This Article Abstract Full Text (PDF) All Versions of this Article: 89/6/517    most recent hh1801.097165v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, S. Articles by Pickering, J. G. Search for Related Content PubMed PubMed Citation Articles by Li, S. Articles by Pickering, J. G. Related Collections Cell biology/structural biology Smooth muscle proliferation and differentiation

(Circulation Research. 2001;89:517.)
© 2001 American Heart Association, Inc.

Cellular Biology

Innate Diversity of Adult Human Arterial Smooth Muscle Cells

Cloning of Distinct Subtypes From the Internal Thoracic Artery

Shaohua Li, Yao-Shan Fan, Lawrence H. Chow, Caroline Van Den Diepstraten, Eric van der Veer, Stephen M. Sims, J. Geoffrey Pickering

From the John P. Robarts Research Institute (Vascular Biology Group) (S.L., L.H.C., C.V.D.D., E.V.D.V., J.G.P.), London Health Science Centre (Y.F., L.H.C., J.G.P.), Departments of Pathology (Y.F.), Medicine (Cardiology) (L.H.C., J.G.P.), Physiology (S.M.S.), Biochemistry (J.G.P.), and Medical Biophysics (J.G.P.), University of Western Ontario, London, Canada.

Correspondence to J. Geoffrey Pickering, MD, PhD, London Health Science Centre, 339 Windermere Rd, London, Ontario N6A 5A5. E-mail gpickering{at}rri.on.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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) -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 -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.

Key Words: vascular smooth muscle • proliferation • migration • gene expression

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

Cytogenetic Analysis
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) -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 -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 1(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 -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).

Statistics
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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 -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.

View larger version (195K): [in this window] [in a new window]   Figure 1. Hoffman-modulated contrast photomicrographs of 12 SMC clones derived from human internal thoracic artery. All clones were cultured in M199 with 10% FBS and photographed at confluence. Nine clones have an epithelioid cell morphology, yielding a cobblestone pattern. Three clones (HITB5, HITC6, HITD5) consist of elongated, spindle-like cells that align with each other when confluent and become multilayered. (HIT, human internal thoracic).

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.

View larger version (48K): [in this window] [in a new window]   Figure 2. Verification of the clones as karyotypically normal human SMCs. A, Ethidium bromide–stained RT-PCR products resolved on 2% agarose gel. The 220-bp PCR product was amplified using primers specific to human smooth muscle calponin cDNA (GenBank accession numbers U37019, D14437; 5`-AAAGGACGCACTGAGCAACG-3` [forward] and 5`-CGCCACTGTCACATCCACATAG-3` [reverse]). B, Karyograms of an epithelioid (HITA2, 26th subculture) and spindle-shaped (HITC6, 17th subculture) SMC clone. Chromosomes were prepared from SMCs in metaphase and banded using trypsin-Giemsa staining. The karyotype for both is 46,XY. Photomicrographs are representative of 5 to 10 metaphase cells for each SMC clone.

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 -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-ß, similar to the response documented for nonclonal cultures of human internal thoracic artery SMCs.³¹

View larger version (41K): [in this window] [in a new window]   Figure 3. Expression of smooth muscle markers by human SMC clones. A, Western blot analysis of 2 epithelioid clones (HITA2 and HITD6) and 2 spindle-shaped clones (HITB5 and HITC6) for expression of SM -actin, SM myosin heavy chain, calponin, high- and low-molecular-weight caldesmon, vinculin, and metavinculin. B, Western blot showing extracellular accumulation over 48 hours of pro1(I) and 1(I) collagen chains by cloned SMCs. Bands detected by the antibody LF-67 are the full-length pro1(I) chain, partially processed pro1(I) collagen chains lacking either the N- or C-terminal propeptide, and mature 1(I) collagen lacking both terminal propeptides. SMCs were treated with vehicle or transforming growth factor-ß (2.5 ng/mL) for 48 hours. Blots are representative of 4 experiments.

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

View larger version (16K): [in this window] [in a new window]   Figure 4. Growth curves of human SMC clones. A, SMCs from each of 12 clones were plated at 3.5x103 cells/cm2 and cultured for up to 9 days in M199 with 10% FBS. Cell number was counted daily from quadruplicate wells. B, SMCs from epithelioid (HITA2, HITD6) and spindle-shaped (HITB5, HITC6) clones were rendered quiescent in M199 with 0.5% FBS for 48 hours and then incubated with PDGF-BB (10 ng/mL) or FGF-2 (25 ng/mL) and counted serially.

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.

View larger version (26K): [in this window] [in a new window]   Figure 5. Migration of epithelioid (HITA2, HITD6) and spindle-shaped (HITB5, HITC6) human SMC clones on type I collagen-coated culture dishes determined by digital time-lapse video microscopy. Cells were treated with 2 ng/mL PDGF-BB or vehicle for 8 hours and migration speeds were determined over the subsequent 8 hours. Ten cells for each group were studied and the results are representative of 3 experiments. *P<0.05 vs control treatment for the same cell clone; P<0.05 vs the corresponding treatment of HITA2 and HITD6 SMCs.

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.

View larger version (32K): [in this window] [in a new window]   Figure 6. A, Agonist-induced calcium transients in single cells of human SMC clones. Cells were grown on glass coverslips and [Ca2+]i was measured using fura-2. Agonists were applied to individual cells using pressure ejection from a micropipette. Lines under each [Ca2+]i trace indicate the duration of agonist application. Results are representative of 10 to 20 experiments. B, Phase-contrast photomicrographs of a HITC6 SMC before (left panel) and after (right panel) application of angiotensin II (1 µmol/L) showing contraction. The cell was driven to a mature state by serum withdrawal for 3 days and loosened from the substrate with trypsin-EDTA as described in Materials and Methods.

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 -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).

View larger version (67K): [in this window] [in a new window]   Figure 7. SMCs sequentially released from the media of an inverted fragment of human internal thoracic artery by serial enzymatic digestion. A, Hoffman-modulated contrast images of the released SMCs after 20 hours of culture. Panels I through IV depict cells sequentially released from the abluminal to the deepest layers of the media. B, Frequency histograms of cell area (y-axis, µm2) quantified in the 4 sequentially released SMC cultures. Histograms were fitted to 2 gaussian distributions. C, Paraformaldehyde-fixed SMCs from the first medial digestion showing small (arrows) and large cells. Both are immunopositive for SM -actin (left panel) but only the large cells are positive for h-caldesmon (middle panel). Dividing cells were negative for h-caldesmon (right panel).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 -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 -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.

	Acknowledgments

 
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.

Received March 30, 2001; accepted August 6, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Mosse PR, Campbell GR, Wang ZL, Campbell JH. Smooth muscle phenotypic expression in human carotid arteries, I: comparisons of cells with diffuse intimal thickenings adjacent to atheromatous plaques with those of the media. Lab Invest. . 1985; 53: 556–562.

2. Kuro-o M, Nagai R, Nakahara K, Katoh H, Tsai RC, Tsuchimochi H, Yazaki Y, Ohkubo A, Takaku F. cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis. J Biol Chem. . 1991; 266: 3768–3773.

3. Zanellato A, Borrione AC, Tonello M, Scannapieco G, Pauletto P, Sartore S. Myosin isoform expression and smooth muscle cell heterogeneity in normal and atherosclerotic rabbit aorta. Arteriosclerosis. . 1990; 10: 996–1009.

4. Pickering JG, Chow LH, Li S, Rogers KA, Rocnik EF, Zhong R, Chan BM. ₅ß₁ Integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury. Am J Pathol. . 2000; 156: 453–465.

5. Majesky M, Giachelli CM, Reidy MA, Schwartz SM. Rat carotid neointimal smooth muscle cells rexpress a developmentally regulated mRNA phenotype during repair of arterial injury. Circ Res. . 1992; 71: 759–768.

6. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest. . 1993; 92: 1686–1696.

7. Rocnik E, Saward L, Pickering JG. HSP47 expression by smooth muscle cells is increased during arterial development and lesion formation and is inhibited by fibrillar collagen. Arterioscler Thromb Vasc Biol. . 2001; 21: 40–46.

8. Nikol S, Isner J, Pickering J, Kearney M, Leclerc G, Weir L. Expression of transforming growth factor ß-1 is increased in vascular restenosis lesions. J Clin Invest. . 1992; 90: 1582–1592.

9. Murry CE, Bartosek T, Giachelli CM, Alpers CE, Schwartz SM. Platelet-derived growth factor-A mRNA expression in fetal, normal adult, and atherosclerotic human aortas: analysis by competitive polymerase chain reaction. Circulation. . 1996; 93: 1095–1106.

10. Giuriato L, Scatena M, Chiavegato A, Tonello M, Scannapieco G, Pauletto P, Sartore S. Non-muscle myosin isoforms and cell heterogeneity in developing rabbit vascular smooth muscle. J Cell Sci. . 1992; 101: 233–246.

11. Frid MG, Printesva OY, Chiavegato A, Faggin E, Scatena M, Koteliansky VE, Pauletto P, Glukhova MA, Sartore S. Myosin heavy-chain isoform composition and distribution in developing and adult human aortic smooth muscle. J Vasc Res. . 1993; 30: 279–292.

12. Frid MG, Moiseeva EP, Stenmark KR. Multiple phenotypically distinct smooth muscle cell populations exist in the adult and developing bovine pulmonary arterial media in vivo. Circ Res. . 1994; 75: 669–681.

13. Ko YS, Yeh HI, Haw M, Dupont E, Kaba R, Plenz G, Robenek H, Severs NJ. Differential expression of connexin43 and desmin defines two subpopulations of medial smooth muscle cells in the human internal mammary artery. Arterioscler Thromb Vasc Biol. . 1999; 19: 1669–1680.

14. Wohrley JD, Frid MG, Moiseeva EP, Orton EC, Belknap JK, Stenmark KR. Hypoxia selectively induces proliferation in a specific subpopulation of smooth muscle cells in the bovine neonatal pulmonary arterial media. J Clin Invest. . 1995; 96: 273–281.

15. Lemire JM, Covin CW, White S, Giachlli CM, Schwartz SM. Characterization of cloned aortic smooth muscle cells from young rats. Am J Pathol. . 1994; 144: 1068–1081.

16. Frid MG, Aldashev AA, Dempsey EC, Stenmark KR. Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities. Circ Res. . 1997; 81: 940–952.

17. Bochaton-Piallat ML, Ropraz P, Gabbiani F, Gabbiani G. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Arterioscler Thromb Vasc Biol. . 1996; 16: 815–820.

18. Adams LD, Lemire JM, Schwartz SM. A systematic analysis of 40 random genes in cultured vascular smooth muscle subtypes reveals a heterogeneity of gene expression and identifies the tight junction gene zonula occludens 2 as a marker of epithelioid "pup" smooth muscle cells and a participant in carotid neointimal formation. Arterioscler Thromb Vasc Biol. . 1999; 19: 2600–2608.

19. Benzakour O, Kanthou C, Kanse SM, Scully MF, Kakkar VV, Cooper DN. Evidence for cultured human vascular smooth muscle cell heterogeneity: isolation of clonal cells and study of their growth characteristics. Thromb Haemost. . 1996; 75: 854–858.

20. Li S, Sims S, Jiao Y, Chow LH, Pickering JG. Evidence from a novel human cell clone that adult vascular smooth muscle cells can convert reversibly between noncontractile and contractile phenotypes. Circ Res. . 1999; 85: 338–348.

21. Pickering JG, Weir L, Rosenfield K, Stetz J, Jekanowski J, Isner JM. Smooth muscle cell outgrowth from human atherosclerotic plaque: implications for the assessment of lesion biology. J Am Coll Cardiol. . 1992; 20: 1430–1439.

22. Li S, Chow LH, Pickering JG. Cell surface-bound collagenase-1 and focal substrate degradation stimulate the rear release of motile vascular smooth muscle cells. J Biol Chem. . 2000; 275: 35384–35392.

23. Clark T, Ngai PK, Sutherland C, Groschel-Stewart U, Walsh MP. Vascular smooth muscle caldesmon. J Biol Chem. . 1986; 261: 8028–8035.

24. Fleischmajer R, MacDonals ED, Perlisch J, Burgeson RE, Fisher LW. Dermal collagen fibrils are hybrids of type I and type III collagen molecules. J Struct Biol. . 1990; 105: 162–169.

25. Pickering JG, Uniyal S, Ford C, Chau T, Laurin MA, Chow LH, Ellis CG, Fish J, Chan BM. Fibroblast growth factor-2 potentiates vascular smooth muscle cell migration to platelet-derived growth factor: upregulation of ₂ß₁ integrin and disassembly of actin filaments. Circ Res. . 1997; 80: 627–637.

26. Rocnik EF, Chan BM, Pickering JG. Evidence for a role of collagen synthesis in arterial smooth muscle cell migration. J Clin Invest. . 1998; 101: 1889–1898.

27. Sims SM, Jiao Y, Preiksaitis HG. Regulation of intracellular calcium in human esophageal smooth muscles. Am J Physiol. . 1997; 273: C1679–C1689.

28. Karkanis T, Jiao Y, Hurley BR, Li S, Pickering JG, Sims SM. Functional receptor-channel coupling compared in contractile and proliferative human vascular smooth muscle. J Cell Physiol. . 2001; 187: 244–255.

29. Li S, Fan SX, McKenna TM. Vascular smooth muscle cells on Matrigel as a model for LPS-induced hypocontractility and NO formation. Am J Physiol. . 1997; 272: H576–H584.

30. Stacey GN. Cell contamination leads to inaccurate data: we must take action now. Nature. . 2000; 403: 356.

31. Ford C, Li S, Pickering JG. Angiotensin II stimulates collagen synthesis in human vascular smooth muscle cells: involvement of the AT1 receptor, transforming growth factor ß, and tyrosine phosphorylation. Arterioscler Thromb Vasc Biol. . 1999; 19: 1843–1851.

32. Blank RS, Swartz EA, Thompson MM, Olson EN, Owens GK. A retinoic acid-induced clonal cell line derived from multipotential P19 embryonal carcinoma cells expresses smooth muscle characteristics. Circ Res. . 1995; 76: 742–749.

33. Seidel CL, Helgason T, Allen JC, Wilson C. Migratory abilities of different vascular cells from the tunica media of canine vessels. Am J Physiol. . 1997; 272: C847–852.

34. Dartsch PC, Voisard R, Bauriedel G, Hofling B, Betz E. Growth characteristics and cytoskeletal organization of cultured smooth muscle cells from human primary stenosing and restenosing lesions. Arterioslerosis. . 1990; 10: 62–75.

This article has been cited by other articles:

				J. E. Basford, Z. W.Q. Moore, L. Zhou, J. Herz, and D. Y. Hui Smooth Muscle LDL Receptor-Related Protein-1 Inactivation Reduces Vascular Reactivity and Promotes Injury-Induced Neointima Formation Arterioscler Thromb Vasc Biol, November 1, 2009; 29(11): 1772 - 1778. [Abstract] [Full Text] [PDF]

				T. W. Small and J. G. Pickering Nuclear Degradation of Wilms Tumor 1-associating Protein and Survivin Splice Variant Switching Underlie IGF-1-mediated Survival J. Biol. Chem., September 11, 2009; 284(37): 24684 - 24695. [Abstract] [Full Text] [PDF]

				H. Y. Choi, M. Rahmani, B. W. Wong, S. Allahverdian, B. M. McManus, J. G. Pickering, T. Chan, and G. A. Francis ATP-Binding Cassette Transporter A1 Expression and Apolipoprotein A-I Binding Are Impaired in Intima-Type Arterial Smooth Muscle Cells Circulation, June 30, 2009; 119(25): 3223 - 3231. [Abstract] [Full Text] [PDF]

				M. J. Frontini, C. O'Neil, C. Sawyez, B. M.C. Chan, M. W. Huff, and J. G. Pickering Lipid Incorporation Inhibits Src-Dependent Assembly of Fibronectin and Type I Collagen by Vascular Smooth Muscle Cells Circ. Res., April 10, 2009; 104(7): 832 - 841. [Abstract] [Full Text] [PDF]

				E. van der Veer, C. Ho, C. O'Neil, N. Barbosa, R. Scott, S. P. Cregan, and J. G. Pickering Extension of Human Cell Lifespan by Nicotinamide Phosphoribosyltransferase J. Biol. Chem., April 13, 2007; 282(15): 10841 - 10845. [Abstract] [Full Text] [PDF]

				R. Gros, Q. Ding, S. Armstrong, C. O'Neil, J. G. Pickering, and R. D. Feldman Rapid effects of aldosterone on clonal human vascular smooth muscle cells Am J Physiol Cell Physiol, February 1, 2007; 292(2): C788 - C794. [Abstract] [Full Text] [PDF]

				T. W. Small, Z. Bolender, C. Bueno, C. O'Neil, Z. Nong, W. Rushlow, N. Rajakumar, C. Kandel, J. Strong, J. Madrenas, et al. Wilms' Tumor 1-Associating Protein Regulates the Proliferation of Vascular Smooth Muscle Cells Circ. Res., December 8, 2006; 99(12): 1338 - 1346. [Abstract] [Full Text] [PDF]

				D. G.M. Molin and M. J. Post Do intrinsic arterial wall features determine atherosclerosis susceptibility? Cardiovasc Res, October 1, 2006; 72(1): 3 - 4. [Full Text] [PDF]

				J. Sainz and M. Sata Maintenance of Vascular Homeostasis by Bone Marrow-Derived Cells. Arterioscler Thromb Vasc Biol, June 1, 2006; 26(6): 1196 - 1197. [Full Text] [PDF]

				M. Sata Role of Circulating Vascular Progenitors in Angiogenesis, Vascular Healing, and Pulmonary Hypertension: Lessons From Animal Models Arterioscler Thromb Vasc Biol, May 1, 2006; 26(5): 1008 - 1014. [Abstract] [Full Text] [PDF]

				A. Yaghi and S. M. Sims Constrictor-induced translocation of NFAT3 in human and rat pulmonary artery smooth muscle Am J Physiol Lung Cell Mol Physiol, December 1, 2005; 289(6): L1061 - L1074. [Abstract] [Full Text] [PDF]

				Z. W. Q. Moore and D. Y. Hui Apolipoprotein E inhibition of vascular hyperplasia and neointima formation requires inducible nitric oxide synthase J. Lipid Res., October 1, 2005; 46(10): 2083 - 2090. [Abstract] [Full Text] [PDF]

				E. van der Veer, Z. Nong, C. O'Neil, B. Urquhart, D. Freeman, and J. G. Pickering Pre-B-Cell Colony-Enhancing Factor Regulates NAD+-Dependent Protein Deacetylase Activity and Promotes Vascular Smooth Muscle Cell Maturation Circ. Res., July 8, 2005; 97(1): 25 - 34. [Abstract] [Full Text] [PDF]

				D. R. Greaves and S. Gordon Thematic review series: The Immune System and Atherogenesis. Recent insights into the biology of macrophage scavenger receptors J. Lipid Res., January 1, 2005; 46(1): 11 - 20. [Abstract] [Full Text] [PDF]

				Z. Wang, P. J. Rao, M. R. Castresana, and W. H. Newman TNF-{alpha} induces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H293 - H301. [Abstract] [Full Text] [PDF]

				M. Sata and R. Nagai Origin of Neointimal Cells in Autologous Vein Graft Arterioscler Thromb Vasc Biol, July 1, 2004; 24(7): 1147 - 1149. [Full Text] [PDF]

				C. A. Argmann, C. G. Sawyez, S. Li, Z. Nong, R. A. Hegele, J. G. Pickering, and M. W. Huff Human Smooth Muscle Cell Subpopulations Differentially Accumulate Cholesteryl Ester When Exposed to Native and Oxidized Lipoproteins Arterioscler Thromb Vasc Biol, July 1, 2004; 24(7): 1290 - 1296. [Abstract] [Full Text] [PDF]

				V. Andres Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential Cardiovasc Res, July 1, 2004; 63(1): 11 - 21. [Abstract] [Full Text] [PDF]

				Y. Gui and X.-L. Zheng Epidermal Growth Factor Induction of Phenotype-dependent Cell Cycle Arrest in Vascular Smooth Muscle Cells Is through the Mitogen-activated Protein Kinase Pathway J. Biol. Chem., December 26, 2003; 278(52): 53017 - 53025. [Abstract] [Full Text] [PDF]

				Y. Tintut, Z. Alfonso, T. Saini, K. Radcliff, K. Watson, K. Bostrom, and L. L. Demer Multilineage Potential of Cells From the Artery Wall Circulation, November 18, 2003; 108(20): 2505 - 2510. [Abstract] [Full Text] [PDF]

				K. Tanaka, M. Sata, Y. Hirata, and R. Nagai Diverse Contribution of Bone Marrow Cells to Neointimal Hyperplasia After Mechanical Vascular Injuries Circ. Res., October 17, 2003; 93(8): 783 - 790. [Abstract] [Full Text] [PDF]

				L. Stiebellehner, M. G. Frid, J. T. Reeves, R. B. Low, M. Gnanasekharan, and K. R. Stenmark Bovine distal pulmonary arterial media is composed of a uniform population of well-differentiated smooth muscle cells with low proliferative capabilities Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L819 - L828. [Abstract] [Full Text] [PDF]

				H. Hao, G. Gabbiani, and M.-L. Bochaton-Piallat Arterial Smooth Muscle Cell Heterogeneity: Implications for Atherosclerosis and Restenosis Development Arterioscler Thromb Vasc Biol, September 1, 2003; 23(9): 1510 - 1520. [Abstract] [Full Text] [PDF]

				C. Van Den Diepstraten, K. Papay, Z. Bolender, A. Brown, and J. G. Pickering Cloning of a Novel Prolyl 4-Hydroxylase Subunit Expressed in the Fibrous Cap of Human Atherosclerotic Plaque Circulation, August 5, 2003; 108(5): 508 - 511. [Abstract] [Full Text] [PDF]

				T. Karkanis, S. Li, J. G. Pickering, and S. M. Sims Plasticity of KIR channels in human smooth muscle cells from internal thoracic artery Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2325 - H2334. [Abstract] [Full Text] [PDF]

				M. Sata, K. Tanaka, R. Nagai, Y. Hu, H. Dietrich, F. Davison, M. Mayr, Q. Xu, B. Ludewig, M. Erdel, et al. Origin of Smooth Muscle Progenitor Cells: Different Conclusions From Different Models * Response Circulation, April 29, 2003; 107 (16): e106 - e107. [Full Text] [PDF]

				M. H. Laughlin, J. R. Turk, W. G. Schrage, C. R. Woodman, and E. M. Price Influence of coronary artery diameter on eNOS protein content Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1307 - H1312. [Abstract] [Full Text] [PDF]

				G. Gabbiani and G. K. Hansson ATVB In Focus: Smooth Muscle Cells Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 379 - 379. [Full Text] [PDF]

				C. Castro, A. Diez-Juan, M. J. Cortes, and V. Andres Distinct Regulation of Mitogen-activated Protein Kinases and p27Kip1 in Smooth Muscle Cells from Different Vascular Beds. A POTENTIAL ROLE IN ESTABLISHING REGIONAL PHENOTYPIC VARIANCE J. Biol. Chem., February 7, 2003; 278(7): 4482 - 4490. [Abstract] [Full Text] [PDF]

				Y. J. Woo and T. J. Gardner Myocardial Revascularization with Cardiopulmonary Bypass Card. Surg. Adult, January 1, 2003; 2(2003): 581 - 607. [Full Text]

				E. F. Rocnik, E. van der Veer, H. Cao, R. A. Hegele, and J. G. Pickering Functional Linkage between the Endoplasmic Reticulum Protein Hsp47 and Procollagen Expression in Human Vascular Smooth Muscle Cells J. Biol. Chem., October 4, 2002; 277(41): 38571 - 38578. [Abstract] [Full Text] [PDF]

				S.S.M Rensen, V.L.J.L Thijssen, C.J De Vries, P.A Doevendans, S.D Detera-Wadleigh, and G.J.J.M Van Eys Expression of the smoothelin gene is mediated by alternative promoters Cardiovasc Res, September 1, 2002; 55(4): 850 - 863. [Abstract] [Full Text] [PDF]

				H. Hao, P. Ropraz, V. Verin, E. Camenzind, A. Geinoz, M. S. Pepper, G. Gabbiani, and M.-L. Bochaton-Piallat Heterogeneity of Smooth Muscle Cell Populations Cultured From Pig Coronary Artery Arterioscler Thromb Vasc Biol, July 1, 2002; 22(7): 1093 - 1099. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 89/6/517    most recent hh1801.097165v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, S. Articles by Pickering, J. G. Search for Related Content PubMed PubMed Citation Articles by Li, S. Articles by Pickering, J. G. Related Collections Cell biology/structural biology Smooth muscle proliferation and differentiation