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Originally published In Press as doi:10.1074/jbc.M403370200 on June 7, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34849-34855, August 13, 2004
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Stretch of the Vascular Wall Induces Smooth Muscle Differentiation by Promoting Actin Polymerization*

Sebastian Albinsson, Ina Nordström, and Per Hellstrand{ddagger}

From the Division of Molecular and Cellular Physiology, Department of Physiological Sciences, Biomedical Center, Lund University, SE-221 84 Lund, Sweden

Received for publication, March 26, 2004 , and in revised form, May 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system ({alpha}-actin, calponin, SM22{alpha}, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins ({beta}-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins {gamma}-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22{alpha} synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, >15 min; cofilin-P, >1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of smooth muscle phenotype is of major importance for a number of disease states in the cardiovascular, respiratory, and visceral organs, and its molecular mechanisms are rapidly being elucidated (1, 2). Both intrinsic and extrinsic factors contribute to smooth muscle differentiation, marked by the expression of a limited number of proteins, primarily associated with the contractile/cytoskeletal apparatus (3). It is likely that mechanical stress in the walls of hollow organs is one of the key factors that regulate smooth muscle development as well as growth and phenotypic differentiation (4, 5). The signal mechanisms of stretch-dependent gene expression involve cell-cell contacts, the extracellular matrix, and integrins. This indicates that the intact tissue environment is critical for determining cell differentiation.

The effects of wall stress have been extensively studied in blood vessels, which respond to altered transmural pressure with growth and remodeling to normalize the mechanical stress in the tissue (4). In this process the smooth muscle cells are maintained in a contractile phenotype, which contrasts with the loss of differentiation that occurs at sites of vascular injury, as in atherosclerosis and restenosis after angioplasty (6). In organ culture of intact vessels, stretch has been shown to promote the expression of h-caldesmon and filamin in the rabbit aorta (7) and of SM22{alpha}, {alpha}-actin, and calponin in the rat or mouse portal vein (8, 9).

Although several transcription factors are likely to cooperate in promoting the expression of smooth muscle-specific genes (2), serum response factor (SRF)1 is so far the only one that has been shown to regulate nearly all of the smooth muscle specific genes. The promoter region of most smooth muscle specific genes contains one or more sites (CArG boxes) binding SRF (10). It is now clear that additional factors complement SRF binding in conferring smooth muscle specificity (2, 10). These include the recently identified proteins myocardin (11) and myocardin-related transcription factors (12). Molecular interactions between SRF, myocardin, and myocardin-related transcription factors seem to regulate binding to DNA, which suggests that cytoplasmic signals may influence transcriptional activity via nuclear translocation of any of these factors. Specifically, actin polymerization via RhoA activity regulates promoter activity of the prototypical differentiation markers SM22{alpha} and {alpha}-actin (13, 14). This is suggested to be due to nuclear translocation of the myocardin-related transcription factor known alternatively as MAL/MKL1 (megakaryocytic acute leukemia/megakaryoblastic leukemia 1), which is promoted by a decrease in the cytoplasmic concentration of monomeric G-actin caused by actin polymerization (15, 16).

Because extensive evidence suggests that factors influencing actin polymerization regulate smooth muscle differentiation, the present challenge is to determine whether such a mechanism operates in native cells in the intact tissue. The effects of stretch on smooth muscle differentiation suggest that this may be one condition where a physiological stimulus affects gene expression via actin polymerization. One preparation that has been studied with respect to stretch-induced protein synthesis both in vivo and in vitro is the rat portal vein, which rapidly develops hypertrophy with preserved contractility in response to increased pressure (17). Organ culture of the portal vein under maintained stretch (simulating pressure) reproduces many of the in vivo findings, including greater contractility and rate of protein synthesis than those of unstretched vessels cultured in parallel (18). This response involves autocrine production of angiotensin II and of endothelin-1 and is blocked by inhibitors of extracellular signal-regulated kinase 1/2 and of actin polymerization (8, 18, 19). The present study was designed to investigate the pattern of stretch-sensitive protein synthesis and to correlate this with direct determinations of actin polymerization and with the activity of its regulating pathways. The results indicate that stretch promotes actin polymerization in the intact tissue, which activates synthesis of smooth muscle differentiation marker proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Portal Veins and Organ Culture—Female NMRI mice (30–35 g) and Sprague-Dawley rats (200–250g) were killed by cervical dislocation and inhalation of CO2, respectively, as approved by the regional Animal Ethics Committee. The portal vein was removed and dissected free from fat and connective tissue under sterile conditions. Rat portal veins were split longitudinally into two strips, whereas mouse vessels were used intact. The veins were then incubated at 37 °C in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) with 2% dialyzed fetal calf serum and 10 nM insulin as described previously (8). To stretch the vessel wall, one mouse portal vein was loaded with a 0.3-g weight, and one strip of rat portal vein was loaded with a 0.6-g weight. These loads cause extension approximately representing the optimal length for active force development in the respective preparation (9, 18). Matched preparations were kept unstretched during incubation. To inhibit Rho-associated kinase (ROCK), Y-27632 (10 µM; Calbiochem) was added to the culture medium. Jasplakinolide (0.01–0.3 µM; Sigma) and latrunculin B (0.05 µM, Calbiochem) were used to promote and inhibit actin polymerization, respectively. All of the drugs were replaced every 24 h.

Protein Synthesis—Mouse portal veins were kept in organ culture for 48 h and then exposed to [35S]methionine in a low methionine medium for 24 h. Protein synthesis was measured by autoradiography following one- or two-dimensional polyacrylamide gel electrophoresis as described earlier (8, 9). All of the gels and autoradiographs were run and analyzed in pairs (unstretched or drug-treated versus stretched). PD Quest (Bio-Rad) software was used for analysis, and both the gels and the corresponding autoradiographs were normalized by computation of the total optical density of valid silver-stained spots. From a total of about 300 proteins separated under these conditions, those showing stretch sensitivity on the autoradiographs were selected for analysis, together with a number of non-stretch-sensitive proteins that were clearly distinguished and used for comparison (see Fig. 1). The proteins were identified by Western blot using specific antibodies or by mass spectrometry (matrix-assisted laser desorption ionization time-of-flight; Swegene Proteomics Resource Center, Lund, Sweden) of spots cut out from the gels.



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  		FIG. 1.
Identification of stretch-sensitive proteins by two-dimensional gel electrophoresis. Mouse portal veins were cultured either stretched or unstretched for 72 h, with [35S]methionine present during the last 24 h. Silver-stained gels and corresponding autoradiographs are shown, illustrating protein contents and protein synthesis rates, respectively. Squares and circles mark stretch-sensitive and -insensitive proteins, respectively. The proteins marked are actin ({alpha}, {beta}, and {gamma} isoforms), tropomyosin (TM; {alpha} and {beta} isoforms), desmin (Des), vimentin (Vim), heat shock 70-kDa protein 5 (HSPA5), heat shock 70-kDa protein 8 (HSPA8), heat shock 60-kDa protein 1 (HSPD1) (A). Summarized data from silver-stained gels (pH 4–7, 6–11, or nonlinear 3–10) and autoradiographs are shown. The dashed lines represent no change in protein contents or synthesis (n = 3–9; B).

 
Analysis of Rho Activation—A Rho activation assay kit (Upstate Biotechnology, Inc.) was used according to the manufacturer's instructions. Because the assay requires a relatively large amount of protein, rat portal veins were used. One half of a rat portal vein was stretched, and the other half was used as control. Following incubation for 15 min or 24 h, four halves were pooled and homogenized in 225 µl of ice-cold lysis buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1% Igepal CA-630, and 10% glycerol. The homogenate was centrifuged for 15 min at 14,000 x g at 4 °C. A small portion of the supernatant was used for protein determination and analysis of total Rho in the lysate. The Rho-binding domain of rhotekin (RBD) was used to precipitate GTP-bound Rho from the rest of the lysate, which contained ~300 µg of protein. For positive and negative controls, rat aortic extracts were separated into two equal volumes and incubated with GTP{gamma}S or GDP to obtain a maximum or a minimum of GTP-bound Rho. The samples were analyzed by Western blot using an antibody against RhoA, RhoB, and RhoC (3 µ g/ml) to evaluate the fraction of GTP-bound Rho to total Rho. The bands were visualized using ECL detection (West Femto; Pierce).

Cofilin-2 Phosphorylation—Following organ culture, mouse portal veins were quickly frozen, pulverized in liquid nitrogen, and extracted. The same amount of protein was loaded in each lane on one gel for analysis of total cofilin-2, and six times that amount was loaded in each lane on a second gel for analysis of phospho-cofilin-2. Total and phosphorylated cofilin were determined by Western blot using anti-cofilin 2 (1:1000) and anti-phospho-cofilin 2 (Ser3; 1:500) (Upstate Biotechnology, Inc.). The bands were visualized using ECL.

F-actin Sedimentation—Mouse portal veins were kept in organ culture for 24–72 h and then homogenized at 37 °C in a lysis and F-actin stabilizing buffer containing 50 mM PIPES (pH 6.9), 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 1 mM ATP, 5% glycerol, 0.1% Nonidet P-40, 0.1% Triton X-100, 0.1% Tween 20, 0.1% {beta}-mercaptoethanol, and 1:100 protease inhibitor mixture (Sigma). The F-actin and G-actin pools were separated by ultracentrifugation at 100,000 x g at 30 °C. The supernatant was diluted 1:2 with Laemmli buffer, whereas the pellet was resuspended in cold distilled H2O with 1 µM cytochalasin D and sonicated for 10 s. The pellet fraction was kept on ice for 45 min and then diluted 1:4 with Laemmli buffer. Both fractions were then boiled for 5 min and centrifuged at 14,000 x g for 10 min at 4 °C to remove remaining connective tissue. The same relative amounts of supernatant and pellet fractions (2:1) were loaded on 12.5% polyacrylamide gels and analyzed by Western blot using an anti-actin antibody (1:500, Sigma).

Fluorescence Microscopy—Mouse portal veins were cultured for 24 h and then incubated for 5 min at 4 °C in 150 mM NaCl, 10 mM Tris (pH 7.4), 2 mM MgCl2, 0.2 mM dithioerythritol, 0.01% Triton X-100, and 10% glycerol (v/v) (20). The portal veins were then washed in ice-cold PBS (pH 7.4) followed by fixation in 4% formaldehyde in PBS for 1 h. The vessels were washed twice for 10 min in PBS and embedded in Tissue-Tek (Sakura), and frozen. Transverse sections (10 µm) were cut, permeabilized for 15 min in 0.2% Triton X-100, blocked in 2% bovine serum albumin with 0.01% Triton X-100 for 1 h, and stained for G-actin (20 g/ml Texas Red-DNase I; Molecular Probes) and F-actin (2 g/ml fluorescein isothiocyanate-Phalloidin; Sigma) in blocking buffer for 1 h. All of the solutions were prepared in PBS (22 °C). The tissue sections were washed three times in PBS for a total of 45 min and then mounted with Aqua Poly/Mount (Polysciences, Inc.) to prevent photobleaching. The slides were examined using a Zeiss LSM 510 confocal microscope. F-actin was detected by monitoring fluorescein isothiocyanate fluorescence at 505–530 nm for excitation at 488 nm. G-actin was monitored at >650 nm for excitation at 543 nm. To standardize the fluorescence measurements, the settings were optimized before each experiment and then kept constant throughout. Three randomly placed fields (50–150 m2) in each image were analyzed using the Zeiss LSM 510 Pascal analysis software. Fluorescence intensity ratio was calculated by measuring pixel intensity of each dye within the fields. For each portal vein approximately 10 images were used in the analysis.

Statistics—The values are presented as the means ± S.E. Except as stated, unpaired Student's t test was used for evaluation of statistical significance. For multiple comparisons one-way analysis of variance and Dunnett's post test was used. p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Stretch on Protein Synthesis in the Portal Vein— Fig. 1A shows silver-stained two-dimensional gels (pH 4–7) and corresponding autoradiographs of [35S]methionine incorporation in a stretched and an unstretched mouse portal vein. The culture time used here (72 h) is not sufficient to cause more than moderate effects on the relative contents of individual proteins, as seen from the silver-stained gels. However, {alpha}-actin, {alpha}- and {beta}-tropomyosin, and desmin show dramatic stretch sensitivity on the autoradiographs, whereas {gamma}-actin, like the intermediate filament protein vimentin, was weakly stretch-sensitive. The ubiquitous {beta}-actin isoform was not stretch-sensitive. Three proteins that were clearly resolved and did not show any stretch sensitivity were identified as heat shock proteins (HSPA5, HSPA8, and HSPD1).

Summarized data on protein composition and synthesis rates are shown in Fig. 1B. Data for SM22{alpha} and calponin were determined from pH 6–11 or nonlinear 3–10 gels. The results are expressed as relative protein contents and radiolabel incorporation in unstretched versus stretched veins. Most of the proteins with stretch-sensitive synthesis rates, which are predominantly those associated with the contractile/cytoskeletal apparatus, appear in lower amounts by varying degree on the silver-stained gels from the unstretched veins. This does not include any contribution from greater overall protein contents in stretched veins because gels were loaded with equal amounts of total protein. The range of synthesis rates is, however, much wider than that of protein contents, with dramatic decrease of the rate for some proteins (<5% remaining) in unstretched vessels, whereas others were unaffected. Among the known differentiation marker proteins shown in Fig. 1B, the order of stretch sensitivity with respect to synthesis rates is: calponin {approx} SM22{alpha} > desmin {approx} tropomyosin > {alpha}-actin > {gamma}-actin.

Effect of Jasplakinolide on SM22{alpha} Synthesis—To investigate whether stabilization of actin filaments increases the synthesis of SM22{alpha}, we cultured portal veins in the presence of jasplakinolide (0.3–0.01 µM) for 72 h and analyzed the amount of synthesized SM22{alpha} during the last 24 h with one-dimensional gel autoradiography. SM22{alpha} appears as a 22-kDa band, as previously shown using Western blot (8) and knock-out of the SM22{alpha} gene (9). Jasplakinolide caused a concentration-dependent increase in SM22{alpha} synthesis to the same level as seen in stretched veins (Fig. 2).



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  		FIG. 2.
Jasplakinolide increases SM22{alpha} synthesis in unstretched mouse portal veins. The veins were cultured for 72 h, stretched or unstretched as indicated, with [35S]methionine present during the last 24 h. SM22{alpha} synthesis was evaluated from one-dimensional gel autoradiographs and normalized to the value of the unstretched vein in each experiment (n = 3). **, p < 0.01 versus unstretched, calculated by analysis of variance and Dunnett's post test. The location of SM22{alpha} on autoradiographs is indicated by an asterisk. Jasp., jasplakinolide.

 
Role of ROCK in Stretch-dependent Protein Synthesis—Actin dynamics are in part regulated by ROCK via its downstream effectors LIM kinase and cofilin (21). To investigate the role of ROCK in stretch-dependent protein synthesis, we used the specific inhibitor Y-27632 and examined the effect on two-dimensional gel autoradiographs. The synthesis of most contractile proteins was reduced because of inhibition of ROCK (Fig. 3). However, the effect was not as pronounced as in unstretched versus stretched veins.



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  		FIG. 3.
Effects of ROCK inhibition on protein synthesis in stretched mouse portal veins. The veins were cultured for 72 h in the absence and presence of Y-27632 (10 µM), with [35S]methionine present during the last 24 h. Autoradiographs of two-dimensional gels from the basic region (pH 8.0–9.2), resolving calponin and SM22{alpha} are shown (A). The data are summarized from silver-stained gels (pH 3–10, actin isoforms not resolved) and autoradiographs. The dashed lines represent no change in protein contents or synthesis (n = 3; B).

 
Stretch-induced Rho Activation—Because the stretch-induced signaling is partially dependent on ROCK, we sought to determine the effect of stretch on the activation of its upstream regulator Rho. Rho activation was investigated by pull-down of GTP-bound Rho with rhotekin-RBD. The stretch-induced response was analyzed after 15 min or 24 h in rat portal veins. As shown in Fig. 4, there was no significant increase in Rho activation after 15 min of stretch. However, after 24 h a 2-fold greater Rho activation was observed in stretched versus unstretched veins.



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  		FIG. 4.
Stretch-induced Rho activation in rat portal vein. One strip of a longitudinally split rat portal vein was cultured under stretch for 15 min or 24 h, whereas the matching strip was kept unstretched. Four strips were pooled for each assay. The effect of stretch on Rho activation was evaluated by rhotekin-RBD pull-down and Western blot. GTP-bound Rho/total Rho was expressed relative to values for unstretched strips (n = 3–5; A). *, p < 0.05 versus unstretched calculated by paired Student's t test. Western blots with anti-Rho antibody from representative experiments, including the corresponding bands for GTP{gamma}S- and GDP-bound Rho as positive and negative controls, are shown below the summarized data (B).

 
Effects of Stretch and ROCK Inhibition on Cofilin-2 Phosphorylation—Cofilin-2 belongs to a family of actin-severing proteins, which dissociates F-actin filaments at their pointed (-) end. In contrast to cofilin-1, it is a muscle-specific protein (22). The severing activity of cofilins is inhibited by phosphorylation by LIM kinase (21). We determined the time course of the effect of stretch on cofilin-2 phosphorylation by using a phospho-specific antibody (Fig. 5A). There was no change during the first hour of stretch, but then phosphorylation gradually increased during the following 24 h. A maximally 2-fold greater cofilin-2 phosphorylation in stretched versus unstretched portal veins was observed at 24 h. The effect of stretch on phosphocofilin-2/total cofilin-2 ratio at 72 h was reduced compared with the earlier time point. However, at 72 h but not at earlier time points, stretch also affected the levels of total cofilin-2, which was 113 ± 6% greater in stretched than in unstretched portal veins.



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  		FIG. 5.
Effects of stretch and ROCK inhibition on cofilin-2 phosphorylation. Mouse portal veins were cultured for 15 min to 72 h under stretched or unstretched conditions and analyzed by Western blot with anti-P-cofilin-2 and anti-cofilin-2 antibodies. P-cofilin-2/total cofilin-2 is plotted against the time of incubation (n = 4–9; A). The effect of Y-27632 (10 µM) on cofilin-2 phosphorylation in portal veins cultured for 24 h under stretched or unstretched conditions. Representative Western blots are shown below the summarized data (B). *, p < 0.05; **, p < 0.01 versus unstretched.

 
Because cofilin-2 is a downstream effector of ROCK, it is likely to mediate the effect of Y-27632 on stretch-induced protein synthesis. In stretched veins, Y-27632 markedly reduced cofilin-2 phosphorylation after 24 h, whereas there was no significant reduction in unstretched veins (Fig. 5B).

Stretch-induced Actin Polymerization—Phosphorylation of cofilin inhibits its filament severing activity and thereby directly affects the structure of actin filaments. However, other signaling pathways could compensate for the stretch-induced inhibition of cofilin. Therefore, stretch-induced effects on actin dynamics were studied by measurement of F/G-actin ratios in the portal vein. The F- and G-actin pools were separated by ultracentrifugation and analyzed by Western blot. A 40% increase in the F/G-actin ratio was observed after 24 h of stretch (Fig. 6A). However, the effect was reduced in veins stretched for 48 or 72 h (data not shown). The F-actin stabilizing agent jasplakinolide increased the F/G-actin ratio in unstretched veins, whereas the actin depolymerizing agent latrunculin B had the expected opposite effect and decreased the F/G-actin ratio in stretched vessels.



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  		FIG. 6.
Effects of stretch on F/G-actin ratio in mouse portal veins. The veins were cultured for 24 h, stretched, unstretched, with latrunculin B (LB; 0.05 µM), or with jasplakinolide (Jasp, 0.1 µM), as indicated. The F- and G-actin pools were separated by ultracentrifugation and analyzed by Western blot. F/G-actin ratios and representative blots are shown (n = 4–7; A). For confocal microscopy, the veins were cultured for 24 h, stretched (panel a), unstretched (panel b), and stretched in the presence of latrunculin B (LB; 0.05 µM; panel c). Cross-sections were stained with fluorescein isothiocyanate-phalloidin for F-actin (green) and Texas Red-DNase I for G-actin (red). Yellow indicates presence of both F- and G-actin. The outer longitudinal muscle layer (L), inner circular layer (C), and cross-sectioned nuclei (arrow) are identified in panel a. The summary data (panel d) show normalized F/G actin ratios calculated from 6–8 portal veins, 60–80 images, and 180–240 fields for each condition (B). *, p < 0.05; ***, p < 0.001.

 
Measurements of the F/G-actin ratio by ultracentrifugation represent average values that might be affected by tissue orientation, because the portal vein contains, in addition to longitudinal muscle, a thin circular muscle layer that will be differently affected by the longitudinal stretch. We therefore examined actin polymerization by an alternative and independent method, using confocal microscopy after labeling F- and G-actin with, respectively, phalloidin and DNase I conjugated to fluorescent probes. After a 24-h culture, the mouse portal veins were fixed, and slides were prepared for histochemistry. Fig. 6B shows representative images of a stretched (panel a), an unstretched (panel b), and a latrunculin B-treated stretched (panel c) portal vein in transverse sections, demonstrating that the majority of smooth muscle cells are longitudinally arranged, i.e. in the direction of the applied stretch. Analysis indicated a 55% higher F/G-actin ratio in stretched versus unstretched veins (Fig. 6B, panel d). Using both methods, the effects of latrunculin B were quantitatively similar to that in the absence of stretch.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that the synthesis of differentiation marker proteins in native vascular smooth muscle is potentiated by stretch of the vascular wall by a mechanism involving actin dynamics. The steady-state level of actin polymerization was increased by stretch, and a direct influence of stretch on actin treadmilling was demonstrated by the increased cofilin phosphorylation. These effects were prominent after 24 h of stretch but tended to decrease later. This may be due to tissue remodeling or other compensatory mechanisms, such as the increase in total cofilin-2 in stretched versus unstretched veins evident at 72 h but not at earlier time points. Nevertheless, a clear effect of stretch on cofilin phosphorylation, as well as synthesis of differentiation marker proteins, is apparent up to the latest time point studied here (72 h). The effect of stretch was slow in onset, because a clear increase in Rho activity was seen after 24 h but not after 15 min of stretch, and cofilin phosphorylation was unaltered at 1 h. The slow onset of Rho activity suggests that upstream mechanisms coupling to Rho may also be relatively slow in onset. The correlation of differentiation marker expression with alterations in actin dynamics, produced by stretch as well as pharmacological inhibition (Y-27632 and latrunculin B) and potentiation (jasplakinolide) of actin polymerization, strongly suggests regulation of smooth muscle differentiation in the vascular wall by the actin cytoskeleton.

The portal vein contains predominantly longitudinal musculature and in this study was longitudinally distended to approximately the optimal length for force development. The distension can be considered to represent physiological conditions in terms of mechanical stress on the tissue, whereas the nondistended state is clearly nonphysiological. Possible hypertrophy stimulated by an extra load was not investigated here but would be expected to occur in a maintained contractile phenotype. Experimental portal hypertension in rats causes hypertrophy primarily of the longitudinal muscle layer, with increased total protein contents but a composition similar to that of control veins, the most notable difference being an increase of desmin contents (17). In our previous study an approximately proportional increase in SM22{alpha} contents was found in the rat portal vein following pressure-induced hypertrophy in vivo (8). In the present work, the desmin contents in unstretched mouse portal veins following culture were reduced to ~65% compared with stretched veins, whereas its rate of synthesis was <8% of that in stretched veins. A similar reduction in synthesis rate was seen for SM22{alpha}, whereas the total contents were better preserved (~85%). The tissue levels reflect the balance of synthesis and breakdown, but little is known about the effects of mechanical forces on breakdown rates of specific smooth muscle proteins.

All of the stretch-sensitive proteins examined here ({alpha}-actin, {gamma}-actin, SM22{alpha}, calponin, {alpha}-, {beta}-tropomyosin, and desmin) have CArG box containing promoters. Except for {alpha}-tropomyosin, they have been confirmed to be SRF-regulated (for references, see Ref. 10). Smooth muscle {alpha}-tropomyosin has been shown to be regulated coordinately with h-caldesmon and to undergo conversion to fibroblast isoforms in dedifferentiating smooth muscle cells (23). The {gamma}-actin spot was only weakly stretch-sensitive, similar to vimentin, but may represent a mixture of smooth muscle and nonmuscle {gamma}-actin. Considering the diversity of smooth muscle, it is interesting that a limited number of contractile and cytoskeletal proteins seem to be the common defining elements of smooth muscle cells and that these are reversibly regulated by exposure to mechanical forces, the main physiological function of muscle. In the context of the putative role of MAL/MKL1 in transmitting signals from Rho and the actin cytoskeleton to the nucleus (15, 16, 24), it is significant that this ubiquitously expressed protein can activate SRF-dependent expression of endogenous smooth muscle genes in undifferentiated embryonic stem cells (16). The effects of stretch on smooth muscle development (5) may thus involve signals impinging on actin dynamics.

Stretch, in addition to its effect on Rho-dependent signaling, activates the mitogen-activated protein kinase pathway as demonstrated in a number of vascular preparations including the portal vein (18, 25–27). This would be expected to stimulate protein synthesis associated with cell proliferation by virtue of ternary complex factor-dependent c-fos activation by SRF (28). It is therefore notable that the proteins found to be strongly stretch-sensitive are those regulated by SRF binding independent of ternary complex factor. The ternary complex factor Elk-1 has recently been shown to compete with myocardin for binding to SRF and thereby to act as a repressor of differentiation (29). Because MAL/MKL1 is a potent activator of smooth muscle genes and acts synergistically with myocardin (12, 16), it is possible that increased actin polymerization in response to stretch also increases the nuclear translocation of this factor enough to override an inhibitory effect of ternary complex factor. This might, on the other hand, not be the case in other instances of growth factor-dependent signaling, such as cell growth and proliferation in response to vascular injury.

Although SRF is a likely candidate for mediating smooth muscle-specific gene transcription via Rho activation and actin polymerization, it should be pointed out that several other transcription factors are activated in response to stretch (4). With respect to smooth muscle differentiation, one potential mechanism that has been shown to be present in intact vessels is nuclear translocation of the calcineurin-dependent transcription factor NFAT (nuclear factor of activated T cells) in response to pressurization of intact cerebral arteries (30). A calcineurin- and GATA 6-dependent pathway for {alpha}-actin and myosin heavy chain promoter activity in vascular smooth muscle cells has been demonstrated (31).

No previous study has to our knowledge investigated the stretch-induced coordinate effects on Rho activation, actin polymerization, and protein synthesis in intact vessels. Studies in cultured smooth muscle cells of Rho activation and actin polymerization in response stretch present conflicting results (32–36), which may partly be due to differences in stretch protocols and culture dish coating. In intact vascular tissue, suppression of G-actin levels at increased transmural pressure has been reported in cannulated cerebral arteries (37). Administration of the ROCK inhibitor Y-27632 has been shown to lower blood pressure in several hypertensive, but not normotensive, rat models (38), and elevated Rho expression and activation have been demonstrated in vessels from hypertensive rats (39). Rho and ROCK activity are likely to contribute to increased vascular tone by raising Ca2+ sensitivity (38). Our results suggest that stretch-induced Rho signaling also preserves smooth muscle cell differentiation in vascular hypertrophy by influencing actin dynamics.

A role of ROCK downstream of Rho has been shown for stress fiber formation as well as SM22{alpha} and {alpha}-actin promoter activity (14). In the present work Y-27632 eliminated the effect of stretch on cofilin phosphorylation and lowered the synthesis of stretch-sensitive proteins in a pattern similar to that in unstretched vessels. The effects of Y-27632 on protein synthesis were, however, smaller than those caused by the absence of stretch, even though ROCK activity was effectively blocked as demonstrated by the effect on cofilin-2 phosphorylation. This was evident for the rates of SM22{alpha}, calponin, and desmin synthesis, which were reduced to 55, 33, and 25%, respectively. This contrasts with the much greater effects on the synthesis of these proteins (<8% preserved) in unstretched vessels. Also, we have previously found a lower effect of Y-27632 compared with the RhoA inhibitor C3 on SM22{alpha} synthesis in rat portal vein (8). Hence, pathways in addition to ROCK-cofilin seem to contribute to the effects of stretch on gene expression. One candidate is signaling downstream from Rho via mDia-profilin, which stimulates incorporation of actin monomers into G-actin and has been shown to interact with the ROCK-LIM kinase pathway in activating SRF in PC12 cells (40). Even though much evidence supports a role of actin treadmilling in transmitting Rho activity to SRF, there is also evidence that Rho may activate SRF-dependent transcription independently of its effect on actin dynamics (41, 42). Src tyrosine kinase is implicated in the stretch response in intact vascular tissue (18, 26) and has been shown to activate SRF in conjunction with mDia independently of ROCK (43). Thus the mDia pathway may regulate actin dynamics and gene expression partly in response to other signals than those operating on ROCK. The role of these mechanisms for stretch-dependent differentiation remains to be determined.

This study shows that stretch of the blood vessel wall is critical for maintaining the smooth muscle contractile phenotype, expressing contractile and cytoskeletal proteins. The signaling mechanisms of this response include Rho activation and actin polymerization, partly but probably not exclusively mediated via ROCK activity and cofilin phosphorylation.


   FOOTNOTES
 
* This work was supported by Swedish Science Council Grant 71X-28 and the Swedish Heart-Lung Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Molecular and Cellular Physiology, BMC F12, SE-221 84 Lund, Sweden. Tel.: 46-46-222-9585; Fax: 46-46-222-4546; E-mail: Per.Hellstrand{at}mphy.lu.

1 The abbreviations used in this paper are: SRF, serum response factor; ROCK, Rho-associated kinase; RBD, Rho-binding domain of rhotekin; HSP, heat shock protein; GTP{gamma}S, guanosine 5'-O-(3-thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Maria F. Gomez, Karl Swärd, and Asad Zeidan for valuable advice and discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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