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Cardiovascular Research 1997 36(1):118-126; doi:10.1016/S0008-6363(97)00156-9
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

The A10 cell line: a model for neonatal, neointimal, or differentiated vascular smooth muscle cells?

Rohini S Raoa, Joseph M Mianob, Eric N Olsonc and Charles L Seidela,*

aBaylor College of Medicine, Section of Cardiovascular Sciences, Houston, TX 77030, USA
bMedical College of Wisconsin, Department of Physiology, Houston, TX, USA
cUT Southwestern Medical Center, the Hamon Cntr. for Basic Cancer Research, Houston, TX, USA

* Corresponding author. Dept. of Medicine, Section of Cardiovascular Sciences, Room 512C, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: +1-713-798-4977; fax: +1-713- 790-0681.

Received 6 January 1997; accepted 26 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The A10 cell line was derived from the thoracic aorta of embryonic rat and is a commonly used model of vascular smooth muscle cells (VSMC). Despite its wide use this cell line has not been well characterized. This is especially important in light of recent evidence of phenotypically distinct cell populations isolated from rat vascular tissue. Therefore, the present study was undertaken to confirm the VSMC nature of A10 cells and to investigate whether these cells particularly resemble adult, neonatal, or neointimal rat VSMC. Methods: A variety of defining characteristics were used that included immunofluorescent analysis for smooth muscle {alpha}-actin, smooth and non-muscle myosin heavy chains, desmin and vimentin; Western analysis for smooth muscle and non-muscle myosin heavy chains; mRNA analysis for smooth muscle myosin heavy chain, calponin, SM22{alpha}, tropoelastin and PDGF-B peptide; and functional assays of cell migration, proliferation and agonist induced intracellular Ca transients. Results: A10 cells expressed smooth muscle {alpha}-actin, SM22{alpha}, smooth muscle calponin and vimentin, characteristic of in vivo rat VSMCs; however they also resembled de-differentiated smooth muscle cells in that they expressed non-muscle myosin rather than smooth muscle myosin heavy chain. A10 cells resembled cultured rat neonatal smooth muscle cells ("pup cells") in that they had an epithelioid shape and lacked functional PDGF-{alpha} receptors; however they did not express PDGF-B mRNA or proliferate in low serum containing medium as do neonatal cells. A10 cells had several characteristics in common with neointimal cells including the expression of {alpha}-actin, vimentin, and non-muscle myosin and the lack of expression of PDGF-B mRNA as well as the ability to migrate in response to PDGF-BB. Conclusion: In conclusion, A10 cells are nondifferentiated VSMC that differ from neonatal but bear significant resemblance to neointimal cells.

KEYWORDS A10 cells; Rat; Vascular smooth muscle; Cytoskeletal proteins; PDGF; Cell migration; Cell proliferation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Schwartz and colleagues [1–4]have described two distinct subpopulations of rat aortic smooth muscle cells: pup smooth muscle cells (P-SMC) and adult smooth muscle cells (A-SMC). Both types are found in cultures from neonatal rat aorta. P-SMC are epithelioid, grow in a monolayer, express high levels of several extracellular matrix protein genes, and secrete a PDGF-like activity. A-SMC are spindle-shaped, grow in hills and valleys, express reduced levels of extracellular matrix protein genes, and do not secrete a PDGF-like activity. P-SMC and A-SMC also differ in the combination of PDGF peptides and receptor subunit genes expressed. Majesky et al. [4]demonstrated that cells cultured from rat carotid neointima are identical to P-SMC with respect to these described properties. Recently, Bochaton-Piallat et al. [5]have shown that normal rat aorta and the neointimal layer from injured aorta are composed of four clonal cell lines present in different proportions. A clone with spindle morphology predominates in normal vessels while one with epithelioid morphology dominates in the neointimal layer. Taken together, these studies indicate that the rat tunica media is composed of a heterogeneous cell population.

A10 cells were derived from 14–17 day old embryonic BDIX rat thoracic aorta [6]and have been used extensively as models of vascular smooth muscle cells. However, because of the cellular heterogeneity of rat tunica media it is important to identify which vascular wall cell they most closely resemble. The designation of A10 cells as a smooth muscle cell line was based on their anatomical origin, electrophysiological properties, ultrastructural characteristics, the presence of muscle creatine phosphokinase and myokinase [6]and negative immunostaining for cytokeratin and neuron-specific proteins [7]. Since these studies, additional markers have been used to characterize vascular smooth muscle cells, but these have not been applied to A10 cells. The purpose of this work was to use such markers to characterize A10 cells. Because of the embryonic origin and epithelioid shape of A10 cells, the hypothesis of the present work was that A10 cells resembled P-SMC.

A difficulty in defining cell lines as VSMC arises from intrinsic smooth muscle heterogeneity and the extreme plasticity of the differentiated VSMC phenotype. In addition, a single protein that uniquely distinguishes VSMC from other cell types, as von Willebrand factor marks endothelial cells, has not been clearly established [8]. Therefore, a broad panel of markers was used.

Smooth muscle {alpha}-actin, smooth muscle myosin heavy chain and calponin are sequentially expressed during embryonic development of VSMC [9–11]and along with SM22{alpha} [12]are also expressed in differentiated VSMC in the adult. Expression of such proteins by A10 cells would imply their smooth muscle cell origin. Other proteins were selected because of their presence in VSMC under various conditions and therefore their presence or absence would round out the characterization of the A10 cell line. Desmin was originally thought to be a marker for all muscle cells but it has been shown to be absent from some smooth muscle cells in vivo [13]. Its presence, but not absence, in A10 cells would suggest a smooth muscle origin. Vimentin, though not characteristic of muscle cells in general, is present in some VSMC in vivo [14, 15]. Its presence along with VSMC specific proteins would support a VSMC origin of A10 cells. The presence of vimentin in the absence of desmin would show similarity to neointimal cells. The non-muscle form of myosin heavy chain is not expressed in adult rat aortic VSMC in vivo but is in primary and passaged cultures [16]as well as in neointimal cells [17]. Its presence with VSMC markers would suggest that phenotypic modulation had occurred from a more differentiated muscle cell origin. Finally, A10 cells were compared to published properties of P-SMC [1, 4]by determining growth characteristics in low serum, PDGF {alpha} receptor function, and expression of PDGF B and tropoelastin mRNA.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
A10 cells were obtained from ATCC (Rockville, MD) at passage number 18. They were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 150 units/ml penicillin, and 150 µg/ml streptomycin (complete DMEM, cDMEM). Cells were incubated at 37° in 5% CO2 and 95% air. Confluent cells were passaged using 0.05% trypsin/0.53 mM EDTA. Cell culture reagents were all from GIBCO/BRL, Gaithersburg, MD.

A7r5 cells, used for comparison in RNase protection assays, were obtained from ATCC and grown like A10 cells except in 10% FBS cDMEM. They are another putative smooth muscle cell line.

2.2 Immunocytochemistry
A10 cells were seeded at a density of 21,000 cells/cm2 on glass coverslips (No. 1, Corning Glassworks, Corning, NY) in 35 mm culture dishes (Falcon, Becton Dickinson and Co., Oxnard, CA). Indirect immunocytochemistry for smooth muscle {alpha}-actin, nmMHC, desmin, and vimentin was performed as previously described [18]. Non-specific labeling was assessed using only the secondary antibody. All labeling was done in triplicate. The anti-smooth muscle {alpha}-actin antibody (used at a 1:50 dilution) was a mouse monoclonal made against a decapeptide identical to the amino-terminal sequence of smooth muscle {alpha}-actin [19]; it was obtained from Sigma. The anti-desmin antibody (used at a 1:100 dilution) was the mouse monoclonal clone DE-U-10 from ICN Immunobiologicals, Lisle, IL. The anti-vimentin antibody (used at a 1:500 dilution) was the mouse monoclonal clone V9 from ICN Immunobiologicals. The anti-nmMHC antibody (used at a 1:50 dilution) was a polyclonal to platelet nmMHC from Biomedical Technologies, Inc., Stoughton, MA. The 2° antibodies were goat anti-mouse rhodamine-conjugated IgG or goat anti-rabbit rhodamine-conjugated IgG. Labeled cells were photographed using either a 2.5x Nikkormat 35 mm camera attached to a Nikon microscope with a 20x objective or a Zeiss Axiophot microscope with 63x objective. Ektachrome 400 color slide film was used.

2.3 SDS-PAGE and Western blot
4.4x106 A10 cells were removed with trypsin-EDTA from three separate T25 flasks (Corning) and extracted for proteins as previously described [20]. Extracts from canine saphenous vein smooth muscle cells (SV) in culture for 3 days (prepared as in [20]) were used as positive controls for nmMHC. Extracts from freshly dispersed canine carotid artery medial cells previously characterized in this laboratory [18]were used as positive control for smMHC. Total protein content of the extracts was determined either by BCA Assay (Pierce, Rockford, IL) or Coomassie Protein Assay (Pierce). Protein samples from each flask were electrophoresed on a 7.5% acrylamide Porzio polyacrylamide-sodium dodecyl sulfate (SDS) gel [21]designed specifically to resolve high molecular weight myofibrillar proteins. The gels were stained with Coomassie blue to visual the proteins as described previously [20]. For Western analysis, proteins were transferred to nitrocellulose as described by Towbin [22]using the 0.1% SDS buffer and 100v for one hour and labeled with antibody as previously described [20, 23]. Immunological detection of smMHC used a monoclonal antibody (diluted 1:1500) to both SM1 and SM2 developed in this laboratory [23]. This antibody does not exhibit species specificity [23]. The ECL detection system was used (Amersham International plc, Amersham, UK) according to instructions.

2.4 Northern blots
A10 cells were grown in cDMEM until confluent and then growth-arrested with 0.5% FBS DMEM for 48 h. At the end of this time total RNA was extracted from some cultures for a zero time value. Other cultures were left in 0.5% FBS DMEM or placed in cDMEM for an additional 8 and 24 h before RNA isolation. Total RNA was extracted using RNAzol (Cinna/Biotecx Lab Inc., Houston, TX) according to manufacturer's instructions. Control RNAs were from 12d old (P-SMC) and 3 month old (A-SMC) Wistar Kyoto (WKY) rat aorta (both gifts from Dr. Mark W. Majesky, Baylor College of Medicine, Houston, TX) and from canine kidney and heart (gifts of Dr. Julius C. Allen, Baylor College of Medicine, Houston, TX).

Total RNA was electrophoresed and transferred to GeneScreen membranes (Dupont New England Nuclear Research Products, Boston, MA) according to Sambrook et al. [24]. After prehybridization in a solution of 50% formamide, 5x SSC [24], 5x Denhardt's solution [24], 25 mM Na2HPO4, 0.1% SDS, and 250 µg/ml salmon sperm DNA for 3 h at 40°C, membranes were probed for PDGF B peptide (using a 3.0 kb EcoRI rat cDNA fragment from p3-4a [25]), tropoelastin (using a 3.2 kb EcoRI rat cDNA fragment from p56A3 [4]), or SM22{alpha} (using a mouse SM22{alpha} cDNA fragment). The first two cDNAs were a gift from Dr. Mark W. Majesky.

2.5 RNase protection assay
Total RNA was isolated from A7r5 and liver by the acid phenol method [26]. A 308 base pair 3' UTR riboprobe corresponding to the rat smMHC cDNA (nucleotides 1921–2229 [27]) was synthesized with T7 polymerase (Ambion, Austin, TX) in the presence of [{alpha}-32P]UTP (800 Ci/mmol; Amersham). A 225 base pair 5' riboprobe corresponding to the rat smooth muscle cell calponin cDNA (nucleotides 60–285 [28]) was similarly synthesized. Approximately 15 µg of total RNA was hybridized to each of the above riboprobes according to the manufacturer's specifications (Ambion). Protected fragments were precipitated and resolved through a 5% polyacrylamide/7M urea gel, washed in H2O/methanol/acetic acid (80:10:10), dried, and exposed to film (Kodak XAR) for 6 h.

2.6 Growth curve
A10 cells were seeded at a density of 1x10–4/cm2 in T25 flasks in either cDMEM or 0.5% FBS DMEM. After 1, 2 and 4 days, cells were suspended from three flasks at each time point with trypsin and triplicate counts were made of each sample. The results were expressed as a percentage of cells at day one.

2.7 Determination of intracellular calcium concentration
A10 cells were grown to confluence in 8–100 mm dishes (Lux, Nunc, Inc., Naperville, IL) for each experimental set. The Fura-2 procedure of Elliot et al. [29]was used. Briefly, 1.5x107 cells were removed from dishes with trypsin-EDTA, pelleted, resuspended in 10 ml PBS, pelleted, and resuspended in 10 ml HEPES buffered saline (HBS:15 mM HEPES, 5 mM KCl, 140 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM D-glucose, and 0.1% bovine serum albumin). Cell aliquots were incubated at 37°C for 30 min in the dark in HBS containing 20 µM Fura 2/AM (Molecular Probes, Inc., Eugene, OR) then diluted with HBS and incubated for another 30 min. Aliquots were removed prior to measurement, centrifuged and resuspended twice in 2 ml fresh HBS to remove extracellular Fura-2. Excitation wavelength was alternated between 340 and 380 nm every 500 ms and emission fluorescence was measured at 510 nm using an SLM 8000 spectrophotofluorimeter (SLM Instruments, Urbana, IL). PDGF isoforms were added at 2 min. PDGF AA was in a vehicle of 1% BSA/10 mM acetic acid, PDGF AB was in 1% BSA/Hanks' Balanced Salt Solution (GIBCO), and PDGF BB was in 2% BSA/10 mM acetic acid. To control for the effect of vehicle, measurements were also made with vehicle alone added at maximum volume. At 8 min 0.1% Triton-X 100 was added and at 10 min 5 mM EGTA was added to give the maximum fluorescence ratio (Rmax) with Triton-X 100, minimum fluorescence ratio (Rmin) with EGTA, fluorescence at excitation wavelength 380 nm with Triton-X 100 (F380T), and fluorescence at 380 nm excitation with EGTA (F380E). These values were used to calculate intracellular calcium concentration from the following equation, [Ca2+]i=Kd (R–Rmin)/(Rmax–R)*(F380E/F380T), where Kd=224 at 37°C and R signifies fluorescence at 340 nm/fluorescence at 380 nm. Rmax was always 12–18 and Rmin was 0.4–0.7.

PDGF BB (10 ng/ml) was used as a positive control for cell viability for each day's experiments. A minimum of 3 cell aliquots were assessed for each control and reagent concentration. Specific recombinant PDGF isoforms were obtained from Upstate Biotechnology Inc., Lake Placid, N.Y.

2.8 Migration
A wound migration assay was used as described previously [30]. A10 cells were grown to confluence in 12, 35 mm dishes. Culture medium was removed and the dishes washed twice with DMEM. A score was made across the outside of the bottom of the dish. DMEM was added to each dish. The tip of a glass Pasteur pipette was heat sealed and used to make a linear wound through the cell monolayer perpendicular to the score on the dish bottom. Time zero video images of the wound were obtained immediately after wounding using a Nikon microscope with 10x objective, a Cohu solid state video camera and a TARGA M8 video board connected to a 386 microprocessor. Orientation of dishes was defined by wound and score. Images of the same location were again taken after 3 h. Over this time period, cell proliferation does not contribute to wound closure. The area of the wound was determined at the two time points using Optimas image analysis software and plotted as a function of time. The slope was calculated and taken as a measure of the rate of wound closure, that is, cell migration rate.

2.9 Statistics
Analysis of variance (ANOVA) with the student-Newman-Keuls post test was used for Fig. 7 and Fig. 8. Computational assistance was provided by the CLINFO project, supported by the Division of Research Resources of the NIH under grant number RR-00350.


Figure 7
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  		Fig. 7 Ca response of A10 cells to PDGF isoforms. Peak intracellular Ca concentration measured with Fura 2 (see Methods) is plotted as a function of the concentration of added PDGF isoform. A10 cells are most responsive to PDGF BB, and less to PDGF AB. A10 cells do not respond to PDGF AA. PDGF BB points are significantly different from one another and the same is true for PDGF AB points. PDGF AA calcium values are not significantly different from each other. The calcium concentrations for 1 ng/ml PDGF BB and 10 ng/ml PDGF AB are not significantly different. N = at least 3 cell aliquots for each point; the 0 reagent value is the mean of baseline values from each aliquot before reagent was added.

 

Figure 8
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  		Fig. 8 Migratory response of A10 cells. The rate of wound closure (area units/h) in the presence of DMEM (control), 10 ng/ml PDGF-BB, 10% FBS DMEM, and 10–6 M serotonin (5-HT). Values are mean±S.E.M and n = 3 dishes for all but control DMEM, where n = 1; the latter is not used in statistical comparison. Migration in PDGF BB (22.2±2.8 units/h) was found to be significantly greater (P<0.05) than in 5-HT (13.8±0.9 units/h) and neither differed significantly from 10% FBS DMEM (20.4±7.1 units/h).

 

   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Despite the origin of A10 cells, that is, embryonic thoracic aorta, this cell line has not been characterized with markers used to characterize VSMCs in vivo and in culture. Therefore, we first characterized A10 cells in terms of their cytoskeletal protein expression to confirm their smooth muscle cell origin. Markers characteristic of differentiated and non-differentiated VSMC were used (Table 1).


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  		Table 1 Comparison characteristics of cultured A10 cells, neonatal rat aortic smooth muscle cells (P-SMC), and adult rat aortic smooth muscle cells (A-SMC)

 
As illustrated in Fig. 1, smooth muscle {alpha}-actin, non-muscle myosin and vimentin but not desmin are expressed by A10 cells. Gel electrophoresis (Fig. 2A) indicates that A10 cells do not express either the SM1 or the SM2 isoform of smooth muscle myosin heavy chain. A Western blot (Fig. 2B) with an antibody that cross-reacts with both SM1 and SM2 confirms this observation. RNase protection (Fig. 3B) using a probe that detects both SM1 and SM2 also indicates that A10 cells do not express detectable levels of smMHC mRNA. No signal appeared in the A10 lane even after overexposure for 3 days (data not shown). Even though A10 cells do not express smMHC, they do express message for the muscle specific proteins calponin and SM22{alpha}. RNase protection (Fig. 3A) demonstrates the presence of basic smooth muscle specific calponin mRNA while Northern analysis (Fig. 4) shows the presence of SM22{alpha} mRNA (~1.3 kb). Consistent with another report [31], SM22{alpha} transcription is not appreciably altered by serum stimulation (Fig. 4).


Figure 1
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  		Fig. 1 Alpha Actin: Indirect immunofluorescent labeling of A10 cells for smooth muscle {alpha}-actin shows an organized smooth muscle {alpha}-actin cytoskeleton. Objective 50x. Vimentin: Indirect immunofluorescent labeling of A10 cells for vimentin shows a highly organized filamentous structure. Objective 50x. Non-Muscle Myosin: Indirect immunofluorescent labeling of A10 cells for nmMHC shows positive labeling in a filamentous organization. Objective 63x. Background-A: Typical background non-specific labeling seen when cells were labeled with only the secondary rhodamine-conjugated antibody used with the primary antibodies specific for {alpha}-actin, vimentin and nmMHC. Objective 63x. Desmin: Indirect immunofluorescent labeling of A10 cells for desmin is negative. There is no organized desmin structure and the fluorescence intensity is not greater than in the control (Background-B). Objective 50x. Background-B: Typical background labeling seen when cells were labeled with only the secondary rhodamine-conjugated antibody used with the specific desmin antibody. Note the presence of a low level of background labeling. Objective 50x.

 

Figure 2
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  		Fig. 2 SDS-PAGE and Western analysis of myosin heavy chain protein expression. (A): SDS-PAGE of A10 (Lane 1, 10 µg) and canine saphenous vein medial (Lane 2, 10 µg) cell proteins. The SV lane has 204 kd (SM1), 200 kd (SM2), and 196 kd (nmMHC) bands (identified previously by immunoblot [20]) whereas the A10 lane shows the 196 kd nmMHC band and a faint 200 kd band. (B): Immunoblot of A10 (Lane 1, 10 µg) and canine carotid artery medial (Lane 2, 10 µg) cell proteins with an antibody against smMHC (both SM1 and SM2). A10 cells do not express detectable levels of smMHC protein. This Western also indicates that the faint 200 kd band seen on the SDS-PAGE is not SM2.

 

Figure 3
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  		Fig. 3 RNase protection assay for (A) smooth muscle calponin and (B) smMHC. In each case, A7r5 RNA is the positive and liver is the negative control. (A) A10 cells possess smooth muscle calponin mRNA under conditions of 72 h 0.5% FBS (Q, quiescent) as well as 48 h 0.5% FBS +24 h 20% FBS (S, serum stimulated). (B) A10 cells lack smMHC mRNA under the condition of 48 h 0.5% FBS +24 h 20% FBS (S).

 

Figure 4
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  		Fig. 4 Northern blot of total RNA (30 µg) from A10 cells in 0.5% FBS for 72 h (serum deprived, Lane 1), freshly dispersed canine carotid artery medial cells (Lane 2), and A10 cells in 0.5% FBS for 48 h followed by 24 h in 20% FBS (serum stimulated, Lane 3). The membrane was probed for SM22{alpha}. All lanes are positive.

 
A10 cells were further characterized relative to published characteristics shared by P-SMC and cultured neointimal cells, including the expression of PDGF B chain and tropoelastin mRNAs as well as the ability to proliferate in low serum containing medium.

Expression of PDGF B chain and tropoelastin mRNAs were determined after various periods of serum stimulation and deprivation. Serum deprivation was used in an attempt to promote differentiation and serum was used to stimulate the dedifferentiated phenotype. The former simulates conditions of the normal media and the latter mimics the injured vessel and neointima.

The Northern blot in Fig. 5A shows that at no point in serum stimulation or serum deprivation do A10 cells express a detectable level of PDGF B mRNA. A strong signal for PDGF B mRNA appeared in the P-SMC lane of the expected size (~3 kb) though less RNA was loaded. The Northern blot in Fig. 5B shows that A10 cells express tropoelastin mRNA (~3.5 kb) after 56 hr of serum deprivation (Lane 2), 48 h of serum deprivation +8 h of serum stimulation (Lane 3), and at a higher level after 72 h of serum-deprivation (Lane 4). However, none of these levels of expression approached the amount of tropoelastin mRNA in the P-SMC lane.


Figure 5
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  		Fig. 5 Northern blots for PDGF B and tropoelastin mRNAs. A: Northern blot of 30 µg total RNA from A10 cells grown in 0.5% FBS for 48 h (quiescent, Lane 1), 0.5% FBS for 56 h (serum deprived 8 h, Lane 2), 0.5% FBS for 48 h +20% FBS for 8 h (serum stimulated 8 h, Lane 3), 0.5% FBS for 72 h (serum deprived 24 h, Lane 4), and 0.5% FBS for 48 h +20% FBS for 24 h (serum stimulated 24 h, Lane 5) together with total RNA controls of 10 µg P-SMC (Lane 6), 10 µg A-SMC (Lane 7), 21 µg canine kidney (Lane 8), and 30 µg canine heart (Lane 9). The membrane was probed for PDGF B. Only P-SMC was positive for PDGF B mRNA. B: The same membrane probed for tropoelastin. Of the A10 lanes, 0.5% FBS for 56 h (serum deprived 8 h, Lane 2) and 0.5% FBS for 48 h +20% FBS for 8 h (serum stimulated 8 h, Lane 3) show faint bands and 0.5% FBS for 72 h (serum deprived 24 h, Lane 4) shows a stronger signal. All of these signals were weak in comparison to the tropoelastin band of P-SMC (Lane 6).

 
Growth curves in Fig. 6 show that A10 cells do not proliferate in low-serum medium of 0.5% FBS as they do in their normal 20% FBS cDMEM up to day 4. This shows a dependence on serum factors for proliferation in vitro which are reported not to be required for P-SMC proliferation [3].


Figure 6
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  		Fig. 6 Growth curves of A10 cells in 0.5% FBS DMEM and 20% FBS DMEM. Cell number is expressed relative to the number of cells present after one day in culture. One T25 flask was counted in triplicate for each point. Values are mean±S.E.M and n = 3. A10 cells proliferated rapidly between day 1 and 2 in cDMEM and reached a plateau with confluence at day 4. A10 cells did not proliferate well in 0.5% FBS; these cells did not approach confluence even at day 8 (data not shown).

 
The effect of PDGF isoforms on intracellular calcium concentration (Fig. 7) of A10 cells was assessed to determine if functional PDGF receptors were present in A10 cells. The measurement of intracellular Ca2+ transients in response to PDGF AA, AB, and BB showed that A10 cells respond differently to these three ligands. At the concentrations examined, the response to PDGF BB was always the greatest. Responses to the AB isoform were seen at concentrations equal to or greater than 20 ng/ml while no responses were detected to any of the concentrations of AA that were used.

Having demonstrated that PDGF-BB increases intracellular Ca concentration, its ability to stimulate A10 cell migration (Fig. 8) was determined and compared with serotonin (5-HT) and serum, two known chemoattractants. Cells exposed to 10 ng/ml PDGF BB migrated faster than control cells as well as those exposed to 5-HT (P<0.05 relative to 5-HT). 10% FBS, which contains PDGF and 5-HT in addition to many other growth factors and chemoattractants, was used as a nonspecific stimulus. Migration rates in response to PDGF BB and 5-HT did not differ significantly from the 10% FBS rate.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study addresses two questions: (1) Can A10 cells be considered to be VSMCs? and (2) which of the three broad categories of VSMCs — A-SMC, P-SMC and neointimal — best describes the A10 cell? To answer the first question, markers of VSMC — smooth muscle {alpha}-actin, smMHC, desmin, calponin and SM22{alpha} — were used to state more definitively whether or not the clonal A10 cell line is VSMC. Of these markers, A10 cells express smooth muscle {alpha}-actin (Fig. 1), basic calponin (Fig. 3) and SM22{alpha} (Fig. 4) suggesting a VSMC origin. A10 cells can be considered VSMC despite the absence of smMHC (Fig. 2). SM2 only appears after birth in rabbits [32], so it is not expected in the embryonic A10 cells. SM1 would show normal age-dependent differentiation, but smMHC is a marker that is quickly lost by SMC in culture [13, 16]. Cultures of neonatal cells do not lose smooth muscle myosin to the extent that adult cultures do [13], but it is still possible that A10 is a clone of one of the original 15% of smooth muscle myosin negative cells. Frid et al. [33]have cloned cells from adult bovine artery media that are epithelioid shaped like A10 and also express smooth muscle {alpha}-actin and calponin but not smooth muscle myosin in passage 5. The faint 200 kd protein band from A10 cells in Fig. 2A may be a nonmuscle myosin termed SMemb, which is identical to nmMHC-B [34]. This protein is present in embryonic and neointimal rabbit VSMC [34]. The band is not likely to be SM1, either fragmented or a fast-migrating form as reported by Babij et al. [35], unless the nonspecific SM1/SM2 epitope necessary for detection by Western analysis was lost. Interestingly, A7r5 cells, which are spindle-shaped like A-SMC, do have smMHC (Fig. 3). As they were derived simultaneously with A10 cells but from a different aorta, this may show heterogeneity of smMHC expression in embryonic rat aorta, that loss of smMHC is not a necessary event for cell line establishment, or that smMHC is lost by embryonic rat aortic cells grown in 20% FBS but not 10% FBS. The absence of desmin also does not disqualify A10 cells as VSMC. The reason for the absence of desmin may be that the original cell that was cloned was desmin negative. This is highly possible because about 40% of neonatal and adult rat aortic VSMC are desmin negative [13]. Alternatively, the lack of desmin may be a result of de-differentiation in culture. Desmin is lost quickly in primary culture and passage such that by passage 5, there are no longer any cells containing desmin in cultures of neonatal or adult rat aortic VSMC [13]. The combined expression of smooth muscle alpha actin and vimentin (Fig. 1) is also characteristic of myofibroblasts [14]. However, myofibroblasts would not be expected to express basic calponin and SM22{alpha}, characteristics of A10 cells, and therefore, could be differentiated from A10 cells by the absence of these markers. From these data it is concluded that A10 cells are of VSMC lineage but the absence of smMHC means that they can not be considered differentiated adult SMCs.

We next determined if they most closely resembled "pup" (P-SMC) or neointimal cells. A10 cells have a few characteristics in common with P-SMC in that A10 cells have an epithelioid shape (Fig. 1) and have few or no functional PDGF {alpha} receptors as indicated by a lack of change in intracellular calcium concentration in response to PDGF AA (Fig. 7). Welsh et al. [36]also observed that PDGF AA had little or no effect on phospholipase D activation, mitogenesis, or chemotaxis of A10 cells. Since PDGF {alpha} receptors bind PDGF A and PDGF B peptides but PDGF β receptors only bind PDGF B peptide [37], a functional PDGF receptor dimer could only form upon the addition of PDGF AA if PDGF {alpha} receptors were present. Thus if PDGF AA elicits no response from A10 cells, it is suggested that there are few or no functional PDGF {alpha} receptors in the cell membrane of A10 cells. It would also be expected, if there are no functional PDGF {alpha} receptors, that PDGF AB should elicit no response, since a PDGF {alpha} receptors would be necessary to bind the A peptide. Yet, A10 cells responded to PDGF AB with a change in intracellular Ca2+ albeit to a lesser extent than to PDGF BB (Fig. 7). Others, too, have shown that A10 cells respond to PDGF, presumably PDGF AB, by demonstrating a mitogenic activity and stimulation of Na+ influx [38, 39]. A possible explanation for the response to PDGF AB is suggested by Inui et al. [40], who demonstrated that PDGF AB can cause functional rat aortic VSMC PDGF ββ dimerization in the absence of PDGF {alpha} receptors. PDGF AB was found to have a higher KD than PDGF BB [40], which may explain the smaller response of A10 cells to PDGF AB in the present work. A10 cells are concluded to resemble P-SMC in not expressing PDGF {alpha} receptors.

However, A10 cells do not fulfill other criteria of P-SMC. P-SMC proliferate in plasma derived serum [1], while A10 cells do not proliferate in 0.5% FBS (Fig. 6). Although it was not tested directly by the careful removal of PDGF from the culture medium, A10 cells do not seem to have the PDGF independence that was demonstrated in a certain adult rat SMC culture [41]. Secondly, A10 cells do not express PDGF B mRNA (Fig. 5A) at the level that P-SMC do. This may explain the lack of A10 growth in low-serum medium: A10 cells do not secrete PDGF B as a potential autocrine mitogen like P-SMC. Hultgårdh-Nilsson [37]and Sjölund [42]also cultured neonatal rat SMC that did not express PDGF B mRNA. Thirdly, A10 cells do not possess the large amount of tropoelastin mRNA that P-SMC (Fig. 5B) and embryonic rat VSMC [43]do. Tropoelastin expression was increased after extended incubation in low serum medium, perhaps indicating that nonproliferative A10 cells produce more extracellular matrix.

A10 cells have several characteristics in common with neointimal cells. Like cultured neointimal cells described by Majesky et al [4], A10 cells are epithelioid in shape and lack PDGF {alpha} receptors and A10 cells resemble in vivo rat neointimal cells in lacking PDGF B mRNA [25]and expressing smooth muscle {alpha}-actin and vimentin but not desmin [44]. Furthermore, the ability of A10 cells to respond to PDGF with a rise in intracellular calcium (Fig. 7) and with migration (Fig. 8) is significant because PDGF has been shown to be important for neointima formation by stimulating cell migration [45, 46].

In conclusion, A10 cells are VSMC because they contain smooth muscle {alpha}-actin, smooth muscle calponin and SM22{alpha}; they are nondifferentiated VSMC because they lack smooth muscle myosin heavy chain. A10 cells are distinct from P-SMC in that they do not proliferate in low serum medium and express little tropoelastin and no PDGF B mRNA. A10 cells resemble in vivo neointimal cells because they lack PDGF B mRNA, migrate in response to PDGF BB, and express smooth muscle {alpha}-actin, vimentin but not desmin. However, A10 cells differ from cultured neointimal cells in the same way that they differ from P-SMCs. A10 cells can be used as a model system to study the regulation of transcription of VSMC markers and signaling cascades involved in neointimal formation.

Time for primary review 21 days.


   Acknowledgements
 
We thank Dr. Julius C. Allen and Dr. Mark W. Majesky for helpful discussion and for critically reading the manuscript. Dr. William P. Schilling is gratefully acknowledged for his advice and for the use of equipment in the intracellular calcium measurements. Work by RSR and CLS was supported in part by a grant from the American Heart Association (910-13100). RSR is a student in the Graduate Program in Cardiovascular Sciences, Baylor College of Medicine. ENO is funded by the National Institutes of Health, Muscular Dystrophy Association and the Robert A. Welch Foundation. JMM is a recipient of an NRSA Postdoctoral Fellowship.


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

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