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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:821-829

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:821-829.)
© 1996 American Heart Association, Inc.

Articles

Distinct Rat Aortic Smooth Muscle Cells Differ in Versican/PG-M Expression

Joan M. Lemire; Susan Potter-Perigo; Keith L. Hall; Thomas N. Wight; Stephen M. Schwartz

From the Department of Pathology, University of Washington, Seattle, and the Department of Biology (K.L.H.), San Diego State University, San Diego, Calif.

Correspondence to Dr Joan M. Lemire, University of Washington, Department of Pathology, Box 357470, Seattle, WA 98195-7470. E-mail joanlemi@u.washington.edu.


*    Abstract
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 *Abstract
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Abstract Smooth muscle cells (SMCs) with distinct phenotypes are present in blood vessels, and distinct culture types appear when SMCs are maintained in vitro. For example, cultured SMCs from rat adult media grow as bipolar cells, which differ in gene expression from the predominantly cobblestone-shaped SMCs from rat pup aortas and rat neointimas that we call {pi} SMCs. Since proteoglycans are present at different concentrations in the normal intima and media and are elevated in atherosclerotic plaque, we sought to determine whether {pi} and adult medial SMC types synthesize different or unique proteoglycans that are characteristic of each phenotype. [35S]sulfate-labeled proteoglycans were purified by ion-exchange chromatography. An adult medial SMC line synthesized a large proteoglycan (0.2 Kav on Sepharose CL-2B) that was not detectable in a {pi} SMC line. Digestion of this proteoglycan with chondroitin ABC lyase revealed three core glycoproteins of 330, 370, and 450 kD. By Western blot analysis, the two smallest of these reacted with two antibodies to the human fibroblast proteoglycan versican. RNAs hybridizing to versican probes were found only in adult medial–type SMCs, including an adult medial type clone from pup aorta, by Northern blot analysis. Both SMC types synthesize RNAs that hybridize to probes for other proteoglycans, such as perlecan, biglycan, and decorin. We conclude that rat {pi} SMC cultures, unlike monkey, human, and rat adult medial SMC cultures, express little or no versican. This difference in expression may be responsible for the different morphologies and growth properties of the two cell types.


Key Words: PG-M • differentiation • versican • artery • proteoglycans


*    Introduction
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 *Introduction
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As a model system to study the role of SMCs in vascular development and pathology, we have examined the phenotypic diversity of rat aortic SMCs in vitro and have defined a novel phenotype, the {pi} phenotype.1 2 3 4 5 6 Rat SMC cultures derived from pup aorta and adult neointimas share a number of properties, leading to our term {pi}, for pup intimal type. These cultures differ significantly from the more conventionally studied smooth muscle cultures in cell shape, growth properties, and gene expression, including the overexpression of the two extracellular matrix proteins elastin and osteopontin.2 3 5 7 {pi} Phenotype cells have a cobblestone morphology and predominate in cultures derived from rat pup aorta or from neointimas that form after balloon angioplasty of rat carotid arteries.1 2 3 4 5 6 8 Other groups have described SMCs with similar morphology and growth properties,9 10 11 12 although the expression of genes characteristic of the {pi} phenotype has not been examined. Whereas {pi} cells grow as cobblestone-like monolayers, we, like many others, have found that cultured SMCs from adult arterial media grow as multilayers (hills and valleys) of spindle-shaped or bipolar cells.5 9 13 14

Hamati et al15 report that the treatment of adult rat SMC cultures with an inhibitor of proteoglycan synthesis results in a reversible change in cell shape and the loss of multilayered growth. These changes in morphology and cell behavior are similar to the phenotypic differences between adult medial and {pi} SMCs and led to our hypothesis that {pi} and adult medial cells might differ in proteoglycan expression.

The major proteoglycans synthesized by arterial SMCs are versican (CSPG), perlecan (heparan sulfate proteoglycan), and decorin and biglycan (dermatan sulfate proteoglycans).16 17 18 19 Versican was originally cloned from human fibroblast cultures and named for the versatile properties predicted by its cDNA sequence: it contains an amino-terminal hyaluronan-binding region, a large central exon encoding GAG attachment sites, and epidermal growth factor–like, lectin-like, and complement regulatory protein–like domains at its carboxy terminal.20 Versican cDNAs with identical sequences have been amplified and cloned from human and monkey aortas and SMCs.17 A large hyaluronan-binding CSPG derived from chick embryonic mesenchyme, PG-M, has been cloned that appears to be the chicken homologue of versican.21 Several in vitro functions of versican/PG-M have been described: versican from aorta and SMCs form large aggregates with hyaluronan16 ; the chicken form blocks the binding of cells to fibronectin-, collagen-, and vitronectin-coated surfaces22 ; and versican/PG-M is excluded from focal contacts of cultured chick, mouse, and human cells.23

Expression of versican by SMCs in vivo may be significant in vascular disease. Versican has been found in the neointima formed in the rat carotid artery after balloon-catheter denudation.24 Proteoglycans are increased in thickened versus normal intima of rabbits and accumulate in atherosclerotic plaque.25 Antibodies to the large CSPGs isolated from bovine aorta stain the intimal layer of normal nonhuman primate and rabbit arteries and the intima formed after arterial injury more intensely than the underlying media.16 26 CSPGs may contribute to the deposition of lipoproteins in atherosclerosis by binding lipoproteins.27 CSPGs can be coisolated with lipoproteins from plaque and bind to LDL in vitro (for review, see References 25, 27, and 2825 27 28 ).

In light of the changes in cell shape in the presence of a proteoglycan synthesis inhibitor15 and the differential distribution of proteoglycans between the intima and the underlying media,16 25 26 we have extended our analyses of the differences between {pi} and adult medial SMC types to their proteoglycan expression patterns. We show that while both cell types express perlecan, biglycan, and decorin mRNAs, versican mRNA and protein are expressed solely by cultured adult medial phenotype SMCs. The strong difference in levels of expression of versican between adult medial and {pi} cells further supports the concept of diversity among the SMCs of the arterial wall.


*    Methods
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 *Methods
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SMC Cultures
SMC lines WKY12-22, SD12d, WKY3M-22, and SD3M-23 were established by collagenase/elastase digestion of the medial layer of thoracic aortas of 12-day- and 3-month-old WKY and SD rats and were used between passages 10 and 20.14 Based on gene expression, growth properties, and morphology, the former two lines are {pi}-type SMCs.2 3 4 5 7 Clonal pup SMC lines (Pups I through VI) were derived from the WKY12-22 line by dilute plating4 ; clonal adult SMC lines were derived by dilute plating of cultures established as explants of thoracic aortic media.29

Metabolic Labeling
To assess proteoglycan synthesis, subconfluent cells were labeled with 100 µCi/mL Na2[35S]sulfate or 40 µCi/mL [35S]methionine (ICN Biomedical) in Dulbecco's modified Eagle's medium (GIBCO) and 5% calf serum for 24 hours. The media were removed, and the cell layers were harvested by being scraped into 8 mol/L urea buffer (8 mol/L urea, 2 mmol/L EDTA, 0.1 mol/L NaCl, 50 mmol/L Tris-HCl, 5 mmol/L benzamidine, 100 mmol/L 6-aminohexanoic acid, 1 mmol/L phenylmethylsulfonyl fluoride, and 2% Triton-X-100 detergent, pH 7.4). The amount of radioactivity incorporated into proteoglycans and GAGs was determined by using the cetylpyridinium chloride precipitation method.30 The relative amounts of chondroitin/dermatan sulfate and heparan sulfate were determined by cetylpyridinium chloride precipitation of concentrated, dialyzed samples before and after enzymatic digestion with chondroitin ABC lyase (ICN).

Isolation and Analysis of Proteoglycans and GAGs
To determine the size classes of [35S]sulfate-labeled proteoglycans and the types of GAGs synthesized and secreted by the cells, media and cell-layer extracts were purified and concentrated by ion-exchange chromatography on DEAE-Sephacel (Pharmacia Fine Chemicals) in 8 mol/L urea buffer. Aliquots of labeled material were then applied to an 8x1130 mm Sepharose CL-2B molecular-sieve column (Pharmacia) in 4 mol/L guanidine buffer (4 mol/L guanidine, 10 mmol/L EDTA, 0.5% Triton-X-100 detergent, and 50 mmol/L sodium acetate, pH 7.4).31

PAGE and Western Blotting
Metabolically labeled preparations that had been concentrated over DEAE-Sephacel were precipitated in 80% ethanol and 1.3% potassium acetate at -20°C for 2 hours, resuspended in water, and reprecipitated in ethanol with potassium acetate. Chondroitin sulfate and dermatan sulfate were digested by incubation of the pellet with 2.3 U/mL chondroitin ABC lyase in 45 mmol/L Tris, 0.09 mg/mL BSA, and 2.7 mmol/L sodium acetate, pH 8.0, at 37°C for 3 hours.32 Digested and original samples were applied under reducing conditions (1.7% ß-mercaptoethanol in the loading buffer) to 4% to 12% gradient polyacrylamide-SDS gels33 with 3% polyacrylamide stacking gels. [14C]-labeled high-molecular-weight standards (Bethesda Research Laboratories Life Technologies, Inc) were used for estimating apparent molecular weights. [35S]sulfate-labeled proteoglycans were visualized by fluorography of dried gels exposed to DuPont film at -70°C.

For Western blotting, chondroitin ABC lyase–digested samples were subjected to SDS-PAGE. The resolving portion of the gel was transferred to nitrocellulose (Schleicher and Schuell) on a semi-dry transblot apparatus (Bio-Rad), blocked overnight in 0.1 mol/L Tris-HCl and 0.1 mol/L NaCl (pH 7.5) with 1 mg/mL BSA, and then exposed overnight to different antibodies by using a Miniblotter manifold apparatus (Immunetics). Antibodies included anti-human fibronectin,34 a gift from J.A. Madri, Yale University, New Haven, Conn; VC-E, an antibody raised against a fusion protein corresponding to human versican protein sequences 387 through 692 and then affinity purified by using a peptide corresponding to amino acid residues 383 through 408 (containing the E-rich region)35 ; and VC-3, an antibody raised against recombinant human versican and affinity purified against a fusion protein corresponding to the human versican protein sequence 1815 through 2036 (within the GAG attachment domain).36 VC-E and VC-3 were gifts from Dr R. LeBaron, University of Texas at San Antonio. Antibody binding was visualized by sequentially incubating blots in alkaline phosphatase–conjugated goat anti-rabbit IgG diluted 1:20 000 in 0.1 mol/L Tris-HCl and 0.1 mol/L NaCl (pH 7.5) plus BSA for 2 hours followed by the proprietary chemiluminescent substrate CSPD (Western-Light Chemiluminescent Detection Systems, Tropix Inc) in assay buffer (0.1 mol/L diethanolamine, 1 mmol/L MgCl2, 0.02% sodium azide, and 1:20 Nitro-Block [Tropix Inc], pH 10.0) and autoradiographic exposure for 20 minutes.

RNA Isolation and Northern Blot Analysis
For RNA isolation, all lines were cultured in modified Waymouth medium supplemented with calf serum.4 For some experiments, cells were switched to the same medium lacking serum and later refed with serum-containing medium. RNA was isolated, separated on agarose/formaldehyde gels, transferred to a Zetaprobe membrane, prehybridized, and hybridized.4 A 1-kb DNA ladder (GIBCO) was used to determine RNA sizes, and the size determination was corrected for the difference in mobility between DNA and RNA.37 Versican probes were human PCR1 and monkey PCR4 cDNAs,17 which contain versican sequences corresponding to nucleotides 886 through 1588 and 5969 through 7030 of the human versican cDNA sequence20 ; equal counts were mixed for hybridizations. Other probes were HS-1, containing 1.1 kb of human perlecan cDNA38 ; full-length bovine decorin cDNA39 ; a rat biglycan cDNA that was isolated from a rat pup SMC cDNA library2 that has an identical sequence to that published40 ; and a GAPDH probe as a control for loading.41 Probes were labeled by using a random priming kit (Amersham) and [{alpha}-32P]dCTP (DuPont).

Coupled Reverse Transcription–PCR Analysis
Total RNA (2 µg, prepared as described above) from cell lines cultured in serum and harvested at confluence was reverse transcribed as described by the kit manufacturer (Life Technologies, Superscript II Preamplification System for First Strand cDNA synthesis) using oligo dT as the primer. One quarter of the reaction mixture was subsequently subjected to 25 cycles of PCR amplification. Forty-microliter reactions contained 10.4 mmol/L Tris (pH 8.3), 1.5 mmol/L MgCl2, 46 mmol/L KCl, 0.2 mmol/L each dNTP, 0.1 U/µL Taq DNA polymerase (Stratagene), 0.2 µmol/L each primer, 1.25 mmol/L dithiothreitol, 0.08% gelatin, and 0.125 µg/mL BSA. Samples were denatured at 94°C for 5 minutes, subjected to 25 cycles of amplification (94°C, 53°C, and 72°C for 1 minute each), and completed with an additional 4 minutes at 72°C. The primers used in the reaction were designed to amplify a 575-bp sequence (6813 through 7387 of the human versican V1 sequence),20 which is near the carboxy terminus of versican and included in all known splice variants. Sequences of both primers (5'GACTATGGCTGGCACAA and 5'GTCCTTTGGTATGCAGA) are identical in human,20 mouse,42 and rat (J.M. Lemire, K.R. Braun, S.M. Schwartz, and T.N. Wight, unpublished data, 1995) versican/PG-M. Controls for the PCR reaction included amplification of a rat versican plasmid and a reaction-lacking template.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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Versican Proteoglycan Is Synthesized by Adult Medial but Not {pi}-Type SMCs
[35S]sulfate-labeled proteoglycans from media conditioned by adult medial (WKY3M-22) and {pi} (WKY12-22) SMC lines were separated by molecular-sieve chromatography. Like the molecules from medium conditioned by monkey aortic SMCs,18 43 the [35S]sulfate-labeled molecules from the adult medial SMC line were separated into two peaks (Kav{approx}0.2 and {approx}0.58; Fig 1ADown). Surprisingly, however, the peak of larger hydrodynamic size (Kav{approx}0.2) appeared to be totally absent from the medium of the {pi} SMC line (Fig 1ADown). This peak was also missing from the cell-layer proteoglycans from the {pi} cell line (not shown), indicating that the absence of this peak from the medium was not due to retention by the cell layer.




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  		Figure 1. A, Line graph. [35S]sulfate-labeled proteoglycans purified by ion-exchange chromatography from culture medium were separated by Sepharose CL-2B chromatography. {circ} indicates WKY3M-22 adult medial SMCs; {blacktriangleup}, WKY12-22 {pi}-type SMCs; and horizontal bar, the fractions pooled from adult medial SMC–conditioned medium for further analysis in B. B, PAGE and autoradiography. Lanes 1 and 2, analysis of the fractions in A that contained the large [35S]sulfate-labeled proteoglycan. Lanes 3 and 4, [35S]methionine-labeled proteins were prepared from conditioned medium, and the same fractions as those described in A were pooled. Lanes 2 and 4, digested (+) with chondroitin ABC lyase prior to electrophoresis; lanes 1 and 3, undigested (-).

The large [35S]sulfate-labeled molecule secreted by cultured monkey aortic SMCs has been identified as the fibroblast CSPG versican.17 18 We sought to determine whether the large proteoglycan from rat adult medial SMC cultures was also versican. Autoradiography following electrophoresis of the hydrodynamically large peak from rat adult medial SMCs revealed a very large molecule that did not enter the separating gel; this molecule was sensitive to chondroitin ABC lyase digestion (Fig 1BUp). To characterize the protein core of this large molecule, [35S]methionine-labeled proteoglycans were prepared from adult medial SMC cultures, and the hydrodynamically large fraction (Fig 1AUp) was subjected to chondroitin ABC lyase digestion. The undigested material was retained by the stacking gel, in agreement with the finding from the [35S]sulfate-labeled material (Fig 1BUp). Digestion with chondroitin ABC lyase prior to electrophoresis resulted in the disappearance of the material from the stacking gel and the appearance of three new bands at 330, 360, and 450 kD in the separating gel (Fig 1BUp). These are similar in size to the three core proteins of the large CSPG (range, 390 to 500 kD) observed in the monkey.17 18 44

Western blot analysis of the [35S]methionine-labeled hydrodynamically large fraction of proteoglycans showed that the large CSPG is immunologically related to versican. One lane was subjected to autoradiography to indicate the position of the core proteins of the CSPG (Fig 2Down). Western analysis with two antibodies to human versican, VC-E and VC-3, showed that both bound to three bands that had the same mobility as the bands detected by autoradiography (450, 360, and 330 kD; Fig 2Down). The 360- and 330-kD bands were not seen when the undigested large CSPG was reacted with anti-versican antibody, consistent with the retention of those molecules in the stacking gel as part of the large proteoglycan (Fig 1Up). Staining was also seen at 450 kD in the undigested material; however, as there was staining at this point in all lanes, we could not determine whether it was significant or background staining. The relatively high background staining (bands seen in all lanes) in this experiment is probably due to the long exposures needed because of the low cross-reactivity of the antibodies prepared against human versican to rat versican. The regions recognized by these antibodies is poorly conserved between human and mouse20 42 (rat homology is unknown). To determine whether the smaller bands detected by the anti-versican antibodies were specific to the antibody, two controls were used. The chondroitin ABC lyase–treated material was reacted with an unrelated antibody (anti-fibronectin) that detected a molecule of the appropriate size34 as well as the smaller bands seen in the anti-versican lane but not the 360- and 330-kD versican bands. Other bands that were present in both the anti-versican and anti-fibronectin lanes are attributable to the chondroitin ABC lyase preparation, as shown in lane 4, which contains only the enzyme in its buffer (no rat proteins), reacted with a mixture of the anti-versican antibodies.



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  		Figure 2. Lane 35S-met, The hydrodynamically large [35S]methionine-labeled proteoglycan purified from medium conditioned by adult medial SMCs was subjected to chondroitin ABC lyase digestion, PAGE, and autoradiography. Lanes 1-5, Western blot transfer of material electrophoresed on the same gel as the 35S-met lane. Lanes 1, 2, and 5, the same preparation as in the 35S-met lane. Lane 3, The hydrodynamically large [35S]methionine-labeled proteoglycan, undigested. Lane 4 contains chondroitin ABC lyase in the digestion buffer and no rat proteins. Western transfers were reacted with anti-versican antibody VC-E (1:2000 dilution; lane 1), anti-versican antibody VC-3 (1:1000 dilution; lane 2), both VC-E and VC-3 (each 1:1000 dilution; lanes 3 and 4), or anti-fibronectin antibody (lane 5). Note that the stacking gel was retained for autoradiography but not for Western blot analysis. Double-headed arrows indicate bands that are immunoreactive with anti-versican antibodies and have the same mobility as bands detected by autoradiography of the large, chondroitin ABC lyase–digested, [35S]methionine-labeled proteoglycan; single-headed arrow, fibronectin.

We conclude that the adult medial SMC cell line produces a chondroitin ABC lyase–sensitive proteoglycan that appears to be versican. Preparations of medium (Fig 1AUp) and cell layer (not shown) from the {pi} SMC line, however, lacked detectable versican.

Versican RNA Is Detected in RNA Isolated From Adult Medial but Not {pi}-Type SMCs
We examined the versican mRNA levels in {pi} and adult medial SMCs to determine whether versican is regulated at the mRNA level. Fig 3Down shows that RNA isolated from confluent cultures of the two adult medial SMC cell lines (lanes 2 and 4) contains RNAs that hybridized to versican probes. The predominant bands are significantly larger than 28S RNA, in agreement with the large size of human versican RNAs (8, 9, and 10 kb). No hybridization of the versican probe was detected in the RNA from confluent cultures of the {pi} SMC cell line WKY12-22 (lane 1), even upon long exposure. As shown in the protein analysis (above), cultures from this pup SMC cell line lack the large CSPG. In another pup cell line, SD12d, versican RNAs were not detected (lane 3) except upon long exposure (not shown). In the latter line, significant numbers of cells with the adult medial morphology are present among predominant cells having {pi} SMC morphology (J.M. Lemire, unpublished data, 1993). Hybridization to GAPDH is shown as a loading control.



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  		Figure 3. Expression of versican mRNA by adult medial versus {pi}-type SMCs. Total RNA was isolated from cultures within 24 hours of reaching confluence and subjected to Northern blot analysis. Lane 1, WKY12-22 {pi} SMCs; lane 2, WKY3M-22 adult medial SMCs; lane 3, SD12d {pi} SMCs; and lane 4, SD3M-23 adult medial SMCs. Top, Versican probes; bottom, GAPDH control.

Rat {pi} and adult medial aortic SMCs respond differently to cell-cell contact and produce and respond differently to growth factors.5 45 We therefore examined {pi} (WKY12-22) and adult medial (WKY3M-22) SMC cultures in a variety of growth states to determine whether versican mRNA was always found in adult cells and never detected in {pi} cells (Fig 4Down). Cultures were harvested at 50% to 70% confluence (lanes 1 and 6) or at the point of confluence in cells grown in the presence of serum (lanes 2, 3, 7, and 8). In other cultures, serum was removed at confluence, and cells were either harvested 5 days later (lanes 4 and 9) or fed with serum at that time and harvested 2 days later (lanes 5 and 10). Versican mRNA was present in the adult medial SMC line in all cases but was never detected in the {pi} line (Fig 4Down). Four bands (11.5, 8.7, 7.7, and 7.0 kb) were present in the size range characteristic of versican mRNA from monkey SMCs, bovine and monkey aorta, and human fibroblasts.17 18 20 At this exposure, additional small bands of 3.7, 3.2, and 2.5 kb were also found in those samples, as well as an extremely large band in one sample. The latter has a significantly slower mobility than the largest marker (12 kb).



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  		Figure 4. Expression of proteoglycans by SMCs under different growth conditions. {pi}-Type SMC line WKY12-22 (lanes 1-5) and adult medial SMC line WKY3M-22 (lanes 6-10) were cultured in serum and harvested at 50% to 70% confluence (lanes 1 and 6) or confluence (lanes 2, 3, 7, and 8). Upon reaching confluence in serum-containing medium, cells were grown in serum-free medium for 5 days and harvested (lanes 4 and 9) or refed with serum-containing medium and maintained for an additional 2 days (lanes 5 and 10). RNA was isolated at the indicated times, and Northern blots were prepared. Probes are identified at left.

RNAs that hybridize specifically to probes for perlecan heparan sulfate proteoglycan (Fig 4Up) and biglycan and decorin dermatan sulfate proteoglycans (data not shown) were expressed by both WKY12-22 and WKY3M-22 in all growth conditions. Perlecan mRNA is a very large message (12 to 14 kb), and the detection of this molecule indicates that large RNAs are efficiently transferred and that large mRNA molecules are intact in RNA samples lacking versican.

The restriction of versican mRNA expression to adult medial phenotype SMCs was further examined by analyzing the expression pattern in clonal SMC lines derived from pup and adult aorta. Versican mRNA was detected in RNA from confluent cultures of all 10 adult SMC clones, albeit at highly variable levels (Fig 5ADown). These cultures had the adult medial SMC morphology; however, not all grew in the hill-and-valley multilayers characteristic of adult medial cultures.29




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  		Figure 5. Expression of versican mRNA by cloned rat SMC lines. A and B, Northern blot analysis of total RNA of confluent cultures probed with versican (top) and GAPDH (bottom). A, Ten adult medial SMC clones; B, six SMC clones derived from the {pi}-type pup SMC line WKY12-22. Lane 5 RNA was obtained from the pup clone that has the adult medial phenotype. C, Reverse transcription–PCR analysis of RNA from cell and clonal cell lines. cDNA synthesis was primed with oligo dT, and a region in common with all known versican splice forms was amplified. Lane M, 100-bp ladder (size marker); lanes 1 and 3, adult SMC cell lines SD3M-23 and WKY3M-22; lanes 2 and 4, pup SMC cell lines SD12d and WKY12-22; lanes 5-7, Pup I, II, and III clones derived from WKY12-22 that had the {pi} phenotype; lanes 8-10, WKY I, II, and V clones of adult medial SMCs; lane P, PCR amplification of the rat versican plasmid V4b-1; and lane -, PCR reaction lacking cDNA template. The band at 575 bp has the size predicted from the versican sequence. The larger band may be a novel splice variant or a related molecule.

The versican probe hybridized to RNA from only one pup clone, Pup V (Fig 5BUp, lane 5). We have shown4 that this clone, although derived from a 12-day-old pup, has the adult medial phenotype, including cell shape, expression of PDGF-{alpha}R mRNA, low-level expression of elastin, osteopontin, and CYPIA1 mRNAs, and lack of expression of PDGF-B mRNA. The remaining five clones have a cobblestone morphology and exhibit all (Fig 5BUp, lanes 1 through 3) or most (Fig 5BUp, lanes 4 and 6) of the other properties of {pi} SMC cultures.4 We conclude that versican mRNA is characteristic of adult medial–type SMCs.

Differential expression of versican by adult medial versus {pi} SMCs was further examined by coupled reverse transcription–PCR analysis of RNA from confluent cultures. cDNA synthesis was primed with oligo dT, and a region in the carboxy terminus of versican, which contains most of the lectin-binding domain and the complement regulatory–like domain, was amplified by PCR. Although such analysis is only semiquantitative, the results essentially parallel those of Northern blot analysis. The versican product was present in both adult cell lines and the three cloned adult cell lines analyzed (Fig 5CUp). Versican was present at much lower levels in one pup cell line (SD12d), in agreement with the Northern blot analysis. Versican was undetectable in the other pup cell line (WKY12-22) and in two of the three clones derived from that line. A third clone (Pup II) had a trace of versican PCR product after 25 cycles, indicating very low-level expression; this expression was not detectable by Northern blot analysis (Fig 5BUp).

Interestingly, two bands were amplified by PCR analysis from reverse-transcribed SMC RNA, but only the smaller one was amplified from a rat versican plasmid (Fig 5CUp). The smaller band has the size predicted from human, mouse, and rat sequences. The other band is larger by {approx}150 bp and may represent a splice variant of versican or cross-hybridization of the primers to a related molecule. No differential splicing of versican has been described in this region in any species. Cloning of this product is in progress.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We examined the expression of proteoglycans by two distinct phenotypes of SMCs in vitro. We found that both culture types express mRNAs encoding perlecan, biglycan, and decorin proteoglycans. We have also shown that versican proteoglycan is expressed by cultured aortic SMCs showing the adult medial morphology and not by {pi} SMCs. This major difference in proteoglycan synthesis between the {pi} and adult medial cells reflects a difference in mRNA expression: {pi} SMCs fail to express versican mRNA. Versican mRNA was undetectable by Northern blot analysis and by 25 cycles of PCR amplification in pup cell line WKY12-22, which comprises almost entirely cells with the {pi} morphology (cobblestone). It was also absent or barely detectable by PCR analysis (Pup II) from the cobblestone-shaped clones derived from that line but was present in another clone (Pup V) that has a shape and gene expression pattern similar to adult medial cells.4

The abundance of a number of mRNAs differs between {pi} and adult medial cells.2 3 5 7 Three of those RNAs (PDGF-B, PDGF-{alpha}R, and versican) are detectable in only one of the two phenotypes: PDGF-B in {pi} phenotype SMCs and PDGF-{alpha}R and versican in adult medial phenotype SMCs.1 4 5 Adult SMC clones have been screened for the expression of genes that characterize the {pi} phenotype as well as smooth muscle–specific genes, and only the expression of PDGF-{alpha}R and PDGF-ßR mRNAs correlates with versican mRNA levels (J.M.L., S.M.S., unpublished data, 1993).

The finding of high-level expression of versican mRNA in adult clones that also express high levels of PDGF-{alpha}R mRNA and the finding that both RNAs1 4 are expressed at low or undetectable levels by {pi} phenotype cells suggest at least two possibilities. First, versican and PDGF-{alpha}R may be regulated by the same factor in SMC cells. If this is true, however, the factor is unlikely to be a secreted factor (or is rapidly degraded), because culturing pup SMCs in medium conditioned by adult SMCs does not change their morphology (S.M.S., unpublished data, 1991). Preliminary experiments suggest that PDGF-{alpha}R is regulated at the transcriptional level (M.W. Majesky, unpublished data, 1990), and similar experiments will determine whether versican is similarly regulated. If both genes are regulated at the transcriptional level, then comparisons of their promoter regions may lead to the recognition of transcription factors that determine the adult medial phenotype.

Alternatively, it is possible that one gene regulates the other. We have found that PDGF increases the amount of versican mRNA in cultured monkey cells.18 This leads to the possibility that the activity of the PDGF-{alpha}R may be required for versican expression by SMCs. Analysis of versican expression in {pi} phenotype cells transfected with the PDGF-{alpha}R gene under the control of a constitutive promoter may reveal such a regulation.

The core proteins of the large CSPG that is present in rat adult medial but not {pi} SMCs are similar in size to versican core proteins from monkey aorta and human fibroblasts (which are both 400 to 500 kD)17 18 44 and were identified as versican or versican-like by immunoreactivity with two antibody preparations that recognize two nonoverlapping regions of the versican protein.35 36 At the mRNA level, we have found multiple bands (2.5 to >12 kb) that hybridize to versican probes at high stringency. Some or all of these RNA forms may correspond to the splice variants that have been found in versican/PG-M from human, mouse, and chicken.42 46 47 Versican/PG-M splice variants include molecules containing both, either, or neither of two central exons encoding GAG-attachment domains,42 46 47 and they appear to be responsible for the existence of multiple protein cores.46 PCR analysis and cDNA cloning have indicated the presence of at least two of these isoforms in human aorta,17 46 suggesting that the multiple versican RNA forms that we have described may have an in vivo correlate. It seems likely that these variants, which differ significantly in length and are predicted to have different numbers of GAG chains or to lack GAG chains entirely, would have different functions.42 46 47

Synthesis of versican by adult medial–type SMCs may have profound effects on SMC growth and morphology. Hamati et al15 cultured rat adult aortic SMCs in the presence of 4-methylumbelliferyl-ß-D-xyloside, which disrupts proteoglycan synthesis, although other metabolic pathways may also be affected.48 This treatment caused a reversible change in shape from spindle to "rounded or cuboidal" and prevented the characteristic postconfluent multilayered growth. This change resembles a change in shape and growth properties from the adult medial to {pi} phenotype. Our analysis showed that the major difference in proteoglycan content of adult medial and {pi} cells is the expression of versican by adult medial and not {pi} cells. The morphological similarities between adult cells grown in the presence of a proteoglycan synthesis inhibitor and {pi} cells, which fail to synthesize a particular proteoglycan, versican, suggest the possibility that some of the different properties of adult medial and {pi} cells may result directly from differences in the ability to synthesize versican. Indeed, alterations in the matrix supplied to SMCs have been shown to affect differentiation.49 50 51 52

While we feel that versican is a candidate for the molecule responsible for these differences in SMC shape and differentiation properties, other proteoglycans may be involved. Syndecans are cell-surface proteoglycans that bind both extracellular matrix molecules and fibroblast growth factor, are found in focal contacts, and may interact with the actin cytoskeleton via their cytoplasmic tails.53 Members of the syndecan family have been shown to be involved in cell shape regulation.54 55 Although neither we nor other groups have examined the expression of syndecans in {pi}-type SMCs in vitro, syndecans are expressed by adult medial SMCs in vitro and also increase in the rat carotid artery balloon-injury model with kinetics similar to versican.56 57 58 Furthermore, in the experiment by Hamati et al,15 syndecans, like versican, should be synthesized without GAG chains in the presence of ß-D-xyloside, and therefore either could be responsible for the SMC shape difference. Experimental manipulation of the levels of versican and syndecan expression in SMCs are needed to determine whether either or both molecules are responsible for shape differences between SMC types.

The mechanism by which versican might exert an effect on cell behavior is unclear and may be determined by its adhesive or antiadhesive properties. Sequence analysis of human versican cDNAs has revealed an amino-terminal domain resembling hyaluronan-binding motifs. The binding of hyaluronan by versican has been confirmed by using the isolated recombinant amino-terminal domain as well as by the ability of hyaluronan oligosaccharides to release the large CSPG from the cell layer of cultured monkey SMCs.35 59 The carboxy-terminal domains resemble epidermal growth factor, lectin-binding motifs, and complement regulatory protein.20 60 The lectin-binding domain of human versican has been shown to bind tenascin-R, and the carboxy-terminal region of the chicken homologue (PG-M) has been shown to bind D-mannose, D-galactose, L-fucose, and N-acetyl-D-glucosamine.61 62 In the latter case, however, the in vivo ligand has not been determined. Versican may also function as an antiadhesive molecule via the GAG chains.22 Chicken PG-M (versican) blocks adhesion to fibronectin, collagen, and vitronectin and is excluded from focal contacts.22 23 This property may also be important in cell transformation. Yamagata and Kimata63 have shown that transfection of osteosarcoma cells with anti-sense versican changes the nature of their contacts with substrata from the rosette-type adhesive contacts (typical of malignant cells) to more normal focal contacts. At the same time, the disordered microfilaments reorganize into normal stress fibers. This may be contrasted with the role of syndecan-1, which is reduced, rather than increased, in the transformed state.55 64 The antiadhesive properties of versican may facilitate the migration of malignant cells to new sites. It has been proposed that CSPGs may promote migration by steric exclusion.65 The antiadhesive properties of versican/PG-M require both the protein and GAG components.22 When present at a sufficiently high concentration, a large, highly charged CSPG such as versican, which forms even larger aggregates with hyaluronan, might exert a dominant negative effect over other cell-matrix interactions by nonspecifically preventing ligand-receptor interactions. Experiments designed to specifically inhibit versican expression in adult medial cells, to culture {pi} cells in the presence of versican, or to induce the synthesis of versican by {pi} cells should help establish whether versican is responsible for any of the differences in cell shape and gene expression between {pi} and adult medial cells.

Finally, we have proposed that {pi} cells, a type predominating in cultures derived from rat pup aortas and neointimas and which proliferate in the absence of platelet factors, may correspond to a subset of the cells that migrate into the intima,1 66 ie, to that fraction of migratory cells that proliferate and might be considered as "stem-" or "blast-"like. In support of this hypothesis, we have found that mRNAs (ie, elastin, osteopontin, and PDGF-B) that are more abundant in {pi} SMC than adult medial SMC cultures are expressed at elevated levels in the neointima in vivo.2 8 57 67 A corollary to this hypothesis might be that genes whose expression is low or absent in cultured adult medial cells would not be expressed in the neointima in vivo. However, the detection of PDGF-{alpha}R mRNA in both the medial and neointimal SMCs of balloon-injured rat carotid arteries68 and the localization of versican to the neointima24 do not support this corollary.

At least two possibilities may resolve this conflict, whereby genes expressed by the neointima are predicted to be expressed by {pi} SMCs in vitro. First, a factor inducing {pi} cells to express versican in vivo may be lacking in our culture conditions or lost due to the long time in culture. We have shown that versican RNA levels are increased by PDGF and transforming growth factor-ß in monkey SMCs and decreased by interleukin-1 in human fibroblasts cultured on fibronectin.18 69 Second, we have not examined the expression of versican in cultured neointimal SMCs. Although rat SMC cultures derived from pup aorta and adult neointimas share a number of properties,1 2 3 4 5 6 7 intimal SMCs may differ in some properties, such as versican expression, from cultures of SMCs from rat pups. We believe it would be necessary to assay versican expression by using cloned neointimal cells, because we would be seeking evidence of cells that fail to express versican among a population that is heterogeneous in morphology as well as in expression of another molecule, PDGF-B.67

If neither of these explanations is correct, an alternate corollary to the stem-cell hypothesis might be considered, ie, that the other cells present in the neointima, those that migrate to the neointima after balloon injury but do not proliferate, may be responsible for versican synthesis. Cultured adult medial cells may thus model for another population present in the intima, in support of the work of Campbell and Campbell13 and Thyberg and colleagues.50 51 52

Regardless of how the conflict between versican expression in the neointima but not {pi} SMCs from pup aorta is resolved, this piece of data is insufficient to discard either the stem-cell hypothesis or our culture model, which have been useful in predicting molecules that might be elevated in the neointima as well as in human vascular disease.2 8 57 67 70 71 Further tests of the model require determining whether markers of {pi} and adult medial cultures are expressed by the same or different cells in vivo and whether those markers correlate with the proliferating or nonproliferating neointimal cells. The study of elements regulating versican synthesis should be facilitated by the existence of two well-characterized SMC types expressing this gene at dramatically different levels in vitro.


*    Selected Abbreviations and Acronyms
 
BSA = bovine serum albumin
CSPG = chondroitin sulfate proteoglycan
GAG = glycosaminoglycan
GAPDH = glyceraldehyde 3-phosphate dehydrogenase
PAGE = polyacrylamide gel electrophoresis
PCR = polymerase chain reaction
PDGF = platelet-derived growth factor
PDGF-{alpha}R, -ßR = {alpha}-type, ß-type receptor for PDGF
PG-M = hyaluronan-binding chondroitin sulfate proteoglycan derived from chick embryonic mesenchyme
SD = Sprague-Dawley
SD12d, SD3M-23 = SMC lines isolated from 12-day- and 3-month-old Sprague-Dawley rats
SMC = smooth muscle cell
WKY = Wistar-Kyoto
WKY12-22, = SMC lines isolated from 12-day- and
WKY3M-22
3-month-old Wistar-Kyoto rats


*    Acknowledgments
 
This work was supported by grants HL-03174 (S.M.S.) and HL-18645 (T.N.W.) and postdoctoral training grant HL-07312 (J.M.L.) from the National Institutes of Health. We thank T. Bartosek, C. Tsoi, and K. Braun for expert technical assistance. We are also grateful to Dr R.G. LeBaron and Dr J.A. Madri for supplying antibodies and Dr Mark Majesky for sharing unpublished observations.

Received May 24, 1995; accepted March 5, 1996.


*    References
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