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(Circulation Research. 2000;86:1122.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Mechanical Stress–Induced Heat Shock Protein 70 Expression in Vascular Smooth Muscle Cells Is Regulated by Rac and Ras Small G Proteins but Not Mitogen-Activated Protein Kinases

Qingbo Xu, Georg Schett, Chaohong Li, Yanhua Hu, Georg Wick

From the Institute for Biomedical Aging Research (Q.X., C.L., Y.H., G.W.), Austrian Academy of Sciences, Innsbruck, Austria; and the Department of Internal Medicine (G.S.), University Hospital of Vienna, Vienna, Austria.

Correspondence to Dr Qingbo Xu, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail qingbo.xu{at}oeaw.ac.at

	Abstract

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Abstract—Previous studies have documented that acute elevation in blood pressure results in heat shock protein (hsp) 70–mRNA expression followed by hsp70-protein production in rat aortas. In this article, we provide evidence that mechanical forces evoke rapid activation of heat shock transcription factor (HSF) and hsp70 accumulation. In our study, Western blot analysis demonstrated that hsp70-protein induction peaked between 6 and 12 hours after treatment with cyclic stain stress (60 cycles/minute, up to 30% elongation). Elevated protein levels were preceded by hsp70-mRNA transcription, which was associated with HSF1 phosphorylation and activation stimulated by mechanical forces, suggesting that the response was regulated at the transcriptional level. Conditioned medium from cyclic strain–stressed vascular smooth muscle cells (VSMCs) did not result in HSF-DNA–binding activation. Furthermore, mitogen-activated protein kinases (MAPKs), including extracellular signal–regulated kinases, c-Jun NH₂-terminal protein kinases or stress-activated protein kinases, and p38 MAPKs, were also highly activated in response to cyclic strain stress. Inhibition of extracellular signal–regulated kinase and p38-MAPK activation by their specific inhibitors (PD 98059 and SB 202190) did not influence HSF1 activation. Interestingly, VSMC lines stably expressing dominant-negative rac (rac N17) abolished hsp-protein production and HSF1 activation induced by cyclic strain stress, whereas a significant reduction of hsp70 expression was seen in ras N17–transfected VSMC lines. Thus, our findings demonstrate that cyclic strain stress–induced hsp70 expression is mediated by HSF1 activation and regulated by rac and ras GTP–binding proteins. Induction of hsp70 could be important in maintaining VSMC homeostasis during vascular remodeling in response to hemodynamic stimulation.

Key Words: mechanical stress • smooth muscle cells • heat shock proteins • signaling • G proteins

	Introduction

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The passage of blood through the vascular system generates hemodynamic forces. Fluid flow across the cell surface results in shear stress, whereas strain stress, which produces elongational stretch, is caused by circumferential deformations resulting from transmural pressure gradients and vascular smooth muscle tone.¹ Vascular smooth muscle cells (VSMCs) are one of the major constituents of blood vessel walls responsible for the maintenance of vascular structure and functions.² The arterial wall is an integrated, functional component of the circulatory system that is continually remodeling in response to various stressors, such as hemodynamic stress. These stimuli directly or indirectly damage the vessel wall and eventually induce arterial stiffness and obstruction. In fact, VSMCs produce a high level of stress proteins, also known as heat shock proteins (hsps), protecting the host from damage during hemodynamic stress.³

The hsps are subdivided into multimember families based on the molecular weights of the proteins encoded, ie, hsp27, hsp60, hsp70, and hsp90. They are highly expressed in cardiovascular tissues in response to stress stimuli.^{3 4 5 6} The hsp production is primarily mediated by heat shock transcription factors (HSFs) that interact with a specific regulatory element, heat shock element (HSE), present in the hsp gene promoters.⁷ HSF1-null mice exhibit elimination of the classical heat shock response and HSF1 is essential and sufficient for upregulation of hsp70 expression during downregulation of the ubiquitin proteolytic pathway.^{8 9} Although the activation process seems to involve HSF oligomerization from a monomeric to a trimeric state,⁷ stress-initiated signal-transduction pathways leading to HSF activation are largely unknown.

Mitogen-activated protein kinases (MAPKs) are thought to play a pivotal role in transmitting transmembrane signals required for gene expression and cell differentiation.^{10 11} MAPKs comprise a ubiquitous family of tyrosine and threonine kinases and include extracellular signal–regulated kinases (ERKs), stress-activated protein kinases (SAPKs) or c-Jun NH₂-terminal protein kinases (JNKs), and p38 MAPKs. They are highly activated in VSMCs in vivo and in vitro in response to cyclic strain stress,^{12 13} hypertension,¹⁴ and angioplasty,¹⁵ which are related to altered biomechanical or hemodynamic stress.¹⁶

Xu et al^{17 18} have previously shown that acute hypertension induces a rapid expression of hsp70 mRNA followed by elevated hsp70 proteins in rat aorta. The hsp70 induction is blocked by prevention of elevation in blood pressure, ie, administration of the vasodilator agent sodium nitroprusside. However, it is not known whether hsp70 production is initiated by hemodynamic force per se or by cytokines in vivo. In the present study, we examined the possibility that mechanical stress results in hsp70 production in cultured VSMCs. We found that cyclic strain stress induces hsp-protein production and hsp70-mRNA expression mediated by HSF1 activation, which is regulated by rac and ras G proteins.

	Materials and Methods

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Cell Culture and Cyclic Strain Stress Treatment
VSMCs were isolated from rats by enzymatic digestion of the aorta, as described elsewhere.¹⁹ VSMCs were plated on silicone elastomer–bottomed culture plates (Flexcell). Cells were subjected to mechanical stress with the Cyclic Stress Unit, a modification of the unit initially described by Banes et al.²⁰ After treatment at 37°C, cells were harvested for protein preparation or RNA isolation.

Stable Transfection
VSMCs (passages 3 to 5) were transfected with ras N17, rac N17, and neovector plasmids, respectively, by using Superfect Kit (Qiagen) according to the manufacturer’s instructions. Ras N17–transfected, rac N17–transfected, and neotransfected VSMCs were identified by Western blotting analysis with antibodies to H-ras or myc-tagged proteins.

Protein Extractions and Western Blot Analysis
The cells were washed twice with precold (4°C) phosphate-buffered saline and harvested on ice in buffer A, and 50 µg of total VSMC proteins was separated by electrophoresis through a 10% SDS–polyacrylamide gel. The membranes were processed with a monoclonal antibody to hsp70, as described.²¹ For HSF1 analysis, nuclear proteins (20 µg/lane) and antibodies against mammalian HSF1²² were used. Specific antibody-antigen complexes were detected using the ECL Western Blot Detection Kit.

RNA Isolation and Northern Blots
Total RNA was isolated using a standard protocol, as described previously.²³ Hybridizations were performed using a fluorescein-labeled cDNA probe for hsp70, as described previously.²⁴ Accuracy of loading and transfer, as well as RNA integrity, was confirmed by quantitative analysis of the 28S and 18S RNAs.

Gel Mobility Shift Assays
For nuclear protein preparation, the procedure used was similar to that described by Schreiber et al,²⁵ with a slight modification.²⁶ The procedure for gel mobility shift assays has been described previously.²⁶ In short, DNA binding was determined after incubation of 5 µg of nuclear protein extracts with an oligonucleotide containing the heat shock element (HSE) sequence from the Drosophila hsp70 promoter (5'-GCCTCGAATGTTCGCGAAGTTT-3') labeled with ³²P-dCTP. Super-shift assays were performed using antibodies against HSF1, HSF2,²² c-Fos, and ATF2 (Santa Cruz Biotech).

Kinase Assays
For p38 kinase assays, the procedure used was similar to that described previously.²⁷ Briefly, immunocomplexes were incubated with myelin basic protein and -P³²ATP (5 µCi) for 20 minutes. Proteins in the kinase reaction were resolved by SDS-PAGE (15% gel) and subjected to autoradiography.

Cell Viability Assays
VSMCs were plated in the flexible plates and serum starved. VSMCs were then treated with cyclic strain stress at 37°C for 6 hours or treated with heat shock (42°C) for 30 minutes and recovered at 37°C for 6 hours. H₂O₂ or nitric oxide donor sodium nitroprusside was added to the culture and incubated at 37°C for 24 hours. Cells were harvested with trypsin-EDTA solution and counted.

Statistical Analysis
A paired Student’s t test was used to assess differences between 2 groups. A value of P<0.05 was considered significant.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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hsp70 Protein Induction and mRNA Transcription
Figure 1A shows the time course of hsp70 production in response to mechanical stress. The amount of 70-kDa protein increased by 1 hour after treatment, reached a plateau in 6 hours, and declined thereafter. Figure 1B summarizes data of hsp70 protein induction as determined by quantification of optical densities from autoradiograms of 3 experiments. Between 6 and 12 hours, 3- to 5-fold increases in hsp70 proteins of VSMCs were observed. Importantly, this effect seemed specific to hsp70, because protein levels for hsp27 and hsp90 were unchanged in response to cyclic strain stress (Figure 1C). These results are concomitant with a report showing that shear stress does not induce hsp27 protein production in endothelial cells.²⁸ Likewise, VSMCs treated with a variety of stress strengths for 6 hours resulted in significant increases in hsp70 proteins (Figure 1D).

View larger version (32K): [in this window] [in a new window]   Figure 1. Western blot analysis of hsp70, hsp27, and hsp90 proteins in VSMCs treated with cyclic strain stress. Subconfluent cells cultivated on flexible-bottomed plates were subjected to cyclic strain stress at 37°C. Cells were lysed in the buffer (protein extracts separated on 10% SDS–polyacrylamide gel), transferred to membranes, and probed using a monoclonal antibody to mammalian-inducible hsp70. A, Time course of hsp70 induction in VSMCs treated with cyclic strain stress (60 cycles/minute, 15% elongation). B, Data of hsp70 induction from 3 independent experiments. C, Data of hsp27 and hsp90 expression in VSMCs treated with cyclic strain stress. D, Results from VSMCs treated with various elongations of the original size for 6 hours. Ctl- indicates negative control; and Ctl+, positive control treated with 42°C for 30 minutes and 37°C for 5 hours. *Significant difference from untreated controls; P<0.05.

hsp70 mRNA levels in stress-treated VSMCs were analyzed by Northern blots. As shown in Figure 2, cyclic strain stress resulted in an increase of hsp70 mRNA transcription in VSMCs. A strength-response analysis indicated that levels of hsp70 mRNA in VSMCs treated with 5% elongation were higher and significantly elevated with 10% or 20% elongation. The lower panel of Figure 2A shows the amount of 18S and 28S RNA from the corresponding blot. Figure 2B summarizes hsp70 mRNA induction as determined by quantification of optical densities from autoradiograms of 2 experiments.

View larger version (60K): [in this window] [in a new window]   Figure 2. Mechanical stress–induced hsp70 mRNA expression. A, Strength course of mRNA expression in VSMCs treated with cyclic strain stress at 37°C for 1 hour. Integrity and quantity of RNA were verified by analysis of 18S and 28S RNAs (bottom). Northern blots were hybridized with hsp70 cDNA probes. B, Statistical data on hsp70-mRNA induction from VSMCs treated with mechanical stress. *Significant difference from control (Ctl-); P<0.05.

HSF1 Activation
To determine whether mechanical stress induces hsp70 mRNA through HSF activation, nuclear proteins were isolated from VSMCs and assayed for the presence of HSF-binding activity. As shown in Figure 3, levels of binding activity increased in cyclic strain stress–treated cells, with maximum activity at 2 hours after treatment and decline by 6 hours in stress-treated cells. A tensile strength–response analysis of mechanical stress–induced HSF-DNA binding indicates that the increase of HSF-DNA–binding activities corresponded with increased magnitudes of stretch stress of 10% and 20% (Figure 3B).

View larger version (54K): [in this window] [in a new window]   Figure 3. HSF-binding activity in protein extracts of VSMCs. VSMCs were treated with cyclic strain stress at 37°C. Nuclear proteins were prepared as described in Materials and Methods. Gel mobility shift assay was performed in 4% gel. A, Time course of HSF-DNA–binding activation from VSMCs stressed with 60 cycles/minute and 15% elongation. B, Strength-dependent HSF activation (1 hour). Arrows indicate specific HSE-binding complexes; lines, nonspecific binding.

Figure 4A shows the results of a gel mobility shift assay in the presence or absence of either an unlabeled HSE or nuclear factor (NF)-B–binding element. The mechanical stress–induced increase in binding activity was specific for the HSE, as increased concentrations of unlabeled HSE effectively competed for binding to the factor, whereas the NF-B–binding element did not (Figure 4A). Addition of the anti-HSF1 antibody to the binding reaction resulted in a complete shift of the binding complexes to a slower migrating species, whereas the anti-HSF2, anti–c-Fos, and anti-ATF2 antibodies had no effects or moderate effects (Figure 4B).

View larger version (63K): [in this window] [in a new window]   Figure 4. Specificity of HSE-binding activation in VSMCs in response to mechanical stress. A, Nuclear proteins were incubated with 32P-labeled HSE oligonucleotide in the presence or absence of unlabeled oligonucleotide containing HSE-binding or NF-B–binding element. B, Results of no addition (-) or addition (+) of various antibodies specific to HSF1, HSF2, c-Fos, or ATF2. Arrows indicate specific protein-DNA complexes; lines, supershifted DNA-binding complexes of HSF1 protein-containing complexes.

In untreated cells, HSF1 seemed to be present in nuclei at low levels, evidenced by weaker signals and migrated on SDS-polyacrylamide gels as a 70-kDa protein (Figure 5A). Cyclic-strain treatment and heat stress resulted in an increased HSF1 in nuclei and a shift to higher molecular weight species 1 hour after treatment (Figure 5A). The findings indicate that translocation and modification are necessary to HSF activation in response to mechanical stimulation.

View larger version (79K): [in this window] [in a new window]   Figure 5. A, Western blot analysis of HSF1 proteins. Nuclear proteins prepared from VSMCs treated with heat shock (42°C for 30 minutes) or stretch stress for 1 hour were analyzed by Western immunoblotting for HSF1 proteins. Brackets indicate the position of HSF1 proteins. B, Gel mobility shift assay identifying stress-induced HSF-DNA binding activation. Supernatant 1 indicates nonconcentrated medium; supernatant 10, 10-time–concentrated conditioned medium.

There is evidence that mechanical stress results in synthesis of growth factors and cytokines releasing into medium in cultured VSMCs.²⁹ To verify whether the autocrine and paracrine cytokines are involved in HSF activation, conditioned medium from stressed VSMCs were collected, concentrated, and used to treat cells. The data in Figure 5B show that supernatant or conditioned medium from VSMCs stretch stressed for 1 hour did not result in HSF-DNA–binding activation.

MAPK-Independent HSF Activation in VSMCs
Recent studies have demonstrated that ERK1 phosphorylates HSF1 on serine residues and represses transcriptional activation by HSF1³⁰ , and that p38 MAPKs can induce hsp27 phosphorylation, which is necessary for hsp27 function.³¹ It would be interesting to clarify whether mechanical stress–induced HSF1 activation is regulated by MAPK-signal pathways. As shown in Figure 6A, cyclic strain–stress treatment (60 cycles/minute, 15% elongation) resulted in significant increases in ERK phosphorylation. Kinetic analysis indicates that this response occurred as early as 5 minutes, with maximum induction achieved 10 minutes after treatment and declining thereafter (Figure 6A). Similarly, both JNKs/SAPKs and p38 MAPKs were activated in a time-dependent manner (Figures 6B and 6C). PD 98059, a specific inhibitor of ERK kinases, significantly inhibited ERK1 and ERK2 activation in VSMCs in response to mechanical stress but did not inhibit p38 MAPKs (Figure 6D). Likewise, the p38-specific inhibitor SB 202190 abrogated p38 activity stimulated by strain stress, and no effect on ERK phosphorylation was seen (Figure 6E). Surprisingly, both PD 98059 and SB 202190 did not influence HSF-DNA–binding activation induced by mechanical stress in VSMCs (Figure 6F), indicating an MAPK-independent process of HSF1 activation.

View larger version (28K): [in this window] [in a new window]   Figure 6. Time course of 3-MAPK phosphorylation in VSMCs exposed to mechanical stress. Serum-starved VSMCs were treated with cyclic strain stress (60 cycles/minute, 15% elongation). Western blot analysis was performed using antibodies to phosphorylated ERK1 and ERK2 (A), phosphorylated JNK1 and JNK2 (B), and phosphorylated p38 MAPK (C). VSMCs were pretreated with PD 98059 (50 µmol/L), SB 202190 (5 µmol/L), or PD 98059+SB 202190 (PD+SB) for 1 hour. Cells were stressed for 10 minutes (D and E) and 1 hour (F), respectively, and harvested for protein extracts or nuclear protein isolation. p38 MAPK activities were measured based on phosphorylation of myelin basic protein (MBP) (E). F, Results of gel mobility shift assay.

Rac- and Ras-Regulated hsp70 Expression
To investigate the role of the small GTPase–binding proteins ras and rac in hsp70 expression in stress-stimulated VSMCs, we established VSMC lines stably expressing ras or rac encoding a dominant-negative form (ras N17 or rac1 N17). Rac1 N17–transfected VSMCs expressed a high level of this gene product (Figure 7A). Interestingly, overexpression of rac1 N17 completely inhibited hsp70 protein production stimulated by strain stress and partially blocked heat shock–induced hsp70 expression (Figure 7B). We then assessed the effects of rac1 N17 overexpression on HSF activation in the cell lines treated with mechanical stress. As seen in Figure 7C, strain stress–induced HSF-DNA–binding activities in rac1 N17 cell lines were not detectable. Similarly, HSF1 translocation and phosphorylation stimulated by cyclic strain stress were blocked by overexpression of dominant-negative rac (Figure 7D).

View larger version (42K): [in this window] [in a new window]   Figure 7. Involvement of rac in hsp70 expression. Rat VSMCs were stably transfected with constructs expressing dominant-negative rac (pEF-rac1 N17) or vector (pEF-neo). VSMC lines expressing rac1 N17 were identified with anti–myc-tag antibody, as determined by Western blotting (A). VSMC lines were treated with mechanical stress for 6 hours (60 cycles/minute, 15% elongation) (B) or 1 hour (C and D). Western blot analysis was performed using anti-hsp70 (B) or anti-HSF1 (D) antibodies. C, Results of gel mobility shift assay.

Likewise, ras protein was at a lower level in vector-transfected controls and at a higher level in ras-transfected cells (Figure 8A). Ras-N17 expression largely blocked hsp70-protein induction in the cell lines stimulated by mechanical stress (Figure 8B) and partially inhibited HSF-DNA–binding activation in stressed VSMCs (Figure 8C). Therefore, ras and rac play a role in regulation of hsp70 expression in VSMCs.

View larger version (48K): [in this window] [in a new window]   Figure 8. Involvement of ras in hsp70 expression. The procedures for establishing stably expressing dominant-negative ras (pEF-ras N17) cell lines are similar to those described in the legend to Figure 7 and in Materials and Methods. A, Results of Western blot analysis using anti–H-ras antibody. VSMC cell lines were treated with mechanical stress for 6 hours (15% elongation) (B) or 1 hour (C). Western blot analysis was performed using anti-hsp70 antibody (B) or gel mobility shift assay (C). Data represent similar results from 3 independent experiments.

Effect of Cyclic Strain Stress on VSMC Survival After Free Radical Exposure
To investigate the potential physiological role of cyclic strain stress–induced hsp70 in mediating the protective response to free radical stimulation, we undertook a comparative analysis of cell survival after H₂O₂ or sodium nitroprusside exposure in VSMCs with or without cyclic strain stress treatment or preincubation at 42°C for 30 minutes. Data shown in Figure 1 online (available at http://www.circresaha.org) provide evidence that cyclic strain stress or heat shock significantly increased VSMC survival from H₂O₂ or sodium nitroprusside–induced cell death.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we provide the first evidence that mechanical stress induces hsp70 expression in VSMCs and demonstrate that this effect relies on HSF1 activation. The nature of the primary signal that activates HSF1 in response to heat shock or stress is not fully elucidated. Current studies support a model for regulating heat shock response in which the hsps themselves negatively regulate heat shock gene expression via an autoregulatory loop.⁷ During mechanical stretch stimulation, multiple and complex pathophysiological changes occur in VSMCs, including PDGF-receptor activation,¹² cytoskeletal rearrangement,³² and apoptosis.³³ Thus, mechanical stress–induced and heat shock–induced hsp70 productions share many similarities in activation of HSF1 and regulation of hsp70 gene expression.

Much evidence suggests a role for phosphorylation in the conversion of HSF1 from this intermediate state into a transcriptionally active form.⁷ Recent studies have demonstrated that ERK1 phosphorylates HSF1 on serine residues and represses transcriptional activation by HSF1³⁰ and that p38 MAPKs can induce hsp27 phosphorylation, which is necessary for hsp27 function.³¹ Our findings that mechanical stress rapidly activates 3 members of MAPKs do not support the role of MAPKs in HSF activation or phosphorylation in VSMCs. Thus, mechanical stress–induced hsp70 expression is independent of MAPKs in VSMCs.

What are signal-transduction pathways between mechanical stress stimuli and HSF1 activation? Recently, we demonstrated that cyclic strain stress rapidly activates PDGFR-ERK-AP-1–signal pathways.^{12 13} Suramin, a growth factor receptor antagonist, inhibited phosphorylation of the PDGF receptor³⁴ but did not inhibit cyclic strain stress–induced HSF activation (data not shown). These results indicate that mechanical stress–induced hsp70 expression is independent of PDGF receptor activation. A possible primary mechanosensor candidate may be the G protein. Recent reports by Gudi et al³⁵ indicate that G proteins may act as primary mechanosensors in shear-stressed endothelial cells. Treatment of endothelial cells with antisense Gq oligonucleotides inhibited shear stress–induced ras-GTPase activity. Our recent study demonstrated that small GTP–binding protein ras and rac are activated by cyclic strain stress, which mediates MAPK phosphatase-1 expression.¹³ This hypothesis is supported by our results (Figures 7 and 8) that show that expression of dominant-negative ras and rac in VSMCs exposed to mechanical stress could completely or significantly inhibit hsp70 production and HSF1-DNA–binding activation. These observations suggest that increases in the elongational and transitional mobility in cell membranes activate membrane-bound G proteins by facilitating exchange of GDP to GTP, subsequently leading to HSF1 activation and hsp70 expression.

With regard to the involvement of growth factors and cytokines released by stressed VSMCs in the mechanical stress–induced activation of HSF1, our data do not support ligand-binding activation. The conditioned medium from stressed cells did not result in HSF1 activation. However, the effects of the factors or cytokines on hsp70 production stimulated by mechanical stress cannot be absolutely excluded because of the possible presence of unstable or rapidly inactivated factors in the conditioned medium. In addition, a single myocardial stretch was shown capable of inducing hsp70 expression in isolated perfused rabbit heart,³⁶ and volume overload produced experimentally by banding of the aorta was sufficient to elicit hsp70 induction in the heart.³⁷ However, an in vitro study showed a lack of stretch-induced expression of hsp70 gene in cultured cardiac myocytes.³⁸ In the present study, we demonstrated that VSMCs do express hsp70 in response to stretch stress. These results suggest the significance of potential cell-type specificity in hsp70 induction in the process of cell stretching because of heterogeneous cell compositions of the heart.

Recent studies have demonstrated the presence of VSMC apoptosis in the arterial wall.^{33 39} Proliferating VSMCs show more apoptotic cell death than nonproliferating VSMCs.⁴⁰ There is evidence that free radicals, including H₂O₂ and nitric oxide, involved in the development of vascular diseases can lead to VSMC apoptosis or death.⁴¹ Transgenic mice overexpressing hsp70 show enhanced resistance to ischemic injury,^{42 43} and increased production of hsp70 in atherosclerotic lesions may be beneficial for arterial smooth muscle cell survival.⁴⁴ HSF1-deficient mice exhibit increased mortality after endotoxin challenge.⁸ Our studies demonstrate the role of mechanical stress–induced or heat shock–induced hsp expression in protecting VSMCs against free radical–induced death. Thus, hsps might influence the process of vascular remodeling or hypertrophy via their effects on VSMC apoptosis and proliferation in response to hemodynamic stress and may exert a role in maintaining cellular homeostasis of the vessel.

	Acknowledgments

 
This work was supported by grants P13099-BIO (to Q.X.) and P12213-MED (to G.W.) from the Austrian Science Fund.

Received December 29, 1999; accepted April 5, 2000.

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