Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice

Experimental Cell Research 311 (2005) 317 – 327 www.elsevier.com/locate/yexcr

Research Article

Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice Augusto Orlandi a,*, Amedeo Ferlosio a, Giulio Gabbiani b, Luigi Giusto Spagnoli a, Paul H. Ehrlich c a

Anatomic Pathology, Dept. of Biopathology and Image Diagnostics, Tor Vergata University of Rome, Via Montpellier 1, 00133 Rome, Italy b Department of Pathology and Immunology, University of Geneva, Switzerland c Division of Plastic Surgery, Hershey Medical Center, Hershey, PA, USA Received 21 May 2005, revised version received 16 September 2005, accepted 13 October 2005

Abstract Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less a2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of a-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express a-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Antia2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC – PLCs; anti-a1 and anti-a2 integrin antibodies induced a similar slight inhibition in TI-SMC – PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs. D 2005 Elsevier Inc. All rights reserved. Keywords: Lattice contraction; Extracellular matrix; Arterial wall; Proliferation; a-smooth muscle actin; a2 integrin; a1 integrin

Introduction Accumulation of smooth muscle cells (SMCs) in the intima is a fundamental event in the pathogenesis of atherosclerosis and of restenosis after angioplasty [1,2]. The original hypothesis that all SMCs of the tunica media Abbreviations: Ab, antibody; a-SMA, a-smooth muscle actin; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; NBSMC, newborn rat; FBS, fetal bovine serum; O-, 16 – 18 month old rats; PDGF, platelet-derived growth factor; PCL, populated collagen lattice; PDS, plasma-derived serum; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SMC, smooth muscle cell; TEM, transmission electron microscopy; TI, thickened intima 15 days after ballooning; UM, underlying media; YA, young adult rats. * Corresponding author. Fax: +39 06 20902209. E-mail address: [email protected] (A. Orlandi). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.10.008

undergo phenotypic modulation and migrate into the intima [3,4] has been modified. Following the ‘‘monoclonal’’ hypothesis of Benditt and Benditt [5], successive studies report that SMC populations of human atheromatous plaque are oligoclonal [6,7]. This supports the hypothesis that a predisposed intrinsic or extrinsic cell subpopulation is responsible for the development of the intimal hyperplasia of atherosclerotic and restenotic processes [8– 10]. Indeed, these SMC populations display heterogeneous biological properties [8,11]. It was reported that the media of the normal vessel wall contains the contractile as well as the synthetic SMC phenotypes [3,4]. Blood vessel-derived SMC populations can show distinct characteristics in vitro [12 – 14]. Heterogeneity is documented in rat SMCs from balloon injured carotid and aortic vessels. Spindle-shaped cells, showing ‘‘hill-and-

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valley’’ growth patterns are typical of cultured SMCs derived from normal media [15]. Cultured SMCs derived from the thickened intima 15 days after endothelial injury show an epithelioid SMC phenotype and a cobblestone morphology at confluence [9,13]. In addition, a small percentage of epithelioid clones can be obtained from rat aortic normal media [16]. SMCs derived from normal media require serum for growth, while SMCs derived from thickened intima (TI) can grow in the absence of serum [9,13]. SMCs cultured from normal media of arteries from young and old rats show differences in growth patterns [12,17]. Spindle-shaped SMCs growing in a ‘‘hill-andvalley’’ pattern predominate in cell cultures derived from newborn and young adult rat aorta. In contrast, epithelioid SMCs predominate in cell cultures derived from old rats, which also grow in ‘‘hill-and-valley’’ patterns and show greater proliferation than cells derived from young animals [12]. Dermal fibroblasts derived from the same rats show on opposite behavior, i.e., more proliferating in young compared to old animals, supporting the tissue specificity of this proliferative feature [12]. Arterial SMCs are surrounded and delimited in vivo by a complex extracellular matrix (ECM). Arterial SMCs synthesize ECM molecules that include different collagen types [18]. Interaction with collagen and other ECM components influences SMC proliferation. Integrins are a large family of cell adhesion molecules that include the receptors for cell – collagen interactions, which influence cell proliferation and differentiation [19,20]. The a2h1 integrin is the major receptor for cell –collagen fibril interactions, whereas a1h1 integrin mediates cell – monomeric collagen – matrix interactions [21]. SMCs in a freefloating populated collagen lattice (PCL) induce contraction and decrease in size over time [22,23]. We reported [13] that heterogeneous SMCs contract PCL differently. In this work, we have investigated some of the differences in proliferative capacity and cytoskeletal features of SMC populations when placed in collagen lattices.

Material and methods Cell isolation and culture SMCs were obtained by limited enzymatic digestion from the tunica media of thoracic aorta of 4-day-old newborn Wistar rats (NB-SMCs), 8- to 10-week-old rats, young adult (YA-SMCs) and 16- to 18-month-old rats (OSMCs) as previously reported [12]. The endothelium of the thoracic aorta of 15 young adult Wistar rats was removed by ballooning [13]. Fifteen days after ballooning, SMCs were obtained from the thickened intima (TI)-SMCs and the underlying media (UM)-SMCs by enzymatic digestion, as previously reported [13]. Confluent SMC cultures were passed by trypsinization and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO)

supplemented with 10% fetal bovine serum (FBS), and studied in their 3rd – 5th passage. Cell-populated collagen lattice manufacture and contraction Collagen was purified from bovine tendon by limited pepsin digestion. Briefly, tendons were dissected from young calf hooves and cut into small pieces. All procedures were performed at 4-C. The tissue (1 g per 100 ml) was swollen overnight in 0.5 M acetic acid with stirring. The swollen tissue was homogenized, pepsin (Sigma) added at 10 mg per 100 ml and the mixture stirred for 2 days. The digest was centrifuged at 10,000  g for 30 min, the insoluble pellet discarded, NaCl was added 10% w/v to the supernatant and the mixture stirred overnight. The insoluble collagen was collected by centrifugation and redissolved in 1.0 M NaCl, 50 mM Tris –HCl pH 7.5. The collagen solution was cleared of particulate matter by centrifugation and exhaustively dialyzed against 1 mM HCl. The clear viscous solution was frozen, lyophilized, weighted, dissolved in sterile 1 mM HCl at 5.0 mg/ml and stored at 4-C. The purity of the collagen was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), where all stained protein bands corresponded to collagen protein chains. After 24 h of serum starvation, SMCs freed from monolayer culture by trypsinization were counted. Aliquots of 200,000 cells in 3.0 ml of DMEM with 10% FBS (complete DMEM) were rapidly mixed with 1.0 ml of collagen solution (5 mg) and the mixture poured into a 60 mm Petri dishes. The dishes were placed in a humidified cell culture incubator at 37-C, where the collagen polymerized in less than 90 s, trapping cells within the newly polymerized matrix. The lattices were detached from the dish 1 h after casting. Other groups of SMC – PCLs were cast with DMEM supplemented with 2% plasma-derived serum (PDS) as previously reported [13]. To evaluate differences in SMC populations’ capacity to contract free floating SMC – PCLs, the diameters of 2 ml SMC – PCLs cast in each well of 6-well plates were measured with a ruler to the nearest 0.5 mm over a period of 14 days. The area of each PCL was calculated and recorded. At day 5 and day 9, medium was removed and replaced with 2 ml of fresh complete DMEM or PDS supplemented DMEM. To verify the role of a2 and a1 integrins in the contraction, PCLs were cast with either complete or 2% PDS DMEM and 10 Ag/ml hamster anti-a2 integrin antibody (Ab, clone Ha1/29, PharminGen, S. Diego, CA) or control hamster IgG (PharminGen). Contraction of PCLs cast in quadruplicate was measured after 24 h. Smooth muscle cell proliferation, proliferation and cell cycle Following changes in the numbers of SMCs incorporated in PCL in the presence of 10% FBS was measured by releasing cells by collagenase digestion. At days 2 and

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7, 4 lattices from each group were transferred to 15 ml centrifuge tubes and an equal volume of collagenase digest solution was added (4 mg collagenase, Type IV Sigma and 2 mg soybean trypsin inhibitor, Worthington Biochemical Corporation, Lakewood, NJ, per ml of DMEM with 20 mM HEPES buffer). The tubes were sealed and incubated at 37-C with rotation for 10 min. An equal volume of DMEM with 20% FBS was added and the mixture aspirated 10 times with a pipette. After centrifugation (300  g) for 5 min, the supernatant was discarded and the cell pellet washed with PBS containing 0.2 mg EDTA/ml, then centrifuged and cells suspended in 1.0 ml of serum-free DMEM. In preliminary experiments in triplicate, we have observed that the expected recovery of cells from collagen matrices by collagenase digestion is about 50%, possibly due to losses during casting of the collagen lattices, cells detaching and falling out of lattices or the harsh condition of collagenase digestion. To identify a relationship between the inhibition of cell division, when contained in a collagen matrix, aliquots of SMC– PCLs were harvested at days 2 and 7. The proportion of nuclear DNA in G0 –G1, S and G2 – M phases was determined by flow cytometry analysis as previously reported [24]. Differences in the capacity of SMCs to migrate from collagen lattices were examined. SMC –PCLs were placed in 35-mm dishes, where a sterile coverslip was placed on top. The numbers of cells, which migrated from selected random locations on the periphery of lattices contained in a 200 magnification microscopic field, were counted and recorded. To verify that the effects induced by longterm collagen entrapment of SMCs were reversible, SMC released from 7- and 14-day SMC –PCLs by collagenase digestion, counted, plated in 35 mm dishes and allowed to grow to confluence. Differences in cell morphology were determined by phase contrast optics with a Zeiss Axiophot photomicroscope (Carl Zeiss Inc., Oberkochen, Germany). Immunofluorescence staining Changes in the cytoskeletal makeup of SMC harvested from lattices at days 2, 5 and 7 were performed by immunostained cytospin cell preparations. SMC–PCLs subjected to limited collagenase digestion had the isolated cells washed 3 times in DMEM. The cells were counted with a hemocytometer and diluted to a final concentration of 105 cells/ml. The suspended cells were plated on glass slides by a Shandom cytocentrifuge (Shandom Scientific Co., London, UK) at 125  g. The cells were fixed in methanol at 20-C for 5 min and then immunostained with either a monoclonal anti a-SMA (Dako Cytomation, Glostrup, Denmark, 1:10), anti-vimentin (Dako, 1:40) or with rabbit polyclonal affinity purified IgG directed to smooth-muscle myosin heavy chains (myosin, 1:10) or desmin (1:40) [25]. The control slides received IgG from rat diluted 1:40 and rabbit IgG diluted

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1:20. The secondary Abs were tetramethyl rhodamine-labeled goat anti-mouse IgG (Nordic Immunology Laboratory, Tilburg, Netherlands) or anti-rabbit IgG (Nordic). More than 1000 cells were counted for each stained cell population and the procedure was repeated 3 times. Selected SMC–PCLs were stained in situ for a-SMA using the same procedures. Electron microscopy Small pieces of SMC –PCLs were fixed in glutaraldehyde (2% in 0.1 M sodium cacodylate buffer, pH 7.4) for 3 h at room temperature, post-fixed with 1% OsO4 for 1 h and then infiltrated with EPON 812. Ultrathin sections were cut, placed on 200 mesh copper grids, stained with uranyl acetate as well as lead citrate and then examined with a Philips Morgagni transmission electron microscopy (TEM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting For SDS-PAGE, cells isolated from 2- and 7-day-old lattices by collagenase digestion were mixed with 5 concentrated sample buffer and 10 to 40 Ag of protein were electrophoresed in a 5–20% gradient gel and stained with Coomassie Brilliant Blue R-250 as previously reported [13]. Quantification of total actin and myosin was performed with a computerized laser beam densitometer, as previously reported [13]. For Western blotting [26], 10 to 40 Ag of proteins were electrophoresed and transferred to nitrocellulose membrane (0.45 Am, Schleicher and Schuell, Dassel, Germany). The membranes were incubated with a monoclonal anti-a-SMA (1:500), anti-myosin (1:100), anti-vimentin (Dako, 1:200) or a rabbit anti-a2 integrin (PharminGen; 1:50). Specific protein bands were detected by chemiluminescence (Amersham Biosciences, Uppsala, Sweden). Quantification of protein bands was determined by densitometry [24]. Flow cytometry Cells isolated from lattices were washed in Hanks’ balanced salt solution, centrifuged, incubated with control IgG or primary Ab, washed and resuspended in the appropriate fluorescein isothiocyanate-conjugated secondary Ab for 30 min at 4-C in the dark. After washing, viable cells were analyzed by FACScan flow cytometer (Becton Dickinson); x and y axes represent log fluorescent intensity and cell number, respectively. Statistical analysis Results were expressed as arithmetical mean plus or minus standard error of the mean (TSEM). For statistical evaluation, the results were analyzed with Mann – Withney U test, Student’s t test and non-parametric chi-square tests. The differences were considered statistically significant for value of P < 0.05.

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Results Morphological findings On plastic, confluent TI-SMCs demonstrated an epithelioid morphology and grew as a flat cell layer, as previously reported [9]. O-SMCs appeared somehow smaller in size and epithelioid-like, as described previously [12,27]. Remaining SMC lines exhibited the characteristic hill-and-valley growth pattern typical of cultured vascular SMCs. After 2 days, TI-SMCs in collagen matrices appeared thin and very elongated, often

making bipolar contacts with the extremities of neighboring cells (Fig. 1A), resulting in a loose three-dimensional cell arrangement. Mitotic figures were also observed in these cells. YA-SMCs as well as other SMC lines appeared generally elongated, but more ‘‘dendritic’’, with multiple and variable intercellular cytoplasmic connections, resulting in a complex three-dimensional cell network (Fig. 1B). A progressive reorganization of the collagen matrix was observed. After 7 days, the rate and degree of collagen matrix reorganization were similar for all SMC populations, except TI-SMCs, in which they were less (Figs. 1C and D). In the periphery of lattices,

Fig. 1. Phase contrast images of collagen lattices manufactured with different aortic SMC lines (PCL) obtained from different layers 15 days after ballooning and from normal tunica media of rats of different age. After 2 days, (A) thickened intima (TI)-SMCs entrapped in collagen matrices appear thin and very elongated, with bipolar contacts with the extremities of neighboring cells in a loose three-dimensional arrangement. (B) Young adult medial (YA)-SMCs appear generally elongated, but more dendritic, with multiple and variable intercellular cytoplasmic connections, resulting in a complex three-dimensional network. After 7 days, a reorganization of the collagen matrix is observed, more marked in (C) YA-SMC than in (D) TI-SMC – PCLs. (E) TI-SMC – PCLs placed in plastic dishes covered with a coverslip, show after 24 h cells migrating out, adhering to the underlying plastic surface and re-assuming an epithelioid morphology (insert); (F) after 24 h, there are no cells migrating from the YA-SMC – PCLs. (G, H) TI-SMCs and YA-SMCs obtained from 7-day-old PCLs by enzymatic digestion, when plated in plastic dishes re-acquire at confluence an (G) epithelioid monolayered and (H) hill-and-valley growth pattern, respectively.

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collagen remodeling was more evident than in the central region of the lattices. The morphological features of SMCs oriented in parallel arrays at the periphery of lattices were maintained for 14 days. These differences in collagen matrix reorganization reflected those in PCL contraction (see after). To investigate the cell migration from 7- and 14-day SMC – PCLs, lattices were placed in plastic dishes, covered with a coverslip and the cells migrating out from the lattices counted. The cells from TI-SMC – PCL migrated more rapidly and had re-assumed their epithelioid morphology (Fig. 1E). After 24 h, the number of migrating TI-SMCs per microscopic field was 7.1 T 1.1. There were no cells migrating from the other SMC– PCLs at this time. At 2 days, the number of migrating TI-SMCs was 38.8 T 6.3; for O-SMCs, 2.1 T 0.9; for UM-SMCs, 0.2 T 0.2; for NBSMCs, 2 T 0.3 and for YA-SMCs, 1.8 T 0.6. Is the elongated morphology of SMC entrapped within collagen matrices reversible? SMCs obtained by limited collagenase digestion of 7- and 14-day SMC –PCLs were plated in plastic dishes in complete DMEM. TI-SMCs rapidly proliferated and reached confluence. These cells had reacquired an epithelioid shape, when growing as a monolayer (Fig. 1G). After a delay of 1 to 2 days, the other SMC lines proliferated and at confluence reassumed the hill-and-valley growth pattern (Fig. 1H). Also, SMC – PCLs maintained in PDS medium reacquired their original morphology and growth pattern, when released cells were plated on plastic in complete DMEM (data not shown). Cell-populated collagen lattice contraction SMC – PCL contraction was influenced by the origin of the SMC line (see Fig. 2). In the presence of FBS at day 1, UM-SMC– PCLs and O-SMC –PCLs contracted less than

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YA-SMC – and NB-SMC –PCLs. TI-SMC –PCL contraction remained retarded at all time points (Fig. 2A; P < 0.01). After day 7 no further lattice contraction occurred in any of the SMC – PCLs. When lattices were cast with PDS instead of FBS, SMC – PCL contraction rate was retarded and the final degree of lattice contraction reduced (Fig. 2B). In fact, compared to lattices cast with FBS, those with PDS were significantly less contracted (larger) at all time points (P < 0.01). In the presence of PDS the O-SMC, YA-SMC and UM-SMC PCLs contracted faster and to a greater degree compared to TI-SMC and NB-SMC – PCLs (P < 0.01). Nevertheless, no morphological differences were observed in SMC – PCLs cast with either PDS or FBS (not shown). Ultrastructural features TEM studies showed that SMCs incorporated in collagen lattices had a well-developed endoplasmic reticulum, Golgi complexes and a high number of mitochondria consistent with cells having an elevated synthetic state. Collagen fibers were in direct contact with the plasma membrane of the SMCs (Fig. 3). Morphological details documented that TI-SMC cells in collagen lattices (Fig. 3A) were, in general, more elongated with a reduction in contacts with collagen fibrils, than other SMC lines, in particular YA-SMCs (Fig. 3B); the latter had more cytoplasmic projections (Fig. 3C). No basal laminae were evident on cells within SMC –PCLs. Alpha2 and alpha1 integrin expression and lattice contraction Densitometry scanning of the protein bands (Fig. 4A) showed that TI-SMCs from PCL expressed less a-SMA

Fig. 2. SMC-populated collagen lattice (PCL) contraction is influenced by the phenotype of the SMC line. PCLs were manufactured with aortic SMC lines obtained from thickened intima (TI) and underlying media (UM) 15 days after endothelial injury by ballooning and normal tunica media of rats of different age. (A) In the presence of 10% FBS, at day 1 UM-SMC- and O-SMC-contract less than YA-SMC- and NB-SMC – PCLs. TI-SMC – PCL contraction remains retarded at all studied time points. After day 7, no further lattice contraction occurs. (B) When lattices are cast with 2% PDS instead of FBS, SMC – PCL contraction rate is retarded and final lattice contraction reduced. O-SMC – , YA-SMC – and UM-SMC – PCLs contract faster and to a greater degree compared to TI-SMC – and NB-SMC – PCLs; *P < 0.01).

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Fig. 3. Transmission electron microscopy photographs showing SMCs incorporated in collagen lattices 7 days after manufacture in the presence of FBS. (A, B) Cells in collagen lattices demonstrate a well-developed endoplasmic reticulum, Golgi complexes and a high density of mitochondria. (A) thickened intimaSMCs in collagen lattices appear elongated with a reduction in contacts with collagen fibrils, as compared to (B) young adult media (YA)-SMCs; (C) at higher magnification, a detail of YA-SMC cell membrane in collagen lattice with a cytoplasmic projection in contact with collagen fibrils.

and SM-myosin compared to other SMCs lines. Flow cytometry (Fig. 4B) showed that TI-SMCs from PLC expressed less a2 integrin compared to YA-SMCs, whereas a1 integrin expression was similar. Differences in a2 and a1 integrin expression among TI-SMC and other cell lines were confirmed by Western blotting (data not shown). To investigate if the expression of integrins was related to SMC – PCLs contraction, TI-SMCs and YA-SMCs were cast in collagen lattices with anti-a2 or anti-a1 integrin Ab. When YA-SMCs were cast with anti-a2 integrin Ab, lattice contraction was greatly inhibited ( P < 0.001, Figs. 4C and D), whereas casting with anti-a1 integrin resulted in a much more less evident inhibition. In contrast, TI-SMC – PCLs cast with anti-a2 or anti-a1 integrin Ab showed only a modest inhibition of contraction. Incubation with a control hamster IgG did not significantly modify SMC – PCL contraction. Remaining SMC lines and YA-SMC – PCLs had a similar behavior (not shown).

Smooth muscle cell proliferation and cell cycle analysis in collagen lattices The number of cells recovered from collagenase-treated PCLs is presented in Fig. 5. After 2 days, the TI-SMCs within PCL had gone through one cell doubling. In contrast, the other SMC lines incorporated in collagen lattices had not divided during this same period. At 7 days, the number of TI-SMCs within PLC showed that these cells had undergone another doublings. The other SMC lines incorporated in collagen lattices had undergone only one cell doublings. When incorporated in a collagen matrix, the SMC line that expressed the least amount of a2 integrin (TI-SMCs) showed the most proliferation. YA-SMCs showed the fastest rate and greatest degree of lattice contraction. Cell cycle analysis by flow cytometry is reported in Table 1. All SMC lines recovered from collagen lattices showed that the major portions of cells were in G1 phase of the cell cycle. Compared to other SMC lines at 2 days,

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in the S phase of the cell cycle at 7 days had declined to 1/ 3 of its 2-day value, 15% to 5%. Cytoskeletal features of smooth muscle cells from collagen lattices

Fig. 4. (A) Immunoblottings after SDS-PAGE of protein aliquots from cell lysates harvested from 7-day SMC – PCLs by collagenase digestion. Densitometry scanning of the protein bands after Western blotting reveals that thickened intima 15 days after ballooning (TI)-SMCs in collagen lattice express less a-smooth muscle actin (a-SMA) and smooth muscle (SM) myosin compared to young adult media (YA)-SMCs, old media (O)-SMCs, newborn media (NB)-SMCs and underlying media 15 days after ballooning (UM)-SMCs. (B) Flow cytometry of a2 and a1 integrin in TI-SMCs and YASMCs from collagen lattice. YA-SMCs express more a2 integrin compared to TI-SMCs; (C, D) role of a2 and a1 integrins in SMC – PCL contraction, in either (C) DMEM with 10% FBS and (D) 2% PDS. After 24 h, the YASMC – PCL contraction is inhibited more in the presence of anti-a2 than a1 integrin Ab with either FBS or PDS. TI-SMC – PCLs cast with anti-a2 or a1 integrin Ab demonstrate slight and similar inhibition of lattice contraction; x and y axes correspond to cell number and log fluorescence intensity, respectively.

TI-SMCs released from collagen lattices showed a higher proportion of cells in S and G2/M phases of the cell cycle. At day 7, the proportion of TI-SMCs recovered from collagen lattices in the Go – G1 phase of the cell cycle was the greatest, but that difference was reduced compared to that seen at 2 days. The proportion of TI-SMCs appearing

Differences in the percentage of total actin and myosin per total protein [13] in SMCs isolated by limited collagenase digestion from 2-day SMC – PCLs are presented in Table 2. Densitometry analysis of SDS-PAGE revealed, when TI-SMCs and O-SMCs were maintained in collagen or on plastic, they contained less total actin and myosin compared to other SMC lines (Table 2). In all cell lines, there was more total actin and myosin in SMCs incorporated in collagen lattices compared to the same cells growing on plastic. At 7 days, the percentage of total actin and myosin within the TI-SMCs had declined more than other SMC lines, which retained the same percent of actin and myosin measured at day 2. Cytospin-immunostained preparations of SMCs isolated from PCLs were viewed. All SMCs were vimentin-positive and desmin-negative; moreover, a dramatic decrease in the expression of a-SMA isoform was observed. At day 2, no a-SMA-positive cells were detected in any of the SMC – PCLs. At 5 days, less than 1% of YA-SMCs, UM-SMCs and NB-SMCs were positive for a-SMA. At 7 days, when no further lattice contraction occurs, immunostaining (Fig. 6) showed 11 T 2% of O-SMCs were positive for a-SMA compared to 6 T 1% of the TI-SMC stained PCLs. The percentage of a-SMA-positive cells for YA-SMCs was 21 T 2%; 35 T 3% for UM-SMCs; and 29 T 2% for NB-SMCs. The percentage of SMCs expressing a-SMA was much greater for the same cell lines growing on plastic, where TISMCs had 20 T 3; O-SMCs had 28 T 1 which were the lowest values of all the SMC lines. In monolayer cultures, the NB-SMCs had the highest percentage of a-SMA (89 T 2); followed by UM-SMCs (76 T 4) and YA-SMCs (70 T 3), according to that previously reported [12,13]. Densitometry scanning of immunoblots confirmed immunohistochemical findings. After 2 days in collagen lattices, a-SMA was almost absent in all SMC lines. After 7 days, a partial

Fig. 5. SMC proliferation within collagen lattice is influenced by the origin of the SMC population. The number of recovered TI-SMCs after 2 and 7 days is twice that of all other SMC lines (P < 0.01).

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Table 1 Flow cytometry analysis of DNA content and cell cycle of aortic SMC populations recovered from collagen lattices by enzymatic digestion SMC population

G0 – G1 phase

S phase

G2 – M phase

2 days

7 days

2 days

7 days

2 days

7 days

Thickened intima 15 days after injury by ballooning Underlying media 15 days after injury by ballooning Young rat normal media Newborn rat normal media Old rat normal media

77.58 95.9 95.78 89.87 89.29

87.85 94.15 94.1 91.67 92.03

14.69 0.33 1.37 4.76 5.74

4.61 1.45 2.30 3.66 3.65

5.73 2.97 1.35 3.49 2.27

6.74 3.25 2.60 3.27 2.92

The number of cells in G0 – G1, S and G2/M phases is indicated by the percentage of total events (10,000 cells).

recovery was observed (Fig. 4A). However, the TI-SMCs contained significantly less a-SMA compared to all the other SMC lines (P < 0.01). Evaluation of SM myosin isoform-positive cells showed similar results comparing TISMCs and other SMC lines, although the percentage of positive cells was reduced compared to that of a-SMA in both collagen and plastic conditions (data not shown).

Discussion Our findings clarify some important aspects of the behavior of vascular SMCs entrapped in collagen matrices. SMCs derived from different layers of arterial wall and from rats of different ages showed variations in the rate and degree of lattice contraction, which extends the observations of an earlier report [13]. In particular, the SMC lines displaying a spindle-shaped phenotype or growing in hilland-valley pattern on plastic cultures share a similar capacity to contract SMC – PCLs. In contrast the TI-SMCs, which have an epithelioid phenotype and grow in monolayer, had a limited capacity to contract collagen lattices. This was consistent with the TI-SMCs reduced expression of a2 integrin. It was also noted that the ability of SMCs to proliferate in a collagen lattice is inversely proportional to their capacity to contract that collagen lattice. Like fibroblasts, SMC suspended in a collagen matrix initially show a reduction in a-SMA expression [28]. By 7 days, the vascular-derived SMC – PCL showed a partial recovery in the expression of a-SMA. Vascular SMCs contracted collagen lattices, similarly to what is reported for dermal fibroblasts and intestinal SMCs

[22,29,30]. The retraction of a collagen matrix by resident cells requires the translocation of collagen fibrils and their reorganization into more organized, thicker fibers [31]. Like fibroblasts, SMCs retained an elongated morphology, when residing in a floating collagen matrix [32]. Lattice contraction implies the organization of collagen fibrils, which is independent of cell contraction [33]. This is supported by the finding that lattice contraction started soon after casting collagen lattices, a time when a-SMA expression is minimal. As a consequence, factors other than the presence of a-SMA in cytoskeletal stress fibers promote collagen lattice contraction. Integrin expression is critical for lattice contraction [34]. The integrin family contains different collagen receptors that are structurally related [35]. Whereas these receptors share the subunit h1 integrin, there are 4 unique alpha subunits, resulting in four heterodimers. The a2h1 integrin is the major type I collagen receptor as well as other interstitial collagen types [36]. Our results demonstrate that differences in the capacity to contract floating collagen lattices among SMC populations have a relationship to the expression of a2h1 integrin. In addition, the failure of anti-a2 integrin antibody to totally inhibit SMC – PCL contraction suggests that other receptors may contribute to SMC –collagen interactions. Our results suggest that phenotype also influences a1h1 integrin-mediated SMC cell adhesion, migration and collagen lattice contraction [21,37]. It is worth noting that the PCLs studied here were at a moderate cell density, which induces lattice contraction through a collagen organization process [38]. The finding that antibodies blocking a2 and a1 integrins did not interfere with the proliferation of SMC lines in collagen

Table 2 Percentages of total actin and myosin per total protein of smooth muscle cell (SMC) populations after 2 days of culture on plastic dishes or from collagen lattices in the presence of 10% FBS, calculated by densitometric analysis after SDS-PAGE in triplicate, T SEM SMC population

Thickened intima 15 days after injury by ballooning Underlying media 15 days after injury by ballooning Young rat normal media Newborn rat normal media Old rat normal media

Total actin (%) in SMCs from PLC

Total actin (%) in SMCs in monolayer culture

Total myosin (%) in SMCs from PLC

Total myosin (%) in SMCs from monolayer cultures

8.2 T 0.4

6.9 T 0.4

1.6 T 0.4

1.1 T 0.2

15.3 T 0.5

13.5 T 0.4

3.3 T 0.4

2.8 T 0.3

13.1 T 0.6 14.7 T 0.5 8.7 T 0.6

7.2 T 0.4 8.8 T 0.6 6.6 T 0.4

3.6 T 0.5 3.8 T 0.4 1.5 T 0.2

1.6 T 0.3 1.8 T 0.3 1.2 T 0.3

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Fig. 6. (A, B) Fluorescent microscopy photographs of cytospin preparations and SMCs in collagen lattices from different SMC lines isolated from 7-day SMC – PCLs. (A) The numbers of a-SMA-positive TI-SMCs is markedly less compared to that of (B) YA-SMCs. Immunostaining in collagen lattices for a-SMA shows (C) rare positive elongated TI-SMCs and (D) more frequent positive YA-SMCs, with a dendritic profile.

shows the disconnection between cell proliferation and lattice contraction. Our results confirm that TI-SMCs maintain unusual characteristics when entrapped in floating PCLs. Epithelioid SMCs grow out from explants of normal media from the luminal part of the rat aorta [39] as well as from the thickened intima [13,16]. In SMC cultures from rat aorta and porcine coronary artery, spindle-shaped SMC clones predominate over epithelioid ones [16,40]. Vascular SMC clones that exhibit spindle-shaped or epithelioid phenotypes are also described in mice [41]. These studies support the possibility that TI-SMCs develop essentially from a distinct, medial subpopulation that exhibits an epithelioid phenotype when grown in culture [42]. A proportion of these epithelioid SMCs may exist within the media throughout the life span and phenotypic changes can be induced by circulating or microenvironmental factors. Recent studies suggest that circulating stem cell populations may migrate from bone marrow and contribute to the neointimal SMC population hyperplasia after injury [43]. Independently from their origin, epithelioid SMCs can be cloned from human arterial media [44], suggesting that the expansion of an SMC subset in atherosclerotic lesions is conceivable. Documentation of alteration in the physiology of vascular SMC subpopulations in monolayer cultures shows increasing SMC differentiation, promoting proliferation and/or retarding differentiation [42]. Here, serum deprivation did not eliminate differences seen in SMC lattice contraction as shown between TI-SMC – PCLs and other SMC – PCLs. When suspended in a collagen matrix, epithelioid TI-SMCs showed a greater proliferative activity than other SMC lines. Primate dermal fibroblasts suspended in collagen lattices show a 4-day delay in cell division and these cells are trapped in the G2-M phase of the cell cycle [45]. However,

rodent fibroblasts in collagen lattices do divide and are mostly in the G0 – G1 phase of the cell cycle. All rat SMC lines with the exception of TI-SMC are retarded at cell proliferation, when suspended in a collagen matrix. Autocrine production of platelet-derived growth factor (PDGF)-BB, which is a potent mitogen, supports TI-SMC proliferation on plastic in the absence of serum [9]. Although TI-SMCs did not contract collagen lattices as well as other SMC lines, they divided and migrated from collagen lattices better than other SMC lines. The inhibition of SMC proliferation in PCL does not alter the degree of lattice contraction [30], which confirms that mechanisms unrelated to cell proliferation promote PCL contraction. Moreover, TI-SMCs retained a high capacity of migration from collagen lattice after 14 days. A striking difference between epithelioid or rhomboid shaped and spindle-shaped SMCs is their high migratory capacity [16,40]. It is noteworthy that the collagen matrix failed to induce a permanent change in activated epithelioid cells. At 14 days, TI-SMCs in PLC retained the capacity to rapidly migrate from their surrounding collagen matrix and reacquire their original epithelioid phenotype upon making contact with the plastic surface. High tissue plasminogen activator activity and high levels of metalloproteinase-2 may contribute to this enhanced migratory activity of rat TISMCs [46]. In conclusion, our results confirm vessel SMCs obtained from different layers or from different aged donors display heterogeneous features, when entrapped in a floating collagen lattice. The TI-SMCs appear quite different from other SMC lines and some of these differences are retained, when these cells are maintained in collagen matrices. Our results may be specific for rat SMCs and further studies are needed to verify if these properties are shared from SMC of other species, including

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humans. In any case, the investigation of SMC lines suspended in collagen lattices appears as a promising model for gaining insights into the role of SMC heterogeneity in vascular pathobiology and the microenvironmental influences on cell phenotype.

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