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Cardiovascular Research 2000 47(4):749-758; doi:10.1016/S0008-6363(00)00129-2
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Low density lipoproteins in human plasma make vascular smooth muscle cells resistant to growth inhibition by heparin

P.A. Underwood* and Sue M. Mitchell

CSIRO Molecular Science, 2 Richardson Place, Riverside Corporate Park, Delhi Rd, PO Box 184, N. Ryde, NSW 2113, Australia

* Corresponding author. Tel.: +61-2-9490-5000; fax: +61-2-9490-5005 anne.underwood{at}molsci.csiro.au

Received 15 November 1999; accepted 15 May 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Vascular smooth muscle cell hyperplasia plays a role in atherosclerosis and restenosis. While heparin has shown promise as an inhibitor of smooth muscle cell proliferation in vitro and in some animal models, it has failed to reduce restenosis in clinical trials. We have previously shown that human smooth muscle cells grown in the presence of human serum are heparin resistant, whereas in the presence of bovine serum they are heparin sensitive. In this report, we demonstrate that the heparin resistance factor is present in human plasma as well as serum, and characterise it further. Methods: Human vascular smooth muscle cells were cultured as explants from the media of redundant adult internal mammary or umbilical cord arteries. They were tested for sensitivity to heparin at 100 µg/ml in the growth medium, in the presence of foetal bovine serum, human-plasma-derived serum, human whole blood serum, or fractions derived from these. Results: In the presence of foetal bovine serum, heparin inhibited cell proliferation, while human-plasma-derived or whole blood sera conveyed heparin resistance. This activity was contained within the fraction of plasma/serum which bound to heparin Sepharose, and the sub-fraction which was retained by a membrane filter of molecular weight cut off of 100 000. All the heparin resistance in this latter fraction was supplied by lipoproteins. LDL prepared directly from human plasma conveyed similar heparin resistance to the lipoproteins from the above sub-fraction. Conclusion: LDL in human plasma/serum conveys resistance to the anti-proliferative effects of heparin upon vascular smooth muscle cells. This activity may interfere with potential therapeutic effects of heparin as an anti-restenosis agent.

KEYWORDS Atherosclerosis; Cell culture/isolation; Growth factors; Lipoproteins; Restenosis; Smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
It is widely accepted that vascular smooth muscle cell (VSMC) hyperplasia plays a role in the development of atherosclerosis and in the more acute response of restenosis following procedures such as angioplasty, stent deployment or vascular grafting. Restenosis is a significant clinical problem and extensive research effort has been expended to discover inhibitors of VSMC proliferation which will be clinically effective. Of these heparin has been extensively studied, and is widely reported to limit VSMC proliferation in vitro where bovine serum is used as a growth supplement (see [1] for references). Heparin has also been found to inhibit vascular hyperplasia in animal models (see [1] for references). Clinical trials of heparin as an anti-restenotic agent have been disappointing [2–4], and many explanations have been proposed for the clinical failure of heparin in this regard.

In a previous report, we described the partial isolation of factors present in human serum, which effectively made cultured human VSMC resistant to the inhibitory effects of heparin [1]. These factors were isolated from human serum by heparin Sepharose affinity chromatography and were retained by a filtration membrane of 100 000 molecular weight cut off. Heparin resistance was absent from VSMC cultured in bovine sera but this effect could be induced by the addition of heparin-binding factors isolated from human serum. Thus one explanation for the discrepancy between the sensitivity of cultured VSMC to heparin and the lack of success of heparin in clinical trials as an anti-restenosis agent, may be the choice of serum supplement used in cell culture. There are many heparin-binding proteins in human serum [5,6] but few of these are in the >100 000 molecular weight range. In this report, we demonstrate that heparin-resistance is conferred by human plasma as well as human serum, and that it is largely contained within the low density lipoprotein (LDL) fraction which is associated with apolipoprotein B100. These findings raise important issues in the prevention of clinical restenosis.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Medium 199 containing Earles salts, and L-glutamine were purchased from Gibco BRL. Penicillin and streptomycin were purchased from ICN Biomedicals. YM100 filters were purchased from Amicon. Ultracentrifuge quick seal tubes were purchased from Beckman. Foetal bovine serum (FBS), low endotoxin was purchased from Commonwealth Serum Laboratories, Australia. Human serum (HS) was prepared from samples of whole blood obtained from polycythaemic patients at Royal North Shore Hospital, Sydney. Sera were pooled (eight samples per pool), filter sterilized and stored in aliquots at –20°C. Sterile fresh-frozen plasma was obtained from The Red Cross Blood Bank, Sydney. Three separate samples were thawed and pooled and stored frozen at –20°C in aliquots. Phenazine methosulphate (PMS), heparin (H3149) and the peroxidase substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) were purchased from Sigma. (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent powder was purchased from Promega. Gelatin Sepharose, heparin Sepharose CL-6B and protein A Sepharose CL-4B were purchased from Amrad Pharmacia. [3H(G)] Heparin sodium salt was purchased from NEN-Dupont. Insta-Gel Plus was purchased from Packard Instrument. All other chemicals were of analytical grade. Tissue culture plastic-ware was purchased from either Nunc or Corning. Polyvinyl chloride U-shaped ELISA plates were from Dynex Industries.

2.2 Antibody reagents
Rabbit IgG to Factor VIII antigen was purchased from Dakopatts. Monoclonal antibody 1A4 to human smooth muscle cell {alpha}-actin was purchased from Sigma. FITC-labelled anti-rabbit and anti-mouse Ig were purchased from Amersham. Goat IgG to apolipoprotein (a) antigen was purchased from Fitzgerald Industries International and goat anti-apolipoprotein E serum from Calbiochem. Monoclonal antibody 4C11 to apolipoprotein-B was purchased from Biodesign International. Biotinylated anti-goat Ig and anti-mouse Ig and peroxidase-conjugated streptavidin were from Amersham.

2.3 Preparation of plasma-derived serum
Frozen plasma was thawed and then heated to 56°C for 30 min to precipitate fibrinogen which was removed by centrifugation at 1600g for 10 min. The resultant plasma-derived serum was dialysed against basal culture medium (Medium 199 containing 4 mM glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin) overnight at 4°C, filter sterilized and stored at –20°C. It was added to VSMC in the standard proliferation assay (see Section 2.9).

2.4 Extraction of heparin-binding factors from serum
Foetal bovine serum was depleted of heparin-binding factors as previously described [1]. In brief, serum was first depleted of vitronectin and fibronectin and then applied to a heparin Sepharose column pre-washed with buffer (100 mM NaCl/20 mM Hepes pH 7.5). The serum depleted of vitronectin, fibronectin and heparin-binding factors was collected and stored at –20°C. Following a post-wash with buffer, heparin-binding factors were eluted from the column with 1.0 M NaCl/20 mM Hepes pH 7.5. Fibronectin and human heparin-binding factors were removed from human whole blood serum or plasma-derived serum as described above for depletion of foetal bovine serum, to yield depleted human sera. Human heparin-binding factors were eluted from the heparin Sepharose column as described for the bovine, dialysed against basal medium overnight at 4°C and stored at –20°C. Human depleted sera, and sera reconstituted with heparin-binding factors, or fractions there-from, were included as test sera in VSMC proliferation assays.

2.5 Fractionation of heparin-binding factors from human sera
Heparin-binding factors from either of the human sera (whole blood or plasma derived) were passed through a membrane with molecular weight cut off of 100 000 (Amicon YM100) using a stirred cell (Amicon model 52) at 10 psi. The retained material was washed with basal medium and reconstituted to its original volume with basal medium and referred to as Fraction 100. The serum volume equivalent of this fraction was tested in the presence or absence of 100 µg/ml heparin in the VSMC proliferation assay using basal medium plus serum fractions of various types. A typical yield of heparin-binding factors from human whole blood serum was 2.25 mg protein per ml original serum. Of this 1.89 mg/ml was retained in Fraction 100. The fractionation of foetal bovine serum and human serum are summarised in Fig. 1. Fractions used in the present report are shown in bold type.


Figure 1
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  		Fig. 1 Summary of fractionation of sera. Fractions of foetal bovine serum and human serum (derived from either whole blood or plasma) used in the present experiments are shown in bold.

 
2.6 Lipoprotein and LDL isolation
Potassium bromide (KBr) density gradient ultracentrifugation was used to separate lipoproteins from Fraction 100 from human whole blood serum. The starting material was adjusted to a density of 1.25 by adding solid KBr and mixed at 4°C before dispensing into ultracentrifuge quick seal tubes. The tubes were centrifuged in a Beckman Ti70 rotor at 50 K rpm (190 000g) for 20 h at 10°C with slow acceleration and deceleration. The lipoproteins floated to the top of the tube and were collected by gentle suction into a syringe after puncturing the tube wall above the interface. The heavy bottom zone of the tube was collected as lipoprotein-depleted material and the intermediate zone was discarded. Both fractions were dialysed extensively against phosphate buffered saline (PBS) containing 1 g/l EDTA, plus a final change of basal medium, filter sterilized and stored for short periods at –20°C. A typical yield was 0.198 mg/ml of lipoprotein (original serum equivalent) and 1.19 mg/ml of protein in the lipoprotein-depleted Fraction 100 (original serum equivalent).

LDL from human plasma was a gift from Dr A Brown at the Heart Research Institute, Sydney. It was collected between densities 1.02 and 1.05 by serial ultracentrifugation as described [7]. Briefly, the plasma was first adjusted to a density of 1.02 with KBr and centrifuged in a VTi 50 rotor at 50 K rpm (206 000g) for 2.5 h at 10°C to float all lipoproteins with densities <1.02. The infranatant plasma was removed and readjusted to a density of 1.05 with KBr and centrifuged in a Ti70 rotor at 50 K (190 000g) for 20 h at 10°C. The LDL-containing supernatant was removed, extensively dialysed against PBS, with a final change of basal medium, filter sterilized and stored in the dark at 4°C. This material contains apolipoprotein B100 as the only lipoprotein moiety [8].

Lipoprotein-depleted Fraction 100, lipoprotein fractions, or LDL were added to VSMC in the standard proliferation assay at concentrations equating to 15% of the starting serum or plasma (final concentration of LDL=0.1 mg/ml). LDL and lipoprotein preparations were not stored under conditions which protected against oxidation, and the role of oxidation state on activity of these fractions was not investigated in this study.

2.7 ELISA for apolipoproteins
Plasma or serum fractions to be tested were serially diluted in PBS and coated onto wells of polyvinyl chloride ELISA plates, overnight at 4°C. The ELISA was completed as described in Ref. [9] using primary antibodies to apolipoprotein (a), apolipoprotein E and apolipoprotein B100 at 1/1000 dilution, in conjunction with biotinylated second antibodies and streptavidin-conjugated peroxidase.

2.8 Culture of human vascular smooth muscle cells
Human adult and neonatal VSMC were isolated by explant from adult internal mammary arteries or umbilical arteries as described [1,10]. Cultures were grown and maintained in Medium 199 containing 4 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (basal medium), supplemented with 20% FBS. Cells were passaged at a 1:2 or 1:3 split ratio after disaggregation with 0.125% trypsin/0.02% EDTA, and used between passages 4 and 6. Cultures were routinely checked for cell type by immunofluorescence using a monoclonal antibody specific for smooth muscle {alpha}-actin, with polyclonal anti-factor VIII antibody as a negative control.

2.9 Human vascular smooth muscle cell proliferation assay
Proliferation of VSMC over a 6-day period in test media containing various combinations of heparin or antibodies, diluted in basal medium supplemented with specific additives (15% foetal bovine serum, human whole blood serum, human-plasma-derived serum, or fractions thereof) was estimated as described [1]. The cell number in six replicates of each treatment was estimated after 0 and 6 days, using the MTS/PMS metabolic colorimetric assay which gives a linear response with VSMC number [11]. (Cultures were visually checked daily to ensure that controls did not approach confluence by day 6, which would invalidate the assay). The absorbance of converted MTS was read at 490 nm with 655 nm as reference on a Bio Rad 3550 ELISA plate reader.

The growth index (GI) for each treatment was calculated as GI=A6/A0–1, where A6 represents the MTS absorbance at day 6 for that particular treatment and A0 represents the MTS absorbance at 0 days (common to all treatments). The variance of this measurement was calculated as the variance of the ratio A6/A0 taking A6 and A0 as independent variables [12]. Percent inhibition of proliferation by a particular treatment was calculated as %I=100x(1–GIt/GIc), where GIc represents the control growth index, and GIt the growth index in the presence of inhibitors.

Percent restoration of proliferation by a particular treatment was calculated as (GIt–GId)/(GIc–GId)x100, where GIc represents the control growth index observed in the presence of 15% foetal bovine serum, GId represents the growth index observed in the presence of depleted foetal bovine serum and GIt represents the growth index when the various test fractions were added to the depleted serum.

Statistical significance of particular treatments was determined using the Analysis of Variance and Student Newman Keul's test for multiple comparisons, or Student's t-test where appropriate. At least three different isolates were included in most experimental treatments and the experiments were repeated at least twice with different sets of isolates, in order to determine the consistency of responses. Where analyses of several different experiments were combined, percent changes were used to avoid confounding effects due to the variation in growth index observed between different isolates and between experiments done at different times.

2.10 Binding of tritiated heparin to VSMC
[3H]-Heparin was bound to VSMC as described in Ref. [13]. VSMC were seeded at 1x105 per well in 1 ml of growth medium into 24-well plates in replicates of three–five. After overnight incubation at 37°C, the cells were washed three times with ice-cold phosphate-buffered saline (PBS) and then cooled to 4°C for 5 min in ice-cold PBS. The PBS was removed and 250 µl per well of pre-cooled solutions of either basal medium plus 15% foetal bovine serum, basal medium +15% human serum, or basal medium containing 0.1 mg/ml LDL were added, each containing 2x106 cpm of [3H]-heparin per well. The final concentration of heparin was 17 µg/ml. The wells were incubated at 4°C for 30 min and washed four times in PBS at 4°C. The cell layer was solubilized in 300 µl 0.2 M NaOH overnight at 37°C, neutralized with 2 M HCl and added to Insta-gel Plus for scintillation counting to determine the amount of bound [3H]-heparin. Wells that did not contain cells were also exposed to [3H]-heparin in basal medium, to provide an estimate of non-specific background binding which was subtracted from the experimental data.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Heparin resistance is conveyed by human plasma as well as serum
We have shown previously that heparin at 100 µg/ml inhibited VSMC proliferation in the presence of foetal bovine serum by 50–75% but had no effect on VSMC in the presence of human serum [1]. It was necessary to determine whether the factors present in human serum which render VSMC heparin resistant, were also present in human plasma (and hence not of platelet origin). Previous experiments had shown that adult and neonatal VSMC grew equally well in the presence of bovine or human sera [1], and either cell type showed the same serum-differential responses to heparin [1]. Preliminary experiments in the present study showed that growth of adult VSMC in medium containing human-plasma-derived serum was very poor whereas neonatal VSMC isolates grew in the presence of this serum at rates approaching those obtained with bovine or human whole blood sera (data not shown). This is in agreement with studies on rat neonatal and adult vascular smooth muscle cells where the ability of neonatal cells to grow in plasma-derived serum was correlated with expression of PDGF-B [14]. In consequence, neonatal cells were used for experiments investigating growth in plasma-derived serum whereas either adult or neonatal cells were used for investigating growth in whole blood sera. The results in Fig. 2 show that human-plasma-derived and whole blood sera were equally effective in preventing the inhibitory effects of heparin on neonatal cells. When heparin-binding factors were removed from plasma-derived serum, the proliferation of neonatal VSMC in the presence of this depleted serum was significantly reduced (Fig. 2). Reconstitution of the depleted plasma-derived serum with its heparin-binding factors restored growth and maintained heparin resistance (Fig. 2). The fraction of heparin-binding factors from human-plasma-derived serum responsible for both growth promotion and heparin resistance was retained by a membrane with a molecular weight cut-off of 100 000 (Fig. 2), as we had previously found for whole blood serum [1].


Figure 2
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  		Fig. 2 Resistance to the growth-inhibitory effects of heparin is conveyed by human plasma as well as human serum. Neonatal VSMC were seeded at 1.4x103 per well in 96-well plates in Medium 199+15% foetal bovine serum. After 24 h, the medium was replaced with test media ±100 µg/ml heparin containing: ({blacksquare}) foetal bovine serum; (blk34) human whole blood serum pooled from eight polycythaemic donors; (blk12) human-plasma-derived serum; ({image}) heparin Sepharose-depleted human-plasma-derived serum (no heparin added); ({image}) heparin Sepharose-depleted human-plasma-derived serum with heparin-binding factors added back; ({square}) heparin Sepharose-depleted human plasma-derived serum with added Fraction 100. Percent inhibition was calculated as given in Methods. In treatment ({image}) plasma-derived serum depleted on heparin Sepharose was compared, in contrast to plasma-derived serum with heparin added to the growth medium. Bars are means±S.E.M. from seven experiments using a total of four neonatal VSMC isolates and six replicates per treatment in each experiment. *Significant decrease in inhibition of VSMC growth compared to control foetal bovine serum treatment (P<0.001, Analysis of Variance and Student Newman Keul's test).

 
3.2 The effect of depletion of (and reconsititution with) lipoproteins from human serum heparin-binding factors upon proliferation and heparin sensitivity of VSMC
Both human-plasma-derived and whole blood sera appeared to convey growth promotion and heparin resistance via fractions which bound heparin Sepharose and were retained by a 100 000 molecular weight cut-off membrane. Further characterisation of these activities was restricted to human whole blood serum so that experiments could utilise both adult and neonatal VSMC.

Depletion of foetal bovine serum with heparin Sepharose removes most of the growth-promoting activity from the serum [1]. This activity is largely contained within the fraction of heparin-binding factors which are retained by a molecular filter with molecular weight cut-off of 100 000 (Fraction 100) [1]. The extent to which this growth-promoting activity can be restored by various components of Fraction 100 from human serum is shown in Fig. 3. Similar results were obtained with adult and neonatal cells. Complete Fraction 100 was able to restore the growth-promoting activity of depleted bovine serum by almost 100%. Fraction 100 depleted of lipoproteins also restored growth-promoting activity to a similar level (P>0.05, Analysis of Variance and Student Newman Keul's test). Lipoprotein-depleted Fraction 100 which was reconstituted with the corresponding lipoprotein fraction did not result in any additional increase in growth-promoting activity over that seen with lipoprotein-depleted Fraction 100 alone (P>0.05, Analysis of Variance and Student Newman Keul's test). The lipoproteins from Fraction 100 added alone to heparin Sepharose-depleted bovine serum restored 30% of its growth-promoting activity. This suggests that the majority of the growth-promoting activity of Fraction 100 lies in the non-lipoprotein moiety.


Figure 3
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  		Fig. 3 Restoration of proliferation of VSMC in heparin Sepharose-depleted foetal bovine serum by addition of lipoprotein-deficient heparin-binding factors from human serum. Fractions were prepared as in Methods (see Fig. 1). Adult or neonatal VSMC were seeded in 96-well plates as for Fig. 2. Cells were grown in Medium 199 supplemented with 15% serum additives as follows: ({blacksquare}) Heparin Sepharose-depleted foetal bovine serum+Fraction 100 from human serum; (blk34) depleted foetal bovine serum+Fraction 100 depleted of lipoproteins; (blk12) depleted foetal bovine serum+Fraction 100 depleted of lipoproteins+lipoproteins added back; ({image}) depleted foetal bovine serum +lipoproteins from Fraction 100. Percent restoration was calculated as the increase in cell proliferation due to the added fraction(s), measured as a percentage of the difference between proliferation in heparin Sepharose-depleted foetal bovine serum and that in complete foetal bovine serum. Bars are means±S.E.M. from seven experiments including a total of three neonatal and two adult VSMC isolates and six replicates per treatment in each experiment. *Significant decrease in percent restoration of VSMC growth compared to restorative effect of Fraction 100 (P<0.001, Analysis of Variance and Student Newman Keul's test).

 
Since human Fraction 100 with its lipoproteins removed could restore full growth-promoting potential to heparin Sepharose-depleted bovine serum, the role of lipoproteins in resistance of VSMC to inhibition by heparin could be directly tested by depletion and reconstitution experiments. The role of LDL isolated from whole human plasma could be tested concurrently. The results are shown in Fig. 4. Similar results were obtained with adult and neonatal cells. Heparin inhibited VSMC proliferation in the presence of undepleted foetal bovine serum by 40–50%. When heparin Sepharose-depleted bovine serum was used with added human Fraction 100, the inhibitory effect of heparin was significantly reduced (P<0.001, Analysis of Variance and Student Newman Keul's test). When the Fraction 100 was depleted of lipoproteins, heparin resistance was lost and the cells showed a similar level of sensitivity to heparin as those in undepleted bovine serum (P>0.05, Analysis of Variance and Student Newman Keul's test). The resistance to heparin was completely restored by reconstitution of lipoprotein-depleted Fraction 100 with either its own lipoproteins or purified LDL from whole human plasma


Figure 4
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  		Fig. 4 Lipoproteins from human Fraction 100 and LDL from human plasma convey heparin resistance to VSMC. VSMC were assayed as in Fig. 2. Percent inhibition due to 100 µg/ml heparin was calculated for cells grown in Medium 199 supplemented with 15% serum/plasma additives as follows: ({square}) foetal bovine serum; ({blacksquare}) heparin Sepharose-depleted foetal bovine serum+Fraction 100; (blk34) depleted foetal bovine serum+Fraction 100 depleted of lipoproteins; (blk12) depleted foetal bovine serum+Fraction 100 depleted of lipoproteins+lipoproteins added back; ({image}) depleted foetal bovine serum+Fraction 100 depleted of lipoproteins+LDL from human plasma. Bars are means and S.E.M. from seven experiments including a total of three neonatal and two adult VSMC isolates and six replicates per treatment in each experiment. *Significant decrease in inhibition of VSMC growth by heparin, compared to control foetal bovine serum treatment (P<0.001, Analysis of Variance and Student Newman Keul's test).

 
The identity of the lipoproteins present in human plasma LDL and Fraction 100 was determined by ELISAs using antibodies specific for apolipoproteins (a), B and E. The results are shown in Table 1. Apolipoproteins (a), E and B were all present in the lipoproteins extracted from Fraction 100 while, as expected, apolipoprotein B was the only apolipoprotein present in LDL. Thus the resistance to heparin conveyed by LDL and Fraction 100 lipoproteins correlated with the presence of apolipoprotein B but not with apolipoproteins (a) or E. This ELISA also served to demonstrate the efficiency of lipoprotein removal from Fraction 100 by density gradient centrifugation. We were unable to test apolipoprotein B alone for its effect on heparin resistance, as when separated from LDL the pure apolipoprotein is completely insoluble in physiological media.


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  		Table 1 Detection of apolipoproteins (a), B and E in the lipoprotein fraction of fraction 100, lipoprotein-depleted fraction 100 and LDL from human plasmaa

 
3.3 Binding of [3H]-heparin to VSMC in the presence of bovine or human sera, or LDL
We have previously reported that the specific binding of [3H]-heparin to the surface of VSMC was reduced in the presence of human serum compared with foetal bovine serum [1]. To determine if LDL played a significant role in this reduced binding, the effect of LDL was directly compared with that of bovine and human serum on binding of [3H]-heparin to VSMC at 4°C. The results for a representative neonatal isolate are shown in Fig. 5. The binding of [3H]-heparin in the presence of human serum was significantly lower than with foetal bovine serum (P<0.01, Analysis of Variance and Student Newman Keul's test). The binding of [3H]-heparin in the presence of LDL, however, was not significantly different from that bound in the presence of foetal bovine serum (P>0.05, Analysis of Variance and Student Newman Keul's test). These results suggest that components in human serum other than LDL interfere with the binding of heparin to VSMC. They also suggest that interference with heparin binding to the cell surface does not play a significant role in heparin resistance, since LDL could provide all the heparin resistance conferred by human serum (cf. Fig. 4).


Figure 5
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  		Fig. 5 The effect of foetal bovine serum, human serum or LDL on the binding of [3H]-heparin to VSMC. VSMC were prepared and [3H]-heparin binding measured as given in Methods. Pre-cooled solutions of basal medium contained 1.2x106 cpm of [3H]-heparin per well, plus either 15% foetal bovine serum ({blacksquare}), 15% human serum (blk34), or 0.1 mg/ml LDL final concentration (blk12). [3H]-Heparin bound to VSMC for each treatment was corrected for non-specific binding to basal medium-incubated cell-free controls. Bars are means and S.E.M. from a representative experiment including one neonatal VSMC isolate and four replicates per treatment. *Significant decrease in [3H]-heparin bound to VSMC compared to the foetal bovine serum treatment (P<0.01, Analysis of Variance and Student Newman Keul's test).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We have previously described the occurrence in human whole blood serum of factors which rendered VSMC resistant to the antiproliferative effects of heparin [1]. This activity, together with most of the growth-promoting activity, was included within a fraction of heparin-binding proteins [1]. In the present study, we demonstrated that resistance to heparin was also conferred by human plasma and was contained within the lipoprotein fraction of the heparin-binding proteins. Three major types of lipoproteins have been described to bind heparin — those containing apolipoproteins B100, (a) and E [15]. The heparin Sepharose chromatography conditions used in the present study were likely to bind these forms quantitatively [5]. The ELISA results in our study showed that although apolipoproteins (a) and E were present in the heparin-binding factor preparation, both were absent from LDL. LDL conveyed as much heparin-resistance as the heparin-binding factor preparation, strongly suggesting that LDL (containing only apolipoprotein B100), is the active moiety in human serum/plasma. This is consistent with the fact that human sera have a concentration range of LDL of 0.6–1.6 mg/ml while that of bovine sera (which do not convey heparin-resistance) is five–ten fold lower [16].

Although the protective activity of the lipoproteins from Fraction 100 and LDL correlated with the presence of apolipoprotein B and not apolipoprotein (a), a positive contribution of the latter to the activity of the lipoprotein fraction cannot be definitively ruled out. Lipoprotein (a) has been identified as an independent risk factor for atherosclerosis and the level of circulating lipoprotein (a) is strongly correlated with development of restenosis [17–19]. Lipoprotein (a) has been described to stimulate proliferation of cultured human VSMC via the LDL portion of the lipoprotein (a) particle [20]. This may contribute to the activity we observed for the lipoproteins in Fraction 100. Thus we are suggesting that LDL in human plasma is the major component responsible for conveying heparin-resistance to VSMC.

The mechanism of action of the protective activity of LDL upon VSMC proliferation in the presence of heparin remains hard to define. One possibility is interference with the binding of heparin to VSMC surfaces as previously suggested [1]. In the present study, we show that while significantly less heparin bound to VSMC in the presence of human serum compared with bovine serum, physiological levels of LDL had no significant effect. Since we have shown that similar levels of LDL can account for all of the heparin resistance in human serum, prevention of binding to cells is unlikely to be the mechanism.

The induction of an alternative growth factor pathway by LDL is a more likely mechanism of heparin resistance. The finding that proliferation of either neonatal or adult VSMC in bovine serum could be inhibited by antibody to bFGF while that in human serum was unaffected [1] lends support to this hypothesis. It is also supported by reports that VSMC display LDL receptors for which the binding of LDL and subsequent downstream signalling is not inhibited by heparin [21,22]. It has been reported that treatment with LDL results in upregulation of growth factor receptors on VSMC [23,24], upregulation of PDGF-AA production [23,25] and increased cellular permeability allowing secretion of aFGF and bFGF [26,27]. A combination of these effects could readily change the sensitivity of VSMC proliferation to anti-bFGF antibody. LDL has been reported to exhibit low mitogenic activity itself when used in the absence of other growth factors [8,23,26,28]. Our finding that the lipoproteins from the heparin-binding fraction of human serum promoted a low level of VSMC growth compared to that induced by the remaining heparin-binding proteins is consistent with these reports. The major effect of LDL appears to be in concert with other growth factors when it results in synergistic increases in VSMC mitogenic/proliferative responses to individual growth factors such as EGF, IGF, and bFGF [8,16,29,30], and particularly to PDGF [8,16,23,28,31]. We did not detect any synergistic effects when lipoproteins from Fraction 100 were added to the growth-promoting remaining proteins of this fraction. The mix of growth factors present in this fraction is likely to have been complex compared to the by-and-large single growth factor additions described in the above studies, and the presence of the lipoproteins could have influenced the growth factor pathway to promote heparin-resistance without detectable quantitative change in proliferation.

Although we have demonstrated that LDL conveys heparin resistance in human serum, manifestation of this effect is also dependent on growth-promoting factors present in the non-LDL portion of Fraction 100 from human serum. While the addition of the whole heparin-binding Fraction 100 from human serum (containing both LDL and growth-promoting elements) to bovine serum, results in heparin resistance (Ref. [1] and present results), if human LDL is added alone to bovine serum, no heparin resistance is conferred (Mitchell and Underwood, unpublished observations). This suggests that the combination of LDL and growth-promoting elements from human serum is necessary to manifest heparin resistance, and that there is therefore a fundamental difference between the growth-promoting elements of human and bovine sera. Further work is needed to elucidate the mechanism of growth promotion in these human vs. bovine fractions, in the presence/absence of LDL.

Restenosis has been correlated with high circulating levels of LDL [32,33] as well as lipoprotein (a) [17–19]. Removal of LDL and lipoprotein (a) from patients’ plasma before and after angioplasty, by the process of apheresis, has been reported to reduce the incidence of restenosis [32,33]. Apheresis, which commonly involves passage of the plasma over solid phase dextran sulphate, removes clotting factors as well as LDL [34]. Its effectiveness in reducing restenosis is probably due to combined effects of reduction of each. Furthermore heparin is commonly given as a bolus at the start of the procedure and continued as an infusion to prevent plasma clotting [34,35]. As a result, the patient's vasculature is simultaneously exposed to high concentrations of heparin and lowered LDL levels. Our findings suggest that part of the success in reduction of restenosis using this treatment may be due to an increased ability of heparin to inhibit VSMC proliferation, as well as the removal of growth-promoting activity of LDL.

There have been a number of reports in the literature which classify VSMC on the basis of heparin sensitivity. VSMC isolated from patients at the time of angioplasty or vein grafting, whose vessels subsequently restenosed, tended to be relatively resistant to heparin while those from patients whose vessels remained patent were sensitive to heparin [36,37]. Naturally occuring differences in heparin sensitivity have also been described for VSMC from animal models and heparin-resistant clones have been induced by various selective techniques [38]. We did not observe any naturally occurring heparin-resistant VSMC among the more than 100 isolates from the internal mammary arteries of by-pass patients, which we used over the course of this study. All isolates were heparin-sensitive (>30% inhibited by heparin in medium containing FBS [36,38]). It is not known at present whether heparin resistance induced by LDL is manifest by a similar or different mechanism compared to that occuring in ‘resistant’ isolates

In conclusion, resistance of VSMC to the antiproliferative effects of heparin may be achieved in a number of ways. We have identified one of these to be manifest by the exposure of VSMC to human serum, particularly the LDL component. High levels of circulating LDL are likely to interfere with potential therapeutic regimes of heparin. Much still remains to be done to elucidate the mechanism of this process.

Time for primary review 24 days.


   Acknowledgements
 
We are indebted to Drs Andrew Brown and David van Ryk, of the Heart Research Institute, Sydney, for help and advice with lipoprotein preparation and to the former for gifts of LDL. We are also indebted to the staff of the Department of Cardiovascular Surgery and of the Maternity Unit of the Royal North Shore Hospital, Sydney, for ongoing supply of redundant vascular tissue for isolation of VSMC, to the Department of Haematology at the same hospital, and the Australian Red Cross, Sydney, for human blood and plasma samples. We also thank Dr John Whitelock for ongoing discussions and for critically reading the manuscript and Dr Meg Evans also for the latter.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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I. GOUNI-BERTHOLD and A. SACHINIDIS
Does the coronary risk factor low density lipoprotein alter growth and signaling in vascular smooth muscle cells?
FASEB J, October 1, 2002; 16(12): 1477 - 1487.
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