Skip Navigation

Cardiovascular Research 2000 45(2):503-512; doi:10.1016/S0008-6363(99)00357-0
© 2000 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Templeton, D. M
Right arrow Articles by Fan, M. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Templeton, D. M
Right arrow Articles by Fan, M. Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Heterogeneity in the response of vascular smooth muscle to heparin: altered signaling in heparin-resistant cells

Douglas M Templeton*, Yong Zhao and Mei Ying Fan

Department of Laboratory Medicine and Pathobiology, University of Toronto, 100 College St., Toronto, Canada M5G 1L5

* Corresponding author. Tel.: +1-416-978-3972; fax: +1-416-978-5650 doug.templeton{at}utoronto.ca

Received 23 April 1999; accepted 29 August 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Vascular smooth muscle cells show phenotypic heterogeneity in vivo that affects the extent to which they respond to the antimitogenic effects of heparin. In vitro, heparin-resistant cells are readily selected. This study was undertaken to determine whether differences in the antiproliferative response to heparin involve differences in activity of heparin-sensitive signal transduction pathways. Methods: Rat thoracic aorta smooth muscle cells (ASMC) at early passage together with two established vascular smooth muscle lines, PAC-1 and A10, were examined before and after selection for growth in the presence of heparin (10 µg/ml). Cells were rendered quiescent and then stimulated with serum. Results: The three cell types showed different sensitivities to the antimitogenic effects of heparin. With respect to [3H]thymidine incorporation, A10 cells were insensitive to 1 µg/ml heparin whereas PAC-1 cells responded down to 0.05 µg/ml and ASMC were of intermediate sensitivity. ASMC and PAC-1 cells but not A10 showed a decrease in c-fos mRNA in response to 1 µg/ml heparin, and a decrease in the c-Fos content of AP-1 DNA binding activity. None of the cells had decreased c-jun mRNA in the presence of heparin. Although induction of c-fos by serum is thought to signal through the Erk mitogen activated protein kinase family, Erk activity was decreased more by 1 µg/ml heparin in A10 cells than in PAC-1 or ASMC. When cells were selected by growth in the presence of 10 µg/ml heparin, A10 cells were unaffected but PAC-1 and ASMC showed a blunted effect of heparin on serum stimulation. In contrast to A10 and their controls not exposed to continuous heparin, heparin-selected PAC-1 and ASMC showed a diminished ability to induce c-fos in response to serum. Conclusions: Smooth muscle cell lines show different responses to the antimitogenic effects of heparin that correlate with the heparin sensitivity of c-Fos/c-Jun expression. Although Erk is implicated in c-fos induction, cells comparatively resistant to heparin still show heparin-dependent inhibition of Erk activation, suggesting that other pathways may be more important for heparin resistance. Furthermore, cells selected for heparin resistance may develop c-fos-independent pathways for proliferation.

KEYWORDS ASMC, Aortic smooth muscle cell; FBS, Fetal bovine serum; FGF-2, Basic fibroblast growth factor; MAPK, Mitogen-activated protein kinase; PDGF, Platelet-derived growth factor; PKC, Protein kinase C; VSMC, vascular smooth muscle cell


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Restenosis is the most important barrier to the long-term success of percutaneous transluminal coronary angioplasty, accounting for failure in at least 30% of cases [1]. While the mechanisms of restenosis are poorly understood [1,2], many processes contribute, including thrombus formation, vascular smooth muscle cell (VSMC) migration and proliferation, acute recoil, and chronic remodeling with extracellular matrix accumulation [3,4]. Each component is a potential target for therapy. Postmortem studies reveal that restenosis is due at least in part to neointimal formation [5], and many current attempts to resolve the problem are thus directed at controlling VSMC phenotype using agents found to inhibit their proliferation in various animal models or in vitro. Prominent in restenosis, VSMC proliferation also contributes to other vascular pathologies, notably maturation of the atherosclerotic plaque [4,6].

Following the demonstration that continuous i.v. heparin infusion almost completely abolished intimal VSMC proliferation in injured rat carotid artery [7], numerous studies were initiated on the antiproliferative effects of heparin in vivo and in vitro. The balloon-injured rat carotid artery consistently responds to heparin with inhibited VSMC proliferation [8,9]. A single bolus injection given at the time of surgery suppressed VSMC proliferation by 55%, whereas a 6 h delay in heparin injection proved ineffective [9], indicating that heparin acts on early events in the VSMC response in this model. Protein kinase C (PKC)-dependent signaling to induce c-fos is selectively inhibited by heparin [10], suggesting a molecular basis for decreased proliferation. PKC signals through the Erk family of mitogen-activated protein kinases (MAPKs) to activate c-fos transcription, and heparin inhibits activation of MAPK in rat VSMC [11]. Upstream of Erk, heparin decreases phosphorylation of Raf-1 in response to PDGF, without affecting PDGF receptor phosphorylation [12]. This suggests a post-receptor point of action of heparin in the proximal part of the signaling pathway. However, in a different smooth muscle cell line, heparin decreased MAPK (and MEK-1) activation in serum-stimulated but not PDGF-stimulated cells [13], so the precise heparin-sensitive point(s) in this pathway remains unclear.

Clinical trials of heparin in restenosis have been disappointing [14,15], but the reasons may reveal more fundamental information on VSMC biology. One possible reason for failure of heparin therapy is bioactivation of heparin-binding growth factors that could actually enhance hyperplasia. Heparin stabilizes such growth factors and can release them in active form from matrix reservoirs [16]. Another possibility for failure is phenotypic heterogeneity giving rise to variable responses to heparin. VSMC in the neointimal lesion have lost their differentiated phenotype [17]. It is not known whether this represents true phenotypic change, or selection of a subpopulation of pre-existing cells. However, recent work indicates phenotypic heterogeneity of arterial SMC clones, with both spindle and more heparin-resistant epithelioid clones being obtained from both uninjured media and neointima [18]. VSMC cultured for multiple passages in high heparin concentrations (200 µg/ml) become resistant to the antiproliferative effects of heparin [19]. Interestingly, these cells take on the epithelioid appearance of cells cultured from neonates [20], which themselves are more resistant to heparin. San Antonio et al. [21] selected VSMC for heparin resistance by growing them in high concentrations of heparin, and concluded that effects of heparin on proliferation and differentiation were independent. Heparin sensitivty has also been altered by overexpressing various oncogenes [22]. These manipulations may mimic changes in injured cells.

The present studies were undertaken in order to examine the role in heparin resistance of heparin-sensitive signaling pathways potentially involved in proliferation, using three phenotypically homogeneous cultures of VSMC that display different sensitivities to low concentrations of heparin.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
A10 cells from embryonic rat thoracic aorta [23] were obtained from the American Type Culture Collection (Manassas, VA, product no. CRL-1476) at passage 17. PAC-1 cells from rat pulmonary artery [24] were obtained at passage 40. Arterial smooth muscle cells (ASMC) were also prepared from the descending thoracic aortas of male Sprague-Dawley rats (150–175 g) by collagenase/elastase digestion and characterized as described previously [25]. These were obtained from Dr. Catherine Prody (Hospital for Sick Children, Toronto) and used at early passage. All cells were maintained in 10 cm petri dishes in a 5% CO2 environment at 37°C, in medium containing 10% fetal bovine serum (FBS), penicillin, and streptomycin (all from Gibco/BRL, Burlington, Ont.), and passaged by trypsinization. The basal medium for A10 and ASMC was DMEM. PAC-1 cells were maintained in medium 199, in which they were originally isolated [24]. It contains additional non-essential amino acids, vitamins, and nucleic acid derivatives for optimal growth of this cell line. A10 cells were chosen as a commonly used VSMC line, and compared with PAC-1 cells as an example of non-aortic vascular smooth muscle and with early passage primary cells (ASMC).

To examine effects of serum stimulation, cells were rendered quiescent by growth for 48 h in 0.4% FBS and then stimulated at near confluence by addition of the appropriate medium containing 5% NuSerum IV (Collaborative Research Products, Bedford, MA) with or without bovine lung heparin (Sigma; St. Louis, MO; lot #36 H0659). NuSerum IV contains a 25% FBS base with a standardized formulation of growth factors and other nutrients. Five percent NuSerum is sufficient to give a near-maximal response of [3H]thymidine incorporation and we have found the use of NuSerum to give highly reproducible results of Ca2+ signaling, kinase activation, and mitogenic response [26]. To select cells for growth in heparin, cultures were passaged five times at a 1:5 split ratio in the presence of 10 µg/ml heparin.

2.2 [3H]-thymidine incorporation
Cells were grown in 12 well plates, made quiescent as above, and stimulated with 5% NuSerum. After 16 h they were pulse-labeled for 1 h with 2 µCi of [3H]-thymidine (ICN; Mississauga, Ont.; 6.7 Ci/mmol) in 1 ml of complete medium. Acid-precipitable material was then fixed to the plate by three washes with ice cold trichloroacetic acid and subsequently dissolved in 0.25 M NaOH/0.1% SDS for liquid scintillation counting.

2.3 RNA measurement
For Northern blotting, total RNA was isolated using TRIzol Reagent [27]. RNA was separated by electrophoresis on agarose-formaldehyde gels and transferred to Hybond-N nylon membrane for hybridization with a c-fos cDNA [28] that was labeled with [{alpha}-32P]dCTP by the random primer method. Levels of mRNA were quantitated by densitometry of the Northern blot autoradiographs and normalized to 18S rRNA after probing with labeled cDNA to rat 18S rRNA.

For RT-PCR, first strand cDNA synthesis was carried out with 2 µg of RNA and Moloney murine leukemia virus reverse transcriptase (Gibco/BRL) at 37°C for 1 h. This cDNA was then used as a template for PCR (28–35 cycles) with β-actin and either c-fos or c-jun primers. The primers were:

β-actin:

Formula

Amplified fragment size 578 bp

c-fos:

Formula

Amplified fragment size 432 bp

c-jun:

Formula

Amplified fragment size 459 bp

2.4 AP-1 gel shift and supershift assay
A double stranded 21 base deoxyoligonucleotide containing the AP-1 consensus binding site was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and labeled with 32P using [{gamma}-32P]ATP and T4 polynucleotide kinase (New England Biolabs; Mississauga, Ont.). Nuclear protein extract was prepared from 0.5 to 1x106 cells by scraping into 10 mM Tris–HCl, pH 7.5, containing 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40 in a microcentrifuge tube. After 10 min on ice followed by centrifugation, the nuclear pellet was resuspended in 20 µl of 20 mM HEPES, pH 7.5, containing 5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, and 20% (v/v) glycerol with protease and phosphatase inhibitors (1 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4). After 30 min on ice the mixture was centrifuged at 14 000 r.p.m. and the supernatant containing nuclear extract was stored in aliquots at –70°C. Nuclear extract containing 5 µg of protein was incubated on ice for 15 min with 2 µg poly(dI–dC) in 20 mM HEPES, pH 7.9, containing 1 mM MgCl2, 20 mM KCl, 0.5 mM dithiothreitol, and 4% Ficoll. Labeled DNA probe (105 cpm) was added to a final volume of 20 µl and the mixture let stand at room temperature for 20 min before electrophoresis in a 5% non-denaturing polyacrylamide gel followed by autoradiography. Controls omitting nuclear extract and with a 100 fold excess of unlabeled probe were included on most gels. Both conditions resulted in a loss of signal. For supershift experiments, the specific antibody (either c-Fos goat polyclonal IgG or c-Jun rabbit polyclonal IgG, both from Santa Cruz Biotechnology) was preincubated with the nuclear extract for 20 min at room temperature prior to addition of the probe in DNA binding solution.

2.5 MAPK assay
Quiescent cells were treated with 5% NuSerum with or without heparin for 30 min, scraped into lysis buffer, and processed for immunoprecipitation with anti-Erk-2 antibody (Santa Cruz Biotechnology) as previously described [26]. Immunoprecipitates were recovered by incubation with protein A-Sepharose and MAPK activity was determined by phosphorylation of myelin basic protein in the presence of [{gamma}-32P]ATP. Incorporation of 32P into myelin basic protein was determined by electrophoresis and autoradiography.

2.6 Statistical methods
Values are expressed as mean±s.d. and differences between arithmetic means were assessed for statistical significance using the unpaired Student's t test. Differences with P<0.05 are considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 [3H]-thymidine incorporation
Smooth muscle cells rendered quiescent by 48 h serum starvation incorporated very little [3H]-thymidine during a 1 h pulse prior to stimulation with serum. Subsequent to stimulation with 5% NuSerum, incorporation increased about 10-fold up to 16–18 h and temporarily declined thereafter. Similar kinetics of incorporation were observed in three cell types studied (ASMC, A10, and PAC-1) and were unaffected by heparin (data not shown). We have confirmed by flow cytometry in identical experiments with mesangial cells that this peak of DNA synthesis corresponds to progression of the cells through S phase [29]. Therefore, experiments were carried out with a 1 h pulse labeling between 16 and 17 h after serum stimulation. Incorporation becomes significantly decreased at 0.05 µg/ml heparin in PAC-1 cells and at 0.1 µg/ml in ASMC, whereas A10 cells are insensitive to heparin below 1.0 µg/ml (Fig. 1). At 5 and 10 µg/ml the response of all cells, including A10, appears to level off at a comparable decrease of about 40% (data not shown). Subsequent studies described below were carried out in the range below 1 µg/ml where differences in the responses are observed.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Suppression by low concentrations of heparin of [3H]-thymidine incorporation in vascular smooth muscle cells. Cells were labeled with the indicated concentrations of heparin for 60 min commencing 16 h after stimulation with 5% NuSerum. Incorporation is expressed relative to the heparin-free control taken as 100%. Values are mean±s.d. (n=9 from 3 independent experiments). *Significantly lower than heparin-free control.

 
3.2 Induction of c-fos and c-jun
No c-fos mRNA is detectable by Northern blotting in quiescent cells (not shown). Thirty minutes after stimulation with NuSerum, a strong signal is observed (Fig. 2) that represents induction of the gene [26]. The effect of heparin on c-fos induction in each of the cell types parallels its effects on [3H]-thymidine incorporation. No effect is seen on induction in A10 cells up to 1 µg/ml, whereas PAC-1 cells show an approximately 40% decrease in transcript level at 0.1 µg/ml and ASMC are of intermediate sensitivity (Fig. 2).


Figure 2
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of heparin on c-fos mRNA expression. Quiescent cells were stimulated with 5% NuSerum containing the indicated concentration of heparin and total RNA was isolated 30 min later. Northern blots were probed with cDNAs for 18S ribosomal RNA and c-fos and quantitated by densitometry. The intensity of the c-fos signal is divided by that of the 18S signal in the same lane to correct for loading and the ratio in the heparin-free cells is taken as 100%. Values are mean±s.d. from three experiments with each cell type. One representative blot from PAC-1 cells is shown as an example. *Significantly lower than heparin-free control.

 
The above experiments examining c-fos induction at 30 min do not rule out a difference in the timing of induction. We also wished to investigate the effect of heparin on another major component of transcriptionally active AP-1, c-jun, whose mRNA was undetectable by Northern blotting. Therefore, to address both issues, we examined time courses of c-fos and c-jun mRNA expression using RT-PCR. In all cells, c-fos mRNA levels peak at 30–60 min after serum-stimulation (Fig. 3). Consistent with the data from Northern blots in Fig. 2, expression of c-fos is significantly decreased by heparin treatment in PAC-1 cells. Transcript for c-Fos is also decreased by heparin at 30 min in ASMC, although to a lesser extent than in PAC-1 cells. Whereas expression of c-fos appeared lower in A10 cells treated with heparin, these differences did not reach significance at any time point. The level of c-jun mRNA shows a similar time course to that of c-fos in ASMC and PAC-1 cells and shows a more sustained expression in A10. In contrast to c-fos, heparin is without affect on c-jun expression in any of the cells.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Time course of c-fos and c-jun mRNA expression. Quiescent cells [PAC-1 (A, D), ASMC (B, E), A10 (C, F)] were stimulated with 5% NuSerum with (filled circles) or without (open circles) heparin (1 µg/ml) and total RNA was isolated 30 min later. RT-PCR was performed as described in Methods with amplification of β actin DNA as an internal control. Intensity of fluorescent bands representing the c-fos (panels A, B, C) or c-jun (panels D, E, F) PCR products is corrected for the intensity of the β actin product. The maximum value is taken as 100% in each experiment. Values are mean±s.e. from three independent experiments. Values in heparin-treated cells that are significantly lower than heparin-free controls are indicated by ‘a’ (P<0.05) or ‘b’ (P<0.01).

 
A decrease in c-fos mRNA in ASMC and PAC-1 cells should result in a decreased AP-1 binding activity, and this suggestion was examined using a gel mobility shift assay to measure this activity in nuclear extracts from the cells (Fig. 4). A single prominent band arising from control cells was absent when nuclear extract was omitted or when a 100-fold excess of unlabeled probe was included (not shown), demonstrating specificity of AP-1 binding activity. AP-1 binding is increased over basal levels by treatment with serum for 4 h, and this increase is blunted with heparin (Fig. 4). A supershift assay was used to dissect out the contributions of c-Fos and c-Jun proteins to AP-1 binding as these heterodimers are among the most potent at transcriptional activation [30]. Again consistent with the mRNA data, c-Fos involvement in AP-1 was decreased by heparin in PAC-1 and ASMC but unaffected in A10 cells (Fig. 5). In contrast to its mRNA levels, which were unaltered by heparin in all the cells, the c-Jun content of AP-1 was significantly decreased by heparin in ASMC (Fig. 4E). This may reflect a greater decrease in c-Fos in these cells (Fig. 4B) resulting in decreased heterodimer formation, but this has not been investigated further.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Gel shift assay of AP-1 binding activity. Nuclear extracts from cells stimulated with 5% NuSerum with or without heparin (1 µg/ml) for 4 h were incubated with 32P-labeled AP-1-binding consensus sequence DNA as described in Methods. The three leftmost lanes represent quiescent cells. The binding assay used either extract alone (E; lanes 1, 4, and 7) or extract pre-incubated with an antibody to c-Jun (E+J; lanes 2, 5, and 8) or an antibody to c-Fos (E+F; lanes 3, 6, and 9). The position of the shifted probe representing total AP-1 activity, and of bands supershifted by antibodies against c-Fos and c-Jun are seen in this autoradiograph and are indicated by arrows to the left. A longer exposure of the top part of the gel is shown to the right in which bands shifted by the c-Fos and c-Jun antibodies can be quantitated. Unbound probe has been allowed to run off the bottom of the gel. Results from PAC-1 cells are shown in this representative example.

 

Figure 5
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effect of heparin on c-Fos and c-Jun content of AP-1. Quiescent cells [PAC-1 (A, D), ASMC (B, E), A10 (C, F)] were stimulated at t=0 with 5% NuSerum with (open bars) or without (solid bars) heparin (1 µg/ml). c-Fos (panels A, B, and C) and c-Jun (panels D, E, and F) were determined in AP-1 binding activity by antibody supershift assay as illustrated in Fig. 4. The signal in control cells at 1 h is taken as 100% in all cases. Values are mean±s.e. from three independent experiments. *Value in heparin-treated cells significantly lower than the corresponding control.

 
3.3 [3H]-thymidine incorporation and c-fos induction after heparin selection
To determine whether cells growing continuously in heparin retained the above phenotypes, cells were subcultured into their usual medium, with or without addition of 10 µg heparin per milliliter. Cells continuously exposed to heparin initially grew slowly but after several passages there was no difference in appearance or growth characteristics with or without heparin. Cells thus selected for growth in heparin were compared with their companion cultures after six passages. Cells were serum starved without heparin for 48 h, then stimulated with 5% NuSerum for 16 h with various amounts of heparin, and then labeled with [3H]-thymidine for 1 h, as in Fig. 1. Following this treatment, heparin-selected PAC-1 cells and ASMC were markedly desensitized in that [3H]-thymidine incorporation was only decreased at higher heparin concentration (1 and 0.5 µg/ml, respectively) as opposed to control cells that responded to 20-fold and 5-fold lower concentrations, respectively (Fig. 6). As expected, heparin selection had no effect on the unresponsiveness of A10 cells to these concentrations of heparin.


Figure 6
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effect of heparin on [3H]-thymidine incorporation in heparin-selected cells. Quiescent cells (PAC-1, filled bars; ASMC, shaded bars; or A10, open bars) previously grown for five passages in the presence of 10 µg/ml heparin were stimulated with 5% NuSerum with the indicated concentration of heparin and labeled with [3H]-thymidine for 60 min 16 h later. Values are mean±s.d. of triplicate wells from three separate experiments and are expressed relative to heparin-free unselected cells taken as 100% (compare Fig. 1). *Significantly lower than the heparin-free control in the same set of cells.

 
These results suggested that c-fos induction might also become heparin-resistant in heparin-selected ASMC and PAC-1 cells. However, surprisingly, both PAC-1 and ASMC cells selected by growth in heparin showed a greatly diminished induction of c-fos by serum, as compared to unselected controls, regardless of the inclusion of 1 µg/ml heparin in the stimulating medium (Fig. 7). Heparin selection was without effect on induction of c-fos in A10 cells.


Figure 7
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Induction of c-fos in control and heparin-selected cells. Cells were previously unexposed to heparin (Control) or passaged five times in the presence of 10 µg/ml heparin (Heparin-selected). Quiescent cells were stimulated with 5% NuSerum with (S+H) or without (S) heparin (1 µg/ml), or left untreated (0), and total RNA was collected 30 min later. Northern blots were probed for c-fos mRNA. Blots were also probed for 18S rRNA (not shown) and the signal intensities shown in the histograms are of c-fos mRNA relative to 18S rRNA, normalized to a value of 1.0 for serum-treated control cells to allow comparison between experiments. Typical blots of c-fos are shown to the left. For PAC-1 and A10 cells the bars are the mean±s.d. from three separate experiments. There are no significant differences among the four serum-stimulated bars in A10. In control PAC-1, (S+H) differs from (S) with P<0.05 (lane 3 vs. lane 2). In heparin-selected PAC-1, (S+H) and (S) do not differ from each other but differ from their control counterparts with P<0.0001 (lane 5 vs. lane 2) or p<0.01 (lane 6 vs. lane 3). Data from ASMC are for the single blot shown.

 
Because heparin-sensitive induction of c-fos by serum is signaled through Erk [26], this result might indicate that this pathway is insensitive to heparin in both control and heparin-selected A10 cells, permitting normal c-fos induction and proliferation, whereas heparin-selected PAC-1 and ASMC cells become heparin-resistant by utilizing alternative pathways to sustain progression to S phase. In fact, Erk is activated by serum in PAC-1, ASMC, and A10 control cells (Fig. 8). Whereas heparin lowers Erk activity to near-basal levels in serum-stimulated ASMC and A10 cells, the effect is diminished with PAC-1, in contrast to the effects on induction of c-fos. Furthermore, Erk is activated to a similar degree in heparin-selected and control ASMC and PAC-1 cells (Fig. 8) but fails to induce c-fos in the heparin-selected cells (Fig. 7). Erk activation by serum may even be increased by heparin selection of A10 cells (though not reaching significance in Fig. 8), and the subsequent response to heparin is diminished.


Figure 8
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Erk activity in control and heparin-selected cells. Cells (PAC-1 top panel, ASMC middle, A10 bottom) were previously unexposed to heparin (Control) or passaged five times in the presence of 10 µg/ml heparin (Heparin-selected). Quiescent cells were stimulated with 5% NuSerum with (S+H) or without (S) heparin (1 µg/ml), or left untreated (0). Immunoprecipitable Erk-2 activity was assayed with myelin basic protein 15 min later and quantitated by densitometry of the autoradiographs as described in Methods. Values are given relative to that of the serum-stimulated activity in control cells in the same experiment taken as 100%. Values are mean±s.e. from three separate experiments. Differences between the indicated pairs by Student's t test are NS, not significant; a, P<0.01; b, P<0.005; c, P<0.001.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Most previous studies on heparin's mechanism of action have been carried out with concentrations in the range 100–200 µg/ml [11–13,31] At these concentrations, heparin may be expected to have multiple points of action in the cell cycle, to demonstrate electrostatic effects, and to displace molecules including heparan sulfates from the extracellular matrix. However, the antimitogenic activity of heparin can be studied at much lower concentrations, comparable to circulating levels of heparan sulfates in vivo [32], as in the present study. Furthermore, at lower concentrations, differential responses to heparin can be observed. At 10 µg/ml, all three VSMC types studied showed comparable suppression of serum-stimulated [3H]-thymidine incorporation (data not shown). The effect on A10 cells was lost at 1 µg/ml but PAC-1 cells showed a response down to 0.05 µg/ml. Comparative studies were therefore carried out at 1 µg/ml where PAC-1 responded with an approximately 50% decrease, A10 cells were unresponsive, and ASMC showed an intermediate response.

It has been suggested that the antiproliferative characteristics of heparin require binding to the cell surface followed by internalization and metabolism [33,34]. We have argued that inhibition of serum-induced mitogenesis in smooth muscle-like mesangial cells does not involve internalization [26,29]. Radiolabeled heparin is internalized slowly (over hours) and degraded, whereas Erk activation is inhibited as early as 30 s after addition of heparin together with a suitable stimulus [26]. Furthermore, dextran sulfate interferes with heparin's antiproliferative action without affecting uptake [35]. Nevertheless, the difference in the responsiveness of PAC-1, ASMC, and A10 cells could be due to differences in the numbers of surface binding sites or putative heparin receptors, whether related to internalization or not. A receptor associated with heparin's antimitogenicity in VSMC has remained elusive, and it may be that if one exists, its observation is masked by the moderate-affinity (Kd{approx}10–8–10–9 M) non-specific heparin binding sites that are found on the surfaces of most cells [19,29]. We have not measured heparin binding in the VSMC in this study and it was not our aim to elucidate proximal events in heparin's mechanism. Rather, we attempted to determine whether identifiable signaling events responded to heparin in a manner that correlated with the degree of the antimitogenic response.

Assembly of the AP-1 transcription factors as heterodimers of Fos and Jun family members or Jun homodimers is a necessary step preceding the induction of AP-1-dependent genes involved in cell cycle progression [36]. Among the family members with the highest biological activity are c-Fos and c-Jun [36]. Induction of the immediate early response genes c-fos and c-jun is thus an early indicator of entry of quiescent cells into the cell cycle, that is followed by increased AP-1 binding activity and permits eventual progression to S phase and incorporation of [3H]-thymidine into DNA. Activation of Erk leads to induction of c-fos by phosphorylation of the transcription factor Elk-1 [37]. Therefore, demonstration that heparin blocks the activation of Erk [11,12,26] can account for the partial suppression of c-fos induction and can potentially account for inhibition of subsequent AP-1-dependent events including [3H]-thymidine incorporation. In the present study, the qualitative correlations among the degree of suppression by heparin of c-fos and c-jun induction, c-Fos and c-Jun content of AP-1, and [3H]-thymidine incorporation in the three cell types strongly supports this temporal sequence of events. In A10 cells, resistance to 1 µg/ml heparin arises at the proximal end of this pathway: heparin has no effect on the initial induction of c-fos and c-jun, again consistent with the view that the usual heparin-sensitive point is in the upstream signaling pathway.

We have previously considered Erk-dependent c-fos induction as pivotal in the effect of heparin on proliferation [26,38]. Evidence in the present study supports the involvement of alternative pathways as well, although we cannot rule out the possibility that proliferation is dependent upon but relatively insensitive to changes in Erk levels under the conditions of our experiments. A high concentration of heparin (100 µg/ml) was found to inhibit VSMC migration stimulated through both Erk-dependent and PI3 kinase-dependent, Erk-independent signals [39]. In control A10 cells in the present study, heparin blocks the activation of Erk to a large extent, without affecting c-fos mRNA levels, whereas the effect of heparin on c-fos mRNA in PAC-1 cells is greater than its effect on Erk activity (compare Figs. 2 and 8Go). A candidate to account for these observations is c-jun. Both c-fos and c-jun mRNA increase transiently following serum stimulation. However, whereas the c-Fos content of AP-1 more or less follows its transcript levels in heparin-treated cells, c-jun is unaffected by heparin. The latter may become important for sustaining proliferation in heparin-selected cells.

Earlier studies to isolate heparin-resistant VSMC with heparin selection pressure [19,21,33,34,40] relied on 200 µg/ml heparin for 10–30 passages. As we show here, significant changes in signaling, gene expression, and heparin sensitivity can be observed after only several passages in a much lower concentration of heparin. However, it is unclear whether the cells in the present study are altered in the same way as those in previous studies. In fact, there appear to be differences among different laboratories in the earlier studies. Some studies found no difference in the rate of binding or internalization of heparin in resistant cells [21,33] but a loss of upregulation of heparin receptors on continuous exposure [33,34], while others found heparin resistance to be associated with decreased heparin binding and internalization [19,40]. The role of binding, internalization, and metabolism in heparin resistance remains undecided [41], but as noted above the early effects of heparin suggest to us that its antimitogenic activity is exerted from an extracellular location.

There is additional support for the idea that altered signaling is involved in heparin resistance. Total PKC activity assayed by phosphorylation of myelin basic protein was decreased by 50% in the heparin-treated cells of Bârzu et al. [19], although if this is also true of the cells in the present study it is without consequence for Erk activity. Forced overexpression of c-myc was shown to confer resistance to heparin and several other antiproliferative treatments in VSMC [42], while another group found that transfection with SV40 large T antigen, but not v-myc, conferred heparin resistance [22]. Pukac et al. [43] did not find any decrease in inducibility of c-myc or c-fos in VSMC after long term growth in 200 µg/ml heparin. In contrast, we find loss of induction of c-fos in PAC-1 and ASMC after heparin selection, but not in relatively heparin-resistant A10 cells. Loss of c-fos expression in the heparin-selected PAC-1 cells is particularly noteworthy in view of the normal level of Erk activation. The failure of Erk to induce c-fos indicates a block in the activation of transcription at the level of the serum response element in the c-fos promoter [37], and indicates that the cells have become independent of c-fos for survival and proliferation. Proliferation is normal in fibroblasts with the c-fos gene disrupted, presumably because of functional redundancy of the Fos/Jun family members [44]. One interpretation of our data is that c-fos-dependent proliferation is heparin-sensitive in control PAC-1 and ASMC cells, while heparin exposure selects cells that underexpress c-fos and utilize alternative AP-1 components whose expression is not sensitive to heparin. This possibility is under investigation.

Time for primary review 25 days.


   Acknowledgements
 
This work was supported by grant T4166 from the Heart and Stroke Foundation of Ontario.


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

  1. Feuerstein G.Z, ed. Coronary restenosis. From genetics to therapeutics. (1997) New York: Marcel Dekker.
  2. O’Brien E.R, Schwartz S.M. Update on the biology and clinical study of restenosis. Trends Cardiovasc. Med. (1994) 4:169–178.[CrossRef][Web of Science]
  3. McBride W, Lange R.A, Hillis L.D. Restenosis after successful coronary angioplasty. Pathiophysiology and prevention. New Engl. J. Med. (1988) 318:1734–1737.[Web of Science][Medline]
  4. Schwartz S.M, deBlois D, O’Brien E.R.M. The intima. Soil for atherosclerosis and restenosis. Circ. Res. (1995) 77:445–465.[Free Full Text]
  5. Ueda M, Becker A.E, Fujimoto T, Tsukada T. The early phenomena of restenosis following percutaneous transluminal coronary angioplasty. Eur. Heart J. (1991) 12:937–945.[Web of Science][Medline]
  6. Weissberg P.L, Grainger D.J, Shanahan C.M, Metcalfe J.C. Approaches to the development of selective inhibitors of vascular smooth muscle cell proliferation. Cardiovasc. Res. (1993) 27:1191–1198.[Free Full Text]
  7. Clowes A.W, Karnovsky M.J. Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature (1977) 265:625–626.[CrossRef][Medline]
  8. Clowes A.W, Clowes M.M. Kinetics of cellular proliferation after arterial injury. II Inhibition of smooth muscle cell growth by heparin. Lab. Invest. (1985) 52:611–616.[Web of Science][Medline]
  9. Lindner V, Olson N.E, Clowes A.W, Reidy M.A. Inhibition of smooth muscle cell proliferation in injured rat arteries. Interaction of heparin with basic fibroblast growth factor. J. Clin. Invest. (1992) 90:2044–2049.[Web of Science][Medline]
  10. Castellot Jr J.J, Pukac L.A, Caleb B.L. Wright Jr. T.C. Karnovsky M.J. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells. J. Cell Biol. (1989) 109:3147–3155.[Abstract/Free Full Text]
  11. Ottlinger M.E, Pukac L.A, Karnovsky M.J. Heparin inhibits mitogen-activated protein kinase activation in intact rat vascular smooth muscle cells. J. Biol. Chem. (1993) 268:19173–19176.[Abstract/Free Full Text]
  12. Pukac L.A, Carter J.E, Ottlinger M.E, Karnovsky M.J. Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells. J. Cell. Physiol. (1997) 172:69–78.[CrossRef][Web of Science][Medline]
  13. Daum G, Hedin U, Wang Y.X, Wang T, Clowes A.W. Diverse effects of heparin on mitogen-activated protein kinase-dependent signal transduction in vascular smooth muscle cells. Circ. Res. (1997) 81:17–23.[Abstract/Free Full Text]
  14. Lehmann K.G, Doria R.J, Feuer J.M, Hall P.X, Hoang D.T. Paradoxical increase in restenosis rate with chronic heparin use: final results of a randomized trial. J. Am. Coll. Cardiol. (1991) 17:181A.
  15. Karsch K.R, Preisack M.B, Baildon R, et al. Low molecular weight heparin (Reviparin) in percutaneous transluminal coronary angioplasty. Results of a rendomized, double-blind, unfractionated heparin and placebo-controlled, multicenter trial (REDUCE trial). J. Am. Coll. Cardiol. (1996) 28:1437–1443.[Abstract]
  16. Jackson R.L, Busch S.J, Cardin A.D. Glycosaminoglycans: Molecular properties, protein interactions, and role in physiological processes. Physiol. Rev. (1991) 71(2):481–539.[Free Full Text]
  17. Shanahan C.M, Weissberg P.L, Metcalfe J.C. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ. Res. (1993) 73:193–204.[Abstract]
  18. Bochaton-Piallat M.-L, Ropraz P, Gabbiani F, Gabbiani G. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler. Thromb. Vasc. Biol. (1996) 16:815–820.[Abstract/Free Full Text]
  19. Bârzu T, Herbert J.-M, Desmoulière A, Carayon P, Pascal M. Characterization of rat aortic smooth muscle cells resistant to the antiproliferative activity of heparin following long-term heparin treatment. J. Cell. Physiol. (1994) 160:239–248.[CrossRef][Web of Science][Medline]
  20. Schwartz S.M, Foy L, Bowen-Pope D.F, Ross R. Derivation and properties of platelet-derived growth factor-independent rat smooth muscle cells. Am. J. Pathol. (1990) 136:1417–1428.[Abstract]
  21. San Antonio J.D, Karnovsky M.J, Ottlinger M.E, Schillig R, Pukac L.A. Isolation of heparin-insensitive aortic smooth muscle cells: Growth and differentiation. Arteriosclerosis Thromb. (1993) 13:748–757.[Abstract/Free Full Text]
  22. Caleb B.L, Hardenbrook M, Cherington V, Castellot Jr J.J. Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin. J. Cell. Physiol. (1996) 167:185–195.[CrossRef][Web of Science][Medline]
  23. Kimes B.W, Brandt B.L. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp. Cell Res. (1976) 98:349–366.[CrossRef][Web of Science][Medline]
  24. Rothman A, Kulik T.J, Taubman M.B, et al. Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures. Circulation (1992) 86:1977–1986.[Abstract/Free Full Text]
  25. Geisterfer A.A.T, Peach M.J, Owens G.K. Angiotensin II induces hypertrophy, not hyperplasia of cultured rat aortic smooth muscle cells. Circ. Res. (1988) 62:749–756.[Abstract/Free Full Text]
  26. Miralem T, Wang A, Whiteside C.I, Templeton D.M. Heparin inhibits mitogen-activated protein kinase-dependent and -independent c-fos induction in mesangial cells. J. Biol. Chem. (1996) 271:17100–17106.[Abstract/Free Full Text]
  27. Chomczynski P, Mackey K. Modification of the TRI Reagent(TM) procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Bio Techniques (1995) 19:942–945.
  28. Curran T, Gordon M.B, Rubino K.L, Sambucetti L.C. Isolation and characterization of the c-fos (rat) cDNA and analysis of post-translational modification in vitro. Oncogene (1987) 2:79–84.[Web of Science][Medline]
  29. Wang A, Templeton D.M. Inhibition of mitogenesis and c-fos expression in mesangial cells by heparin and heparan sulfates. Kidney Int. (1996) 69:437–448.
  30. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. (1995) 270:16483–16486.[Free Full Text]
  31. Halayko A.J, Rector E, Stephens N.L. Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition. Am. J. Physiol. (1998) 274:L17–L25.[Web of Science][Medline]
  32. Volpi N, Cusmano M, Venturelli T. Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma. Biochim. Biophys. Acta (1995) 1243:49–58.[Medline]
  33. Letourneur D, Caleb B.L, Castellot J.J Jr. Heparin binding, internalization, and metabolism in vascular smooth muscle cell. I. Upregulation of heparin binding correlates with antiproliferative activity. J. Cell. Physiol. (1995) 165:676–686.[CrossRef][Web of Science][Medline]
  34. Letourneur D, Caleb B.L, Castellot J.J Jr. Heparin binding, internalization, and metabolism in vascular smooth muscle cells. II. Degradation and secretion in sensitive and resistant cells. J. Cell. Physiol. (1995) 165:687–695.[CrossRef][Web of Science][Medline]
  35. Arroyo-Yanguas Y, Cheng F, Isaksson A, Fransson L.Å, Malmström A, Westergren-Thorsson G. Binding, internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts. J. Cell. Biochem. (1997) 64:595–604.[CrossRef][Web of Science][Medline]
  36. Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr. Opin. Cell Biol. (1997) 9:240–246.[CrossRef][Web of Science][Medline]
  37. Rosen L.B, Ginty D.D, Greenberg M.E. Advances in second messenger and phosphoprotein research. Means A.R, ed. (1995) vol. 30. New York: Raven Press. 225–253.
  38. Miralem T, Templeton D.M. Heparin inhibits Ca2+/calmodulin-dependent kinase II activation and c-fos induction in mesangial cells. Biochem. J. (1998) 330:651–657.[Web of Science][Medline]
  39. Pukac L, Huangpu J, Karnovsky M.J. Platelet-derived growth factor-BB, Insulin-like growth factor-I, and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration. Exp. Cell Res. (1998) 242:548–560.[CrossRef][Web of Science][Medline]
  40. Bârzu T, Pascal M, Maman M, et al. Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: Comparison between heparin-sensitive and heparin-resistant cultures. J. Cell. Physiol. (1996) 167:8–21.[CrossRef][Web of Science][Medline]
  41. San Antonio J.D, Verrecchio A, Pukac L.A. Heparin sensitive and resistant vascular smooth muscle cells: Biology and role in restenosis. Connect. Tiss. Res. (1998) 37:87–103.[Web of Science][Medline]
  42. Bennett M.R, Evan G.I, Newby A.C. Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-gamma, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ. Res. (1994) 74:525–536.[Abstract/Free Full Text]
  43. Pukac L.A, Castellot Jr J.J, Wright T.C, Caleb B.L, Karnovsky M.J. Heparin inhibits c-fos and c-myc mRNA expression in vascular smooth muscle cells. Cell Regulation (1990) 1:435–443.[Web of Science][Medline]
  44. Brüsselbach S, Möhle-Steinlein U, Wang Z.-Q, et al. Cell proliferation and cell cycle progression are not impaired in fibroblasts and ES cells lacking c-Fos. Oncogene (1995) 10:79–86.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. Wang and V. C. Hascall
Hyaluronan Structures Synthesized by Rat Mesangial Cells in Response to Hyperglycemia Induce Monocyte Adhesion
J. Biol. Chem., March 12, 2004; 279(11): 10279 - 10285.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
J. M. Sarjeant, A. Lawrie, C. Kinnear, S. Yablonsky, W. Leung, H. Massaeli, W. Prichett, J. P. Veinot, E. Rassart, and M. Rabinovitch
Apolipoprotein D Inhibits Platelet-Derived Growth Factor-BB-Induced Vascular Smooth Muscle Cell Proliferated by Preventing Translocation of Phosphorylated Extracellular Signal Regulated Kinase 1/2 to the Nucleus
Arterioscler Thromb Vasc Biol, December 1, 2003; 23(12): 2172 - 2177.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
H. Zeng, Y. Liu, and D. M. Templeton
Ca2+/calmodulin-dependent and cAMP-dependent kinases in induction of c-fos in human mesangial cells
Am J Physiol Renal Physiol, November 1, 2002; 283(5): F888 - F894.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Templeton, D. M
Right arrow Articles by Fan, M. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Templeton, D. M
Right arrow Articles by Fan, M. Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?