Innate Diversity of Adult Human Arterial Smooth Muscle Cells
Cloning of Distinct Subtypes From the Internal Thoracic Artery
Abstract— Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105±9 μm in length, and had a doubling time of 39±2 hours. Spindle-shaped clones (n=3) were larger (267±18 μm long, P<0.01) and grew slower (doubling time 65±4 hours, P<0.01). Both types of clones expressed smooth muscle (SM) α-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca2+ transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM α-actin–positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.
Smooth muscle cells (SMCs) in the adult artery wall can manifest a diversity of phenotypes. This is most apparent in diseased or injured arteries where SMCs within the intima can be distinguished from those in the underlying media based on morphology1 and differential expression of contractile apparatus proteins,2,3 integrins,4 extracellular matrix molecules,5–7 and growth factors.8,9 Typically, the differences suggest the presence of relatively less specialized SMCs within the remodeling arterial intima, which is in accordance with their role in expanding and restructuring the vessel wall.
Heterogeneity among SMCs has also been found within the media of normal arteries.10–13 This has led to the suggestion that there may one or more subsets of medial SMCs that are more likely to participate in lesion development.14 Testing such a possibility is difficult because it requires a functional evaluation of definable SMC subsets within a heterogeneous population. In recent years, however, a few laboratories have successfully generated stable, phenotypically distinct SMC populations from a single artery. In rats and cows, unique SMC populations have been isolated by either harvesting SMCs from arterial layers or by cloning SMCs.15–18 Some SMC subsets have been shown to differentially respond to mitogenic factors, consistent with the idea that selective SMC expansion may be a mechanism of lesion formation.15–17
Unfortunately, the extent to which the concepts arising from animal studies can be reliably extrapolated to human arterial behavior is limited. Although there is a small amount of in situ evidence for medial SMC diversity in humans,11,13 there has been little success at isolating stable and clearly distinguishable subpopulations of SMCs from adult human arteries. The difficulty in cloning human SMCs is well recognized and illustrated by a report of cloning that yielded anchorage-independent cell colonies, implying a loss of physiological growth control as a precursor to cloning.19 Recently, however, we successfully cloned an anchorage-dependent SMC line from the human internal thoracic artery.20 This clone could convert from a proliferating, migratory state to a nonproliferating cell that contracted in response to vasomotor agonists. Whether the phenotype plasticity evident in this line is universal to all medial SMCs is unknown. Similarly, the finding does not preclude the possibility of inherently distinct SMC subsets contributing differentially to arterial behavior. In this regard, it is noteworthy that SMC heterogeneity in the media of the internal thoracic artery has recently been documented, based on the distribution of desmin and connexin43.13 However, it is not known if the observed diversity was because of inherently distinct SMC subsets or because of regional variations in the environmental milieu, nor if the SMC differences observed in situ had functional consequences.
In this report, we describe the isolation of 12 stable, human SMC clones derived from the media of a single fragment of adult internal thoracic artery. Based on morphology, these clones could be segregated into 2 discrete groups. Despite relatively subtle differences in the expression of SMC differentiation markers, these 2 groups of clones were strikingly different in their growth rate, collagen expression, response to vasoactive hormones, and response to growth factors. These findings argue for the existence of inherently distinct SMCs in the adult human internal thoracic artery and suggest selective participation of SMCs in the execution of vascular functions.
Materials and Methods
Cell Culture and Cloning
A primary culture of human vascular SMCs was derived from the media of a fragment of distal internal thoracic artery, as described previously.21 Clones were derived from SMCs in the sixth subculture, using a combination of dilute plating and ring isolation.20 Cells plated at 2 cells/cm2 in dishes coated with 100 μg/mL gelatin were cultured in M199/10% FBS that had been conditioned by primary human SMC cultures in log phase growth, plus fresh FBS bringing it to a final concentration of 20%. Individual colonies derived from a single isolated cell were released by localized application of trypsin-EDTA and expanded. One of the clones derived from this artery has been characterized in detail with respect to its capacity to shift from a noncontracting cell to a contracting cell.20
Cells in log phase growth were treated with 2.5 ng/mL ethidium bromide and 0.25 μg/mL colcemid for 5 hours. Chromosome smears were prepared and banded using the trypsin-Giemsa staining method. G-banded chromosomes from 5 to 10 metaphase cells for each clone were analyzed (Perceptive Scientific Instruments).
Western Blot Analysis and Immunostaining
Protein expression was assessed by Western blot analysis of near-confluent cells, as described previously.22 Proteins were resolved by electrophoresis through 12% (for smooth muscle (SM) α-actin and calponin), 7.5% (for heavy [h]- and light [l]-caldesmon and vinculin), or 6% (for SM myosin heavy chains [MHC] and type I collagen) SDS-polyacrylamide gels. Primary antibodies used were mouse monoclonal antibodies raised against SM α-actin (DAKO), calponin (hCP, Sigma), and vinculin (hVIN-1, Sigma), a rat monoclonal antibody to SM myosin heavy chains (G-4, Santa Cruz), rabbit anti-chicken caldesmon polyclonal antisera (kindly provided by Dr M. Walsh, University of Calgary),23 and a rabbit polyclonal antibody to the α1(I) chain of human type I collagen (LF-67, gift of Dr L.W. Fisher, National Institute of Dental Research, Bethesda, Md).24 Cells fixed with 4% paraformaldehyde were immunostained for SM α-actin, h-caldesmon (hHCD, Sigma), and factor VIII–related antigen (F8/86, DAKO), as described previously.4
Cell Proliferation and Migration
To evaluate SMC replication, cells were plated in 24-well plates at a density of 3000 cell/cm2 in M199 with 10% FBS. Cells from quadruplicate wells were dissociated with trypsin-EDTA daily for 9 days and counted using a hemacytometer. To assess the response to growth factors, cells were rendered quiescent in M199 with 0.5% FBS for 48 hours, stimulated with platelet-derived growth factor-BB (PDGF-BB) (10 ng/mL) or fibroblast growth factor-2 (FGF-2) (25 ng/mL), and counted serially over 6 days.
Cell migration was quantified as we have previously described using digital time-lapse video microscopy.25,26 Cells were seeded onto culture dishes precoated with 100 μg/mL type I collagen and motility at 37°C was evaluated over 8 hours before and after addition of 2 ng/mL PDGF-BB.
Measurement of [Ca2+]i
Agonist-induced calcium transients were measured in single SMCs loaded with 0.2 μmol/L fura 2-acetoxymethyl-ester (fura 2-AM), as described previously.27 Whole-cell cytoplasmic calcium concentration ([Ca2+]i) was measured by epifluorescence illumination of cells at alternating 345/380-nm excitation from a Deltascan system (Photon Technology International) and the 510-nm emission signal was detected with a photomultiplier. The [Ca2+]i was determined from the fluorescence intensity at 345 nm divided by that at 380 nm, after subtraction of background fluorescence. Agonists were applied to cells by pressure ejection from a micropipette.
Contraction of SMCs
SMC contraction was studied as described previously.28 Briefly, cells on a glass coverslip were loosened from the substrate using 1/3 concentration of standard trypsin-EDTA solution (final concentration, 0.075% trypsin-0.03 mmol/L EDTA). This did not itself elicit contraction. Contraction, defined as shortening within 20 seconds, was assessed after application of angiotensin II by pressure ejection from a micropipette, or after addition of 100 mmol/L KCl, isosmotically substituted for NaCl in PBS. Cells were dynamically imaged by videomicroscopy.
Serial Enzymatic Dissociation of SMCs From Internal Thoracic Artery
To compare the features of the SMC clones with that of cells freshly dispersed from medial layers, SMCs were dissociated layer by layer from a segment of human internal thoracic artery, as described previously.29 After removal of fat and adventitia, the vessel was inverted and both ends tied off. The preparation was then incubated at 37°C in Ca2+- and Mg2+-free Hanks’ solution (137 mmol/L NaCl, 5.4 mmol/L KCl, 0.44 mmol/L KH2PO4, 4.17 mmol/L NaHCO3, 10 mmol/L HEPES, 5.55 mmol/L glucose, 2 mmol/L l-glutamine, and 0.2% BSA) containing 2 mg/mL collagenase (type III, 100 U/mg, Sigma), 0.14 mg/mL elastase (type III, 4 U/mg, Sigma), and 0.4 mg/mL soybean trypsin inhibitor (Sigma). After a 30-minute digestion period to liberate endothelial cells and other intimal cells, the vessels were rinsed with Ca2+- and Mg2+-free Hanks’ solution and subjected to another four 30-minute digestions. Cells were harvested after each digestion and cultured in M199 supplemented with 10% FBS. The cell area of 250 cells from each digestion was quantified from digital images (Northern Eclipse, Empix ImagingInc).
Results are expressed as mean±SEM. Difference between groups was evaluated by one-way analysis of variance and Student-Newman-Keuls a posteriori testing. Statistical significance was set at P<0.05.
Morphology of Cloned SMCs
Twelve clones of human SMCs were generated from a single fragment of internal thoracic artery. Each clonal line had a homogenous appearance, which was in contrast to the nonuniform appearance typically observed in primary SMC cultures from human internal thoracic artery. Interestingly, each of the 12 lines manifested one of two discrete morphological patterns (Figure 1). Nine clones were relatively small (mean length 105±9 μm) and at confluence assumed a polygonal, epithelioid morphology with a cobblestone appearance. The clones of this morphological appearance (HITA1 [human internal thoracic, HIT], HITA2, HITA4, HITA5, HITB2, HITC1, HITD1, HITD4, and HITD6) were designated epithelioid clones. Another 3 clones (HITB5, HITC6, and HITD5) were larger (mean length 267±18 μm, P<0.01), more elongated and spindle-shaped, and at confluence assumed a hill-and-valley pattern. These clones were designated as spindle-shaped clones. All clones homogeneously stained for SM α-actin and were negative for factor VIII–related antigen (data not shown). The morphology of a given clone was maintained over time and incubation of epithelioid clones with medium conditioned by spindle-shaped SMCs, and vice versa, did not alter their morphology. The stability of the morphological profile was further assessed by recloning of selected clones, which yielded clones of the identical cell characteristics.
Cloned SMCs Are of Human Origin and Display a Normal Karyotype
Successful cloning of human, adult, nonhematopoietic cells has been rare. In view of this, alternative explanations for the clones warranted consideration. Contamination of clones by other cell types has been identified as an important problem.30 To ensure that the HIT clones were not derived from contaminating animal SMCs (which are readily cloned) or from contaminating transformed cell lines (such as the widely used HeLa cell line), we assessed for the expression of the muscle-specific isoform of human calponin by reverse transcriptase–polymerase chain reaction (RT-PCR) using primers specific to this isoform. A product of predicted size was amplified in 3 of 3 clones studied (Figure 2A). To corroborate the human origin of the clones and to assess for chromosome abnormalities, we performed cytogenetic analysis. Each of 4 clones studied (2 epithelioid clones, HITA2 and HITD6, and 2 spindle-shaped clones, HITB5 and HITC6) showed normal male human karyotype, 46,XY (Figure 2B). These independent approaches unambiguously confirm the clones as human SMCs. All spindle-shaped clones had a finite lifespan, reaching senescence between the 36th and the 40th subculture. Senescence of the epithelioid clones has not yet been observed (60th subculture for HITA2 SMCs). Detailed analyses described next were performed using cells within the first 26 subcultures.
Protein Expression Profiles in Epithelioid and Spindle-Shaped SMC Clones
Two epithelioid clones (HITA2 and HITD6) and 2 spindle-shaped clone (HITB5 and HITC6) were chosen for expression profiling. As shown in the Western blots of Figure 3A, all 4 clones contained SM α-actin, SM MHC, calponin, and h-caldesmon, indicating all clones to be relatively mature SMCs. There were differences between the spindle-shaped and epithelioid clones. The ratio of h-caldesmon to l-caldesmon was higher in the spindle-shaped SMCs, and only the spindle-shaped SMCs expressed metavinculin, a muscle-specific splicing variant vinculin, indicating epithelioid clones to be somewhat less differentiated than spindle-shaped clones. Interestingly, although the HITB5 spindle-shaped clone appeared more mature, based on cytoskeletal and contractile apparatus proteins, expression of type I collagen was greater in this cell line (Figure 3B). Expression of type I collagen by both spindle-shaped and epithelioid clones increased in response to transforming growth factor-β, similar to the response documented for nonclonal cultures of human internal thoracic artery SMCs.31
Epithelioid Clones Exhibit Greater Proliferative Responses to Serum and Growth Factors Than Spindle-Shaped SMCs
As shown in Figure 4A, SMC proliferation in M199/10% FBS tightly segregated in accordance with the clone morphology. The average population doubling time of the 9 epithelioid clones was 39±2 hours. The spindle-shaped clones grew significantly more slowly, doubling every 65±4 hours (P<0.01). Epithelioid clones failed to replicate in M199 without serum and died within 72 hours. Spindle-shaped clones also ceased to proliferate in serum-free M199 but, in contrast to epithelioid clones, remained viable for up to 14 days and progressed through a maturation program, as described previously.20
We next determined the proliferative response to growth factors. As shown in Figure 4B, following incubation for 48 hours in media with 0.5% FBS, the epithelioid clones, HITA2 and HITD6, showed a brisk growth response to exogenous PDGF-BB (10 ng/mL) and FGF-2 (25 ng/mL), whereas the spindle-shaped clones, HITB5 and HITC6, did not.
Epithelioid SMCs Migrate Slower Than Spindle-Shaped SMCs but Are More Responsive to PDGF-BB
Using Boyden chamber–type chemotaxis assays, we initially established that both epithelioid (HITA2 and HITD6) and spindle-shaped (HITB5) cells could directionally migrate toward PDGF-BB through a type I collagen (100 μg/mL)-coated porous membrane (data not shown). However, direct comparison of migration speeds between clone types using this approach is confounded by the differences in cell size and shape, which will influence translocation through the pores. We therefore quantified SMCs crawling using digital time-lapse video microscopy. In M199 with 1% FBS, the epithelioid clones HITA2 and HITD6 migrated on a type I collagen-coated substrate at 6.8±0.4 and 6.7±0.4 μm/h, respectively. In contrast, the spindle-shaped clones HITB5 and HITC6 migrated substantially faster at 17.0±0.8 and 24.1±2.3 μm/h (P<0.01), respectively. On stimulation of cells with PDGF-BB (2 ng/mL), the migration speed of HITA2 and HITD6 SMCs increased by 57.3% and 60.2%, respectively, whereas HITB5 and HITC6 migration speeds did not increase significantly (6.7% and 9.4%, respectively; P=NS) (Figure 5). Thus, in M199/1% FBS, the epithelioid clones migrated slower than the spindle-shaped clones, but were nonetheless more responsive to PDGF-BB.
Differences in Response of Spindle-Shaped and Epithelioid SMC Clones to Vasoactive Stimuli
We next determined the responsiveness of the SMC clones to vasoactive stimuli, by quantifying changes in [Ca2+]i in individual cells loaded with the fluorescent indicator fura-2. As shown in Figure 6, application of 20 μmol/L histamine or 1 μmol/L angiotensin II caused a significant increase in [Ca2+]i in spindle-shaped SMCs but elicited only small calcium transients in the epithelioid cells. Spindle-shaped SMCs were also responsive to norepinephrine stimulation but epithelioid SMCs were not. In contrast to their differential response to the vasoactive hormones, spindle-shaped and epithelioid cells showed a similar [Ca2+]i increase after stimulation with PDGF-BB, suggesting that epithelioid clones are more responsive to growth stimuli than vasoactive regulation.
Only Spindle-Shaped SMCs Are Capable of Contracting In Vitro
To determine if the induced calcium transients could be coupled to SMC contraction, cells were deprived of FBS for 3 days in an effort to promote further maturation, then loosened from the substrate with a low concentration of trypsin-EDTA. Cells were then stimulated with angiotensin II applied via micropipette or subjected to membrane depolarization with isosmotic PBS containing 100 mmol/L KCl. Epithelioid SMCs manifested no sudden shape changes with these interventions; however, all 3 spindle-shaped clones appreciably shortened within 20 seconds of delivery of agonist or depolarization medium, as illustrated in Figure 6B.
SMCs Released Sequentially From the Media of the Internal Thoracic Artery Segregate Into Two Phenotypes
To examine whether the type of diversity that we identified through cloning reflected cells in different regions of the arterial media, we performed serial enzyme digestions of an inverted segment of internal thoracic artery. An initial 30-minute digestion released the endothelial cells and any intimal SMCs, leaving the internal elastic lamina and deeper arterial layers intact, as assessed histologically in fragments from 2 arteries fixed and sectioned (data not shown). The first medial digestion liberated a mixture of cells of different sizes (Figure 7A). Deeper into the media, the cell morphology became more homogeneous and generally the cells were larger. As illustrated in Figure 7B, cells released by the first medial digestion had a wide range of sizes that distributed in a bimodal pattern. Gaussian curve fitting delineated a population of smaller cells of mean cell area (±SD) 107±39 μm2 and a second population with a mean cell area of 464±104 μm2. These 2 populations were also identified in cells derived from the deeper medial layers; however, the frequency distribution progressed to that of mostly larger cells. In the first medial digestion, 60.4% of the cells were accounted for by the smaller cells, whereas in the 4th digestion only 0.5% of the cells fell into this statistical category. Both small and large cells immunostained for SM α-actin. However, the smaller cells expressed little to no immunostainable h-caldesmon whereas the larger cells did. Occasional mitotic figures were seen exclusively in the first medial digestion and these cells were also h-caldesmon–negative (Figure 7C).
We have successfully applied a cloning strategy to study SMCs of the human internal thoracic artery. In doing so, we established that a single fragment of arterial media can give rise to clones that can be segregated into 2 distinct categories: (1) a small epithelioid-appearing cell with a relatively fast growth rate, robust proliferative responses to PDGF-BB and FGF-2, and no contractile competence in vitro; and (2) a spindle-shaped cell that grows more slowly and is less responsive to PDGF-BB and FGF-2, yet has the capacity to contract in response to vasoactive hormones. These quantifiable distinctions between the 2 karyotypically normal clone types were maintained over multiple passages, and incubation of one type of clone with the conditioned medium from the other did not lead to convergence of phenotypes. The findings thus provide evidence for innate functional diversity among human SMCs derived from the media of a single, nonatherosclerotic artery.
Both spindle-shaped and epithelioid clones expressed SM α-actin, SM MHC, calponin, and h-caldesmon. However, only spindle-shaped SMCs expressed metavinculin, suggesting a more mature cell phenotype, and the abundance of SM MHC, calponin, and h-caldesmon was greater in the spindle-shaped SMCs. The finding of 2 SMC types that modestly differ in expression of differentiation markers is noteworthy given a recent description of 2 SMC phenotypes evident in the media of the human internal thoracic artery differing in expression of desmin and connexin43, but not by the stainable presence of 5 other SMC-associated proteins including SM MHC.13 Although such subtle distinctions in expression of SMC differentiation markers could be caused by variations in the local microenvironment, the present results indicate that they can also reflect intrinsic differences between SMC subtypes.
Despite the apparent closeness in SMC differentiation state, the 2 populations had very different functional capacities. Particularly striking were differences in short-term responses to vasoactive hormones. We previously determined that the HITB5 SMC clonal line could briskly contract in response to histamine or angiotensin II.20,28 The present study established that this was a unifying feature of all spindle-shaped SMC clones. Associated with this were robust calcium transients in response to histamine, angiotensin II, and norepinephrine. In contrast, epithelioid clones showed no shortening in response to vasoactive agonists and had blunted or no detectable calcium transients in response to these factors. Whether the repressed calcium transients and lack of contractile capacity observed in the human epithelioid clones reflect differences at the receptor level or at a distal location is under study. However, either possibility is consistent with the epithelioid clones being functionally less specialized SMCs.32 Although difficult to test in vivo, the findings raise the possibility that not all SMCs in the media of the internal thoracic artery have equivalent contractile capabilities.
The 2 clone types also differed in their sustained responses to agonists. Epithelioid SMCs displayed a greater proliferative response to serum and to PDGF-BB and FGF-2. This relative growth advantage is similar to that of epithelioid SMCs derived from the intima of injured rat carotid artery, the aorta of newborn rats, and the inner layer of the bovine pulmonary artery.15–17 However, the human epithelioid clones differed from previously studied animal epithelioid SMCs in some ways. First, the human epithelioid SMCs could not autonomously proliferate in the absence of serum. Second, they crawled slower in serum-supplemented media than the spindle-shaped clones. This latter finding was ascertained by direct quantification of crawling path and speed. The effect was not because of increased adhesion, as the epithelioid SMCs in fact released more quickly from the substrate with trypsin or EDTA (data not shown); other components of the migration machinery likely mediate the difference. Importantly, the epithelioid clones were able to migrate up a gradient of PDGF-BB. As well, the relative increase in migration speed after PDGF-BB stimulation was greater for epithelioid clones than spindle-shaped SMCs. Thus, the epithelioid clones manifested greater responsivity to PDGF-BB with respect to both proliferation and migration. Such differential responsivity to PDGF, which has also been noted among cells derived from the bovine pulmonary and canine carotid arteries,16,33 could underlie a pathway for selectively targeting specific SMCs to contribute to human intimal expansion.
By sequentially releasing SMCs from the internal thoracic artery, we found a population of relatively small cells that were liberated preferentially from the layers closest to the vascular lumen, and a second population of larger cells that were released from all layers. Both populations expressed SM α-actin, but the smaller cells expressed little to no h-caldesmon in an immunostainable form. Although we cannot be certain that these 2 populations represent the precise cells that gave rise to the 2 categories of SMC clones, the findings provide support for size- and maturation-dependent diversity of SMCs in this artery and establish a regional basis for the diversity. Interestingly, SMCs of 2 sizes have also been identified in SMCs cultured from human atherosclerotic lesions,34 suggesting that morphological subtypes of SMCs may be represented within both normal and diseased human arteries.
The existence of inherent SMC diversity documented by this study does not preclude environment-induced shifts in SMC behavior as contributing to vascular remodeling. In fact, the spindle-shaped clones that we derived from the internal thoracic artery manifested considerable functional plasticity, dependent on the presence of serum, such that both contractile and synthetic roles could be played by the same cell.20 Furthermore, even though the spindle-shaped clones replicated slower than the epithelioid clones, they had the capacity to migrate quickly and elaborate copious collagen. These characteristics are well suited to cells required to play a role in rapid vascular repair, as would be important after plaque rupture. Conversely, epithelioid-like SMCs might have less of a role in this context. It is possible, therefore, that there is a repertoire of mechanisms by which SMCs restructure the artery wall, and that specific SMC populations are differentially recruited, depending on the circumstances.
In summary, these studies identify inherently distinct subpopulations of SMCs within the media of the human internal thoracic artery. These subpopulations function differently in response to the same environmental stimuli, which is compatible with differential participation of SMC subsets in the regulation of arterial behavior. For a given artery, the profile of innate SMC diversity may be important in determining how that vessel will respond to the various stresses imposed on it.
This research was supported by grants from the Canadian Institutes of Health Research (MOP11715, MOP10019). J.G. Pickering is a Career Investigator of the Heart and Stroke Foundation of Ontario. S. Li was supported by a Premiers Research Excellence Award (to J.G.P.). The authors thank Drs D. Boyd and R. Novick, London Health Sciences Centre, London, Ontario, for providing the surgical specimens and Diana Munavish, Cytogenetics Division, London Health Sciences Centre, for technologic support.
Original received March 30, 2001; revision received July 12, 2001; accepted August 6, 2001.
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