Intraluminal Pressure Is Essential for the Maintenance of Smooth Muscle Caldesmon and Filamin Content in Aortic Organ Culture

From Institut National de la Santé et de la Recherche Médicale, Unité 141, Hôpital Lariboisière, Paris, France (N.B., S.L., R.M., A.T.); and the Laboratory of Molecular Endocrinology, Cardiology Research Center of the Russian Academy of Medical Sciences, Moscow, Russia (K.G.B., V.P.S.).

Correspondence to Alain Tedgui, INSERM U141, Hôpital Lariboisière, 41 Blvd de la Chapelle, 75475 Paris, France. E-mail tedgui{at}infobiogen.fr

	Abstract

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Abstract—Different forms of mechanical stimulation are among the physiological factors constantly acting on the vessel wall. We previously demonstrated that subjecting vascular smooth muscle cells (VSMCs) in culture to cyclic stretch increased the expression of high-molecular-weight caldesmon, a marker protein of a differentiated, contractile, VSMC phenotype. In the present work the effects of mechanical factors, in the form of circumferential stress and shear stress, on the characteristics of SM contractile phenotype were studied in an organ culture of rabbit aorta. Application of an intralumininal pressure of 80 mm Hg to aortic segments cultured in Dulbecco's modified Eagle's medium containing 20% fetal calf serum for 3 days prevented the decrease in high-molecular-weight caldesmon content (70±4% of initial level in nonpressurized vessel, 116±17% at 80 mm Hg) and filamin content (80±5% in nonpressurized vessel, 100±2% at 80 mm Hg). SM myosin and low-molecular-weight caldesmon contents showed no dependence on vessel pressurization. Neither endothelial denudation nor alteration of intraluminal flow rates affected marker protein content in 3-day vessel culture, thus excluding the possibility of any shear or endothelial effects. Maintenance of high high-molecular-weight caldesmon and filamin levels in the organ cultures of pressurized and stretched vessels demonstrates the positive role of mechanical factors in the control of the VSMC differentiated phenotype.

Key Words: stretch • aorta • marker protein • caldesmon • filamin

	Introduction

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The arterial wall is continuously subjected to the action of mechanical forces in the form of tensile stress and shear stress. In addition, the pulsatile nature of blood pressure imposes cyclic stretch on the wall. In vivo studies and clinical observations suggest that decreased values of tensile and shear stress in surgically injured vessels are correlated with activation of cell proliferation and extracellular matrix production, which may result in vessel occlusion.¹ In contrast, reestablishment of baseline tensile stress and shear stress levels is correlated with inhibition of cell proliferation and restoration of vessel wall morphology and cellular ultrastructure characteristic of the differentiated VSMC phenotype.^{1 2} These features and morphology indicate that VSMCs undergo phenotypic changes in regions of altered hemodynamic patterns. Experiments on isolated vessels suggest that appropriate mechanical stimulation is essential for the maintenance of VSMC contractile function and sensitivity to vasoconstrictors,³ as well as morphology of the vessel wall.^{4 5} However, the role of mechanical stimulation in the maintenance of SM marker protein content is still unclear.

Changes in the expression of VSMC marker proteins are usually coordinated during VSMC transition to the synthetic phenotype in primary culture or at the loci of vascular injury.^{6 7} However, under certain conditions, expression of some marker proteins may be regulated independently of each other. For example, in VSMCs maintained in a defined serum-free medium, addition of 10% fetal bovine serum stimulated a marked increase in nonmuscle ß-actin mRNA levels and synthesis, had no effect on SM -actin expression,⁸ and increased SM -tropomyosin.⁹

Studies of cultured cells have shown that mechanical stimulation is able to activate intracellular signaling systems,^{10 11} followed by specific cellular responses.^{12 13 14} Moreover, cyclic stretching of cultured VSMCs has been shown to increase the expression of an SM variant of CaD, h-CaD,¹⁵ as well as SM myosin heavy chains and myosin light-chain kinase.¹⁶ A distinct feature of arterial VSMCs is the plasticity of their phenotype: in culture, they are capable of displaying broad changes in ultrastructure and express a number of marker proteins characteristic of certain phenotypic states, such as SM actin, myosin, CaD, and CN, in reaction to environmental changes (reviewed in References 17 and 18^{17 18} ). Vessel organ culture allows maintenance of VSMCs in undissected tissue and thus discriminates effects of mechanical stimulation on VSMC phenotype from the majority of perturbations associated with the establishment of cell culture. The goal of the present study was to investigate how mechanical factors influence the content of SM marker proteins in medial VSMCs. We studied the effects of transmural pressure and flow on the VSMC content of SMM, CaD, CN, and FIL by using an organ culture model of rabbit aorta.

	Methods

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Artery Preparation
Male New Zealand White rabbits (2 to 2.5 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV). The animals were intubated and artificially ventilated. The abdominal and pleural cavities were opened, and the entire length of the descending thoracic aorta was exposed. Preparations of aorta used for pressurization in organ culture were processed as previously described.¹⁹ In brief, the intercostal arteries were cauterized 2 to 3 mm from the vessel, which was cleaned of adhering periadventitial tissue. A cannula fitted to the internal diameter of the aorta was inserted retrogradely into the distal end of the aorta and connected to a reservoir placed 100 cm above the animal. The proximal end of the aorta was then ligated just below the arch, and a second cannula, pointing distally, was inserted anterogradely. With this procedure, pressure was maintained continuously within the artery and prevented collapse of the vessel wall and endothelial damage.¹⁹ A ligature was tied around the midregion of the aorta, and a cannula was inserted distally to this ligature. The lower part of the descending thoracic aorta was then excised on splints, at in vivo length. A similar procedure was used to excise the upper part of the aorta. The animals were cared for in accordance with the European Community Standards on the Care and Use of Laboratory Animals (No. 00577).

When necessary, the endothelium was removed from aortic segments by gentle scraping of the intraluminal surface without inflicting medial injury with the use of a Fogarty catheter as previously described.¹⁹ The efficiency of deendothelialization was confirmed by the absence of vasorelaxation of precontracted segments in response to 0.1 µmol/L acetylcholine.

Organ Culture
Organ culture of aortic segments was carried out under sterile conditions. Removed aortic segments were immersed in an organ culture bath placed in an incubator and filled with Dulbecco's modified Eagle's medium (Gibco BRL) containing antibiotics (penicillin 100 IU/L, streptomycin 100 mg/L, and amphotericin B 10 mg/L) supplemented with 20% decomplemented fetal calf serum (Boehringer Mannheim France).

A device constructed for application of intraluminal pressure, flow, or both to vessel segments in organ culture has been described in our previous work.¹⁹ Each aortic segment was connected to a perfusion circuit consisting of a three-port glass reservoir, a peristaltic pump (Masterflex 60648, Cole-Palmer Instrument Co), and a pressure chamber. The upper port of the glass reservoir was connected to the pressure chamber, which permitted the application of a controlled hydrostatic pressure to the intraluminal compartment. The two lateral ports of the glass reservoir were used for input and output of the circulating intraluminal medium, which was identical to the extraluminal medium described above. Endothelial integrity in this model has been previously confirmed by examination of scanning electron photomicrographs of aortas maintained for 3 days at various pressure levels.¹⁹ Arteries were pressurized at 80 mm Hg and perfused at 8 or 40 mL/min (producing a cyclic change in vessel diameter of 1.5% or 9%, respectively, calculated by using an ultrasonic echo-tracking microdensitometer). Another (control) group represented vessel segments cannulated and mounted on the perfusion circuit but pressurized at 10 mm Hg and perfused at 1 mL/min. Special care was taken to maintain these latter segments at the zero level of distension to avoid applying additional mechanical stretch on them. Control vessel rings 3 to 5 mm long (relaxed state) were put into Petri dishes and cultured at zero transmural pressure and without flow. Aortic rings cultured under relaxed conditions were shaken twice a day to change a portion of the culture medium in the vessel lumen.

Vessels freshly removed from animals and processed for gel electrophoresis and Western blot served as a reference for the marker protein content in vivo. Segments used for comparison of different pressure or flow regimens were obtained from the same rabbit and processed simultaneously. Six to eight aortic segments were studied under each experimental condition.

Sample Preparation
The medial layer of the vessel strips was separated from the adventitia with fine forceps, briefly washed with cold Hanks' buffer, quickly frozen, and powdered in LN₂ by mortar and pestle. Sample buffer containing 0.2 mol/L Tris HCl, pH 6.8; 32% (vol/vol) glycerol; 6.4% (wt/vol) SDS; 1.2 mol/L 2-mercaptoethanol; and 0.2% (wt/vol) bromophenol blue was added to the minced tissue at a ratio 1 mL to 20 mg wet weight; the sample was then boiled for 3 minutes, and insoluble material was sedimented at 12 000 rpm in a Beckman M-12 microcentrifuge. Protein concentrations of samples for electrophoresis were equalized by using an amido black protein assay.

Gel Electrophoresis and Scanning Densitometry
SDS-PAGE was performed according to Laemmli²⁰ by using 7.5% gels for detection of CN and CaD and 6% gels for detection of SMM and FIL. Myosin heavy-chain isoforms were separated by SDS-PAGE in 5% highly porous gels with 0.065% bisacrylamide as described by Rovner et al.²¹ Scanning densitometry of Coomassie blue R-250–stained gels, performed with an Ultrascan 2002 laser densitometer (LKB), confirmed equal protein loading per track.

Antibodies and Quantitative Immunoblotting Techniques
Polyclonal antibodies to chicken gizzard FIL were elicited in rabbits with five booster injections at 2-week intervals. Antibody production was screened by ELISA. Antibodies were affinity purified from whole serum on FIL–Sepharose 4B. Antibodies recognized a single band with an MW of 250 kDa on the blot from the transferred protein lysate. Rabbit polyclonal affinity-purified antibodies against chicken gizzard CaD, chicken gizzard CN, and chicken gizzard SMM have been characterized previously.^{22 23 24}

Proteins were electrophoretically transferred from polyacrylamide gels onto nitrocellulose according to Towbin et al.²⁵ Quantitative immunoblotting on nitrocellulose membranes was performed as previously described.²⁶ Polyclonal antibodies to CaD, CN, and SMM were used at a concentration of 4 µg/mL; antibodies to FIL were used at a concentration of 8 µg/mL.¹²⁵I-labeled anti-rabbit secondary antibodies at a final concentration of 0.1 µCi/mL (Amersham France SA.) were used for the immunoreactive quantification of marker proteins.

Protein Synthesis Activity
Protein synthesis activity was determined by incorporation of [³⁵S]methionine into proteins of interest. [³⁵S]methionine (Amersham France SA) was added to the culture medium at a final concentration of 5 µCi/mL 6 hours before termination of the experiment. Samples from vessel segments were prepared and subjected to SDS electrophoresis as outlined above. FIL and myosin protein bands were visualized by staining the gels with Coomassie blue R-250, and radiolabel incorporation into proteins of interest was assessed with a PhosphorImager system (Fuji Corp). In parallel, aliquots of tissue lysates were applied to nitrocellulose filters. Filters were washed three times for 10 minutes in 5% trichloroacetic acid, and total ³⁵S incorporation into extractable protein fractions was measured.

Statistical Analysis
Data were expressed as mean±SEM. A two-way ANOVA was constructed with marker protein content data to test the effects of pressure, flow, and endothelial denudation. Comparisons were carried out by use of Bonferroni's t test, and a value of P<0.05 was considered statistically significant.

	Results

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To characterize changes in FIL, SMM, h- and l-CaD, and CN content in the course of culture, aortic segments were cultured in Dulbecco's modified Eagle's medium containing 20% fetal calf serum.

Expression of FIL
FIL is an abundant protein of medial VSMCs. Densitometric analysis of Coomassie blue–stained gels suggested that FIL content in the fresh aorta accounted for 10% of the total extractable protein (Figure 1). Three-day organ culture of rabbit aorta under relaxed conditions or at 10 mm Hg resulted in a 30% and a 20% decrease in FIL content (P<0.05), respectively, whereas pressurizing the segments at 80 mm Hg maintained FIL at a level similar to that observed in freshly isolated segments (Figures 1 and 2).

View larger version (58K): [in this window] [in a new window]   Figure 1. Marker protein content in fresh aorta (1), in 3-day culture of vessel rings (2), of vessels pressurized at 10 mm Hg and perfused at 1 mL/min (3), and of vessels pressurized at 80 mm Hg and perfused at 8 mL/min (4). A, Coomassie blue staining of tissue extracts subjected to SDS-PAGE; B, corresponding Western blots with antibodies against FIL, SMM, CaD, and CN.

View larger version (36K): [in this window] [in a new window]   Figure 2. Quantitative analysis of SM marker protein levels in 3-day organ culture of rabbit aorta in the form of vessel rings (relax) or segments cannulated and perfused at 10 or 80 mm Hg. Results (mean±SEM) represent percent of protein expression in fresh aorta. *P<0.05 versus fresh aorta, n=6 to 8.

Expression of CaD in Aortic Segments Pressurized in Organ Culture
h-CaD content in aortic rings cultured for 3 days under relaxed conditions was decreased and reached 81.5±6.5% of that in freshly isolated vessels (P<0.05) (Figures 2 and 3). Low levels of intraluminal pressure (10 mm Hg) and perfusion (1 mL/min) did not significantly affect h-CaD levels in cultured vessel segments compared with relaxed conditions. However, pressurizing the segments at 80 mm Hg was sufficient to completely prevent the drop in h-CaD content. In contrast, pressurization at neither 10 mm Hg nor at 80 mm Hg modified l-CaD content versus relaxed conditions (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 3. Role of flow and endothelium in pressure-mediated preservation of FIL and CaD contents. Effects of 3-day culture of vessel rings (relax), segments kept at 80 mm Hg and perfused at 8 or 40 mL/min with endothelium, or segments perfused at 40 mL/min without endothelium (endo-). Results (mean±SEM) represent percent of protein expression in fresh aorta. *P<0.05 versus fresh aorta, n=6 to 8.

Expression of CN and SMM
Analysis of CN content in organ culture of rabbit aorta revealed a decrease of 20% in CN levels in both relaxed and 10 mm Hg–pressurized vessels compared with fresh vessels (Figures 1 and 2). Applying an intraluminal pressure of 80 mm Hg maintained CN content at a level similar to that observed in fresh vessels. In contrast, the relative content of SMM in cultured vessels was unchanged compared with that in fresh vessels, irrespective of intraluminal pressure levels (Figures 1 and 2).

Effects of Flow and Endothelium on Marker Protein Content
Thus, the application of intraluminal pressure to aortic segments in organ culture prevents the loss of several proteins abundant in differentiated medial VSMCs. To investigate whether the integrity of the endothelial layer or a specific flow rate was essential for the maintenance of marker protein content in pressurized vessels, we subjected aortic segments with or without intact endothelium to different flow rates. Vessel segments pressurized at 80 mm Hg were perfused at low (8 mL/min) or high (40 mL/min) flow rates, representing a pulsatility of 1.5% or 9% of vessel diameter, respectively. No differences in marker protein levels were observed in these experiments (Figure 3). To evaluate the direct role of the endothelium in the preservation of marker protein content, experiments were performed with deendothelialized segments. Figure 3 shows that removal of the endothelium did not have any effect on marker protein content in pressurized vessels.

Marker Protein Synthesis in Pressurized Vessels
Incorporation of [³⁵S]methionine into particular proteins was measured in segments perfused at 1 mL/min and pressurized at 10 or 80 mm Hg for 3 days. Total protein ³⁵S incorporation and incorporation into myosin were slightly decreased in vessels pressurized at 10 mm Hg, to 78±5% or 81±4%, respectively, of that in vessels maintained at 80 mm Hg (P<0.05). However, specific ³⁵S incorporation into FIL at 10 mm Hg was even lower (63±8% of levels in vessels pressurized at 80 mm Hg, P<0.05). Quantification of these experiments is summarized in the Table.

	Discussion

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Numerous studies have been undertaken to characterize VSMC phenotypic changes in primary culture, as reviewed previously.¹⁷ Isolated rabbit aortic VSMCs in culture transformed from a contractile to a synthetic phenotype within 5 to 7 days. This phenotypic modulation was accompanied by a 2- to 3-fold decrease in h-CaD content, a concomitant 3-fold increase in l-CaD content,^{27 28} a 2-fold decrease in SMM content,²⁷ and 4-fold decrease in CN levels.²³ However, not much is known about changes in protein marker expression in the model of vascular organ culture. Our results suggest that changes in the protein marker content of VSMCs in organ culture have some peculiarities. The CN and h-CaD contents of VSMCs in the organ culture model decreased less than in the cell culture model,^{23 28} even in the absence of mechanical stimulation (relaxed conditions). Furthermore, the decrease in h-CaD content was not accompanied by activation of l-CaD expression. Analysis of factors affecting l-CaD expression in primary VSMC culture has shown that a high density of intercellular contacts, the presence of laminin and collagen type IV surrounding medial VSMCs, and suppression of SMC proliferation are negative regulators of l-CaD expression.²⁷ Thus, the environment of VSMCs in organ culture provides conditions for inhibition of l-CaD expression.

In a previous work,¹⁹ we characterized the metabolic activity of aortic segments maintained in organ culture under relaxed conditions or mounted on a specially designed system that allowed application of hydrostatic pressure to the intraluminal compartment of vessel segments. It was demonstrated that neither under relaxed conditions nor under moderate intraluminal pressure (80 mm Hg) did vessel culture result in activation of [³H] thymidine incorporation in segments perfused with serum-containing culture medium. These findings suggested that activation of proliferation did not occur in relaxed segments or segments pressurized for 3 days in our model. In the present study, in addition to relaxed segments cultured in Petri dishes without pressure and flow, vessel segments pressurized and perfused at subphysiological levels (10 mm Hg, 1 mL/min) were used to further ensure that differences in marker protein content between relaxed and pressurized segments were not due to different conditions of vessel culture (axial tension) and circulation of culture medium.

The results presented here demonstrate that SMM content does not change significantly during a 3-day organ culture, regardless of the level of transmural pressure, as opposed to the marked decrease reported in VSMC culture.²⁹ Because a high density of intercellular contacts and particular types of extracellular matrix may control the expression of SMM in VSMC culture,^{6 30} it is possible that these factors persisting in organ culture are favorable for the maintenance of unchanged SMM levels.

Organ culture of rabbit aortic segments pressurized at 80 mm Hg for 3 days completely prevented the decrease in h-CaD content observed in relaxed segments or in segments pressurized at 10 mm Hg. These results agree with previous work, in which the effects of long-term cyclic stretch on rabbit VSMCs in culture were studied.^{15 31} However, cyclic stretching of VSMCs with a profoundly modulated phenotype was able to maintain h-CaD levels at only 20% to 25% of the level found in freshly isolated medial VSMCs, thus delaying the drop to 7% to 8% observed in unstretched cells.³¹ In comparison, mechanical stimulation of aortic segments in organ culture completely prevented the decrease in h-CaD content caused by culture conditions. Results reported in the current article suggest that the combination of mechanical stimulation with the natural VSMC environment obtained in organ culture is sufficient for the maintenance of in vivo h-CaD levels. Interestingly, in a small series of experiments, h-CaD levels were found to be further reduced in vessels maintained for 6 days at 10 mm Hg, whereas their counterparts maintained at 80 mm Hg showed h-CaD contents close to normal (data not shown). Experiments undertaken by Reckless et al³² in the living animal model showed that application of a rigid collar on the rabbit carotid artery induced a sustained decrease in the h-CaD content, which was observed for 3 weeks after surgery. Limitation of carotid artery distension by a rigid, in situ collar was thus sufficient to downregulate h-CaD expression. Hence, results obtained with cultured VSMCs, vessel organ culture, and animal models clearly suggest that mechanical stimulation is important for the maintenance of h-CaD expression.

An important finding of this study was that pressurizing the vessel segments in organ culture had a pronounced effect on FIL content in medial VSMCs. FIL is a cytoskeletal protein capable of bundling actin filaments and thus, of organizing thin filaments into a three-dimensional network.³³ A nonmuscle form of FIL has been found in macrophages, platelets, and a number of cell lines.^{34 35} However, the content of FIL in nonmuscle tissues is 30-fold less than in SM, as described by Brown and Binder.³⁶ Furthermore, FIL organ content is strictly correlated with the amount of SM tissue, so that FIL can be considered an additional marker of the SM phenotype.³⁶ In SMCs, FIL is localized in the so-called "structural" compartment of the actin network, which is not involved in the process of contraction but is essential for the maintenance of SMC cytoskeletal integrity.³⁷ The ability of FIL to interact with intermediate filaments³⁸ also supports its role as an "integrative" protein of the SMC cytoskeleton. Using a model of experimental hypertension of the rat portal vein, Malmqvist and Arner³⁹ described that increased blood pressure maintained higher levels of FIL content. These results, combined with our data obtained from mechanically stimulated vessels in organ culture, in which applying a pressure of 80 mm Hg was necessary to maintain FIL content in vessel segments, suggest a positive regulation of FIL content by mechanical factors.

Thus, our experiments show that intraluminal pressure and the concomitant vascular stretch directly control h-CaD and FIL contents of medial VSMCs. A possible role for flow, the endothelium, or both in the maintenance of marker protein levels was also evaluated. Deendothelialization of pressurized aortic segments had no effect on marker protein content. In agreement with these data, high and low levels of perfusion had no significant effect on the marker protein content in pressurized vessels. Moreover, in one set of experiments, deendothelialized segments were pressurized in serum-containing or serum-free medium to assess the possible effects of serum factors (data not shown). No significant difference was observed. Interestingly, the pulsatile nature of the induced flow did not contribute to better marker protein preservation either, in spite of the fact that cyclic stretching affects marker protein expression in cultured VSMCs.^{15 16} Thus, taken together, the results suggest that intraluminal pressure is a primary determinant for the maintenance of high levels of h-CaD and FIL and that the endothelium plays no significant role in the maintenance of marker protein content in our model.

To establish whether the decrease in FIL content observed in vessels pressurized at 10 mm Hg resulted from decreased synthesis, [³⁵S]methionine incorporation into FIL and myosin bands was compared. Application of an intraluminal pressure of 10 mm Hg to vessel segments diminished the radiolabel incorporation into cellular proteins. However, the extent of label incorporation was different for SMM and FIL. A 19% decrease in [³⁵S]methionine incorporation into SMM at 10 mm Hg was correlated with the general 22% decrease in protein synthesis activity of pressurized vessel segments, whereas radiolabel incorporation into FIL was below this level and reached 63% of control. Thus, decreased pressure in cultured vessels resulted in a general downregulation of protein synthesis, including marker protein myosin, and particularly reduced FIL synthesis.

In summary, we conclude that h-CaD and FIL contents in SM are regulated by mechanical factors. Hemodynamic forces in vivo may therefore influence not only the cytoarchitecture of the vessel wall but also the expression of particular contractile and cytoskeletal VSMC proteins. Our findings support the idea that restoration of physiological hemodynamic parameters after vascular injury is essential for both the vascular structure and the VSM phenotype.

	Selected Abbreviations and Acronyms

 

CaD = caldesmon CN = calponin FIL = filamin h- = high-molecular-weight l- = low-molecular-weight PAGE = polyacrylamide gel electrophoresis SMM = smooth muscle myosin (V)SM(C) = (vascular) smooth muscle (cell)

	Acknowledgments

 
K.G.B. and S.L. were recipients of fellowships from the Institut National de la Santé et de la Recherche Médicale (INSERM) and INSERM/Fonds de la Recherche en Santé du Québec (FRSQ), respectively. Support of K.G.B. by Russian Fundamental Research Foundation grants 96–04-49274 and 96–04-49106 is also gratefully acknowledged.

Received August 6, 1997; accepted December 22, 1997.

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