving gels [22]. Prior to analysis the lyophilized medium samples were dissolved in 62.5 mM Tris-HCl (pH 6.8) containing 4 M urea, 2% SDS, 10% glycerol, 0.0025% bromphenol blue, and 5% 2-mercaptoethanol. The cellmatrix samples were reduced prior to electrophoresis by addition of 5% 2-mercaptoethanol. The gels were stained with 0.25% Coomassie brilliant blue in 20% TCA and destained in 7% acetic acid-15% methanol mixture. The gels were prepared for fluorography using En3Hance (New England Nuclear, Boston, MA). The gels were dried and exposed to Kodak XAR-5 film at -70°C. For quantitation of FN and TSP in the medium, cultures ELISA. were incubated in MEM supplemented as described for metabolic labeling. After the medium was collected and centrifuged, it was dialyzed against PBS containing 0.05% Tween. FN and TSP levels in the medium were quantitated by indirect ELISA as described by Rennard [23]. Human FN standard was purchased from Biomedical Technologies, Inc. (Cambridge, MA). Rabbit antibody against human FN was purchased from Calbiochem-Behring (San Diego, CA). TSP standard and rabbit anti-human TSP antibody were a gift from Dr. George Tuszynski, Lankenau Hospital (Philadelphia, PA). Goat anti-rabbit IgG conjugated to alkaline phosphatase was purchased from Sigma (A8025, St. Louis, MO) as was p-nitrophenyl phosphate (Sigma 104-O). Radiolabeled FN and TSP were immunoImmunoprecipitation. precipitated from the combined medium (which had been dialyzed
AND
KEFALIDES
against water) and cell-matrix fractions recovered from metabolically labeled EC grown in the absence and presence of 90 pg/ml heparin. For immunoprecipitation, samples (1 X lOa cpm) in 400 ~1 of 23.4 mM Tris-HCl (pH 6.8) containing 1.5 M urea and 0.75% SDS were mixed with an equal volume of immunoprecipitation buffer: PBS (pH 7.4) containing 0.5% Tween 20,0.1% BSA, and 0.02% sodium azide. Rabbit antibody (30 ~1) specific for human FN or for human TSP was added to the samples. To assess nonspecific binding, samples were also treated with normal rabbit serum. Following an overnight incubation at 4”C, antibody-antigen complexes were precipitated using 70 ~1 of swelled protein A-Sepharose. The precipitate was washed twice with immunoprecipitation buffer and then reconstituted for analysis by SDS-PAGE. Cell proliferation assay. During the first passage of EC, approximately 7.0 X lo4 cells were seeded into a 9.5-cm2 well in CCM. Cells to be treated with heparin were allowed to attach after seeding, prior to addition of the reagent (approximately 3 h postseeding), so that potential variations in attachment efficiency would not affect the calculated proliferation. The cells were incubated for 3 days at 37°C in a humidified atmosphere of 5% CO, in air and then trypsinized and counted using a Coulter counter. The trypsinized cultures were examined by microscopy to ensure complete removal of the cells and lack of lysis by the process. The net growth was calculated by subtracting the product of the number of cells seeded and the attachment efficiency from the number of cells counted at the termination of the experiment. The percentage stimulation of proliferation was determined: Percentage
stimulation = 100 x
The attachment CCM was found
of proliferation net growth
in heparin - net growth net growth in control
efficiency of EC plated onto experimentally to be 95%.
a plastic
in control
substrate
in
RNA isolation. For isolation of total RNA, 50 pg/ml ascorbic acid and 50 pg/ml @-aminoproprionitrile fumerate were added to EC cultured in CCM in the absence or presence of heparin concentrations ranging from 0.09 to 90 fig/ml 24 h prior to harvesting. At the time of harvest, the cell layer was removed by trypsinization and the cells were washed with PBS. The cells were then lyzed, homogenized with a glass/Teflon homogenizer, and incubated in 10 mM Tris-HCl (pH 7.5), 5 mMEDTA, and 0.5% SDS buffer containing 125 pg/ml proteinase K (Boehringer-Mannheim Biochemicals, Indianapolis, IN) for 90 min at 42°C. Total cellular RNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1), selectively precipitated with 3 M sodium acetate, 5 mM EDTA, pH 7.0, and then washed with ethanol as adapted from the method described by Rowe et al. [24]. RNA levels were quantitated by spectrophotometry at 260 nm. Approximately 12 pg of total RNA was recovered per 106EC, independent of the presence of heparin. RNA blotting and hybridization. Total cellular RNA (0.16-0.64 pg/ slot) was applied to nitrocellulose using the Minifold II slot-blot apparatus and the procedure of Schleicher and Schuell, Inc. (Keene, NH) [25]. The nitrocellulose filters were baked for 2 h at 78°C under vacuum. For hybridization analysis, cDNA probes were nick translated [26] with [a-32P]dCTP and [cu-32P]dTTP (3000 Ci/mmol; Amersham, Arlington Heights, IL) without excision from the plasmid to a specific activity of 5-8 X 10’ cpm/pg. The filters were prehybridized overnight and then hybridized with the denatured labeled probes for 22 h at 42°C in a solution containing 50% formamide, 5X standard saline citrate (SSC; 0.15 MNaCI, 0.015 Mtrisodium citrate), 1 mMEDTA, 2X Denhardt’s solution [27], 100 Fg/ml salmon sperm DNA, and 0.1% SDS. The filters were then washed with a final stringency of 0.5X SSC containing 0.1% SDS at 65°C. The filters were exposed to Kodak XAR-5 film with a DuPont Cronex Lightening Plus intensifying screen at -70°C for various lengths of time. Radioactivity hybridizing to RNA on the filters was quantitated by scanning densitometry of the autoradiograms, with the band intensities being within the linear response
HEPARIN
EFFECT
TABLE
ON
FIBRONECTIN
1
Heparin Effects on EC Medium FN and TSP Levels
Substrate
Heparin concentration (M/mu
FN (pg/106 cells)
TSP (fig/lo6 cells)
Gelatin
0 30 180
4.63 + 0.40 (4) 1.93 i 0.26 (4) 2.35 i 0.11 (4)
39.3 2 0.75 (2) 40.1 + 2.70 (2) 37.7 k 2.50 (3)
Plastic
0 30 180
9.32 i 0.29 (2) 4.36 i 0.07 (2) 4.51 t 0.10 (2)
55.7 Ifr 5.70 (2) 59.2 t 0.10 (2) 71.0 2 3.60 (2)
Note. EC were grown on gelatin or plastic substrates in the absence or presence of heparin. For determination of FN and TSP levels in the medium, cells were incubated in serum-free medium for 22 h. At the termination of incubation, the medium was removed and processed for competitive ELISA analysis as described under Materials and Methods. The cells were trypsinized and counted. The heparin concentrations shown were present both during the growth of the cells and in the serum-free incubation. The numbers in parentheses indicate the number of experiments. The data represent the mean + standard deviation.
range of the film. The levels of radioactivity were linear with respect to the quantity of total RNA appiied to the filters. CDNA probes. The probes used included the cDNA clone pFHl containing a 2.1-kb insert encoding about 1300 bp of the coding region plus the complete 3’ untranslated region of human FN mRNA, which was obtained from Dr. Francisco Baralle, University of Oxford [28]. The hybridization probe for TSP was a cDNA clone with a 1.9-kb insert which specifies the entire heparin-binding domain and part of the type V collagen binding domain and was a gift from Dr. Vishva Dixit, University of Michigan [29]. Human fibroblast actin and tubulin cDNA clones were provided by Dr. Larry Kedes, Stanford University. The 1.8-kb actin insert includes sequences from the coding region up to amino acid 145 plus the complete 3’ untranslated region of the message. It hybridizes to both the y- and P-actin mRNAs [30]. The fl-tubulin cDNA clone pHFPT-1, with its 2.1-kb insert, is virtually a full-length clone specific for fi-tubulin mRNAs. This clone has the same sequences as the P-tubulin cDNA clone (D/3-1) described by Hall et al. [31].
RESULTS FN and TSP levels in the medium from heparintreated and control EC grown on plastic were analyzed by ELISA. As shown in Table I, EC cultivated in the presence of 90 or 180 pg/ml heparin exhibited a greater than 50% decrease in FN levels, while no change in TSP levels was evident. To eliminate the possibility that the heparin effect reflected heparin interference in the ELISA, FN levels were compared in control culture medium supplemented with heparin (90 pg/ml) and in unsupplemented medium. The presence of heparin did not interfere with the ELISA quantitation since FN levels were similar under both conditions. Both ELISA analyses and metabolic labeling studies demonstrated that heparin decreased FN levels (data not shown). To assess whether the heparin effect on FN levels was modulated by the substrate upon which the cells were
AND
THROMBOSPONDIN
SVNTHESIS
41
cultivated, EC were seeded in culture flasks coated with 1% gelatin in PBS and grown in CC in either the absence or the presence of heparin. Tine levels of TSP in the medium recovered after ~~c~bati~~ serum for 22 h were determined using ELISA. The data are summarized in Table 1. Like be~a~~~-t,reated EC grown on plastic, heparin-treated EC grown cm gelatin exhibited a greater than 50% ease in FN levels, whereas the TSP levels were una cted. The substrate upon which the cells were cultivated did not modulate the capacity of heparin to decrease levels of FN. Ail subsequent analyses were conducted using EC grown on plastic. Studies of the Effect of Heparin Biosynthesis o,fFN
on EC
The kinetics of FN synthesis and been extensively studied using cultured chick embryo fibroblasts 1321. FN has a half-life of 36 h on the surface of these cells. A significant loss of cell surface FN was found to occur by release of the protein into the medium. Once released into the medium, FN accumulates over time since it is not graded by proteolysis and very little (~10%) of the m m FN becomes reassociated with the cell surface. Given these findings, the els of FN in the medium of heparin-treated EC observed following a 22-h incubation is most likely a consequence of lowered biosynthesis of the protein rather ‘than an increased rate of degradation. To verify this assu EC cultures grown in the absence or presence o ml heparin were pulse-labeled for 2 h with [“‘§I nine. Medium and cell-matrix fractions were combined for analysis by SDS-PA . As shown in Fig. 1; the level of radiolabeled FN was reased in the medium-cellmatrix fraction from EC treated with hepa ning densitometry, it was determined that ment resulted in a 50% reduction in r contrast, the level of radiolabeled TS by heparin treatment. ~mmuno~reci~~tai~i~~ using antibodies specific for FN corroborated th’e apparent decrease in total radiolabeled FN in ~~~ari~-treated EC (Fig. ZA). Likewise, immu~opreci~itat~or~ using antibodies specific for TSP showed no decrease in radiolabeled TSP in the combined medium-cell,rix as a conse.quence of heparin treatment (Fig. f, Thus, using pulse-labeling conditions to minimize t effects of heparin on the rates of degradation of FN an TSP, the levels of radiolabeled FN were still decreased i heparin-treated EC cultures, wbereas ~a~~l~beled TSP levels were not. These a further substantiate the premise that heparin dec ses EC synthesis of FN but not that of TSP. ECGF Modulation
oy’ Heparin
Effect
The preceding studies of the effect of heparin synthesis were conducted using cells culture
on FN in the
42
LYONS-GIORDANO,
BRINKER.
AND
KEFALIDES
and ECGF was dependent upon the presence of ECGF in the culture medium (Fig. 3). The difficulty in maintaining EC in culture medium without exogenous ECGF as well as the fact that EC synthesize ECGF [20] pose a problem in assessingthe effects of heparin, independent of ECGF, on EC biosynthetic phenotype. FN -
TSP
Concentration Dependence of the Heparin Effect on FN Synthesis EC synthesis of FN in response to different concentrations of heparin was quantitated using ELISA. EC were incubated for 12 h in serum-free medium in the presence of heparin concentrations ranging from 0.09 to 90 pg/ml. FN levels per lo6 cells in the heparin-treated cultures were normalized as percentage inhibition relative to the levels in untreated cultures. The percentage inhibition of FN synthesis observed with each heparin concentration was plotted as a function of heparin concentration as shown in Fig. 4. By interpolation, a half-maximal response would be expected with 2.5 pg/ml heparin. Maximal heparin-induced reduction of FN levels was observed with a heparin concentration of 90 pg/ml. No increase in suppression was evident in cultures treated with 180 pg/ml heparin as shown in Table 1. No effect on TSP levels was detected by ELISA over the-same range of heparin concentrations (data not shown).
-
Correlation of the Heparin Inhibitory Effect on FN Synthesis and the Heparin Stimulatory Effect on EC Proliferation
HEPARIN FIG. 1. Heparin synthesis of FN and EC was compared by [%]methionine. For and heparin-treated contains 10,000 cpm.
-
+
effects on EC biosynthesis of FN and TSP. The TSP by control and heparin-treated (90 pg/ml) pulse-labeling the cultures for 2 h with 40 &X/ml analysis, total radiolabeledproteins from control cultures were resolved by SDS-PAGE. Each lane
presence of 30 pg/ml ECGF. Heparin is known to modulate the activity of ECGF. Specifically, heparin enhances the mitogenic efficacy of this peptide [2], potentiates its chemotactic activity [31, and increases the affinity of the peptide for its receptor [33]. To assesswhether the effect of heparin on FN synthesis was modulated by ECGF, cells grown in CCM lacking ECGF were treated with 90 pg/ml heparin in the absence and presence of 30 pgjml ECGF for 24 h. Control cells were grown in medium containing ECGF but lacking heparin. After 24 h, the cells were labeled with [14C]proline for 22 h. Radiolabeled proteins recovered in the medium and cell-matrix fractions were resolved by SDS-PAGE and visualized by fluorography. Heparin and/or ECGF did not affect cellmatrix levels of FN (data not shown). The decrease in FN synthesis by cells cultured in the presence of heparin
The concentration dependence of heparin-induced EC proliferation is presented in Fig. 4. The percentage stimulation of proliferation was calculated as described under Materials and Methods. The concentration of heparin expected to yield half-maximal stimulation of proliferation was 2.5 pg/ml. This was the same concentration as that required for half-maximal inhibition of FN synthesis. The correlation coefficient for heparin-induced stimulation of EC proliferation and inhibition of FN synthesis was determined to be -0.94 by correlation analysis. Effects of Heparin on FN and TSP mRNA Levels The rates of synthesis of extracellular matrix components may be regulated through changes in the steadystate level of their mRNAs, effects on the translatability of these mRNAs, or alterations in the rate of intracellular degradation of the newly synthesized proteins. To elucidate the mechanism by which heparin reduced EC synthesis of FN, the effect of heparin on FN mRNA levels was assessed.Total RNA was prepared from subconfluent EC grown in CCM in the absence or presence of concentrations of heparin ranging from 0.09 to 90 pg/ml. For analysis, equivalent amounts of RNA from heparintreated and control EC were applied to nitrocellulose us-
IIEPARIN
EFFECT
ON
FIBRONECTIN
AND
THR~~~~SPQND~N
SYNTHESIS
43
FN
TSP
HEPARIN FIG. 2.
-
+
Immunoprecipitation of newly synthesized or TSP in the medium-cell-matrix fraction from control nine was immunoprecipitated using specific antibodies. respectively, using antibody to FN or TSP. Lanes 3 and using preimmune serum. The amount of radiolabeled FN and by scintillation counting of the immunoprecipitates.
-
+
HEPARlN
-
FN (A) and TSP (B) from control and heparin-treated EC cultures. Radiolabeled FN and heparin-treated cultures following a 2-h pulse-labeling with 40 &i/ml [“%]methioLanes 1 and 2 contain immunoprecipitates from control and heparin-treated cultures, 4 contain immunoprecipitates from control and heparin-treated cultures, respectively, or TSP immunoprecipitated was assessed by scanning densitometry of the fluorogram
ing a slot-blot apparatus and hybridized with a 32P-labeled cDNA probe specific for FN. Concomitantly, hybridizations using radiolabeled cDNA probes for TSP, actin, and tubulin messageswere performed to assessthe specificity of the effects of heparin. Typical autoradiograms of slot-blotted RNA from EC grown in the absence or presence of increasing concentrations of heparin when hybridized with cDNA probes specific for FN or TSP are shown in Figs. 5A and 5B, respectively. Heparin caused a concentration-dependent decrease in FN and TSP messagelevels (Fig. 6). Maximal inhibition of both FN and TSP mRNA levels was observed with EC treated with 9.0 pg/ml heparin. Maximal heparin-induced suppression of FN mRNA was approximately 85% whereas that of TSP mRNA was 40%. By interpolation, the concentrations of heparin expected to yield halfmaximal suppression of FN and TSP mRNA levels were
the same (2.6 pg/ml). This concentration approximated that requisite for half-maximal inh tion of EC FN synthesis by heparin (Fig. 4). Hepari ffected FN protein and mRNA levels in a coordinate manner. fn contrast, heparin suppression of TSP mRNA Ieve accompanied by decreased TSP synthesis i res (Figs. 1 and 2B). The effects of heparin on SP mRNA levels were not a consequence of a nonspecific suppression of RNA since approximately equal a of total RNA/cell were recovered fr beparin and control cultures. Also, actm a -tubuhn levels were not affected by treatment of EC with the same range of heparin concentrations to assay the concentration dependence of heparin s ession of FN and TSP mRNA levels. In addition, the specificity of the hybridization reaction was verified by the lac ization of EC RNA to the cDNA clone
44
LYONS-G10
)RDANO.
BRI :NKER,
Medium
FN
TSP
AND
KEFALIDES
substrate, including adhesion, cytoskeletal organization, migration, proliferation, and differentiation [36]. Enhanced migration and proliferation of the endothelium are fundamental events in the process of angiogenesis [37]. Despite the current view that FN and other extracellular matrix components play a central role in the regulation of these events, limited data are available concerning the effects of angiogenic factors on EC synthesis of extracellular matrix components. The data presented here provide the first evidence that heparin, an angiogenic factor [5], inhibits EC FN synthesis. EC synthesis of TSP, an extracellular matrix component believed to be important in the regulation of smooth muscle cell growth [38] and in the attachment of a variety of cells, including cells of mesenchymal origin [39], is not affected by heparin treatment. The inhibition of FN synthesis by heparin is independent of the substrate upon which the cells are cultivated but is modulated by the presence of exogenous ECGF in the culture medium. A requirement for ECGF has previously been reported for enhancement of EC migration and proliferation by heparin [2, 31. The possibility that the growth regulatory effects of heparin in the presence of ECGF
-
-
120
250
200 .-5 5 $ z 150
+
ECGF
+
-
E 60
Heparin
-
+
procu2(1) collagen mRNA [34] since EC do not synthesize collagen type I. Moreover, a Northern blot of the same RNA was included in each hybridization reaction to further ensure the specificity. Finally, hybridization probe
PA,
a genomic
clone
E 100
+
FIG. 3. ECGF modulation of heparin effect. EC grown in CCM in the absence and presence of heparin (90 rig/ml) or grown in CCM lacking ECGF but supplemented with heparin (90 pg/ml) were labeled for 22 h with [14C]proline. Cells grown in the presence of heparin were labeled in the presence of heparin. ECGF was not present during the labeling of the cultures. Medium proteins visualized by SDS-PAGE and fluorography are shown. Each lane contains 10,000 cpm.
to the
encod&?
g
most
of the
human 28s rDNA gene [35], confirmed that equal amounts of RNA from control and heparin-treated cultures were bound to the nitrocellulose filters. DISCUSSION
Fibronectin has been implicated in the regulation of activities involving the interaction of the cell with its
;
40 50
20 .o 1
I
1”“‘1
.I
1
Heparin
r “““I
“““I 10
100
r *-
0 1,000
(vg/ml)
Concentration dependence of heparin effects on EC FN synthesis and proliferation. EC cultivated in the absence or presence of heparin concentrations ranging from 0.09 to 90 pg/ml were incubated for 12 h in serum-free medium supplemented with the same concentration of heparin used to culture the cells. The FN level in the medium was then quantitated by ELISA and the cells were counted. Values for FN levels in control and treated cultures were compared after normalization by the cell number in the respective cultures. To assay the concentration dependence of heparin-augmented proliferation, cells were cultured in the presence of heparin concentrations ranging from 0.09 to 90 pg/ml for 3 days and then trypsinized and counted. Data are presented as a percentage of control cell proliferation (+) and FN (U) synthesis.
HEPARIN
5
%
EFFECT
b
ON
FIBRONECTIN
C
AND
THROMBOSPONDIN
45
SYNTHESIS
affected by heparin treat centration response ana nate, concentration-depe and TSP messages. The of the FN steady-state m than that of the TSP mRNA level. The s FN mRNA levels by heparin correlated tion of FN synthesis with respect to the ~o~~e~tratio~ dependence of the phenomena. that heparin decreases EC expres 1 part by decreasing the amount of for translation. This decrease ma e a consequence of reduced transcription and/or increased degradation of FN mRNA. These results do not preclude the possibility that heparin lowers FN synthesis by reducing the eEiciency of translation of FN effect of heparin on steady-state message levels for FN and TSP suggests that heparin may control the activity of cellular elements which regulate their RNA levels in a concerted manner. At present little is known about the gene expression Previous work in our Iaborat demonstrated that heparin increases human vascular smooth muscle cell synthesis of increased synthesis is reflected TSP mRNA [44]. The failure of expression, alth h it reduces TS points to the pos lity that the rate of EC! synthesis of
120
FIG. 5. Heparin effects on FN and TSP mRNA levels in EC. Total cellular RNA from cells grown in the absence (row 1) or presence ofO.09 (row Z), 0.9 (row 3), 9 (row 4), and 90 (row 5) pg/ml of heparin was applied to nitrocellulose filters using a slot-blot apparatus. The RNA was hybridized with either the 32P-labeled cDNA probe for (A) FN or (B) TSP (see Materials and Methods). The autoradiograms are representative of hybridizations from which the data in Fig. 6 were obtained. RNA/slot: (a) 0.16, (b) 0.32, and (c) 0.64 pg.
are related to the biosynthetic modulatory effects of the combined factors is further suggested by the high correlation between the concentration dependence of the two phenomena. Other studies provide precedents for translational as well as transcriptional regulation of FN gene expression [40-421. Previous work from our laboratory has demonstrated that heparin increases human vascular smooth muscle cell synthesis of FN 1431 and that this increase is independent of changes in steady-state FN mRNA levels [44]. To elucidate the mechanism by which heparin suppresses EC synthesis of FN, the steady-state levels of FN mRNA in control and heparin-treated cells were determined. For comparison, the levels of TSP transcripts were likewise assessed since EC TSP synthesis was not
100 I
100
Heparin
(q$mE)
FIG. 6. Quantitation of the concentration dependence of beparin suppression of FN and TSP mRNA levels in EC. The mRNA levels of FN (m), TSP (@), actin (Cl), and P-tubulin (0) in EC grown in the presence of increasing concentrations of heparin were determined by scanning densitometry of autoradiograms following hybridization of total cellular RNA to the specific 32P-labeled cDNA probes. The percentage of each message relative to that in control cells is plotted as a function of heparin concentration present in the culture medium.
46
LYONS-GIORDANO,
BRINKER,
AND
KEFALIDES
TSP is regulated at the translational or post-translational level. The data presented herein demonstrate that heparin, in the presence of ECGF, induces changes in EC secretory phenotype concomitantly with its stimulation of EC growth in uitro. In uivo the combined effects of ECGF and heparin on EC growth and matrix protein biosynthesis may be particularly important in the repair of the vessel wall following injury or in events leading to neovascularization. Local increases in the concentrations of heparin or heparin-like molecules may increase the availability of matrix-associated ECGF to EC near the damaged area and facilitate the restoration of vessel wall integrity.
17.
Gospodarowicz, D., Greenburg, G., Foidart, (1981) J. Cell. Physiol. 107,171-183.
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The expert technical assistance of Helen Conaway is gratefully acknowledged. This work was supported in part by NIH Grants AR20553, HL-29492, and HL-07502 and an award from the W. W. Smith Charitable Trust.
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