Modulation of smooth muscle phenotype in vitro by homologous cell substrate

F. Tao1,2, S. Chaudry1, B. Tolloczko1, J. G. Martin 1, and S. M. Kelly1

1 Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada H2X 2P2; and 2 Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have developed a novel cell culture system that supports the shortening of smooth muscle cells. Primary rat airway smooth muscle cells were plated on an ethanol-fixed, confluent monolayer of homologous smooth muscle cells (homologous cell substrate, HCS). Cells grown on HCS exhibited morphological and functional characteristics consistent with a differentiated phenotype. Cells on HCS were spindle shaped with a well-defined long axis, whereas cells grown on glass were larger and irregularly shaped. Smooth muscle-specific alpha -actin immunostained diffusely in cells on HCS, whereas it appeared as stress fibers in cells on glass. Agonists recruited a greater fraction of HCS cells to contract, resulting in greater changes in cell area or length on average, but the maximal capacity of shortening of individual cells was similar between the groups. Unlike cells on glass, cells on HCS shortened to methacholine. HCS was reversible and persisted over several passages. Agonists stimulated intracellular Ca2+ oscillations in cells on HCS, whereas they elicited biphasic peak and plateau transients in cells on glass. HCS modulates smooth muscle cell phenotype in vitro.

cell contraction; extracellular matrix; intracellular calcium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MECHANICAL AND BIOCHEMICAL controls of smooth muscle contraction are important to study for understanding normal smooth muscle physiology as well as smooth muscle pathophysiology in diseases such as hypertension, diabetes, and asthma. For example, in the airways, hyperreactive smooth muscle is the effector of the reversible airways obstruction that is characteristic of asthma. There is some debate, however, as to whether the airway smooth muscle is intrinsically more contractile in asthmatic patients (2, 6, 24) or whether extrinsic mechanisms, such as airway wall thickening (23, 51) or diminished airway-parenchymal interdependence (32), allow for bronchoconstriction. The ability to study isolated smooth muscle is necessary for determining whether intrinsic properties of the muscle are responsible for the excessive contraction observed in disease. However, dissecting out behavior that is attributable exclusively to smooth muscle under either normal or abnormal conditions has been difficult because of limitations in obtaining pure and fully functional preparations for experimental testing. Smooth muscle in situ, whether intact in the animal or excised with its surrounding tissues, is subject to modulation by other tissues or humoral factors. In contrast, smooth muscle cells in culture are a pure preparation that can be easily manipulated experimentally, but they undergo a number of phenotypic changes in culture that impair their ability to contract. For example, in airway smooth muscle cells (ASMC) the expression of smooth muscle isoforms of actin, myosin, and myosin light chain kinase decreases during the proliferative phase after initial cell seeding (11). Although expression of these contractile proteins partially recovers once the cells become confluent, they do not return to levels present in freshly isolated smooth muscle. Other investigators have also noted a decrease in smooth muscle-specific contractile proteins during proliferation and with each subsequent passage (34, 46). Despite such drawbacks, ASMC in culture do respond to contractile agonists by increasing intracellular Ca2+ concentration (34) and stiffness (18, 37) and even by decreasing length (1). These studies suggest that under appropriate conditions cultured cells can be used to study the contractile properties of smooth muscle.

A dynamic relationship exists between cells and the extracellular matrix (ECM) because cells in culture either proliferate or differentiate depending on the type and density of ECM proteins on which they grow (5, 21, 28). For example, vascular smooth muscle cells (VSMC) grown on laminin and collagen IV express smooth muscle-specific contractile proteins (13, 44) whereas those cultured on rat tail collagen (25, 50) or Matrigel (30) shorten when exposed to contractile agonists. In contrast, VSMC grown on collagen I or fibronectin proliferate (3, 13, 16, 30, 44, 52). In addition, prolonged serum deprivation of confluent ASMC induces a subpopulation of cells to become contractile after elongation and migration on neighboring cells (31). We hypothesized that contact with other smooth muscle cells and/or the specific matrix secreted by a confluent cell layer causes smooth muscle cells in culture to become contractile. To test this hypothesis, we developed a novel substratum on which to culture smooth muscle cells. Homologous cell substrate (HCS) is a confluent monolayer of smooth muscle cells that is fixed with ethanol. Live smooth muscle cells are then plated on the biochemically inert HCS and can be studied shortly thereafter. We report that HCS modulates the morphology, enhances the contractility, and alters the intracellular Ca2+ signaling of smooth muscle cells in culture.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Primary cultures of tracheal smooth muscle cells were prepared as previously described (45). Once confluent, the cells were detached with a 0.25% trypsin-0.02% EDTA solution, resuspended in DMEM-F-12 containing 10% FBS, and plated onto round glass coverslips, some of which were coated with HCS. Coverslips of 12-mm in diameter were used for contraction measurements, and coverslips of 20-mm diameter were used for anti-smooth muscle alpha -actin staining and intracellular Ca2+ measurements. Twenty-four hours later, the cells were growth arrested in DMEM-F-12 containing 1% FBS. All the outcomes in this study were measured after 2 days of growth arrest unless otherwise stated.

HCS preparation. An inert layer of homologous smooth muscle cells was used as the substrate upon which the cells were cultured. This substrate was prepared by growing first-passage smooth muscle cells to confluence on glass coverslips placed inside 35-mm diameter wells of six-well plates. At confluence, the cells were fixed with 1 ml of 70% ethanol for 10 min. After the ethanol was removed, the plates were exposed to ultraviolet light under laminar flow for 30 min to ensure sterility and complete evaporation of residual ethanol. The plates were used immediately or stored at 4°C before use. Because preliminary experiments showed no differences in cell morphology or contraction between glass or collagen substrata, glass was used as the control substratum.

Contractility measurements. Coverslips were transferred from the tissue culture well to a glass-bottomed dish containing 800 µl of Hanks' balanced salt solution (HBSS) warmed to 37°C. The dish was then placed on the heated stage of an inverted microscope (Olympus Optical, Tokyo, Japan), and the cells were visualized with Nomarski optics. Images of each field of cells were captured with a CCD-200-R camera (Videoscope International, Washington, DC) and recorded on an optical disk (Panasonic, Osaka, Japan).

An initial recording was made to obtain the size of quiescent cells. To measure contractile responses to specific agonists, 200 µl of HBSS or agonist [serotonin (5-HT) or methacholine (MCh)] was added to the dish and images were recorded at regular intervals for 10 min. The images were digitized and the surface area and length of individual cells were analyzed with a software package (Scion, NIH Image). Only those cells with a distinct longitudinal axis and clearly visible cell-substrate boundaries were selected for analysis. Surface area was obtained by tracing the outer boundary of the cell, and length was measured by drawing a line through the cell along its longest visible axis. The extent of contraction was calculated as the ratio of the change in surface area or length to the initial value of the parameter (Delta A/A0 or Delta L/L0).

Immunofluorescent staining for alpha -actin. After the cells were fixed with acetone, they were treated with murine anti-smooth muscle-specific alpha -actin followed by FITC-conjugated rabbit anti-mouse IgG. Fluorescence was visualized under an IMT-2 inverted microscope (Olympus, Tokyo, Japan) equipped with an InSight Plus confocal attachment (Meridian Instruments, Okunos, MI) at 40× magnification. Images were captured with a progressive scan charge-coupled device camera and digitized with custom software (Alex Informatics, Lachine, QC, Canada).

Cell proliferation measurements. When the cells were growth arrested on the day after subculture, the supernatant of DMEM-F-12 with 10% FBS was collected to determine the number of cells that did not attach. The cells were pelleted, resuspended in HBSS, and counted with a hemacytometer. The supernatant of DMEM-F-12 with 1% FBS was similarly collected and processed when proliferation was assessed. To measure proliferation, cells were detached with 500 µl of a 0.025% trypsin solution and an aliquot was immediately counted with a hemacytometer. HCS was not visibly affected by trypsinization.

Spectrofluorimetric measurement of intracellular Ca2+. Intracellular Ca2+ was detected with fura 2-AM dye as previously described (45). Cells were loaded with 5 µM fura 2-AM for 30 min at 37° C. The coverslips were mounted in a Leiden chamber (Medical Systems, Greenville, NY), which was then placed onto the stage of an epifluorescence-equipped inverted microscope with a 40× oil-immersion objective (Nikon, Tokyo, Japan). Fura 2 was excited at 340 and 380 nm with a PTI Deltascan 1 dual monochromator illuminator (Photon Technology International, Princeton, NJ), and its emission was detected at 510 nm by a PTI D104 microphotometer. Ca2+ responses to 5-HT were tracked in single cells for 5 min and were analyzed with PTI software. The cells were maintained at 37°C throughout.

Chemicals. Elastase, collagenase, 5-HT, MCh, anti-alpha -actin, and anti-mouse IgG were purchased from Sigma (St. Louis, MO). DMEM, Ham's F-12, FBS, penicillin, and streptomycin were supplied by GIBCO Canada (Mississauga, ON, Canada). RGD peptide (Peptite 2000) was supplied by Telios Pharmaceutical (San Diego, CA). Fura 2-AM and pluronic acid were obtained from Molecular Probes (Eugene, OR).

Statistics. Unpaired t-tests or ANOVA were used to test morphological and functional differences between cells grown on glass or on HCS. The chi 2-test was used to compare the frequency of positive responders between the two groups.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell morphology. Cells grown on HCS were morphologically distinct from cells grown on glass when viewed under the light microscope. Cells on glass (Fig. 1A) were large [surface area 3,901 ± 204 µm2, length 129 ± 5 µm (means ± SE); n = 97], flat, and spread out. Their nuclei were prominent, and secretory granules and stress fibers were evident. Many of the cells had irregular shapes and no well-defined long axis. In contrast, the cells grown on HCS (Fig. 1B) were significantly smaller (surface area 966 ± 33 µm2, length 74 ± 2 µm; n = 343) and were spindle shaped with a well-defined long axis. The differences in surface area and length between the cells grown on glass and the cells grown on HCS are depicted in Fig. 1, C and D, respectively. The boundaries of the cells on HCS were better delineated because of the enhanced contrast in optical density between the cells and their surroundings. This suggested that these cells might be thicker than those grown on glass. Neither secretory granules nor stress fibers were visible in the cells grown on HCS under the light microscope.


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Fig. 1.   Airway smooth muscle cell (ASMC) morphology is substratum dependent. A and B: photomicrographs of ASMC growing on glass coverslips (A) or homologous cell substrate (HCS; B). Cells grown on glass manifest irregular shapes, whereas cells grown on HCS are spindle shaped. C and D: comparison of surface area (C) and length (D) of ASMC grown on glass or HCS. E and F: confocal photomicrographs of smooth muscle-specific alpha -actin immunofluorescence of ASMC grown on glass (E) and HCS (F). Bar: 100 µm. * P < 0.05 HCS vs. glass.

Differences in morphology were also evident from the distinct arrangement of smooth muscle alpha -actin in the cells grown on the two substrates. alpha -Actin was arranged as stress fibers in the cells on glass (Fig. 1E), whereas it was diffusely distributed in the cells on HCS (Fig. 1F).

Cell proliferation. A similar number of cells attached to glass or HCS after 3 days of culture in DMEM supplemented with 1% FBS (Table 1). These cells did not appear to have divided because their numbers after 3 days of growth matched their initial seeding density. When the cells were grown in DMEM supplemented with 10% FBS for 3 days, the cells grown on glass significantly increased in number, indicating that the cells had proliferated. In contrast, the cells grown on HCS did not increase in number above their initial seeding density. Under this growth condition (i.e., 10% FBS), a similar number of dead cells was found in the supernatants from culture wells supporting glass and HCS substrates (data not shown). The different number of cells attached to glass or HCS thus did not appear to arise from differences in cell detachment or inability of cells to attach after initial seeding.

                              
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Table 1.   Number of cells on glass or HCS after 3 days of growth in DMEM supplemented with 1% or 10% FBS

Cell shortening. Figure 2 illustrates the shortening of cells on glass and on HCS to 10 µM 5-HT. Figure 2, A and B, depicts cells grown on glass before and after 10 min of 5-HT stimulation, respectively. Only the cell marked C has shortened. Figure 2, C and D, shows cells grown on HCS before and after 10 min of 5-HT stimulation, respectively. Ten cells (labeled a-j) have shortened. Although the extent of shortening varied widely from cell to cell, a greater fraction of the cells grown on HCS shortened than cells grown on glass.


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Fig. 2.   ASMC grown on glass or HCS contract in response to serotonin (5-HT). A and B: cells grown on glass: quiescent cells (A) and cells 10 min after 10 µM 5-HT stimulation (B). The cell marked C has contracted. C and D: cells grown on HCS: quiescent cells (C) and cells 10 min after 10 µM 5-HT stimulation (D). Cells marked a-j have contracted. Note particularly cells b, d, e, g, and h. Bar: 100 µm.

Quantitation of cell shortening confirmed these observations. The changes in surface area (Delta A/A0) and length (Delta L/L0) for all cells after addition of HBSS, 5-HT, or MCh to the medium are depicted in Fig. 3, A and B, respectively. These data represent the average responses of all the cells, whether they shortened or not. Both parameters show that on average cells grown on HCS are more responsive to agonists than cells grown on glass. The cells grown on glass did not undergo any marked change in area or length with either HBSS or agonists. In contrast, the cells grown on HCS shortened ~5% in response to HBSS alone and this was significantly greater than the HBSS response of the cells grown on glass (P < 0.01 for both area and length). In response to agonists, the cells grown on HCS shortened significantly more than to HBSS (P < 0.01 for both area and length). The changes in area were the same in response to MCh or 5-HT, regardless of concentration, but the changes in length were significantly greater in response to 10 µM MCh than to 1 or 10 µM 5-HT. Length changes in response to 1 and 10 µM 5-HT were similar.


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Fig. 3.   ASMC grown on HCS contract more than cells grown on glass. A: change in cell surface area normalized to surface area of the cell at rest (Delta A/A0). B: change in cell length normalized to resting length (Delta L/L0). n = 20-23 Cells grown on glass; n = 23-106 cells grown on HCS. #P < 0.01 compared with glass; *P < 0.01 compared with Hanks' balanced salt solution (HBSS). MCh, methacholine.

The frequency distribution of responsive cells also differed under the two culture conditions (Fig. 4). In light of previously reported shortening of adherent smooth muscle cells in culture (25, 26, 30) as well as our own data showing that cells would change their area by 4.08 ± 1.54% or length by 5.07 ± 1.23% in response to buffer alone, we considered >10% change in area or length to be a positive response. With this criterion, the fraction of cells grown on glass that shortened was modest compared with the fraction of shortening cells grown on HCS. Less than 10% of the cells on glass responded positively to HBSS or agonists in terms of changes in surface area (Fig. 4A) or length (Fig. 4B). In contrast, 20% (HBSS) to 80% (agonists) of the cells on HCS were positive responders. Using changes in surface area as the index of shortening provided a higher fraction of positive responders (averaging 70-80% of the cells depending on the stimulus) than using changes in length (averaging 40-60% positive responders). For the cells grown on HCS, the fraction of the population that shortened >10% was significantly greater in response to agonists than to HBSS (P < 0.005).


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Fig. 4.   Fraction of positive responders (contractile ASMC) is higher on HCS than on glass. A: fraction of cells undergoing a change in surface area >10%. B: fraction of cells undergoing a change in length >10%. *P < 0.005 between glass and HCS with each treatment.

It should be noted that in all treatment groups of cells on HCS, changes in area or length of individual cells ranged from 10% to 80%. That is, it was possible to find one or two cells in the control group that shortened by 80% in response to HBSS alone and it was possible to find nonresponsive cells in the agonist-treated groups. Thus the maximal shortening capacity was similar among all groups. The differences between glass and HCS shown in Fig. 3 result from averaging all the cells in each group and therefore reflect the higher number of responsive cells to agonists.

When the cells grown on HCS were treated with 10 µg/ml RGD peptide for 10 min before shortening was measured, the cells lost their spindle shape and rounded up, indicating that they were attached to the HCS via focal contacts (data not shown). Shortening could not be measured under these conditions.

Phenotype persistence. To further explore the effects of HCS, we tested whether the cells grown on HCS would still shorten under culture conditions adverse for cell contraction. First, we tested whether third-passage smooth muscle cells could still shorten on HCS, because myocytes have been shown to manifest decreased expression of contractile proteins with successive passages (11, 34, 46). Second, we maintained these cultures in 10% FBS to promote proliferation rather than differentiation. Our results show that the third-passage cells grown on HCS and sustained in 10% FBS shortened in response to 5-HT or MCh (Fig. 5A). When the responses of all the cells (i.e., responsive and nonresponsive) were averaged, shortening was significantly greater in response to 5-HT or MCh than to HBSS (P < 0.05). Shortening was similar between 5-HT and MCh. In addition, these agonists recruited significantly more cells to shorten >10% of their original length than HBSS (Fig. 5B; P < 0.005). There were no significant differences in the shortening capacity or in the fraction of responding cells between first-passage cells maintained in 1% FBS and third-passage cells maintained in 10% FBS.


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Fig. 5.   Contractile phenotype of ASMC grown on HCS is maintained after several passages and while grown in 10% FBS-supplemented DMEM. A: change in cell length normalized to resting length after 10 min of HBSS, 10 µM 5-HT, or 10 µM MCh. B: fraction of cells that underwent a change in length >10%. *P < 0.005.

Intracellular Ca2+ signaling. To determine whether the mechanical differences observed between the cells grown on glass and the cells grown on HCS were paralleled by signaling differences inside the cell, we compared Ca2+ responses to 10 µM 5-HT in individual cells grown under the two culture conditions. The two sets of cells displayed similar baseline Ca2+ levels as well as similar peak responses to 5-HT (Fig. 6A). However, cells grown on the different substrates exhibited considerably different Ca2+ oscillations during the sustained phase of the signal. The plateaus of 12 of 13 cells grown on glass were generally flat (Fig. 6B), whereas the cells on HCS manifested a variety of spiking oscillations of variable frequency and amplitude (Fig. 6C). The cells did not respond with Ca2+ transients to 10 µM MCh (data not shown).


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Fig. 6.   Intracellular Ca2+ mobilization in ASMC grown on glass and HCS substrates. A: initial peak Ca2+ transient in response to 10 µM 5-HT. [Ca2+]i, intracellular Ca2+ concentration. B: tracings of [Ca2+]i oscillations in ASMC grown on glass in response to 10 µM 5-HT. C: [Ca2+]i oscillations in individual ASMC grown on HCS in response to 10 µM 5-HT. Numbers in upper right-hand corners indicate the fraction of cells manifesting that particular type of oscillation.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to develop a simple cell culture model of smooth muscle shortening. There are several obstacles to smooth muscle cells shortening in culture. One impediment is that smooth muscle cells dedifferentiate and acquire a proliferative phenotype in culture, although the expression of contraction-specific proteins can be partially recovered under certain culture conditions (17, 40). Another impediment is that cultured smooth muscle cells are constrained from shortening by their attachments to the substratum. Several approaches to overcome these problems have been reported. VSMC in culture have been shown to shorten in response to agonists when grown on rat tail collagen (26) or Matrigel (30, 47, 48), whereas chicken gizzard smooth muscle cells have been shown to shorten when grown on laminin (12). Alternatively, confluent ASMC subjected to prolonged serum deprivation shorten when exposed to agonist if they are gently loosened from underlying cells by mild collagenase and papain digestion (10). Cultured ASMC have also been shown to shorten after they are delicately and mechanically lifted from their substratum (39). In our hands, rat ASMC plated on rat tail collagen did not shorten to 5-HT any more than cells grown on glass.

VSMC in culture have been shown to secrete an ECM composed of a complex mix of fibronectin, nidogen, heparan sulfate-proteoglycans, laminin, as well as collagen types I, III, IV, V, and VI (42). We inferred that ASMC in culture secrete a similar ECM network of mixed composition. Given that the ECM secreted by VSMC is less supportive of endothelial cell adhesion and proliferation than the ECM secreted by endothelial cells (49), we further reasoned that different cell types secrete specific ECM mixtures that influence the behavior of the attached cells . We therefore postulated that the complex ECM secreted by the ASMC themselves would be a superior substrate to promote myocyte differentiation than individual ECM proteins or Matrigel. We developed a system in which we seeded live cells on top of a confluent layer of dead cells. The first layer of cells, termed homologous cell substrate (HCS), was fixed with 70% ethanol to render it biochemically inert while leaving its secreted ECM intact. Ethanol was used as the fixative because it is routinely used for fixation in immunohistochemistry. Because the present study aimed to describe the validity of the HCS technique for measuring cell shortening, we did not characterize the composition of the HCS substrate further.

The first striking effect of the HCS was that the cells grown on it visually resembled freshly isolated smooth muscle cells. Unlike the expansive, irregularly shaped appearance of cells grown on glass, the cells on HCS were small, elongated, and spindle shaped. Cell shape is determined by the spatial distribution of ECM contacts that are available for attachment, and shape in turn can regulate cell phenotype (5). A large area for expansion has been reported to promote cell spreading and to support proliferation, a confined space restricted cell size and induced apoptosis, and intermediate-sized attachment areas caused cells to differentiate (8). In addition, the geometric configuration of the ECM network regulates cell proliferation and migration as well as intracellular signaling and architecture (4, 36). Therefore, the distinct ultrastructure of cells grown on HCS might have been regulated by a distinct spatial distribution of contact sites and a complex geometric arrangement of the HCS's ECM network. In contrast, cells in culture adhere to glass or plastic because these materials nonspecifically adsorb ECM proteins such as fibronectin and vimentin, which are solubilized in serum (19). The spatial distribution and arrangement of the adsorbed ECM proteins would be expected to be random under these circumstances, so the ECM network would consequently have a less specific influence on cell phenotype. Other regulators, such as growth factors or the availability of expansion space, may then have greater control over cell phenotype.

Differences in ECM architecture between HCS and glass might also account for the substrate-specific ability to support cell differentiation as evidenced by the significantly greater proportion of contractile cells grown on HCS than on glass. Differentiation persisted in cells that had been passaged several times and then plated on HCS, even though the high serum concentration we used normally favors proliferation rather than differentiation. It appeared that HCS prevented cell proliferation because after 3 days of culture in DMEM supplemented with 10% FBS the number of cells on HCS was the same as the initial number seeded whereas the number of cells grown on glass significantly increased. Although ECM-integrin interactions and soluble growth factors coordinately determine cell phenotype and share some downstream signal transduction pathways (9), the ECM has been shown to be capable of dominating this relationship (20, 22). Our data concur with this latter observation.

HCS also affected the arrangement of smooth muscle-specific alpha -actin inside the overlying live cells. In the ASMC on glass, alpha -actin was arranged as stress fibers. These are typically observed in smooth muscle cells in culture (10, 29). In contrast, alpha -actin in the cells on HCS was diffusely distributed. Freshly isolated VSMC do not contain stress fibers and only begin to develop them after 4 days of culture under conditions that favor cell proliferation (15). This suggests that stress fibers may not exist in differentiated smooth muscle cells but may develop as a result of tensional adhesion in culture. Stress fibers radiate from focal contacts (38). If the number of focal contacts is limited, then the formation of stress fibers may also be restricted. Fewer focal contacts could also result in weaker adhesion and reduced cell tension, thus decreasing the need for internal stress fibers to resist the external tensional load. We postulate that the absence of stress fibers in the cells grown on HCS may be partly due to reduced focal contacts between the cell and the HCS.

Additionally, the interaction of the live cells with the HCS may have signaled alpha -actin to organize as a mesh network rather than bundle into fibers. In VSMC, individual ECM proteins have been shown to have specific effects on intracellular signaling. Fibronectin and collagen type I stimulate VSMC proliferation, whereas laminin stimulates differentiation (16, 52). Compared with laminin, fibronectin supports greater tyrosine phosphorylation at the focal adhesion complexes (FACs), and this appears to determine the degree of cell spreading, the number of FACs, and the arrangement of cytoskeletal actin (14). The unique network of ECM presented by HCS might similarly modulate intracellular signaling pathways in a highly precise manner that would define cell morphology and behavior.

We postulate that shortening of the myocytes on HCS could have only occurred if the cells had detached to some degree from the substratum. Furthermore, because detachment occurred without experimental intervention (e.g., enzyme digestion, mechanical disruption), it would have been actively coordinated by the cells themselves. We observed a few cases in which a cell on HCS was quiescent for a short period after agonist exposure and then shortened in a snap. This type of instantaneous response occurs with freshly isolated ASMC floating freely in suspension, presumably because they are not restricted by any external load (41). In contrast, cells in culture are subjected to the external load imposed by their attachments to their substratum. The few rapidly shortening cells we observed may have reflected a rapid loss of external load through rapid detachment from the HCS. Most of the cells, however, required minutes to shorten. We speculate that this delay is caused by the time required to disengage focal contacts. In support of this idea, other investigators studying smooth muscle cell contraction in culture must mechanically or enzymatically detach the cells from their substrates before detecting shortening (10, 39). Cells manipulated thus also take minutes to reach maximal shortening. Whether the cell generates excessive mechanical tension to detach itself from the HCS, focal contacts are biochemically disrupted, or both mechanisms contribute remains uncertain but of interest for future investigation.

It is interesting to note that 20% of the cells grown on HCS shortened in response to HBSS. This was likely due to the mechanical perturbation of introducing additional liquid to the sample well and suggests that the HCS might also increase mechanoreceptor sensitivity in a subpopulation of cells. The increased sensitivity might arise from phenotypic changes of the cells growing on HCS and/or from variability in the number and type of focal contacts the cells make with the HCS according to location.

Intracellular Ca2+ signals to 5-HT were affected by growing smooth muscle cells on HCS. On glass, cells had a biphasic Ca2+ response to 5-HT consisting of a rapid, transient peak followed by a flat plateau that remained elevated above baseline for several minutes. Cells grown on HCS still displayed the initial peak but manifested heterogeneous spiking oscillations rather than a level plateau during the secondary phase. It is unclear how growth on HCS altered the pattern of Ca2+ signals to 5-HT inside the cell. However, because transformation of cell shape has been reported to impair agonist-mediated Ca2+ mobilization by altering the spatial relationship between PLC and the inositol 1,4,5-trisphosphate receptor (35), the modified morphology of cells grown on HCS may have concomitantly modified the spatial localization of Ca2+-regulating proteins. A related possibility is that the Ca2+ spikes resulted from the dynamic changes in cell shape during shortening because cytoskeletal F-actin remodeling and intracellular Ca2+ transients are interdependently regulated via membrane phosphatidylinositol 4,5-bisphosphate (PIP2) turnover (43).

The cells on HCS shortened to MCh. Typically, cultured myocytes are unresponsive to MCh (7, 34) because muscarinic receptor type 3 (M3) expression is downregulated in culture (33). A contractile response to MCh suggested that M3 expression was recovered by growing the cells on HCS. Cholinergic responses were also recovered in prolonged serum-deprived, confluent ASMC grown on top of other live cells (10), corroborating our findings that growth on an appropriate substrate might induce the expression of M3 in cultured myocytes. However, the cells on HCS did not have Ca2+ transients in response to MCh, suggesting that M3 expression was not recovered by HCS. If M3 expression was not recovered, it might have been possible that the MCh activated M2 receptors to activate nonspecific cation channels that led to cell shortening, except that this requires Ca2+ release via M3 activation (27). Our data are inadequate to reconcile the dissociation between shortening and Ca2+ transients to MCh in cells grown on HCS, although these observations suggest that the shortening of cells on HCS may not require Ca2+. By extension, the 5-HT-dependent Ca2+ oscillations may be unrelated to the cell shortening but may trigger other events. More thorough receptor subtyping will be necessary to clarify the mechanism of action of MCh on the cells on HCS and resolve the issue between Ca2+ oscillations and shortening of cells on HCS.

In response to agonists or depolarization, freshly dissociated, freely floating canine ASMC shortened between 20% and 65% from their original length whereas normal human bronchial myocytes shortened an average of ~25% (41). Maximal shortening of VSMC cultured on rat tail collagen was ~16-22% of their resting length in response to angiotensin II or 5-HT (25, 26). On Matrigel, VSMC shortened between 28% and 41% in response to agonists, depending on the animal species and strain from which the cells were derived (47, 48). Prolonged serum-deprived, confluent canine ASMC maximally shortened between 20% and 90% after they were enzymatically semidetached (10). The rat ASMC grown on HCS shortened between 10% and 80%. In terms of the fraction of positively responding cells (i.e., >10% shortening in response to agonist), 55% of canine vascular myocytes shortened on Matrigel (30) whereas ~62% of prolonged serum-deprived, confluent canine ASMC shortened after partial enzymatic detachment (10). Between 46% and 56% of the cells grown on HCS shortened >10% in response to agonist. Thus cells grown on HCS display shortening capacities and frequencies comparable with data reported in the literature for other substrates. It is worth noting that among responding cells grown on HCS, saline or agonist elicited the same degree of contraction. This observation suggests that agonists recruit more cells to contract rather than increasing the contractile capacity of individual cells.

One limitation of our model is the selection bias introduced by sampling cells on the basis of shape. Cells were required to have a distinct long axis suitable for analysis. This concern was of greater pertinence for the cells grown on glass, which tend to be less elongated and are often more irregularly shaped cells than the cells grown on HCS. The cells on HCS that we analyzed had long to short axis ratios that were at least 2. We did not systematically examine the influence of cell shape on the measured contractions, nor do we know the error that may be introduced by imprecise recognition of the edges of the cells. Another limitation is the protracted, nonphysiological time course of cell shortening, but this may be a general intractable problem with adherent cell remodeling. Clarifying the composition of the HCS ECM would also be important to understand how HCS enables cells to shorten.

In conclusion, we have described a simple and novel method with which to study contractile smooth muscle cells in culture. The present data demonstrate that HCS might be useful for investigating the interaction among 1) the effect of an endogenous ECM network on cell phenotype, 2) the mechanics of smooth muscle cell shortening, and 3) the intracellular signal transduction events that transpire during shortening. The mechanisms of its action remain to be clarified, but studies using this system could potentially improve understanding of how mechanical and biochemical systems integrate within the myocyte to regulate cell phenotype and function.


    ACKNOWLEDGEMENTS

We thank S. Shore and J. Fredberg for discussions about the data.


    FOOTNOTES

These studies were supported by the Canadian Institutes of Health Research (Grant MOP/36334).

Present address of S. M. Kelly: Novartis Pharmaceuticals Canada Inc., Dorval, Quebec, Canada H9S 1A9.

Address for reprint requests and other correspondence: F. Tao, Physiology Program, Dept. of Environmental Health, Harvard School of Public Health, 665 Huntington Ave., II-223, Boston, MA 02115 (E-mail: ftao{at}hsph.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published March 5, 2003;10.1152/ajpcell.00264.2002

Received 7 June 2002; accepted in final form 10 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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