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(Circulation Research. 1996;79:1046-1053.)
© 1996 American Heart Association, Inc.

Articles

Mechanical Strain Increases Smooth Muscle and Decreases Nonmuscle Myosin Expression in Rat Vascular Smooth Muscle Cells

Peter Reusch, Hisham Wagdy, Regina Reusch, Emily Wilson, Harlan E. Ives

the Division of Nephrology, Cardiovascular Research Institute, University of California, San Francisco.

Correspondence to Harlan E. Ives, MD, PhD, Director, Division of Nephrology, 672 Health Sciences East, Box 0532, University of California–San Francisco, San Francisco, CA 94143. E-mail ives@itsa.ucsf.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The effect of cyclic (1-Hz) mechanical strain on expression of myosin heavy chain isoforms was examined in neonatal rat vascular smooth muscle cells cultured on silicone elastomer plates. Myosin heavy chain isoforms were identified by immunoblot using antibodies recognizing (1) smooth muscle myosin heavy chain isoforms SM-1 and SM-2, (2) SM-1 exclusively, and (3) nonmuscle myosin heavy chains A and B. In response to 36 to 72 hours of strain, SM-1 and SM-2 increased by fourfold to sixfold, whereas nonmuscle myosin A decreased to 30% of control. Nonmuscle myosin B was unaffected by strain. SM-1 mRNA increased by twofold to threefold after 12 hours of strain but decreased toward control levels thereafter. SM-2 mRNA was only barely detectable. Nonmuscle myosin A mRNA decreased to 50% of control after 3 hours of strain and then returned to the control level. Since these cells secrete platelet-derived growth factor (PDGF) in response to strain, we assessed the effects of PDGF on myosin isoform expression. Exogenous PDGF (10 ng/mL) decreased SM-1 expression by 35% and increased nonmuscle myosin expression twofold, opposite the effect of strain. In cells exposed to strain with neutralizing antibodies to PDGF-AB, the strain-induced increase in SM-1 was enhanced 10-fold, and nonmuscle myosin A was reduced to 40% of control. Finally, the effect of extracellular matrix on transduction of the strain signal was studied. Forty-eight hours of cyclic strain increased SM-1 by twofold in cells cultured on collagen type I and threefold in cells cultured on laminin. In fibronectin-cultured cells, strain elicited no increase in SM-1. Thus, mechanical strain, sensed through specific interactions with the matrix, can alter myosin isoform expression toward that found in a more differentiated state.

Key Words: vascular smooth muscle • myosin • mechanical stress • platelet-derived growth factor • extracellular matrix

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Repetitive physical deformation is a prominent feature of the environment of VSM cells in situ. However, little is known about the role that cyclic mechanical strain may play in determining the phenotype of VSM cells. Interest in the mechanisms by which smooth muscle cell phenotype is altered in disease states in which mechanical forces may change (hypertension and atherosclerosis) has led to extensive study of the VSM cell phenotype both in situ and in vitro.¹

Although cultured VSM cells retain the ability to contract for a number of days in culture,² they rapidly change from this "contractile" phenotype into a "synthetic" phenotype characterized by increased proliferation, protein secretion, and diminished staining for smooth muscle myosin.^{3 4} It has been proposed that this synthetic phenotype of cultured VSM cells may be similar to that seen in vivo in atherosclerotic lesions.^{5 6 7} Recent data suggest that the phenotype of VSM cells in vitro may be more plastic than previously assumed. Rovner et al⁸ found that subconfluent, rapidly growing VSM cells express predominantly nonmuscle myosin but that postconfluent quiescent cells exhibit enhanced expression of SM-1 and SM-2 smooth muscle myosin isoforms.⁸ Thus, it appears possible that selection of appropriate in vitro conditions may allow more detailed study of the factors that determine VSM phenotype.

Cyclic mechanical strain has been found to exert important effects on phenotype and growth of a number of cultured cell types. Sumpio et al⁹ found altered synthesis of cytoskeletal proteins in aortic endothelial cells, and both Sumpio et al¹⁰ and Upchurch et al¹¹ found suppression of prostacyclin secretion by vascular endothelial cells in response to strain. Banes et al¹² found that fibroblasts from intact chicken tendons showed reduced synthesis of tubulin in response to cyclic tension.

A growing body of work has begun to examine the effects of cyclic strain on the phenotype of VSM cells. Leung et al¹³ found that cyclic strain increased synthesis of collagen, hyaluronate, and chondroitin-6-sulfate in arterial smooth muscle cells. Kollros et al¹⁴ and Sumpio et al,¹⁵ using a similar model system, also found increased collagen synthesis in VSM cells. Smith et al¹⁶ demonstrated increased DNA synthesis and increased myofilament content, as determined by electron microscopic examination. Finally, Kanda and Matsuda¹⁷ found that VSM cells in stress-loaded three-dimensional gels demonstrate increased content of myofilaments and dense bodies reminiscent of the contractile phenotype.

Previous work in this laboratory has focused on the effects of cyclic strain on the growth of VSM cells. We have found that strain stimulates the proliferation of VSM cells through production and autocrine action of PDGF.¹⁸ Strain also causes synergistic enhancement in the growth response to other mitogens, including thrombin¹⁸ and angiotensin II.¹⁹ More recent work shows that strain is sensed by specific interactions with the extracellular matrix and that the response to strain may vary significantly, depending on which matrix proteins are present.²⁰

The potential importance of mechanical strain in determining the phenotype of VSM cells and the observation that strain enhances myofilament content in cultured VSM cells led us to examine the effects of strain on the expression of myosin isoforms in these cells. We find that the application of cyclic strain to neonatal rat VSM cells can substantially increase the expression of smooth muscle myosin heavy chain protein and mRNA and can concomitantly decrease expression of NM-A. Neutralization of the growth factors secreted in response to strain enhance these changes in myosin expression even further. As previously described for the growth response,²⁰ the signal for the strain-induced change in myosin expression depends importantly on the presence of specific extracellular matrix proteins. However, the extracellular matrix proteins that best support the altered myosin isoform expression in response to strain differ from those that support the proliferative response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
All materials were purchased from Sigma Chemical Co unless otherwise indicated. Recombinant PDGF-AB was purchased from Boehringer-Mannheim.

Antibodies
Anti–SM-1/SM-2 antibody was a polyclonal antiserum raised against the peptide Cys-Asp-Ala-Asp-Ser-Asn-Gly-Thr-Cys-Ala-Ser-Cys. This antibody was kindly provided by Berlex Pharmaceuticals. Anti–SM-1 antibody was a polyclonal serum raised against an isoform-specific peptide, Cys-Arg-Arg-Ser-Gly-Gly-Arg-Arg-Val-Ile-Glu-Asn-Ala. This antibody was kindly provided to us by A.F. Martin, University of Illinois, Chicago. This polyclonal antibody does not recognize SM-2.²¹ Anti–NM-A antibody (Biomedical Technology Inc) was a rabbit polyclonal antibody against human platelet myosin. Secondary antibodies for immunoblots were goat anti-rabbit and rabbit anti-mouse antibodies (Amersham). Anti-SMemb (anti–NM-B) was an antiserum raised against Ser-Asp-Val-Asn-Glu-Thr-Gln-Pro-Glu-Ser-Glu from the deduced sequence of SMemb.²² This antibody was kindly provided by Drs K. Itoh and R.S. Adelstein (National Institutes of Health). Anti–PDGF-AB was polyclonal goat anti-human PDGF-AB (Upstate Biotechnology Research).

Control Myosin Preparations
Crude extract of human platelets and rat aorta were prepared as previously described.²³ Outdated human platelets were obtained from the University of California–San Francisco, blood bank. Skin fibroblasts were cultured from explants of human skin biopsies (University of California–San Francisco).

Cell Culture
Primary cultures of VSM cells from newborn rat were established as previously described.² The cells were maintained in MEM with 10% fetal bovine serum, tryptose phosphate broth (20 mg/mL), penicillin (50 U/mL), and streptomycin (50 U/mL) in a humidified atmosphere of 5% CO₂/95% air at 37°C. Culture medium was changed every other day until cells were confluent. For passaging, cells were released from culture dishes with trypsin-versene and pancreatin (2 mg/mL), and cells from passages 10 to 15 were used for the present studies.

Application of Cyclic Strain to Cultured Cells
Cells were plated on six-well silicone elastomer–bottomed culture plates (Flexcell Corp) coated with collagen type I, laminin, or fibronectin as indicated. Cells were maintained in complete medium for 3 days, achieving 80% confluence. Medium was then changed to "quiescence" medium, containing MEM with 0.5% fetal bovine serum. After 3 days in quiescence medium, cells were subjected to mechanical deformation with the Flexercel Stress Unit (Flexcell Corp). The stress unit is a modification of the unit initially described by Banes et al¹² and consists of a computer-controlled vacuum unit and a baseplate to hold the culture dishes. Vacuum (15 to 20 kPa) is repetitively applied to the rubber-bottomed dishes via the baseplate, which is placed in a humidified incubator with 5% CO₂ at 37°C. The computer system controls the frequency of deformation and the negative pressure applied to the culture plates. Cyclic deformation (1 Hz) was used for comparability with previous work^{18 20} and is the highest cycling frequency this instrument is capable of producing without damaging the silicone plates.

Protein Electrophoresis
VSM cells were scraped from the silicone elastomer dishes in PBS at 4°C. Harvested material was centrifuged for 4 minutes at 3000g, and supernatants were discarded. Cells were lysed in 0.5% SDS in PBS, and protein was quantified by the BCA method (Pierce). Aliquots were boiled in electrophoresis sample buffer (0.125 mol/L Tris, pH 6.8, 2% SDS, 10% glycerol, and 0.75 mol/L ß-mercaptoethanol) for 5 minutes. Ten micrograms of protein per lane was loaded onto 4% SDS-polyacrylamide gels and electrophoresed in 8x10-cm minigels (Bio-Rad) at 4°C using the Laemmli buffer system. Each gel was run in duplicate for protein staining or Western blot.

Immunoblots
Proteins separated by SDS-PAGE were electroblotted for 2 to 3 hours at constant current (200 mA) onto nitrocellulose paper (Hybond-ECL, Amersham). Transfer buffer was Tris (25 mmol/L), glycine (192 mmol/L), and methanol (20%) at 4°C. After electroblotting, gels were stained with Coomassie blue to ensure that transfer of proteins was complete. To reduce nonspecific binding, filters were blocked with 10% nonfat dry milk in TBS (20 mmol/L Tris, pH 7.5, 50 mmol/L NaCl, and 0.1% Tween 20) and then incubated with the primary antibody of interest for 1 to 3 hours at room temperature. After washing, blots were incubated with the required horseradish peroxidase–conjugated secondary antibodies, washed, and incubated with enhanced chemiluminescence reagents (Amersham). Blots were exposed to x-ray film for between 30 seconds and 5 minutes to obtain ideal exposure. Quantification was by densitometry scanning of bands on the developed film.

RNA Templates
cRNA used for RNAse protection assay of SM-1 and SM-2 was a 380-bp Pst I–HincII fragment from plasmid RAMHC15 (a generous gift from Dr Philip Babij, University College, London),²⁴ which includes 80 bp of common coding sequence and the unique 39 bp specific to SM-2. Restriction fragments were subcloned into pTZ18R (Pharmacia) and linearized with HindIII. Protection of the full 380-bp fragment was due to SM-2 mRNA, whereas protection of two fragments (261 and 80 bp) corresponds exclusively to SM-1 mRNA. A 650-bp rat genomic clone (CH4a#14), specific for NM-A, was also kindly provided by Dr P. Babij. This fragment, subcloned into pTZ18R, yielded a protected fragment of 195 bp with nonmuscle myosin mRNA.²⁵ An 816-bp cDNA (clone 9L3), specific for NM-B (SMemb), was kindly provided by Kazuyuki Itoh and Robert Adelstein from the National Heart, Lung, and Blood Institute. A 500-bp fragment of this clone was subcloned into pTZ18R. A commercially available cDNA (Ambion) probe for rat GAPDH was used as a control. These templates were all used to generate ³²P-labeled probes with [³²P]UTP using in vitro transcription with the Maxiscript kit (Ambion). Full-length transcripts were separated from prematurely cleaved transcripts on denaturing 8% urea gels. Full-length transcripts were then cut out of the gels and eluted.

RNase Protection Assays
Total cellular RNA was isolated from VSM cells by using commercially available RNA-stat 60 (Tel Test Co), a protocol based on the phenol/guanidine thiocyanate RNA isolation initially described by Chomczynski and Sacchi.²⁶ Determination of the RNA concentration was achieved by measuring absorption in a spectrophotometer at 260 nm. Purity was checked by monitoring the optical density (at 260 nm/280 nm) ratio. Various amounts of total RNA were hybridized overnight at 42°C with the probes described above. After digestion of nonhybridized fragments with a 1:250 dilution of RNase mixture (RPA II kit, Ambion), the remaining protected fragments were separated on a denaturing 8% urea gel and visualized by exposure to Amersham Hyperfilm for 2 to 24 hours at -80°C. Quantification of the bands was performed by liquid scintillation of the removed bands.

Statistics
Dimensionless quantities (band densities, etc) from multiple similar experiments were combined by calculation of the fold increase (or decrease) versus control under each experimental condition. Combined data were expressed as mean±SD (n3 in all cases). P values were calculated using SD from the mean for each experimental condition.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibody Specificity
To study changes in the distribution of myosin heavy chain isoforms in response to mechanical strain, it was first necessary to determine the specificity of the antibodies used for immunoblotting. Lysates of human platelets, cultured neonatal VSM cells, skin fibroblasts, and crude extract of rat aorta were subjected to gel electrophoresis. All four preparations exhibited pronounced protein bands at the molecular mass of myosin heavy chain, 200 to 205 kD (Fig 1). The intact aorta preparation exhibited a doublet at this molecular mass, most likely SM-1 and SM-2. Indeed, immunoblotting using an antiserum recognizing both SM-1 and SM-2 (Fig 1) showed significant staining of both bands. In the cultured VSM cells, expression of SM-1 and SM-2 could also be demonstrated when gels were loaded with 20-fold higher quantities of protein than for intact aorta (see "Discussion"). None of the other preparations exhibited expression of SM-1 or SM-2. Similarly, a polyclonal antiserum specific for SM-1²¹ recognized a band in the cultured VSM cells and intact aorta but not in the other preparations. Immunoblotting with a polyclonal antibody to NM-A showed heavy staining of a band in platelets, the cultured VSM cell preparation, and skin fibroblasts, but not aorta. Finally, a polyclonal antibody raised to a peptide from the deduced sequence of NM-B (also termed SMemb²⁷ ) recognized a protein found only in the cultured neonatal VSM cells. Thus, the VSM cells used for the present study express at least four different isoforms of myosin heavy chain. Despite the potential for some cross-reactivity of the antibodies (see "Discussion"), the use of all four antisera greatly reduced the potential for errors in interpreting the effects of mechanical strain on the various myosin isoforms.

View larger version (23K): [in this window] [in a new window]   Figure 1. Comparison of immunoblot staining patterns of myosins from various sources. Cell lysates (10 µg) from platelets (P), cultured neonatal aortic VSM cells (C), human skin fibroblasts (F), intact rabbit aorta (A), or a skeletal muscle myosin marker (S) were electrophoresed on 4% SDS-polyacrylamide gels as described in "Materials and Methods." Coomassie stain reveals a densely staining band (doublet in the aorta) at a molecular mass of 200 to 205 kD in each preparation. Immunoblotting was performed on similar gels, in which each lane was loaded with 10 µg of protein except where indicated. In lanes labeled SM-1/SM-2, immunoblotting was performed with a polyclonal anti–smooth muscle myosin antiserum. The lane containing aorta was loaded with 0.5 µg of protein. In lanes labeled SM-1, immunoblotting was performed on a gel loaded with 10 µg (P, C, and F) or 0.5 µg (A) of protein. Blotting was performed using a polyclonal anti–SM-1 antiserum. In lanes labeled NM-A, immunoblotting was performed using a polyclonal IgG against nonmuscle myosin A. In lanes labeled NM-B, immunoblotting was performed with a polyclonal antibody against nonmuscle myosin heavy chain B. Blots shown are representative of four similar experiments.

Cyclic Mechanical Strain Alters Expression of Myosin Isoforms in VSM Cells
To determine the effects of cyclic mechanical strain on the expression of the four myosin isoforms in cultured neonatal rat aortic smooth muscle cells, cells were exposed to 1-Hz cyclic mechanical strain for times between 12 and 72 hours. Control cells were plated in identical dishes for the full 72 hours but were not subjected to strain. Cell lysates were subjected to electrophoresis and immunoblotting with the four anti-myosin antibodies (Fig 2). Mechanical strain caused significant increases in labeling by both the smooth muscle myosin (SM-1/SM-2, Fig 2) and the SM-1–specific (SM-1, Fig 2) antibodies. Simultaneously, immunolabeling with NM-A antibody was decreased to 30% of control values (Fig 2). NM-B was unaffected by strain (Fig 2). Scans averaged from multiple similar experiments (Fig 3) indicated that SM-1 and SM-2 increased over 72 hours by 4.5- and 3.5-fold, respectively (Fig 3A). Using the SM-1–specific antibody, SM-1 increased by 6-fold after 72 hours (Fig 3B). NM-A decreased to 30% of baseline values (Fig 3C), whereas NM-B was unaffected by strain (Fig 3D). Thus, mechanical strain significantly increases both SM-1 and SM-2 and concomitantly decreases NM-A in neonatal rat VSM cells.

View larger version (20K): [in this window] [in a new window]   Figure 2. Time course of changes in distribution of myosin isoforms during mechanical strain. VSM cells grown on collagen-coated Flex plates were subjected to cyclic strain as described in "Materials and Methods." Control cells not subjected to strain for 72 hours (C) or cells harvested from the stretch plates at the indicated times (12 to 72 hours) were lysed and subjected to electrophoresis. Immunoblotting was performed with antibodies as described in the legend to Fig 1.

View larger version (24K): [in this window] [in a new window]   Figure 3. For each antibody in Fig 2, densitometric scans of bands at each time point were averaged from four independent experiments (two experiments for SM-1/SM-2). Bar graphs show mean±SD fold increase over control. A, SM-1 (closed bars) and SM-2 (open bars) antibody (P<.05 for both antibodies at all times >24 hours). B, SM-1 antibody (12 hours, P=NS vs control; 24 hours, P<.2 vs control; P<.05 vs control for all other times). C, NM-A antibody (P<.05 vs control for all times). D, NM-B antibody (P=NS for all times).

Strain Alters Expression of SM-1 and Nonmuscle Myosin mRNA
We next determined whether the changes in myosin isoform protein content described above were accompanied by changes in mRNA expression. Total cellular RNA from cells exposed to strain for various periods were analyzed by RNase protection assays using specific probes for the myosin isoforms. A single probe yielding protected fragments of different size was used to analyze SM-1 and SM-2 mRNA (Fig 4A). In three similar experiments, SM-1 mRNA increased significantly by 3 hours and achieved a maximum twofold increase after 12 hours of mechanical strain (Fig 4B). Exposure to strain for additional time did not yield further increases, and mRNA fell to control level by 36 hours (data not shown). SM-2 mRNA was barely detectable under these conditions.

View larger version (27K): [in this window] [in a new window]   Figure 4. Effect of strain on myosin isoform mRNA. RNase protection assays were performed on total RNA extracted from controls (C) and from cells exposed to strain (S) on collagen-coated Flex plates for the indicated times with RNA probes as described in "Materials and Methods." Aliquots of total RNA were hybridized with a probe for GADPH and a probe for either SM-1 or SM-2 (A) or a probe for NM-A (C). Bar graphs (B and D) depict the mean±SD fold increases and decreases (strain over control) from three separate experiments. Individual counts were obtained by scintillation counting of the appropriate bands. Values are shown for SM-1 mRNA (B; 1 hour, P=NS vs control; P<.001 vs control for all other times) and NM-A mRNA (D; P<.005 vs control for all times).

In contrast, nonmuscle myosin (NM-A) mRNA decreased in response to strain (Fig 4C). In three similar experiments, nonmuscle myosin mRNA fell significantly after 1 hour and achieved a maximal decrease to 40% of control at 3 hours. mRNA returned toward baseline subsequently (Fig 4D). As found for the protein, mechanical strain did not alter steady state mRNA for NM-B (data not shown). Although the time courses of these changes in both smooth muscle and nonmuscle myosin mRNA do not match those for the corresponding protein expression (see "Discussion"), they are directionally identical.

Effect of Strain on Myosin Isoform Expression Is Not Mediated by PDGF
We previously found that application of cyclic mechanical strain leads to increased growth of cultured VSM cells via secretion of PDGF into the culture medium.¹⁸ To determine whether this secreted PDGF affects the pattern of myosin isoform expression, VSM cells were exposed to either mechanical strain or PDGF-AB (10 ng/mL) for 36 hours. Western blots showed that PDGF-AB caused a change in myosin isoform expression qualitatively opposite that observed after application of mechanical strain (Fig 5A). Although strain increased smooth muscle myosin by 3-fold, PDGF-AB decreased smooth muscle myosin by >50% (P<.001, Fig 5B). On the other hand, strain decreased nonmuscle myosin by 35% (P<.001), whereas PDGF-AB increased nonmuscle myosin by 1.6-fold (P<.02, Fig 5C).

View larger version (14K): [in this window] [in a new window]   Figure 5. Effect of strain and PDGF on myosin isoform expression. VSM cells grown on collagen-coated Flex plates were exposed for 36 hours to strain, PDGF (10 ng/mL), or anti–PDGF-AB (3 µg/mL) as indicated. A, Representative immunoblot using SM-1 antiserum (SM-1) or anti–NM-A antibodies (Abs) as indicated. B and C, Densitometric scans of bands using anti–SM-1 (B) or anti–NM-A (C) averaged from three similar experiments. Bar graphs show mean±SD fold increase (strain and/or PDGF) over control for smooth muscle myosin Ab (B; P<.001 vs control for all conditions) and nonmuscle myosin Ab (C; P<.02 vs control for all conditions).

Since PDGF-AB altered the pattern of myosin isoform expression in a direction opposite that observed with mechanical strain, we hypothesized that the response to strain might be enhanced if the PDGF produced by mechanical strain could be neutralized. VSM cells were therefore exposed to mechanical strain for 36 hours in the continuous presence of neutralizing antibodies to PDGF-AB (Fig 5). The neutralizing antibodies to PDGF-AB greatly enhanced the increase in smooth muscle myosin after strain. Smooth muscle myosin increased 10-fold over control in the presence of both strain and antibodies compared with only 3-fold over control with strain alone (Fig 5B). Furthermore, the decrease in nonmuscle myosin was also enhanced by neutralizing anti–PDGF-AB antibodies. Nonmuscle myosin decreased to 40% of control (P<.001) with strain and anti-PDGF antibody compared with 65% of control with strain alone (Fig 5C). In control cells, PDGF-AB antibody had no effect on myosin isoform expression (data not shown). Thus, PDGF produced in response to mechanical strain does not mediate the strain-induced change in myosin isoform pattern. In fact, PDGF secreted in response to mechanical strain actually blunts the effects of strain on myosin isoform expression.

Effect of Strain Involves Specific Interactions With the Matrix
Secretion of PDGF in response to strain is exquisitely sensitive to alterations in the extracellular matrix, with the largest responses occurring on matrices of fibronectin or vitronectin.²⁰ To determine whether altered myosin isoform distribution after strain is also dependent on interactions with the matrix, VSM cells were grown on silicone elastomer dishes coated with collagen type I, laminin, or fibronectin and subjected to strain for 48 hours (Fig 6). As shown in Figs 2 and 5 above, strain increased expression of SM-1 by 2-fold in cells cultured on collagen type I (Fig 6). When cells were cultured and exposed to strain on laminin, this response increased significantly, to 3.2±0.3-fold (P<.002 compared with collagen). Strikingly, when cells were cultured on fibronectin, there was no increase in expression of SM-1. Basal expression of SM-1 was increased 1.3±0.1-fold in cells cultured on fibronectin (compared with collagen). However, this effect of fibronectin was small compared with the effect of strain in cells cultured on laminin. The highest overall expression of SM-1 was consistently found in cells cultured on laminin and exposed to mechanical strain.

View larger version (16K): [in this window] [in a new window]   Figure 6. Effect of extracellular matrix on strain-induced SM-1 expression. VSM cells were cultured on Flex plates coated with collagen (Col), laminin (Lam), or fibronectin (Fib) and subjected to strain (S) or no strain (C) for 48 hours. Immunoblots were performed with the SM-1–specific antibody (SM-1). A, Blot shown is representative of four similar experiments. B, Densitometric scans were averaged from four experiments. Bar graphs show mean±SE fold-increase strain over control on each of the indicated matrix proteins (P<.002 for collagen or laminin vs control; P=NS for fibronectin vs control). Basal expression of SM-1 was also determined by densitometry. Assigning a value of 1 arbitrary density unit to expression of SM-1 in cells on collagen, expression was 0.9±0.1 with laminin (P=NS) and 1.3±0.1 with fibronectin (P<.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell shape and cell deformation play an important role in the biology of many cell types. In particular, VSM cells are subjected to repetitive mechanical deformation caused by pulsatile blood flow. Conventional in vitro culture systems cannot simulate this potentially important factor. There is considerable evidence that mechanical forces can substantially alter the growth and phenotype of cells in culture (see the introductory section). The recent development of a system for growing cells on deformable silicone elastomer membranes¹² has allowed much progress in this area.

Our previous work focused on the effects of mechanical strain on proliferation of VSM cells.^{18 19} Strain induces secretion of the A and B chains of PDGF, which in turn cause proliferation.¹⁸ This induction of PDGF secretion is highly sensitive to alterations in the extracellular matrix or to blockade of specific integrins.²⁰ In the present study, we asked whether strain causes the cells to develop phenotypic characteristics of the proliferative state. Specifically, we examined the effects of strain on the expression of smooth muscle and nonmuscle myosin isoforms.

Since all myosin isoforms are highly homologous, it was important to ascertain the specificity of the various anti-myosin antibodies to be used. As expected, the SM-1/SM-2 and the SM-1 antibodies recognized the appropriate myosin heavy chains in protein extracts from aorta. Despite the well-documented decreased expression of smooth muscle myosin isoforms (and increased nonmuscle myosin) in cultured VSM cells,^{28 29} our cultured neonatal VSM cells (passages 10 to 15) still clearly express SM-1 (and to a lesser degree, SM-2) in gels loaded with 20-fold greater quantities of protein than for intact aorta (Fig 1). However, SM-1 is obviously not the predominant myosin isoform in these cells, on the basis of the Coomassie blue staining of a single band at the molecular mass of nonmuscle myosin in Fig 1. We did observe a slight discrepancy in molecular mass of the protein detected by the SM-1 antibody in aorta and VSM cells. This could be due to a cross-reactivity of this antibody with a lower molecular mass myosin not found in platelets or fibroblasts (eg, NM-B) or to the effect of differences in protein loading on migration in gels. The former possibility seems unlikely in view of our finding that NM-B does not respond to mechanical strain (Figs 2 and 3D), whereas SM-1 does respond (see below). Antibodies to platelet myosin (NM-A) exhibited the expected specificity in platelets, cultured VSM cells, and fibroblasts. Finally, an antibody to NM-B recognized a protein in the neonatal VSM cells but not in the other preparations. By using all four of these antibodies, it was possible to determine the effect of mechanical strain on all of the major myosin isoforms presently described in VSM cells.

In VSM cells subjected to mechanical strain for up to 72 hours, immunoblots with the antibodies described above clearly indicated that the expression of smooth muscle myosin isoforms is substantially increased by strain. This change was accompanied by a >50% decrease in the expression of NM-A but no effect on NM-B. These findings may help explain earlier work showing that VSM cells strained on collagen gels¹⁷ or silicone elastomer membranes¹⁶ exhibit increased concentrations of microfilaments when examined by electron microscopy.

To determine whether this change in pattern of expression is accompanied by similar changes in mRNA for the relevant genes, RNase protection assays were performed. Although changes in mRNA for both SM-1 and nonmuscle myosin were directionally the same as the changes in protein expression, there were several important differences in the time course of these changes. SM-1 protein rose steadily for up to 72 hours of strain, whereas mRNA for this gene increased maximally by 12 hours and returned to control levels after 36 hours. Similarly, nonmuscle myosin protein decreased maximally after 12 hours of strain, whereas mRNA decreased for only 3 to 6 hours compared with control. These data suggest that the observed changes in protein expression following exposure to strain were probably due in part to changes in message abundance but most likely also involve regulation at the level of protein synthesis or half-life.

Although the effects of mechanical strain on myosin expression we describe are highly reproducible, they may not be observable under all conditions. For example, we (data not shown) and others^{30 31} have found that smooth muscle myosin is increased by strain in medium containing 0.5%, but not 10%, serum. This effect of serum may be due to presence of PDGF or other growth factors in serum. We also found that strain exerted little to no effect on smooth muscle myosin expression in passaged aortic smooth muscle cells from adult Brown-Norway rats. Although the present observations may thus apply only to certain specific conditions, they are nonetheless important in view of the present limited knowledge of factors leading to differentiation of VSM cells.

One surprising feature of these findings is that SM-1 mRNA and protein were increased under conditions similar to those in which we previously found enhanced proliferation mediated by secretion of PDGF.³² This secreted PDGF can amount to at least 2 to 3 ng/mL in the medium after 48 hours of exposure to strain.³² The present work (Fig 4) and work by others³³ indicate that 10 ng/mL PDGF alters smooth muscle myosin³¹ and other smooth muscle markers³⁴ in the direction opposite that observed with strain. When these effects of strain-induced secretion of PDGF were blocked with neutralizing antibodies to PDGF, the increase in smooth muscle myosin expression following strain was greatly enhanced (Fig 5). This suggests that secreted PDGF actually blunts the effect of strain on smooth muscle myosin expression and that a greater response can be unmasked by neutralization of the secreted PDGF.

Rovner et al⁸ reported that smooth muscle myosin expression is increased as cell density increases in confluent cultures of VSM cells. We previously found that mechanical strain causes VSM cells to proliferate and increase in density,^{18 20} providing a possible explanation for the strain effect on myosin expression observed in the present study. However, other data suggest that the effect of strain on myosin expression is probably not mediated by increased cell density. Anti-PDGF antibody, which reduces cell proliferation in response to strain,¹⁸ enhanced the effect of strain on smooth muscle myosin expression, as discussed above. In addition, the effect of strain on myosin expression was greater when the cells were cultured on plates coated with laminin compared with fibronectin (Fig 6), whereas in previous work,²⁰ we found that the increase in cell density was greater with fibronectin. Therefore, we believe that the effect of strain on myosin expression is mediated by a signal other than cell density or PDGF expression. On the other hand, the 1.3-fold increase in basal expression of SM-1 on fibronectin-coated plates (Fig 6) may be due to increased cell density on these plates.

Why does strain cause VSM cells to both secrete PDGF (thus increasing proliferation) and increase expression of smooth muscle myosin? One explanation is that proliferation and altered myosin isoform expression are occurring in distinct cell populations. VSM cultures are probably inherently heterogeneous, with differential expression of PDGF receptors.³⁵ Alternatively, a heterogeneous response could arise from the heterogeneous strain profile produced by the strain apparatus we are using.¹² This latter explanation seems less likely, particularly because the proliferative response to strain is nearly homogeneous on such plates, presumably because of rapid diffusion of the secreted PDGF.¹⁸

Examination of the role played by the extracellular matrix in transducing the signal of mechanical strain has begun to unravel the seeming paradox of simultaneous increases in proliferation and differentiation of cells from the same culture. When VSM cells were cultured on fibronectin, the response to strain was purely a proliferative one,²⁰ with no increase in SM-1 expression. On the other hand, when cells were cultured on laminin, there was no significant increase in DNA synthesis²⁰ but a substantial increase in SM-1 myosin (Fig 6). Type I collagen appears to support both responses to a smaller extent. One possible explanation for the matrix dependency of myosin isoform expression in response to strain is the production of PDGF by these cells when exposed to strain with fibronectin or collagen.²⁰ With laminin, little PDGF is made,²⁰ allowing full expression of the alterations in myosin isoform content in response to strain.

The differential effects of strain in cells on various matrices raise the possibility that VSM cells may possess several different types of mechanoreceptors. Those that bind fibronectin signal a proliferative response to strain, whereas those that recognize laminin signal a differentiation response to strain. Since the time course of these experiments would allow for endogenous production of extracellular matrix proteins, we cannot state with certainty that fibronectin or laminin per se are responsible for generation of the key signals in each case. However, culture of VSM cells on these proteins clearly results in different responses to strain. Future work on this question may lead to the identification of specific mechanoreceptors associated with specific phenotypic responses to strain.

	Selected Abbreviations and Acronyms

 

NM-A, NM-B = nonmuscle myosin heavy chains A and B PDGF = platelet-derived growth factor SM-1, SM-2 = smooth muscle myosin heavy chains 1 and 2 SMemb = nonmuscle myosin heavy chain B VSM = vascular smooth muscle

	Acknowledgments

 
This study was supported by National Institutes of Health (NIH) grants HL-41210 and HL-48474 and a Grant-in-Aid from the American Heart Association. Dr Wagdy was supported by NIH training grant T32-DK-07219. Dr P Reusch was supported by a grant from the Deutsche Forschungsgemeinschaft. During part of this study, Dr. Ives was an Established Investigator of the American Heart Association. Drs Wagdy and P Reusch contributed equally to this study.

Received May 14, 1996; accepted September 20, 1996.

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