Smooth Muscle Cells Isolated From Discrete Compartments of the Mature Vascular Media Exhibit Unique Phenotypes and Distinct Growth Capabilities
Abstract Heterogeneity of smooth muscle cell (SMC) phenotype and function is rapidly emerging as an important concept. We have recently described that phenotypically distinct SMC subpopulations in bovine pulmonary arteries exhibit unique proliferative and matrix-producing responses to hypoxic pulmonary hypertension. To provide better understanding of the molecular mechanisms contributing to this phenomenon, experimental studies will require a reliable in vitro model. The purpose of the present study was first to determine if distinct SMC subpopulations, similar to those observed in vivo, could be selectively isolated from the mature arterial media, and then to evaluate whether select SMC subpopulations would exhibit heightened responses to growth-promoting stimuli and hypoxia. We were able to reproducibly isolate at least four phenotypically unique cell subpopulations from the inner, middle, and outer compartments of the arterial media. Differences in cell phenotype were demonstrated by morphological appearance and differential expression of muscle-specific proteins. The isolated cell subpopulations exhibited markedly different growth capabilities. Two SMC subpopulations grew slowly in 10% serum and were quiescent in plasma-based medium. The other two cell subpopulations, exhibiting nonmuscle characteristics, grew rapidly in 10% serum and proliferated in plasma-based medium and in response to hypoxia. Certain colonies of the nonmuscle-like cell subpopulations were found to grow autonomously under serum-deprived conditions and to secrete mitogenic factors. Our data, demonstrating that phenotypically distinct cells with enhanced growth potential exist within the normal arterial media, support the idea that these unique cells could contribute selectively to the pathogenesis of vascular disease.
- pulmonary hypertension
- smooth muscle cell proliferation
- smooth muscle cell heterogeneity
For many years, the arterial media was believed to be composed of a phenotypically homogeneous population of SMCs. However, the possibility that the arterial media might be composed of phenotypically and functionally diverse subpopulations of SMCs is raised by the fact that vascular SMCs, especially in response to pathological stimuli, are required to perform numerous functions, including contraction, extracellular matrix protein synthesis, and replication. In disease, essential functions of the vessel, such as contraction, must be maintained while, at the same time, the reparative process is carried out. It seems likely that these diverse functions could best be served by multiple cell subpopulations with different functional capabilities rather than by modulation of a single SMC phenotype into functionally different phenotypes.
Recent in vivo studies in the systemic circulation have demonstrated that morphologically and immunohistochemically distinct SMC phenotypes exist within the arterial media of various mammalian species.1–6 Within the pulmonary circulation, we have recently reported that the normal media of developing and mature bovine arteries is also composed of multiple unique SMC subpopulations based on differential expression of muscle-specific markers7 and diverse profiles of tropoelastin mRNA expression.8 In a series of developmental studies, analysis of the pattern of muscle-specific cytoskeletal protein expression demonstrated that the identified SMC subpopulations progressed along unique developmental differentiation pathways, suggesting the existence of distinct developmental lineages for medial SMC subpopulations.7 We have also shown that the phenotypically distinct SMC subpopulations exhibit markedly different proliferative and matrix-producing capabilities in response to the stimuli associated with hypoxia-induced pulmonary hypertension.9,10 Although it is clear now that the arterial media is a heterogeneous organ composed of SMCs with distinct phenotypes and functional properties, the molecular mechanisms contributing to the existence of distinct SMC subpopulations remain uncertain. To provide better understanding of this phenomenon, experimental studies will require a reliable in vitro model in which the isolated cell subpopulations can maintain unique characteristics of interest over time in culture.
The purpose of the present study was to selectively isolate the phenotypically distinct cell subpopulations (both smooth muscle and “nonmuscle-like”) identified in vivo7 and then to evaluate whether the nonmuscle-like cell subpopulations would exhibit heightened responses to growth-promoting stimuli as well as to hypoxia. Using selective isolation techniques and selective media, we were able to reproducibly isolate from mature bovine arterial media (both pulmonary and systemic) four phenotypically distinct medial cell subpopulations. Two cell subpopulations could be generally classified as smooth muscle, and two could be classified as nonmuscle-like, based on their morphological and biochemical differences. The isolated cell subpopulations were found to exhibit markedly different growth capabilities under various conditions and to maintain these properties over multiple passages in culture. These data raise the possibility that nonmuscle-like cells with enhanced growth capabilities exist in the normal arterial media, exhibit unique responses to pathophysiological stimuli, and thus contribute selectively to the pathogenesis of vascular disease.
Materials and Methods
Isolation of Cell Subpopulations
Main pulmonary arteries and aortic arches were obtained from adult (2-year-old) cows (n=8). Segments of the main pulmonary artery (proximal to its bifurcation) and the aortic arch (immediately distal to the subclavian artery) were used for cell isolation. These vascular segments were cut open and mechanically stripped of adventitia. To ensure complete removal of the adventitia, a thin portion of the outermost media was also discarded. Endothelium was removed by gentle scraping of the luminal surface of the vessel with a scalpel blade.
We have previously demonstrated that the intact mature bovine arterial media is composed of at least four phenotypically unique cell subpopulations that reside in distinct medial layers.7 The subendothelial media is predominately populated by nonmuscle-like cells; the middle media, by SMCs; and the outer media, by two phenotypically distinct cell subpopulations, one smooth muscle and the other nonmuscle-like. In order to isolate the four cell subtypes identified in vivo, we first separated the arterial media into the three previously described layers: (1) a very thin subendothelial layer (termed here L1), (2) an intermediate-sized middle layer (termed L2), and (3) a thick outer layer (termed L3) (Fig 1A⇓, and Reference 77 ). We found that these three medial layers could be mechanically separated from one another because of distinct mechanical properties of each layer, apparently due to specific patterns of cell arrangement and elastic lamellar distribution (Fig 1B⇓). After separation of the media into three layers, cells were grown from each layer by explant techniques as previously described.11 Tissue explants were maintained in complete DMEM (Sigma Chemical Co) supplemented with 200 U/mL penicillin, 0.2 mg/mL streptomycin, and either 10% CS (HyClone Laboratories) or 10% plasma-derived serum (Cocalico Biologicals, Inc). Plasma-derived serum was used to selectively obtain cells with unique growth capabilities as previously described12 and, therefore, was carefully screened before use to make certain it lacked PDGF-derived mitogenic activity.
Since our goal was to obtain pure subpopulations of smooth muscle and nonmuscle-like cells previously identified in the intact mature vascular media,7 we first selectively isolated individual cell colonies with a distinct, although uniform, morphological appearance from primary culture using “ring-based” techniques (see below). We then examined expression of smooth muscle–specific markers in each isolated cell subpopulation. Only cell subpopulations with uniform morphological appearance and uniform patterns of expression (or lack thereof) of smooth muscle markers were selected for subsequent experiments.
To isolate individual cell colonies growing from tissue explants in primary culture, plastic rings (5 to 10 mm in diameter, greased on the bottom) were placed over each cell colony of interest. Cells within the ring were trypsinized and transferred to a 24-multiwell plate for expansion. Simultaneously, a small portion of cells was plated for immunostaining analysis as described below.
All studies were carried out using cells at passages 1 to 8. Cell cultures were tested for mycoplasma contamination using a Gen-Probe Mycoplasma T. C. Rapid Detection System (Gen-Probe Inc) and were negative.
Bovine endothelial cells were obtained from mature main pulmonary artery as previously described.13
Cells isolated from distinct medial layers were assessed for expression of muscle-specific contractile and cytoskeletal proteins, α-SM-actin, smooth muscle myosin heavy chains (termed here SM-myosin), and metavinculin. Indirect single- or double-label immunofluorescence staining techniques were used. Monoclonal anti–α-SM-actin antibodies (clone 1A4) were purchased from Sigma. Rabbit antibodies against bovine aortic SM-myosin were kindly provided by Dr R. S. Adelstein (National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md).14 We have previously shown that these antibodies (termed anti–SM-myosin antibodies in this article) react strongly with smooth muscle SM-1 and SM-2 isoforms of myosin heavy chains in bovine arterial tissue and do not recognize nonmuscle isoforms.7 Affinity-purified rabbit anti-metavinculin antibodies were previously described and shown to react specifically with metavinculin but not vinculin.7 As a control, monoclonal anti-vinculin antibodies15 that react with both vinculin and metavinculin were used. Monoclonal anti-BrdU antibody (Becton Dickinson) was used at a dilution of 1:200. Biotinylated horse anti-mouse IgG and avidin/biotin–horseradish peroxidase complex (Pierce) were applied at dilutions recommended by the supplier. For assessment of endothelial marker expression, affinity-purified rabbit antibodies against von Willebrand factor (DAKO Corp) were used.
Cells grown on Tissue-Tek chamber slides (Nunc) in 10% CS to confluence were then growth-arrested (0.1% CS) for 48 hours, fixed in absolute methanol at −20°C for 10 minutes, and processed for indirect immunostaining as follows. For double-label immunofluorescence staining of α-SM-actin and SM-myosin, fixed cells were incubated with a cocktail of monoclonal α-SM-actin and polyclonal anti–SM-myosin antibodies (diluted 1:100 and 1:1000, respectively) for 1 hour at room temperature. After 3 washes in PBS, cells were incubated with a cocktail of biotinylated anti-mouse IgG and FITC-conjugated anti-rabbit IgG (both diluted at 1:100, and both purchased from Sigma) for 1 hour at room temperature. The staining was accomplished by incubation with streptavidin–Texas Red (1:50, Amersham Corp). For double-label immunofluorescence analysis of Ki-67 and SM-myosin expression, staining with Ki-67 antibody was accomplished first by using a biotin/streptavidin–Texas Red detection system and then by staining with polyclonal anti-metavinculin antibodies and FITC-conjugated anti-rabbit IgG as secondary antibodies. For single-label immunostaining, affinity purified rabbit anti-metavinculin antibodies or monoclonal anti-vinculin antibodies were used at dilutions 1:10 and 1:50, respectively. FITC-conjugated anti-rabbit or anti-mouse IgGs (1:40, Sigma) were used as secondary antibodies. Controls were performed in which primary antibodies were replaced by nonimmune rabbit serum or nonimmune mouse ascites (Sigma) at the same dilutions as primary antibodies.
The stained cells were examined with a Nikon Optiphot epifluorescence photomicroscope. Colored photomicrographs were taken with Ektachrome 160T film. Black and white micrographs were taken on Kodak T-MAX 400 film.
SDS-PAGE and Western Blotting Assays
Protein extraction and SDS-PAGE (using 12% polyacrylamide gels) were performed according to the method first described by Laemmli.16 Twenty micrograms of protein was loaded onto each lane. Protein transfer to a nitrocellulose membrane was carried out according to Towbin et al.17 To detect α-SM-actin in protein extracts, monoclonal anti–α-SM-actin antibodies (clone 1A4, Sigma) at a dilution of 1:2000 were used as primary antibodies, followed by anti-mouse IgG conjugated with horseradish peroxidase (diluted 1:4000, Sigma). Detection of the bound antibodies was performed using an ECL kit (Amersham Corp). The NIH Image program (National Institutes of Health) was used for quantitative densitometric scanning of the blots. Results of three separate Western blots were analyzed. Data were expressed in relative scan units; the content of α-SM-actin in L1-cells equals 100 relative scan units.
Cell Growth Assays
Cell Growth in the Presence and Absence of Serum
Cells (at passages 3 to 7) were plated onto 24-multiwell plates at a density of 20×103 cells per well in DMEM supplemented with 10% CS. On day 1, four wells were trypsinized and counted in a standard SPotlite hemacytometer (Baxter). The remaining wells were rinsed with PBS, and 0.5 mL of DMEM supplemented with either 0.1% CS or 10% CS was added. Cells (in four wells) were trypsinized and counted at different time points. Data were expressed as follows: cell number×104/well. Population doubling time was calculated in exponentially growing cells as described elsewhere.18
Cell Growth in Plasma-Derived Serum
Cells from distinct subpopulations were assessed for their capability to proliferate in medium supplemented with 10% plasma using the same method as described above for growth in 10% serum.
Response to Purified Mitogens (DNA Synthesis Assay)
DNA synthesis of distinct cell subpopulations in response to various purified mitogens was determined by measuring [3H]thymidine incorporation under serum-free conditions. Cells were plated at 20×103 cells/well onto 24-multiwell plates in complete DMEM supplemented with 10% CS. The next day, cells were rinsed with PBS and growth-arrested in 0.1% CS for 72 hours. For additional 24 hours, serum-free DMEM supplemented with 0.5 μCi/mL [3H]thymidine (ICN Biochemicals, Inc) and the following purified mitogens were added: PDGF-BB (10 ng/mL, Bachem Fine Chemicals), IGF-I and IGF-II (both at 100 ng/mL), and bFGF (30 ng/mL, Bachem). Mitogen doses used in the present study were found to elicit maximal responses in bovine SMCs (data not shown). As positive and negative controls, [3H]thymidine incorporation in 10% serum and in 0.1% serum was assessed. Four wells with cells of the same subpopulation were assessed for each mitogen. Measurement of [3H]thymidine incorporation was performed as described elsewhere.19 Cell counts were concurrently obtained from four additional wells. Incorporation of [3H]thymidine into DNA was expressed as disintegrations per minute (dpm) per cell and/or per 1000 cells.
Effects of Heparin on Cell Growth
The effect of heparin on cell growth in different cell subpopulations was assessed as previously described.20 Briefly, cells were plated onto 24-multiwell plate at 1×104 cells/well in 10% serum–supplemented DMEM. Heparin (1 to 1000 μg/mL) was added on day 1 and again on day 3. Cells were counted in four wells on days 1 and 5.
Effects of Hypoxia on DNA Synthesis and Cell Growth
Cells were plated at 20×103 cells/well onto 24-multiwell plates in complete DMEM supplemented with 10% CS. After reaching confluence, cells were rinsed with PBS, and 0.5 mL of DMEM containing 0.1% CS was added to each well. Cells were then placed in sealed humidified gas chambers (Bellco Glass). Chambers were purged with 21% or 3% O2 with 5% CO2 balanced with N2. After gassing for 20 minutes, chambers were placed in the 37°C incubator for 48 hours. After this incubation period, chambers were open, [3H]thymidine (0.5 μCi/mL) was added to wells, and the chambers were sealed and gassed again. After an additional 24 hours of incubation, measurement of [3H]thymidine incorporation was performed as described elsewhere19 and expressed as dpm per cell. To determine not only DNA synthesis but also growth, cells were plated at 20×103 cells/well in 24-well plates, four wells were counted on day 1, and the rest were incubated under normoxic (21% O2) or hypoxic (3% O2) conditions for 3 more days and then counted.
To determine if any of the isolated cell subpopulations secreted growth-promoting and/or growth-inhibiting factors, coculture experiments using “source” and “target” cells were performed as previously described by R. Majack (Cook et al21). Briefly, target cells were plated within a 10-mm plastic ring placed in the center of 35-mm tissue culture dish. On the next day the source cells of interest were plated around the periphery of the ring. After reaching subconfluence, both source and target cells were incubated in serum-deprived medium for 72 hours. Medium from target cells was then withdrawn, the ring was removed, and conditioned serum-free medium from the source cells was allowed to spread over the target cell population. The remaining ring of grease prevented the cells from migrating. After coculture for 48 hours, BrdU was added for additional 24 hours. The percentage of BrdU-positive nuclei in the target cell population was assessed by immunocytochemistry.
Cells Isolated From Distinct Medial Layers Exhibit Unique Morphological Appearances and Patterns of Cytoskeletal Protein Expression
In order to determine whether multiple SMC subpopulations with unique phenotypic characteristics similar to those observed in vivo could be isolated from a single specific vascular site, the arterial media was first separated into distinct layers (Fig 1B⇑ and Reference 77 ), and then tissue explants derived from each medial layer were incubated in culture medium with either 10% complete calf serum or with 10% plasma-derived serum. The cells growing from tissue explants of subendothelial, middle, and outer media were then assessed for morphological appearance and expression of contractile and cytoskeletal proteins as described below.
Since previous in vivo studies had demonstrated the presence of nonmuscle-like cells in the subendothelial compartment of the mature bovine arterial media,7 our objective was to selectively isolate these cells in culture. When subendothelial tissue explants were incubated in 10% complete calf serum, two morphologically distinct cell subpopulations were observed. Cells of one subpopulation (hereafter termed L1-cells), constituting <10% of all cell colonies, appeared small and “rhomboidal” in shape and, at confluence, formed a dense multilayer network (Fig 2A⇓). These L1-cell colonies were selectively isolated from primary culture using plastic rings (see “Materials and Methods”) and assessed for contractile protein expression. Immunofluorescence analysis demonstrated that L1-cells expressed little to no α-SM-actin and virtually no SM-myosin (Fig 3A⇓). Cells in the other subpopulation identified in primary cultures from the subendothelial media appeared bipolar or spindle-shaped. At confluence, these cells formed the “hill-and-valley” pattern traditionally described for SMCs (Fig 2B⇓). Immunofluorescence analysis of these cells demonstrated intense staining with both anti–α-SM-actin and anti–SM-myosin antibodies similar to that observed for SMCs derived from the middle (L2) media (Fig 3B⇓).
Because small rhomboidal cells lacking expression of smooth muscle markers represented only a minority of cell colonies in primary cultures from subendothelial media grown in 10% serum, attempts were made to selectively increase the number of this cell type in primary culture. Since use of plasma instead of serum had previously been shown to allow selective growth of unique SMC subpopulations,12 we incubated subendothelial tissue explants in medium supplemented with 10% plasma-containing medium. Under these conditions, rhomboidal cell colonies were observed to predominate (>90%). Immunofluorescence analysis demonstrated the same pattern of α-SM-actin and SM-myosin expression as seen in L1-cell colonies selectively isolated from primary cultures grown in serum. When rhomboidal L1-cells initially grown in 10% plasma were subsequently subcultured in 10% serum–containing medium, their characteristic rhomboidal appearance and pattern of immunostaining did not change. Thus, using selective isolation techniques and/or culture medium supplemented with 10% plasma, we were able to isolate and expand in culture a subpopulation of nonmuscle-like cells similar to those identified in the subendothelial media of mature bovine arteries in vivo.7
Virtually all cell colonies isolated from the middle (L2) media explants grown in 10% serum exhibited uniform morphological characteristics. These cells (hereafter termed L2-SMCs) exhibited a spindle-shaped bipolar appearance and, at confluence, showed the hill-and-valley pattern traditionally described for vascular SMCs (Fig 2B⇑). No cell growth was observed when middle media explants were incubated in 10% plasma. Immunofluorescence analysis demonstrated intense staining of these cells with both anti–α-SM-actin and anti–SM-myosin antibodies (Fig 3B⇑). The phenotype of this cell subpopulation was consistent with that described in the middle media of the mature bovine arteries in vivo.7
Primary cultures of cells isolated from the outer (L3) media in serum-based medium were observed to be composed of two morphologically distinct cell subpopulations (Fig 2C⇑). One cell subpopulation (constituting ≈70% of all cell colonies) consisted of spindle-shaped cells (hereafter termed L3-“S” cells), which formed at confluence a hill-and-valley pattern (Fig 2e⇑). Another cell subpopulation (constituting ≈30% of all cell colonies observed) consisted of cells that exhibited a “rounded,” cobblestone, or epithelioid morphology (termed L3-“R” cells) and which at confluence formed a monolayer (Fig 2d⇑). Colonies of spindle-shaped L3-“S” cells and rounded L3-“R” cells were selectively isolated from primary culture using plastic rings (see “Materials and Methods”) and processed for immunofluorescence analysis of contractile protein expression. The two cell subpopulations exhibited markedly different patterns of contractile protein expression: spindle-shaped L3-“S” cells stained intensely with both anti–α-SM-actin and –SM-myosin antibodies (Fig 3C⇑), whereas rounded epithelioid L3-“R” cells demonstrated only a moderate intensity of staining of stress fibers with α-SM-actin antibodies but virtually no SM-myosin staining (Fig 3D⇑).
When explants from the outer (L3) media were incubated in 10% plasma, >90% of cell colonies exhibited a rounded epithelioid morphological appearance. When these cells were subsequently subcultured in 10% serum–based medium, they maintained their rounded morphological appearance and biochemical characteristics and never acquired bipolar appearance or expressed SM-myosin.
Because the two muscle cell subpopulations, L2- and L3-“S” SMCs, isolated from the middle and outer media, respectively, exhibited similar morphology and both expressed α-SM-actin and SM-myosin, we questioned whether they could be differentiated on the basis of expression of other muscle-specific proteins. Since metavinculin expression differentiated two SMC subpopulations in vivo,7 we analyzed the expression of this protein by the two morphologically spindle-shaped cell subtypes (L2 and L3-“S” SMCs) in vitro. We found that only the subpopulation of spindle-shaped cells from the outer media (L3-“S” SMCs) expressed metavinculin in culture (Fig 4⇓). These findings are again consistent with the previous in vivo observations.7
To make certain that the isolated nonmuscle-like L1- and L3-“R” cell subpopulations were not contaminated with endothelial cells derived from either the intimal surface of the vessel (for subendothelial L1-cells) or from the vasa vasorum (for outer media L3-cells), immunofluorescence analysis with antibodies against the endothelial cell marker, von Willebrand factor, was performed. No von Willebrand factor staining was noted in nonmuscle-like L1- and L3-“R” cultures (data not shown).
The unique morphological characteristics of all four cell subpopulations were maintained over multiple passages in culture (experiments presented in the present study were carried out on cells at passages 1 to 8). Never were any of the four cell subpopulations observed to revert to another morphological phenotype under similar culture conditions. Cells at early passages could be frozen and thawed for further use without causing changes in morphological appearance.
Quantitative Analysis of α-SM-Actin Content in Distinct Cell Subpopulations: Western Blotting Assay
Because immunofluorescence analysis demonstrated that all four cell subpopulations stained positively with antibodies against α-SM-actin, although with markedly different levels of intensity, we sought to quantitatively assess the relative content of α-SM-actin in different cell subpopulations using Western blotting analysis. Relative content of α-SM-actin was the lowest in L1-cells, 1.82-fold higher in L3-“R” cells, 4.4-fold higher in L2-SMCs, and 6.6-fold higher in L3-“S” SMCs compared with L1-cells (data not shown).
Phenotypically Distinct Cell Subpopulations Exhibit Markedly Different Growth Capabilities Under Identical Conditions
To determine if morphologically and biochemically distinct cell subpopulations exhibited different growth capabilities in response to growth-promoting and growth-inhibitory stimuli, the rate of cell proliferation was measured in response to serum and/or plasma stimulation after serum withdrawal as well as in response to various concentrations of heparin. The effect of heparin and hypoxia on cell growth was also assessed. Additionally, DNA synthesis was determined by measuring [3H]thymidine incorporation under conditions of serum stimulation and serum withdrawal and in response to various purified mitogens as well as in response to hypoxia. Finally, the effect of coculture of different cell phenotypes on DNA synthesis of a specific cell subset was determined.
Cell Growth in Response to Stimulation by Serum and/or Plasma
As shown in Fig 5A⇓, cells isolated from distinct layers of arterial media and exhibiting distinct morphological and biochemical characteristics demonstrated markedly different growth responses to 10% serum stimulation. Subpopulations of L1- and L3-“R” cells grew more rapidly than did the L2- and L3-“S” SMC subpopulations. Population doubling times during log-phase growth for the colonies of L1- and L3-“R” cells were 40.7±2.4 hours and 42±2.8 hours, respectively, whereas subpopulations of L2- and L3-“S” SMCs exhibited doubling times of 87±4.7 hours and 109±8.2 hours, respectively. Interestingly, we found that two cell colonies isolated from the subendothelial (L1) media of two different adult animals (termed here colonies L1–1 and L1–5) grew in 10% serum–containing medium faster than other L1-cell colonies and showed a population doubling time of only 29.5 hours and 32 hours, respectively (data not shown). At day 10, the saturation density of L1-cells was 3.6-fold higher than that of L2-SMCs and 4-fold higher than that of L3-“S” SMCs. The saturation density of L3-“R” cells was 2.1-fold and 2.6-fold higher than that of L2- and L3-“S” SMCs, respectively.
In medium supplemented with 10% plasma instead of serum, L1- and L3-“R” cell subpopulations demonstrated an ability to proliferate, although at a slower rate than in 10% serum. In contrast, L2- and L3-“S” SMC subpopulations were quiescent under these conditions (Fig 5B⇑).
In serum-deprived (0.1% CS) medium, only two cell colonies derived from subendothelial media were observed to proliferate autonomously over prolonged periods in culture and hereafter will be termed L1-AUT cells. Cell counts in 0.1% serum over a 10-day period demonstrated that the number of L1-AUT cells increased ≈8-fold, whereas cell number from other colonies of L1-cells as well as L3-“R”, L2-, and L3-“S” cells remained virtually unchanged (Fig 5C⇑). After 72 hours of serum deprivation, DNA synthesis (assessed by both BrdU and [3H]thymidine incorporation) was low in all cell subpopulations, except for L1-AUT cells, in which DNA synthesis was found to be markedly increased (Fig 6B⇓).
DNA Synthesis in Response to Purified Mitogens
Cell subpopulations were found to differ in their response to peptide mitogens (Fig 6A⇑). PDGF-BB markedly increased DNA synthesis in L3-“R” and L1-cell subpopulations and, to lesser extent, in L2-SMC and especially in L3-“S” SMC subpopulations. IGFs (both -I and -II) stimulated DNA synthesis in L1- and L3-“R” cells but not in L2- or L3-“S” SMCs. No significant differences between IGF-I and -II were noted. When bFGF was tested, it was found to increase DNA synthesis in all cell subpopulations but to lesser extent than did PDGF. Interestingly, L1-AUT cells, in which DNA synthesis under serum-deprived conditions was markedly elevated, were found to be quite unresponsive to the mitogens used in the present study: PDGF-BB had little, although distinct, stimulatory effect, whereas bFGF and IGFs had no stimulatory effect on DNA synthesis in these cells (Fig 6B⇑).
Relationship Between Proliferation and Expression of Muscle-Specific Markers in L2- and L3-“S” SMC Subpopulations
Although L2- and L3-“S” SMC subpopulations ex-hibited somewhat similar growth responses to serum (Fig 5⇑), their markedly different expression of a muscle-specific differentiation-related cytoskeletal protein metavinculin (Fig 4⇑) led us to investigate the correlation between cytodifferentiation and proliferation in response to 10% serum stimulation in these two cell subtypes. We found marked differences in the relationship between cytodifferentiation and proliferation (as defined by the expression of smooth muscle myosin heavy chains [SM-myosin] and the proliferation-associated nuclear marker, Ki-67) in these two SMC subtypes. We observed that under growth-arrested conditions (0.1% CS, 72 hours), the majority of cells in both L2- and L3-“S” SMC cultures expressed SM-myosin. However, after serum stimulation, L2-SMC cultures retained a high percentage of SM-myosin–positive cells, whereas in cultures of L3-“S” SMCs the number of SM-myosin–positive cells decreased by approximately half. Furthermore, we found that serum-stimulated L2-SMCs simultaneously expressed both Ki-67 and SM-myosin antigens at a high frequency (78.9% of all replicating cells expressed SM-myosin), whereas in L3-“S” SMCs the majority (90.8%) of replicating cells were SM-myosin–negative (data not shown). It is important to note that after serum stimulation, the majority of both L2- and L3-“S” SMCs continued to express α-SM-actin.
Cell Growth in Response to Heparin
Because we observed marked differences in responsiveness to promitogenic stimuli among distinct medial cell subpopulations, we also tested whether differences in response to growth-inhibitory factors would be detectable. We examined the effect of heparin on cell growth of two cell subpopulations (L1-cells and L2-SMCs) that exhibited significant differences in serum-stimulated growth. We found that heparin did not affect the growth rate of slow-growing L2-SMCs under serum-stimulated conditions even when applied in high concentrations (1000 μg/mL). In contrast, heparin exerted dramatic growth-inhibitory effects on fast-growing L1-cells even at concentrations as low as 1 μg/mL (Fig 7⇓).
Inhibition of L1-Cell Growth by Coculture With L2-SMCs
Because L1- and L3-“R” cell colonies were observed quite infrequently in actively proliferating primary cultures yet exhibited high growth rates when grown after selective isolation (and therefore somewhat “purified”), we attempted to study whether their growth in primary cultures was inhibited by factors secreted by other cell subpopulations. For this purpose, we chose to assess growth potential of the fastest growing L1-cell colonies, L1-AUT (population doubling time, 29.5 hours and 32 hours, respectively) in coculture with L2-SMCs under various serum concentrations (0.1%, 1%, 3%, and 5% serum). Under all the conditions tested, DNA synthesis in L1-AUT cells was significantly inhibited by coculture with L2-SMCs (Table 1⇓). Even when stimulated by 5% serum, DNA synthesis in L1-cells cocultured with L2-SMCs was inhibited to the level of that in serum-deprived (0.1% CS) medium.
DNA Synthesis in Response to Hypoxia
Because in previous in vivo studies9 we demonstrated differential proliferative responses of phenotypically distinct medial SMC subpopulations to hypoxia-induced pulmonary hypertension, we attempted to evaluate the proliferative response of the isolated distinct medial cell subpopulations to hypoxia. We found that under serum-stimulated conditions, DNA synthesis was increased in L1- and L3-“R” SMCs in response to hypoxia, whereas it was decreased in L2- and L3-“S” SMCs (Fig 8A⇓). Growth assays performed on autonomously growing L1-AUT cells demonstrated a significant increase in cell number under hypoxia (3% O2) in serum-deprived medium compared with normoxia (21% O2) (Fig 8B⇓).
Selected Colonies of L1- and L3-“R” Cells Secrete a Mitogenic Factor
The possibility that certain vascular SMC subpopulations secrete mitogenic factors and that others do not has been raised by previous studies.22 Thus, we were interested in determining whether any of the isolated cell subpopulations secreted mitogenic factors. We hypothesized that those cell subpopulations that were able to proliferate in plasma-based medium were most likely to secrete mitogenic factors. Using coculture techniques, we assessed the effect of serum-free medium conditioned for 48 to 72 hours by different colonies of L1-cells, L2-SMCs, L3-“R” cells, and L3-“S” SMCs on growth-arrested L2-SMCs. As shown in Fig 9⇓, L2-SMCs had low rates of DNA synthesis (as defined by BrdU nuclear incorporation) when cocultured with other L2-SMCs or with L3-“S” SMCs. In contrast, when cocultured with certain colonies of L1- and L3-“R” cells (two of seven L1-cell colonies tested, and three of nine L3-“R” cell colonies tested), DNA synthesis in L2-SMCs increased at least 5-fold, suggesting the secretion of a mitogenic factor(s) by these selected colonies of L1- and L3-“R” cells. We also found that serum-deprived medium, conditioned by these cell colonies for 48 to 72 hours and then applied to the culture of growth-arrested L2-SMCs, had the same mitogenic effect as that observed in live cocultures. Freezing and thawing of this conditioned medium did not affect its growth-promoting effect.
Previous studies have demonstrated that certain phenotypes of vascular SMCs secrete a PDGF-like mitogenic activity.22–24 However, our preliminary results show that the mitogens present in the conditioned medium from selected L1- and L3-“R” colonies are not any of the PDGF isoforms but rather are two unique members of heparin-binding family of growth factors (A.A. Aldashev, unpublished data, 1996).
The present study demonstrated that at least four distinct cell subpopulations, each bearing a strong resemblance to a specific arterial cell subpopulation previously observed in vivo,7 could be reproducibly isolated from the media of mature bovine main pulmonary arteries and aortas. The cell subpopulations with unique characteristics (see Table 2⇓) were obtained from specific compartments of the arterial media (subendothelial, middle, and outer layers), which could be separated mechanically. Our view that these cell subpopulations represented phenotypically distinct cell lines and were not the result of “phenotypic modulation” in culture was based on the following: (1) characteristic morphological, immunobiochemical, and proliferative properties observed at early passages in a specific cell subpopulation were maintained over time in culture (ie, conversion from one phenotype to another was not observed); (2) each cell subpopulation was isolated from a specific medial layer of the same vascular segment and exhibited biochemical and functional characteristics similar to those of cells observed in the corresponding medial layer in vivo; (3) the unique morphological, biochemical, and growth characteristics of each cell subpopulation were maintained under different culture conditions (in complete serum or in plasma); and (4) the findings were consistent among 8 different animals.
The findings regarding arterial SMC diversity reported in the present study in a large mammalian species support and extend previous experimental in vitro data that came primarily from avian and rodent species.12,25–30 The present study, however, provides the first link between in vitro and in vivo observations by demonstrating that phenotypically and functionally unique vascular SMC subpopulations observed in vivo could be isolated in culture and retain, at least to a certain degree, similar biochemical and functional characteristics. For instance, in vivo one specific SMC subpopulation, residing in the outer media, was found to express the cytoskeletal protein metavinculin and to be resistant to the growth-promoting effects of hypoxic pulmonary hypertension. In vitro, only one cell subpopulation (L3-“S” SMCs), isolated from the outer media, expressed metavinculin (Fig 5⇑). This SMC subpopulation also exhibited minimal responses to exogenous mitogens, and its growth capability was suppressed by hypoxia. In addition, previous in vivo studies demonstrated the existence of cell subpopulations in the normal mature vascular media that failed to express smooth muscle–specific markers.7 In vitro, two nonmuscle-like cell subpopulations were derived from the subendothelial and outer media (L1- and L3-“R” cells, respectively). Additionally, we found that L1- and L3-“R” cells exhibited a highly proliferative phenotype and, unlike “traditional” SMCs, proliferated under hypoxic conditions, a response similar to that observed during hypoxic pulmonary hypertension in vivo. Thus, cell subpopulations described in the present in vitro study appear representative of resident cell subpopulations in the vascular media, raising the possibility that molecular mechanisms conferring unique cellular responses to pathological stimuli in vivo can be addressed in vitro.
The idea raised by others that the normal arterial media is composed not only of differentiated SMCs but also of nonmuscle-like cells is supported by our results.12,25–33 In general, the distinct cell subpopulations identified could be subdivided into two major categories: muscle and nonmuscle-like (Table 2⇑). However, our data also suggest that unique cell subtypes exist within each of these two general categories. For instance, within the general category of nonmuscle-like cells, differences in morphology, in growth pattern, and in mitogen secretion were observed. Within the “muscle” group, differences in the expression of cytoskeletal proteins (ie, metavinculin) and in the regulation of contractile protein (SM-myosin) expression during proliferation were noted. Similarly, in vivo these two SMC subpopulations exhibited different biochemical characteristics, as well as distinct proliferative and matrix-producing responses to hypertensive stimuli.7–9 It appears possible, or even likely, that distinct cell subtypes, existing within the general categories of muscle and nonmuscle-like cells, will be found to contribute in unique ways to vascular wall homeostasis under normal and pathological conditions.
Recent in vitro experiments in the systemic circulation have demonstrated the existence of nonmuscle-like cells with enhanced proliferative capabilities in the subendothelial space of normal aortas and suggest a distinct role for these cells in the pathogenesis of intimal thickening.27,31,33,34 Villaschi et al33 isolated relatively undifferentiated cells from the intimal aspect of normal adult rat aortas, very similar to the L1-cells described here. Holifield et al34 demonstrated that a subpopulation of nonmuscle-like cells contributes selectively to the intimal thickening following balloon injury in canine arteries. The nonmuscle-like cells described in the present study were isolated from both the subendothelial and the outer compartments of the media. They exhibited heightened growth potential, unique proliferative responses to hypoxia, and the capacity (in at least certain colonies) to secrete mitogens and thus influence the proliferative phenotype of other SMC subpopulations. The existence of functionally unique cell subpopulations within distinct compartments of the vessel wall suggests that the vascular response to a given pathological stimulus could be cell specific, could be localized, and could differ significantly depending on the inciting injury.
The observation of unique cell subsets with markedly enhanced growth capabilities in animal species leads to speculation about the potential role similar cells (if they exist) could play in human vascular diseases, such as arteriosclerosis, postangioplasty restenosis, and hypertension. At least certain stages in the pathogenesis of these vascular diseases are characterized by accelerated proliferation of SMCs.35,36 However, whether all medial cells can contribute equally to pathophysiological processes or, alternatively, whether there exist specific cell subpopulations that contribute selectively is still not clear. Studies in atherosclerosis, initially by Benditt and Benditt37 and later by other investigators,38–40 as well as experimental studies in primary pulmonary hypertension by Tuder et al41 suggest selective participation of unique cell subtypes in the formation of pathological lesions in these diseases. Another possibility, raised by the present in vitro data, is that injury-induced stimulation of a vascular cell subpopulation with enhanced proliferative potential could contribute to vascular wall thickening not only through selective expansion of a specific cell subpopulation but also through secretion of mitogens by these cells and recruitment of other medial SMCs into the proliferative phenotype.
The isolation of cell subpopulations with markedly enhanced growth capabilities in vitro from the normal, mature, quiescent vascular media (for instance, L1-AUT cells) raises questions as to what controls the growth state of these cells in the intact vessel wall. In primary cultures, these cells were observed rather infrequently and were never found to “overgrow” other cell subtypes. We found that DNA synthesis of L1-AUT cells was dramatically inhibited by coculture with L2-SMCs even under serum-stimulated conditions (Table 1⇑), suggesting that in vivo L1-AUT cells could be maintained quiescent by other SMC subtypes. This finding may account for the fact that bovine arterial SMCs have been cultured by investigators for over 20 years, yet to our knowledge, no other reports have described in culture phenotypically and functionally unique SMC subpopulations. We also found that even very low concentrations of heparin (1 μg/mL) dramatically inhibited serum-stimulated growth of L1-cells. The secretion of heparin-like molecules by endothelial or SMCs has been previously reported,42 suggesting that this can be an additional candidate factor in maintaining the quiescent state of L1-cells in the normal intact vessel wall. Decreased production of inhibitory factors, such as might occur in vascular injury, could allow selective expansion of a specific cell subpopulation with enhanced proliferative potential.
If, as we suggest, the phenotypically and functionally distinct cell subpopulations we have isolated indeed represent cells of unique genetic lineages, the question arises as to the embryological origin of these cells. In avian embryonic arteries, unique cell types, which maintain distinct functional differences in culture, have been shown to be derived from either splanchnopleural mesoderm or cardiac neural crest.28,43–45 In these studies, however, unlike ours, phenotypically and functionally unique arterial cell subpopulations were isolated from different segments of the systemic circulation (aortic arch versus abdominal aorta) rather than from the same arterial segment. A study by Bergwerff et al29 demonstrated, based on the pattern of α-SM-actin expression, a clear phenotypic segregation of cell types, derived from neural crest versus splanchnic mesoderm, within the avian vascular media. We observed a similar developmental process of phenotypic segregation of distinct arterial cell types in the bovine species.7 To our knowledge, however, no lineage analysis of SMCs in the vascular media of large mammals has been performed. An intriguing possibility that the cells observed in the subendothelial space of the avian arteries may arise from endothelial cells has recently been raised by DeRuiter et al46 and discussed by Majesky and Schwartz.47 Our findings of distinct cell subpopulations within arterial media at the same vascular site in the large mammalian species emphasize the need for further studies to determine the precise embryological origins of these cells.
The observation of multiple phenotypically and functionally distinct vascular SMC subpopulations raises important questions as to why cellular diversity exists and how it is maintained during normal vascular development. Numerous functions, including contraction, proliferation, and synthesis of extracellular matrix proteins, are required of vascular SMCs under both normal and pathological conditions. Diverse vascular functions may require different SMC phenotypes. Accordingly, it is highly likely that in response to pathological stimuli, some medial SMC subpopulations maintain, at least initially, their normal contractile function, whereas others exhibit increased proliferation and/or increased synthesis of extracellular matrix proteins. The current in vitro and previous in vivo studies strongly support the concept that numerous phenotypically unique subpopulations of cells exist in the vascular media, may perform different functions, and thus may contribute in unique ways to vascular homeostasis both under normal conditions and in vascular disease.
Selected Abbreviations and Acronyms
|α-SM-actin||=||α-smooth muscle actin|
|bFGF||=||basic fibroblast growth factor|
|IGF||=||insulin-like growth factor|
|L1, L2, L3||=||layers of arterial media|
|PDGF||=||platelet-derived growth factor|
|SMC||=||smooth muscle cell|
|SM-myosin||=||smooth muscle myosin|
- Received May 16, 1997.
- Accepted September 19, 1997.
- © 1997 American Heart Association, Inc.
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