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Cardiovascular Research 1997 34(2):418-430; doi:10.1016/S0008-6363(97)00030-8
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture

Anna Hultgårdh-Nilssona,*, Cecilia Lövdahla, Karin Blomgrena, Bengt Kallinb and Johan Thyberga

aDepartment of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden
bDepartment of Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden

* Corresponding author. Tel.: +46 (8) 7287307; fax: +46 (8) 301833; e-mail: Anna.Hultgardh@cmb.ki.se

Received 6 September 1996; accepted 13 January 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a synthetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and the in vitro system has been established as a useful model in which to study the control of SMC differentiation. The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture. Methods: SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin–collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy. Results: The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days. Especially in the serum-free groups, there was also early activation of the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2). In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in collagen type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern. Among the matrix receptors investigated, the β1 integrin subunit showed a high and persistent expression, whereas the {alpha}5 and {alpha}1 integrin subunits showed lower and more variable mRNA levels. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF {alpha}-receptor gene were noted. Conclusion: It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.

KEYWORDS Gene expression; Fibronectin; Laminin; Collagen; Immunofluorescence; Rat, vascular smooth muscle cells; Smooth muscle cell proliferation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Vascular smooth muscle cells (SMCs) are unusual in their ability to revert from a differentiated state characteristic of the adult to a proliferative and secretory state analogous to that existing in the fetus. Such a modulation from a contractile to a synthetic phenotype is an essential element in the formation of atherosclerotic and restenotic lesions [1]. A similar shift occurs when SMCs are grown in culture and the in vitro system has been much used to explore this process [2]. Basically, it can be divided into two phases. First, the freshly isolated cells attach to and spread out on the substrate. In parallel, a marked change in cell structure takes place with loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex [3]. Moreover, the RNA and protein synthetic activities are raised and the cells become competent to enter a replicative cycle after growth factor exposure [4]. Once in a cycling state, they start to produce mitogens on their own and continue to divide in the absence of external stimuli for a number of generations [5]. As a result of the change in phenotype, secretion of extracellular matrix components is also initiated [6–8].

Studies of early primary cultures have indicated that the transition of the SMCs into a synthetic phenotype is promoted by a substrate of the adhesive plasma proteins, fibronectin and vitronectin, whereas a substrate of the basement membrane proteins, laminin and collagen type IV, holds them back in a contractile phenotype [9–11]. It has further been established that platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) are two mitogens that potently stimulate the newly modified cells to replicate [12–14]. Although there exists a comprehensive literature dealing with the phenotypic modulation and proliferation of vascular SMCs, an integrated view of these processes is still lacking [2, 15]. A problem in this context is that the cells are usually incubated in the presence of serum. Since no strict synchronization of the cultures is possible under these conditions, an overlap of events related to the initial change in phenotype of the cells and the subsequent onset of cell growth will occur. In addition, different functions and regulatory principles are usually analyzed separately or only a few at a time, making it difficult to define how they relate to each other and interact in the process as a whole.

Here, we have studied the coordinated expression of a number of phenotype- and proliferation-related genes in rat aortic SMCs during the first 6 days in primary culture. The freshly isolated cells were seeded on substrates of fibronectin or laminin plus collagen type IV in a serum-free medium. Under these circumstances, the cells convert from a contractile to a synthetic state but remain quiescent [9]. For comparison, cells were seeded directly in plastic dishes in a serum-containing medium. In this case, the shift in phenotype is followed by rapid cell proliferation until confluence is reached and the cells stop growing [3]. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for the expression of specific gene transcripts. In part, the expression of the corresponding proteins was also examined by Western blotting and indirect immunofluorescence microscopy.

Our efforts were focused on the following products: smooth muscle {alpha}-actin, a differentiation marker for SMCs [16]; osteopontin, a glycoprotein secreted by SMCs in a phenotype-dependent pattern [17]; collagen type I, a major matrix component in both the normal media and neointimal lesions [18]; the {alpha}5, {alpha}1 and β1 integrins, subunits of receptors for fibronectin, laminin, and collagen type IV [19]; type IV collagenase (MMP-2) and stromelysin (MMP-3), matrix metalloproteinases engaged in SMC migration and proliferation [20]; the PDGF A- and B-chains as well as the PDGF {alpha}- and β-receptors, ligand and receptor subunits for a set of proteins that strongly promote SMC migration and proliferation [21]. Additional interest was paid to c-fos, c-jun, c-myc, and c-ets-1, proto-oncogenes expressed by SMCs after mitogenic stimulation [22–24]. The results direct attention to a few specific genes that are strongly expressed in the early phases of culture. Further and more mechanistically oriented studies should be able to clarify the role of these genes in the phenotypic modulation of the SMCs.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Ham's medium F-12, newborn calf serum (NCS), and collagenase were obtained from Gibco Brl (Paisley, UK), bovine serum albumin (BSA) from Sigma Chemical Co. (St. Louis, Mo., USA), trypsin from Difco (Detroit, MI, USA), and cell culture plastics from Nunc (Roskilde, Denmark). The medium was supplemented with 10 mM Hepes/10 mM Tes (pH 7.3), 50 µg/ml L-ascorbic acid, and 50 µg/ml gentamycin sulfate. Fibronectin was isolated from human plasma by affinity chromatography on gelatin-Sepharose [9]. Laminin (mouse EHS sarcoma) was purchased from Becton Dickinson Labware (Bedford, MA, USA) and collagen type IV (human placenta) from Sigma. To prepare cell culture substrates, the proteins were diluted in phosphate-buffered saline (PBS, pH 7.3) to a concentration of 10 µg/ml and used to coat the bottom of the plastic Petri dishes (laminin and collagen type IV were mixed to produce a basement-membrane-like layer). After 16 h at 20°C, the dishes were rinsed twice with PBS and then incubated with medium F-12/0.1% BSA for 30 min to block unspecific binding.

2.2 Immunological reagents
The following primary antibodies were used in the immunoblot and cytochemical analyses: mouse anti-smooth-muscle {alpha}-actin (Sigma), rabbit anti-c-Fos (Santa Cruz Biotechnol., Santa Cruz, CA, USA), mouse anti-c-Myc (Santa Cruz Biotechnol.), mouse anti-c-Ets-1 (Transduction Labs., Lexington, KY, USA) goat anti-rat SMC osteopontin [25](C. Giachelli, University of Washington, Seattle, WA, USA), rabbit anti-rat bone osteopontin (D. Heinegård, Lund University, Lund, Sweden), goat anti-human collagen type I (Southern Biotechnol., Birmingham, AL, USA), rabbit anti-rat β1 integrin subunit [26](S. Johansson, Uppsala University, Uppsala, Sweden), rabbit anti-human fibronectin receptor (Telios, San Diego, CA, USA), sheep anti-human stromelysin (The Binding Site, Birmingham, England), rabbit anti-PDGFR-{alpha} and anti-PDGFR-β (Santa Cruz Biotechnol.). Horseradish-peroxidase-conjugated antibodies to mouse and rabbit immunoglobulins were from Amersham (Little Chalfont, UK). Rhodamine- and/or fluorescein-labeled rabbit anti-mouse IgG, swine anti-rabbit IgG, rabbit anti-goat IgG, and rabbit anti-sheep IgG were from Dako (Glostrup, Denmark), and goat anti-mouse IgG and sheep anti-rabbit IgG from Sigma.

2.3 Cell culture
SMCs were isolated from the aorta of 350–400 g male Sprague-Dawley rats by digestion with collagenase in medium F-12/0.1% BSA [27]. After rinsing, the cells were seeded on substrates of fibronectin or laminin–collagen IV in medium F-12/0.1% BSA (50 000 cells/cm2) or in plain plastic dishes in medium F-12/10% NCS (30 000 cells/cm2) and incubated at 37°C in an atmosphere of 5% CO2 in air. Medium was changed after 1 day and then every second day. The experiments were repeated at least once. The investigation was performed with permission from the local ethical committee and conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.4 RNA isolation and analysis of gene expression
RNA was extracted as described [28]. The SMCs were washed twice with PBS at 4°C, lysed in 4 mM guanidine isothiocyanate, 0.03 mM sodium acetate (pH 6.0) and 1% mercaptoethanol, and scraped off the dishes. The lysates were loaded on 4 ml of 5.7 mM cesium chloride and centrifuged at 33,000 rpm for 20 h at 20°C in a SW40 Ti rotor. The supernatant was removed and the RNA pellet resuspended in Tris-EDTA buffer (pH 8.0). The solubilized RNA was precipitated overnight in 0.3 mM sodium acetate and 2.5 volumes of ethanol at –70°C. The isolated RNA was centrifuged at 15 000 rpm for 30 min at 4°C, washed with 80% ethanol, dried, and finally resuspended in sterile water. Quantity and purity were determined by spectrophotometry at 260 and 280 nm. After electrophoretic separation (20 µg RNA/lane) on 1.4% agarose gels containing 2.2 mM formaldehyde [29]the RNA was transferred to Hybond-N filters (Amersham).

Hybridizations were done in 50% formamide, 5x SSC (43.8 g/l sodium chloride and 22 g/l sodium citrate), 5x Denhardt's solution (1 g/l polyvinylpyrrolidone, 1 g/l BSA, 1 g/l Ficoll 400), 0.1% sodium dodecyl sulfate (SDS), 0.1 mg/ml salmon sperm DNA, and 10% dextran sulfate for 20 h at 42°C with cDNA probes labeled with [32P]dCTP by the random priming technique (Stratagene, La Jolla, CA, USA) or the nick translation technique (Amersham). After hybridization the filters were washed for 60 min at 55°C in 0.1x SSC with 0.5% SDS and exposed to Fuji RX-L film for 1–24 h. Between successive hybridizations, the filters were boiled in 0.1x SSC, 0.1% SDS. The following probes were used: smooth muscle {alpha}-actin, pRAo{alpha}A-3'UT [30]; c-fos [31]; c-jun [32]; c-myc [33]; c-ets-1 [34]; osteopontin [35]; collagen type I, pro-{alpha}1(I) chain [36]; {alpha}5 integrin [37]; {alpha}1 integrin (a 32-nucleotide primer end-labeled with T4 polynucleotide kinase) [38]; β1 integrin [39]; stromelysin [40]; type IV collagenase [41]; PDGF-A [42]; PDGF-B (c-sis) [43]; PDGFR-{alpha} [44]; PDGFR-β [45]; and ribosomal RNA [46]. The size of the transcripts was determined relative to rat 18S and 28S RNA. Densitometric analysis of the blots was performed using an LKB GelScan XL.

2.5 Protein extraction and immunoblot analysis
SMCs were grown in 90-mm dishes in medium F-12/10% NCS or on a substrate of fibronectin in medium F-12/0.1% BSA for 2, 4, and 6 days. They were scraped in 5 ml ice-cold PBS, pelleted, and lysed in 100 µl sample buffer (pH 6.8) containing 76 mM Tris-HCl, 10% mercaptoethanol, 5% SDS, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 0.1% dithiothreitol. After drawing repeatedly through a 24-gauge syringe and sonication, the lysates were centrifuged at 13 000 rpm for 15 min at 4°C. Protein concentration was measured spectrophotometrically using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). A total of 0.2–5 µg protein from each sample was mixed with sample buffer to a final volume of 15 µl and heated to 100°C for 5 min. The samples were electrophoresed at 110 V for 1 h on 8–10% SDS-polyacrylamide gels in 25 mM Tris, 192 mM glycine, and 1% SDS [47]. Proteins were transferred onto Hybond-C nitrocellulose membranes (Amersham) by electroblotting at 400 mA for 60 min in 25 mM Tris-HCl (pH 8.3), 150 mM glycine, and 20% methanol. Nonspecific binding sites were blocked by immersing the membranes in PBS with 0.1% Tween 20 (PBS-T) and 7% milk protein for 60 min. After rinsing 2x10 min in PBS-T, they were exposed to primary antibodies diluted in PBS/1% milk protein for 15 h at 4°C, rinsed in PBS-T, and incubated for 60 min with horseradish-peroxidase-conjugated secondary antibodies (Amersham) diluted 1:1000 to 1:8000 in PBS. After extensive washing in PBS-T, the immunoreactive protein bands were finally visualized by enhanced chemiluminescence according to the manufacturer's protocol (Amersham).

2.6 Immunofluorescence microscopy
SMCs were grown on plain or fibronectin-coated glass coverslips in medium F-12 with and without 10% NCS. The cells were fixed in 2% formaldehyde for 20 min, treated with 50 mM NH4Cl for 15 min, and permeabilized with 0.2% Triton X-100 for 3 min (all dissolved in PBS). They were then exposed to primary antibodies for 1–2 h followed by fluorescein- or rhodamine-labeled secondary antibodies for 1–2 h (diluted in PBS/0.1% BSA, which was also used for rinsing). The coverslips were finally mounted in a polyvinyl alcohol medium with triethylenediamine as anti-fading agent and examined in a Nikon Labophot microscope with epifluorescence optics. Controls without or with unrelated primary antibodies were negative.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Changes in mRNA expression during primary culture
3.1.1 Smooth muscle {alpha}-actin
Freshly isolated SMCs were seeded on substrates of fibronectin or laminin–collagen IV in a serum-free medium or in plain dishes in a serum-containing medium. Total cellular RNA was isolated after different times and analyzed by Northern blotting using a cDNA probe specific for smooth muscle {alpha}-actin. The results demonstrate that the levels of {alpha}-actin mRNA (1.7 kb band) were high in freshly isolated cells and decreased to lower but still detectable levels after 2 days in culture. A similar trend was noted in all groups (Fig. 1; Table 1). Later, the {alpha}-actin transcripts increased in abundance again (most clearly in the serum group), but did not return to the levels existing before seeding of the cells (Fig. 1; Table 1).


Figure 1
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  		Fig. 1 Expression of mRNA for smooth muscle {alpha}-actin in freshly isolated SMCs (fresh) and cells grown in primary culture on substrates of laminin–collagen IV (lam/coll) and fibronectin (fn) in serum-free medium or in uncoated dishes in medium with 10% serum (ncs) for the indicated times. Ribosomal RNA (18S and 28S) was visualized as a check of the equality in loading between the different lanes. This control also applies to Fig. 2A and Figs. 3–5GoGo, which show hybridizations made on the same filter. The experiment was repeated once with similar results.

 

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  		Table 1 Changes in relative mRNA levels during primary culture of rat aortic smooth muscle cells as studied by Northern blotting

 
3.1.2 c-fos, c-jun, c-myc, and c-ets-1
Newly prepared SMCs expressed c-fos mRNA (2.2 kb band) at high levels, but a decline then occurred in culture (Fig. 2A; Table 1). After 2 days the highest levels remained in the laminin–collagen IV group, intermediary levels in the fibronectin group, and low levels in the serum group. After 6 days a further reduction in the number of c-fos transcripts was evident, but they were still most numerous in the laminin–collagen IV group. The expression pattern of c-jun (2.7 and 3.2 kb bands) resembled that of c-fos, although the variations between the culture conditions were less pronounced (Fig. 2A; Table 1). Control experiments revealed that the abundance of c-fos and c-jun mRNA in freshly isolated cells did not reflect the presence of these transcripts in the intact aorta. Moreover, the activation of the c-fos and c-jun genes was partly inhibited by adding 50 µM of the antioxidant, butylated hydroxytoluene, to the collagenase solution, suggesting a role for oxidative stress in this phenomenon (Fig. 2B). In contrast, the levels of c-myc mRNA (2.4 kb band) were low in newly prepared SMCs, grew somewhat higher early in culture, and then stayed more or less constant. Yet another pattern was presented by c-ets-1 mRNA (2.3 and 5.3 kb bands), which was expressed at high levels in freshly isolated cells and cells grown in culture for 2 days and then leveled off (Fig. 2A; Table 1).


Figure 2
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  		Fig. 2 (A) Expression of mRNA for c-fos, c-jun, and c-ets-1 in freshly isolated SMCs (fresh) and cells grown in primary culture on substrates of laminin–collagen IV (lam/coll) and fibronectin (fn) in serum-free medium or in uncoated dishes in medium with 10% serum (ncs) for the indicated times. (B) Expression of mRNA for c-fos and c-jun in the intact aorta (in vivo) and in freshly isolated SMCs (fresh) prepared in the absence (–) or presence (+) of 50 µM butylated hydroxytoluene (BHT).

 
3.1.3 Osteopontin, collagen type I, and integrin receptor subunits
Osteopontin transcripts (doublet ~1.6 kb) were expressed at low levels in freshly isolated SMCs and thereafter increased severalfold during the first 2 days after seeding, most strikingly in the serum group. Even if the number of transcripts decreased at the end of the culture period, high levels were still found in all groups (Fig. 3; Table 1). On the other hand, no or only very low levels of osteopontin mRNA were found in the intact aorta (data not shown). Collagen type I transcripts (5.1 and 6.4 kb bands) were present in newly isolated SMCs, but displayed only low levels during incubation on substrates of fibronectin or laminin–collagen IV under serum-free conditions. Conversely, increasing levels were noted in the serum group (Fig. 3; Table 1). The {alpha}5 (1.6 kb band) and {alpha}1 (1.8 kb band) integrin receptor subunits showed low or undetectable mRNA levels before seeding of the cells. In culture, an increased expression of the {alpha}1 integrin gene was observed in the fibronectin and laminin–collagen IV groups. Otherwise, the mRNA levels for the {alpha}5 and {alpha}1 integrins remained low and varied little between the groups. The β1 integrin transcripts (3.2 kb band) were more abundant but again varied little between the groups (Fig. 3; Table 1).


Figure 3
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  		Fig. 3 Expression of mRNA for osteopontin, collagen type I, integrins {alpha}5, {alpha}1, and β1 in freshly isolated SMCs (fresh) and cells grown in primary culture on substrates of laminin–collagen IV (lam/coll) and fibronectin (fn) in serum-free medium or in uncoated dishes in medium with 10% serum (ncs) for the indicated times. The experiment was repeated once with similar results.

 
3.1.4 Stromelysin and type IV collagenase
The genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2), were already expressed in freshly isolated cells and an upregulation then occurred in culture, especially in the case of the first-mentioned enzyme (Fig. 4; Table 1). A 10-fold increase in stromelysin mRNA (1.9 kb band) was seen 2 days after seeding on fibronectin and a 17-fold increase on laminin–collagen IV. After 6 days the levels were lower but still high. In the serum group, a small growth in the number of stromelysin transcripts was noted after 2 days but then dropped to almost undiscernible levels. In the intact aorta, no stromelysin mRNA was detected (data not shown). Type IV collagenase (doublet ~3.5 kb) showed a different pattern with elevated but more stable mRNA levels in the fibronectin and laminin–collagen IV groups and gradually increasing levels in the serum group (Fig. 4; Table 1).


Figure 4
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  		Fig. 4 Expression of mRNA for stromelysin and type IV collagenase in freshly isolated SMCs (fresh) and cells grown in primary culture on substrates of laminin–collagen IV (lam/coll) and fibronectin (fn) in serum-free medium or in uncoated dishes in medium with 10% serum (ncs) for the indicated times. The experiment was repeated once with similar results.

 
3.1.5 PDGF-A, PDGF-B, PDGFR-{alpha} and PDGFR-β
Freshly isolated SMCs expressed no or only small amounts of PDGF A- and B-chain mRNA. Likewise, no PDGF B-chain transcripts could be detected in cultured cells. In contrast, an enhanced expression of the PDGF A-chain gene (1.9, 2.3, and 2.8 kb bands) was observed early during in vitro growth, particularly in the fibronectin and laminin–collagen IV groups (Fig. 5; Table 1). There was also a distinct activation of the PDGFR-{alpha} gene (6.5 kb band) in all groups of cultured cells, but in this case the effect was most prominent at the end of the experimental period. A similar tendency was noted with the PDGFR-β gene (5.3 kb band), although the variations in the mRNA levels were of smaller magnitude (Fig. 5; Table 1).


Figure 5
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  		Fig. 5 Expression of mRNA for PDGF-A, PDGFR-{alpha}, and PDGFR-β in freshly isolated SMCs (fresh) and cells grown in primary culture on substrates of laminin–collagen IV (lam/coll) and fibronectin (fn) in serum-free medium or in uncoated dishes in medium with 10% serum (ncs) for the indicated times. The experiment was repeated once with similar results.

 
3.2 Changes in protein expression during primary culture
Immunoblotting was used to study expression of a few of the genes described above at the protein level. As in the cytochemical stainings, these analyses were confined to SMCs grown on a substrate of fibronectin under serum-free conditions or in plain dishes in the presence of serum. Smooth muscle {alpha}-actin was most abundant in newly isolated cells. In the fibronectin group, the levels of this protein decreased during the first 2 days in culture and then changed only little. In the serum group, a further reduction was seen first, but when the cells became confluent, the content of {alpha}-actin again increased (Fig. 6). In accord with the results of the RNA analyses, the c-Fos protein was present in appreciable amounts in freshly isolated cells and then declined. During growth on fibronectin, the protein disappeared almost entirely at the end of the experiment, whereas more stable levels were maintained in the presence of serum (Fig. 7). Although absolute levels were not measured, the c-Myc and c-Ets-1 proteins appeared to be less abundant than c-Fos in freshly isolated cells and changed less with time and culture conditions than the latter protein (Fig. 7).


Figure 6
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  		Fig. 6 Expression of smooth muscle {alpha}-actin in freshly isolated SMCs (fresh) and cells grown in primary culture in uncoated dishes in medium with 10% serum (ncs) or on a substrate of fibronectin (fn) in serum-free medium for the indicated times. Positions of molecular weight markers (kDa) are shown to the left. The experiment was repeated twice with similar results.

 

Figure 7
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  		Fig. 7 Expression of c-Fos, c-Myc, and c-Ets-1 proteins in freshly isolated SMCs (fresh) and cells grown in primary culture in uncoated dishes in medium with 10% serum (ncs) or on a substrate of fibronectin (fn) in serum-free medium for the indicated times. Positions of molecular weight markers (kDa) are shown to the left. The experiment was repeated twice with similar results.

 
3.3 Immunocytochemical localization of proteins
Due to the lower plating efficiency and incomplete spreading of the SMCs on a substrate of laminin–collagen IV [9], the cytochemical analyses were restricted to cells seeded on fibronectin-coated glass coverslips in a serum-free medium or on plain coverslips in the presence of serum. In the fibronectin group, most of the cells (>90%) remained positive throughout the test period (Table 2). After 2 days an intense staining was observed, reflecting the abundance of myofilaments in this early phase of phenotypic modulation. Later, the cells took on a more flattened shape and {alpha}-actin was redistributed to stress fibers (Fig. 8A–C). A similar picture was first seen in the serum group, but a rapid proliferation then occurred and within 4 days a partly overlapping cell layer formed. In parallel, about two thirds of the cells lost the reactivity for {alpha}-actin (Table 2). However, these cells were stained with rhodamine–phalloidin (a general probe for filamentous actin), indicating that a shift from smooth muscle {alpha}-actin to non-muscle β-actin had taken place (data not shown).


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  		Table 2 Changes in fraction of smooth muscle {alpha}-actin positive cells during primary culture of rat aortic smooth muscle cells as studied by immunofluorescence microscopy

 

Figure 8
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  		Fig. 8 Immunocytochemical demonstration of smooth muscle {alpha}-actin in SMCs grown in primary culture on a substrate of fibronectin in serum-free medium for 2, 4, and 6 days (A–C). Bar = 20 µm.

 
Reactivity for osteopontin was seen after 2 days in culture and then increased slowly with time. In the fibronectin group, the staining was limited to the perinuclear cytoplasm and no extracellular deposition was observed during the 6-day-period studied (Fig. 9A). In the serum group a Golgi-like staining evolved, but only a few of the cells were positive (Fig. 9B). The highest fraction of reactive cells was noted in dense regions, while other parts of the cultures contained no or only a few stained cells. With the antibodies raised against bone osteopontin, extracellular reactivity was low (Fig. 9B). On the other hand, the antibodies against SMC osteopontin gave both a Golgi-like staining and a fibrillar extracellular staining (Fig. 9C). Similar results were obtained for collagen type I, but almost all cells were positive and large amounts of protein were deposited extracellularly. The cellular staining again showed a widespread perinuclear pattern in the fibronectin group (Fig. 10A) and a more Golgi-like pattern in the serum group (Fig. 10B). Especially in the latter group, a network of fibrils was also laid down outside the cells as confluence was reached.


Figure 9
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  		Fig. 9 Immunocytochemical demonstration of osteopontin (opn) in SMCs grown in primary culture on a substrate of fibronectin in serum-free medium for 6 days (A) or directly on glass coverslips in medium with 10% serum for the same time (B,C). Antibodies against rat bone osteopontin were used in A and B, and antibodies against rat SMC osteopontin in C. Bar = 20 µm.

 

Figure 10
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  		Fig. 10 Immunocytochemical demonstration of collagen type I (coll) in SMCs grown in primary culture on a substrate of fibronectin for 6 days (A) or directly on glass coverslips in medium with 10% serum for 4 days (B). Bar = 20 µm.

 
In the matrix receptor studies, similar findings were made with the β1 integrin and fibronectin receptor antibodies and no major differences were noted between the cells in the fibronectin and serum groups. Hence, the description will be limited to cells seeded on fibronectin and stained with β1 integrin antibodies. In the early stages of extension over the substrate, a complex pattern was recorded. Concurrent staining for smooth muscle {alpha}-actin revealed a partial overlap with forming stress fibers (Fig. 11A,D). After 4 (Fig. 11B,E) and 6 days (Fig. 11C,F), the cells had spread further and a closer overlap between the β1 integrin and actin filament patterns was evident. A perinuclear staining for stromelysin was seen after 2 days on fibronectin (Fig. 12A). Later, the reactivity declined and only a faint staining was detected after 6 days. Initially, no distinct reaction was seen in the serum group, but a weak Golgi-like pattern appeared as the cells became confluent (Fig. 12B).


Figure 11
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  		Fig. 11 Double immunocytochemical demonstration of integrin β1 (A–C) and smooth muscle {alpha}-actin (D–F) in SMCs grown in primary culture on a substrate of fibronectin in serum-free medium for 2 (A,D), 4 (B,E), and 6 (C,F) days. Bar = 20 µm.

 

Figure 12
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  		Fig. 12 Immunocytochemical demonstration of stromelysin (sml) in SMCs grown on a substrate of fibronectin in serum-free medium for 2 days (A) or directly on glass coverslips in medium with 10% serum for 6 days (B). Bar = 20 µm.

 
With regard to the PDGF receptors, only the cells in the fibronectin group showed a positive staining (perhaps due to receptor downregulation in the serum group). The PDGFR-{alpha} antibodies gave a weak perinuclear reaction (Fig. 13A) and the PDGFR-β antibodies a stronger, fine punctate staining (Fig. 13B). With both antisera, the reactivity was weak after 2 days of culture and then remained similar in strength at the subsequent intervals.


Figure 13
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  		Fig. 13 Immunocytochemical demonstration of PDGFR-{alpha} (A) and PDGFR-β (B) in SMCs grown on a substrate of fibronectin in serum-free medium for 4 days. Bar = 20 µm.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This report describes the expression of a number of differentiation- and growth-related genes in rat aortic SMCs during the first week in culture under various conditions. The results demonstrate that the transition of the cells from a contractile to a synthetic phenotype is associated with marked changes in gene expression, including a reduced expression of smooth muscle {alpha}-actin, an early rise in expression of c-fos, c-jun and c-ets-1, a more extended increase in expression of stromelysin and osteopontin, and an elevated expression of the PDGF A-chain and the PDGF {alpha}-receptor.

Smooth muscle {alpha}-actin is a major cytoskeletal protein in differentiated SMCs and the content of this protein is reduced both in culture and during formation of neointimal lesions [16, 48]. The mechanism is not known, but mitogens may be involved. Thus, treatment of quiescent SMCs with PDGF has been found to destabilize {alpha}-actin mRNA [49]. Nevertheless, the transcripts declined to a similar extent in cells grown with and without serum, although immunostaining disclosed clear differences between the cultures. In the fibronectin group, {alpha}-actin was redistributed to stress fibers as the cells spread and most of them then remained positive. This suggests that {alpha}-actin is only slowly degraded as long as the cells do not divide. In the serum group, a similar picture was seen first, but after 4–6 days about two thirds of the cells were negative for {alpha}-actin, evidently due to a shift to non-muscle β-actin [50]. However, the net content of {alpha}-actin increased again as the cultures became confluent and cells with a strong positive staining partly re-appeared. Expression of other cytoskeletal proteins, including smooth muscle myosin heavy chains and desmin, has also been reported to be suppressed in connection with the phenotypic modulation [2, 15].

The transcription factor genes, c-fos and c-jun, were activated during the isolation of the SMCs and high mRNA levels persisted after 2 days of culture (i.e., during the structural reorganization of the cells). The transcripts were later lost, but the c-Fos protein persisted in the serum group, perhaps reflecting the growing state of these cultures. The levels of c-ets-1 mRNA were high in freshly isolated cells, increased during the first 2 days in culture, and then decreased slowly. On the other hand, c-myc mRNA was of low abundance both in freshly isolated and cultured cells, and small variations in the expression of the c-Myc protein were detected with time. In accord with these findings, a rapid induction of c-fos and c-jun mRNA and protein has been observed in rat aortic SMCs after endothelial denudation [51, 52]. It was further demonstrated that the c-ets-1 gene is activated in the cells of the arterial media after balloon injury [53]. In growth-arrested subcultures, induction of early response genes is also seen after mitogen exposure [22–24, 53]. These results suggest that transcription factors encoded by the c-fos, c-jun, and c-ets-1 genes take part in the control of differentiated properties and proliferation of SMCs both in vitro and in vivo.

After balloon injury, SMCs in the media are rebuilt structurally before they invade the intima and start to multiply and deposit an extracellular matrix [54]. In the migratory phase, the cells have to penetrate the matrix and much interest has been paid to the role of matrix metalloproteinases in this process [20, 55, 56]. Here, the stromelysin (MMP-3) gene was found to be activated early in culture on substrates of fibronectin and laminin–collagen IV. Concerning type IV collagenase (MMP-2), a weaker but more extended rise in the mRNA levels was noted (also in the serum group). Hence, production of MMPs is likely to be part of the phenotypic modulation. Inhibitor studies further indicate that these enzymes are engaged in both movement and proliferation of SMCs [57–59]. In addition, several mitogens and cytokines have been reported to enhance the expression of MMPs in cultured SMCs [60–62]. Together, these results reflect an inability of the cells to move and divide as long as they are firmly attached to the surrounding matrix.

Fos, Jun and Ets-1 are involved in the transcriptional regulation of the genes for stromelysin and other MMPs [63]. Accordingly, these two groups of genes were activated sequentially in the SMCs, the proto-oncogenes first and the MMPs thereafter. In the case of stromelysin, the mRNA levels were particularly high in cells seeded on fibronectin or laminin–collagen IV, implying a role for matrix constituents in the control of MMP gene expression. In a similar manner, it was shown that fibroblasts seeded on fibronectin and tenascin upregulate synthesis first of c-Fos and then of MMPs [64]. It was further demonstrated that this response is mediated via the fibronectin receptor and that the increase in c-Fos is required for the increase in MMPs [65].

In contrast to the matrix-degrading enzymes, the osteopontin and collagen type I genes showed a higher expression in the serum group and peaked later. Moreover, it was only in the presence of serum that a fibrillar network of these proteins was seen, indicating that the SMCs need to divide and establish contact to be able to deposit an extracellular matrix. As shown in subcultures, serum mitogens may also influence collagen and osteopontin secretion [66, 67]. The expression of the latter protein in primary cultures (this study), its ability to promote cell adhesion and migration [25], and its appearance in atherosclerotic and restenotic lesions [68, 69]point to a function in the regulation of SMC phenotype. Conceivably, collagen type I may also have such a role [70].

The substrate molecules used here bind to SMCs via receptors of the β1 integrin family [71, 72]. At the mRNA level, the β1 integrin subunit was expressed in freshly isolated cells and then changed little in culture. A positive immunostaining was observed at all times and a co-alignment with stress fibers containing {alpha}-actin developed as the cells spread. Thus, the pool of β1 integrins was large enough to allow the cells to interact with the substrates and to create a linkage between the latter and the actin cytoskeleton. The {alpha}5 integrin (fibronectin receptor subunit) showed lower mRNA levels, but the receptor number was evidently sufficient to enable the cells to spread on fibronectin. The {alpha}1 integrin (laminin and collagen receptor subunit) revealed increasing mRNA levels during culture on both laminin–collagen IV and fibronectin. In the latter group, this may be related to production of basement membrane components by the cells [9]. In contrast, only low levels of {alpha}1 integrin transcripts were found in the serum group, possibly reflecting the fact that serum contains fibronectin and vitronectin, adhesive proteins using other receptors [19].

In intact arteries, the SMCs are normally quiescent and cells in culture have to shift into a synthetic phenotype before they are able to proliferate [2]. After entering the cell cycle, they promote their own growth and this is associated with production of a PDGF-like mitogen and expression of the PDGF A-chain gene [5]. Recent studies have shown that PDGF-BB is a potent inducer of DNA synthesis in freshly modulated SMCs [12–14]. Accordingly, the PDGFR-β gene was expressed at higher levels than the PDGFR-{alpha} gene, and the cells have earlier been found to bind more PDGF-BB than PDGF-AA early in culture [73]. Two days after seeding PDGF A-chain mRNA appeared in the fibronectin and laminin–collagen IV groups. In support of the existence of an autocrine or paracrine loop, this was accompanied by activation of the PDGFR-{alpha} gene. A less marked increase was noted in PDGFR-β gene expression (perhaps to ensure a response to B-chain containing PDGF molecules). In accord with these findings, studies on atherosclerotic lesions and neointimal lesions formed after arterial injury have shown that modified SMCs also in vivo express the A-chain of PDGF, while endothelial cells and macrophages express the B-chain [74–77].

Summing up, the results indicate that the phenotypic modulation of arterial SMCs in primary culture includes an integrated series of changes in the expression of genes for transcription factors, extracellular matrix components, matrix receptors, matrix-degrading enzymes, growth factors, and growth factor receptors. This may be part of a predetermined genetic program, normally activated in response to endothelial damage and serving the function to enable SMCs to migrate, proliferate, and deposit extracellular matrix. Such a wound-healing reaction fulfils an important role in body homeostasis. However, if the initiating stimulus is maintained, an excessive proliferative and secretory activity will contribute to formation of atherosclerotic and restenotic lesions. Effective means for prevention and treatment of these disease states are still lacking. Further insight into the biology of vascular SMCs will be an essential part in the struggle for this goal.

Time for primary review 32 days.


   Acknowledgements
 
Dorotee Wurtz is thanked for help with preparation of cDNA probes. Financial support was obtained from the Swedish Medical Research Council, the Swedish Heart Lung Foundation, the Swedish Society of Medical Sciences, the King Gustaf V 80th Birthday Fund, the Loo and Hans Osterman Fund, the Jeansson Foundations, the Åke Wiberg Foundation, the Lars Hierta Fund, the Tore Nilsson Fund, the Janne Elgqvist Fund, and the Karolinska Institute.


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 1 Introduction
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 4 Discussion
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