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,1
* Department of Internal Medicine/Cardiology and
Department of Biochemistry, Giessen University, Giessen, Germany; and
Department of Internal Medicine/Cardiology, Dresden University of Technology, Dresden, Germany
1Correspondence: Internal Medicine/Cardiology, Dresden University of Technology, Fetscherstrasse 76, D-01307 Dresden, Germany. E-mail: r.braun-dullaeus{at}mailbox.tu-dresden.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: signal transduction • remodeling • p53 • restenosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Changes in wall tension as produced by changes in flow or by barotrauma during angioplasty cause apoptosis especially of medial vascular smooth muscle cells (VSMCs; refs. 4
, 5
). The occurrence of apoptosis after balloon angioplasty has been well established in both animal models and humans (6
7
8
9
10
11)
. However, the mechanism of mechanically induced vascular apoptosis and its role for the development of neointima and restenosis have not yet been elucidated conclusively. It has been hypothesized the early wave of apoptosis, occurring within hours of the injury and resulting in a marked decrease in vessel wall cellularity, may exacerbate late neointima formation by provoking a greater wound healing response (1)
. Apoptosis at later time points seems confined to VSMCs of the developing neointima (2)
. This late apoptosis may limit lesion growth.
Although the contribution of cellular aspartate-specific cysteinyl proteases (caspases) in vascular programmed cell death has clearly been demonstrated, not much attention has been given to the cysteine protease calpain, even though its role in apoptosis has been well established also (12
, 13)
. For example, calpain is able to influence the level of the transcription factor p53 (14
, 15)
. p53 has been suggested to regulate vascular cell apoptosis in vitro after mechanical stretching (16)
and in vivo, as p53 is reported to accumulate in atherosclerotic lesions (17)
. Furthermore, forced expression of p53 induces VSMC apoptosis in vitro and inhibits neointima lesion formation in vivo (18
, 19)
. This transcription factor promotes apoptosis by functioning, at least in part, as a positive regulator of Bax expression (20)
and a negative regulator of Bcl-2 expression (21)
. In addition, p53 negatively regulates cell growth through its ability to induce the cyclin-dependent kinase inhibitor p21Cip1 (22)
and may this way limit lesion growth.
In the present study, we hypothesized that calpain is a regulator of mechanical force-induced apoptosis in VSMCs in vitro and after balloon injury in vivo. We were able to demonstrate that calpain is rapidly activated in VSMCs exposed to cyclic stretching in vitro and after balloon angioplasty of the rat carotid artery in vivo. Calpain is able to degrade p53, thereby counteracting medial VSMC apoptosis by preventing excessive p53 up-regulation and transcriptional activity. Consequently, inhibition of initial calpain activation augments apoptosis of medial VSMCs after balloon injury and results in a marked increase in late neointima formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cells were seeded (
10,000 cells/ml) onto fibronectin-coated six-well FlexI plates (Flexcell; McKeesport, PA, USA) and cultured in 10% FBS/DMEM/F12 to obtain confluency. Studies were conducted on confluent VSMCs after serum withdrawal for 2 days to achieve quiescence. On the day of the experiment, fresh serum-free medium was substituted and cyclic uniform uniaxial stretching was applied with a flexercell apparatus (FX-3000; Flexcell; 125% resting length, 0.5 Hz) in a tissue culture incubator.
Balloon angioplasty of the rat common carotid artery
Adult male Wistar rats (300 g body weight, Harlan Winkelmann, Borchem, Germany) were anesthesized (35 mg/kg ketamine, Inresa, Freiburg, Germany; 5 mg/kg xylazine, AstraZeneca, Wedel, Germany), and the common carotid artery was isolated. A Fogarty 2F embolectomy catheter (Edwards Lifesciences, Unterschleissheim, Germany) was introduced into the common carotid artery through the external carotid branch, advanced, inflated, and withdrawn three times. Finally, the balloon catheter was removed, solutions containing antisense oligodesoxynucleotides (ODNs) or control substances were installed as described above, and the proximal external carotid branch suture was tied using a 4-0 silk ligature (Johnson & Johnson, Brussels, Belgium). Wounds were closed and buprenorphine (0.5 mg/kg, Essex Pharma, Munich, Germany) was given for analgesia. On full recovery, animals were returned to the animal care facility and provided standard rat chow and water ad libitum. At the indicated time points, the animals were euthanized by an isofluran overdose (Baxter, Unterschleissheim, Germany). Six to eight animals were used at each time point. All exprimental protocols complied with guidelines of the institutional animal care and use committee. For a detailed description of the vessel harvesting and the morphometric analysis, please refer to the Supplemental Data online.
Vessel harvesting and morphometric analysis
After perfusion-fixation with 4% paraformaldehyde, carotid arteries were embedded in Tissue Tek OCT (Miles Laboratories, Naperville, IL, USA), snap-frozen, and stored at –80°C until use. Samples were sectioned on a Leica cryostat (6 µm; Leica, Wetzlar, Germany) and placed on poly-L-lysine (Sigma, St. Louis, MO, USA) -coated slides for immunohistochemical analysis. For morphometric analyses, hematoxylin and eosin staining was performed according to standard protocols. All sections were examined using the Leica DMRB microscope. For morphometric analyses, KS300 software (Carl Zeiss, Hallbergmoos, Germany) was used to measure external elastic lamina, internal elastic lamina, and lumen circumference, as well as medial and neointimal area of 6 sections per artery, obtained throughout the whole length of the excised artery.
In vitro and in vivo antisense ODN treatment
Cells were treated with 2 µmol/L phosphorothioate-antisense ODNs targeting p53 or calpain I 24 h before and during exposure to cyclic stretching (MWG Biotech, Ebersberg, Germany). The oligonucleotides were designed to hybridize with the initiation codon of calpain I (59-ACTCCTCTGTCATCCTGGGG-39) or p53 (20-CTGTGAATCCTCCATGAC-38) with reverse and scrambled ODNs serving as controls. For ODN transfection, a commercially available lipid formulation (Fugene, Roche, Mannheim, Germany) was used. For a 35-mm dish, 30 µl ODNs was added to 20 µl Fugene and mixed with 100 µl Opti-MEM (GIBCO Life Technologies, Inc., Karlsruhe, Germany) before being added to the cells.
For in vivo experiments, ODNs were instilled 24 h before injury and again immediately after injury to the common carotid artery. Briefly, a catheter was inserted through the left external carotid artery to the common carotid artery. After placement of a temporary ligature around the catheter, the common carotid artery was flushed, and a second temporary ligature was placed toward the aortic arch. One hundred microliters of Opti-MEM containing 20 µmol/L ODNs in Fugene was instilled to the ligated segment of the common carotid artery at 100 mmHg for 20 min.
Quantifcation of VSMC apoptosis
Rubber membranes of FlexI plates (Flexcell) containing cultured VSMCs were cut and mounted on glass slides. VSMCs were fixed using 2% paraformaldehyde, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was performed according to the supplier’s instructions (in situ cell death detection kit, Roche). After all nuclei were stained with DAPI, samples were viewed with an inverted fluorescence microscope (Leica), and two independent investigators, blinded to the treatment, evaluated the relative number of apoptotic cells per well by counting four randomly selected high-power fields. Since TUNEL staining after oligonucleotide transfection revealed unspecific staining of transfected VSMCs, annexin V staining (annexin V-Alexa-568; Roche) was used in these experiments to determine the relative number of apoptotic cells according to the supplier’s instructions.
Preparation of cell lysates and immunoblot analysis
Semiquantitative analysis of proteins in cell lysates was performed by Western blotting and antibody detection as described previously (24)
. Briefly, the cleared supernatant from lysates was run on polyacrylamide gel and blotted onto nitrocellulose (Hybond-ECL, Amersham, Freiburg, Germany) by wet electroblotting. After being blocked, blots were incubated with primary antibody (dilution 1:1000 for antip53; 1:500 for antip21Cip1; 1:500 for anti-Mdm2; 1:2000 for anti-Cdk4; 1:500 for anti-Bax; 1:500 for anti-Bcl; all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); 1:500 for anticalpain I (Calbiochem, Ober der Roeth, Germany) for 1 h at room temperature. Proteins were then detected by enhanced chemiluminescence (ECL+, Amersham) after labeling with horseradish peroxidase-labeled secondary antibody (1:2000 for 1 h) according to the manufacturer’s instructions. ImageJ densitometry software (Version 1.6, National Institutes of Health, Bethesda, MD, USA) was used for gel band quantitative densitometric analysis. Selected bands were quantified based on their relative intensities.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
RNA was extracted from VSMCs using the RNeasy kit (Quiagen, Hilden, Germany). Reverse transcription was carried out by incubation of 0.25 µg of total RNA in a reaction buffer containing 20 mmol/L Tris/HCl, 50 mmol/L KCl, 5 mM MgCl2 at pH 8.3 supplemented with 1 mmol/L of each oligonucleotide, 10 U/ml RNase inhibitor (Perkin Elmer, Wellesley, MA, USA), 2.5 µmol/L random hexamers, and 26 U of avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc., Karlsruhe, Germany) for 1 h at 42°C. Specific primers were directly added to the RT-reaction product, and PCR was carried out with 2.5 U Taq polymerase (Perkin Elmer) in a total volume of 50 µl. Twenty-eight cycles were used with cycle times of 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C. Ten microliters of each PCR product were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide. The following oligonucleotide primers were used with the resultant PCR product given: p53 (674 bp), 5'-CCACAAGGCTACATGAGGGT-3' (left), 5'-TGCCAATTTCCTCCCTTAAA-3' (right); p21Cip1 (161 bp), 5'-AGCAAAGTATGCCGTCGTCT-3' (left), 5'-ACACGCTCCCAGACGTAGTT-3' (right); mdm2 (233 bp), 5'-GCCAAGAAAGTGGCAAAGAG-3' (left), 5'-AATCATTTGGATCGGCTGTC-3' (right). No products were obtained in control reactions in the absence of template. Primers encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as a positive control.
Real-time PCR
Real-time measurements of PCR amplification were performed with the Stratagene MX3000 qPCR System with the SYBR green dye method (Brilliant SYBR Green Mastermix Kit, Stratagene, La Jolla, CA, USA). p53-specific primers used for real-time PCR were as follows: (5'-TCT-GTC-ATC-TTC-CGT- CCC-TTC-TC-3' (sense) and 5'-CCG-TGC-ACA-TAA-CAG-ACT-TGG-CT-3' (antisense); using the following cycling parameters: 95° for 10 min for 1 cycle; 95° for 30 s, 60° for 1 min, 72° for 1 min, for a total of 40 cycles. A melting curve analysis was performed after amplification to verify the accuracy of the amplicon. For quantification, mRNA expression of the target gene was normalized to the expressed housekeeping gene GAPDH. In addition, products were separated by electrophoresis on a 1.5% agarose gel and visualized with ethidium bromide.
Calpain activity assay
Three different assays using different calpain-specific substrates in a complementary manner were used to assess calpain activity in VSMCs. The first assay is based on fluorimetric detection of cleavage of the calpain substrate Ac-LLY-AFC (calpain activity assay kit, EMD Biosciences, Inc., San Diego, CA, USA) according to the supplier’s instructions. Briefly, 50 µg of cleared supernatant from lysed VSMCs or carotid arteries was exposed to 5 µl calpain substrate (Ac-LLY-AFC) for 1 h at 37°C in the presence of a calpain reaction buffer. Cleavage of the substrate resulted in the release of AFC, which was followed in a fluorescence plate reader at excitation of 400 nm and emission at 505 nm.
To measure calpain activity in single cells, we utilized the "Boc" assay (25)
. Briefly, VSMCs were incubated for 20 min in the presence of 50 mM t-butoxycarbonyl-leu-met-chloromethyl aminocoumarin (Boc-LM-CMAC; Molecular Probes, Poortgebouw, The Netherlands) and then exposed to cyclic stretching for 15 min as described above. Boc-LM-CMAC is retained within the cells by conjugation with intracellular thiol groups. Cleavage of the substrate results in retention of the chloromethylaminocoumarin portion of the molecule within the cell and results in increased fluorescence. At the end of the experiment, the elastic "flexer cell" membranes were cut and wet mounted on glass slides, and chloromethylaminocoumarin fluorescence was measured using a Leica fluorescence microscope (DMRB). Representative images of each slide were captured using a SPOT CCD camera.
The third assay uses the specific substrate succinyl-leu-leu-val-tyraminomethylcoumarin (AMC, Bachem, Heidelberg, Germany; ref. 26
). Briefly, SLLVT-AMC was added to the supernatant of all cultures and cells were exposed to cyclic stretching for 15 min at 37°C and 5% CO2. One hundred microliters of the supernatant was then added to a 0.2 ml quartz cuvette, and fluorescence was measured immediately at excitation of 360 nm and emission of 460 nm using an Aminco-Bowman spectrofluorimeter (FA-357, Thermo Electron Corporation, Dreieich, Germany).
p53 transcriptional activity
p53 transcriptional activity was quantified using a TransAM p53 assay kit (Active Motiv, Rixensart, Belgium). Briefly, nuclear extracts of VSMCs were incubated in 96 wells precoated with an oligonucleotide that contained a p53 consensus binding site (5'-GGACATGCCCGGGCATGTCC-3'). The amount of p53 bound to the oligonucleotides was determined using a p53-specific antibody that recognizes an accessible epitope on p53 after DNA binding. After the addition of a HRP-conjugated antibody, the extent of the colorimetric reaction of the added substrate was quantified at 450 nm using a 96-well plate reader. To control specificity of p53 binding, consensus oligonucleotides or mutated consensus oligonucleotides were added to some nuclear extracts before incubation.
p53 degradation assay
Degradation experiments were carried out at 37°C in the presence of 1 mM CaCl2. Human calpain I (Calbiochem) was added at a final concentration of 5 µg/ml to pure recombinant human p53 (5 µg/ml; Santa Cruz). Aliquots of the reaction mixture were sampled at various time points, and the reaction was stopped by addition of electrophoresis loading buffer containing 1% sodium dodecyl sulfate. In addition, separate aliquots of the p53 solution were supplemented with calpeptin (10 µM) or EGTA (1 mM) and were incubated separately for 60 min. All samples were then electrophoresed on 12% gels and electrotransferred onto nitrocellulose for detection by immunoblot analysis using p53-specific antibodies.
Statistical analysis
Data are means ± SE. Statistical analysis was performed by ANOVA. Posttest multiple comparison was performed by the method of Bonferroni. All experiments were independently repeated at least three times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). After cells were subjected to cyclic stretching of 125% of resting length (0.5 Hz) for the indicated time intervals, we observed a rapid increase in calpain activity within 5 min that slowly decreased over time but was significantly elevated for up to 8 h (Fig. 1B
). The specific calpain inhibitor PD150606 (50 µmol/L) was able to prevent the cleavage of Ac-LLY-AFC, indicating specificity of the assay.
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Two additional assays were used to determine calpain activation (Fig. 1C
). While T-Boc allowed visualization of calpain activity on a single cell level, the substrate SLLVT-AMC was released into the supernatant and spectrophotometrically quantified. Depletion of intracellular Ca2+ by administration of BAPTA-AM (30 µmol/L) as well as blocking the release of intracellular Ca2+ using gadolinium chloride (50 µmol/L) prevented stretch-induced calpain acitvation, indicating that stretch-induced calpain activation is dependent on intracellular calcium concentrations.
Stretch-induced apoptosis is markedly augmented by calpain inhibition
Apoptotic rate increased in VSMCs exposed to cyclic stretching for 24 h compared to cells grown under similar conditions without exposure to mechanical stress (8.6±1.3 vs. 2.1±1.9%, respectively; Fig. 2
A). The additional application of the calpain inhibitors calpeptin (10 µmol/L) or PD150606 (50 µmol/L) resulted in a further significant increase in the number of apoptotic cells exposed to mechanical stress (12.1±1.4 and 16.6±2.1%, respectively; Fig. 2A
) but not in cells kept quiescent (demonstrated in
Fig. 5
A). Controls using vehicle alone (DMSO) indicated a specific effect of calpain inhibitors. Further substantiation of these results was provided by using calpain antisense ODNs, which strongly reduced calpain expression 24 h after transfection (Fig. 2B
). This led to a dramatic increase in stretch-induced apoptosis of VSMCs (19.2±1.9 vs. 8.1±0.7%) in stretched cells only (Fig. 2C
), whereas reverse or scrambled ODNs had no effect.
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Inhibition of calpain up-regulates and activates p53
Since it has been suggested that cyclic stretch-induced VSMC apoptosis is, at least in part, mediated by p53 (27
, 28)
, we determined p53 protein expression levels and transcriptional activity in VSMCs exposed to cyclic stretching. p53 protein levels were found elevated within 2 h, reaching a peak at 8–12 h (Fig. 3
A; for additional densitometric quantification see Supplemental Data online). The expression levels of the p53-dependent genes, p21Cip1 and Mdm2, served as determinants of p53’s transcriptional activity. Mdm2 and p21Cip1 levels were found elevated with a similar kinetic as p53, suggesting that the increased p53 expression resulted in an increase in p53 transcriptional activity (Fig. 3A
).
We next examined whether calpain inhibition would affect p53 expression and function under conditions of cyclic stretching. The elevated p53 expression level observed after 2 and 12 h was further dramatically augmented after addition of the calpain inhibitors calpeptin or PD150606 (2 h: Fig. 3B, C
; 12 h: Fig. 3D, E
). Calpain inhibition also augmented the expression of the p53 responsive genes, p21Cip1 and Mdm2, indicating increased p53 transcriptional activity, with respective consequences for downstream-related genes. Furthermore, the calpain inhibitors calpeptin or PD150606 augmented the stretch-induced expression of the proapoptotic protein Bax, resulting in an increase of Bax/Bcl-2 ratio (Fig. 3F, G
).
In accordance with these findings, the direct assessment of transcriptional activity of p53 revealed that the stretch-induced increase in p53 activity was significantly augmented in the presence of the calpain inhibitors calpeptin or PD150606 (2.3±0.26 or 2.89±0.26, respectively, vs. 1.01±0.04, respectively) in stretched cells (Fig. 4
A). The addition of ODNs resembling the p53 consensus binding site for competition to the coated or mutated ODNs served as control for specificity of p53 binding.
Mechanism of calpain-dependent p53 regulation
To further determine the mechanisms regulating p53 protein levels under conditions of cyclic stretching, we examined transcriptional regulation of p53 by RT-PCR and real-time PCR. p53 mRNA levels did not differ within the time span of the experiments, and administration of the calpain inhibitors calpeptin or PD150606 also had no effect on p53 mRNA levels (Fig. 4B
). The semiquantitative mRNA measurements were further quantified by real-time PCR for p53 mRNA expression, as shown in the Supplemental Data online; Fig. 1B). These data suggested a rather posttranscriptional mechanism of force-induced p53 protein regulation and augmentation by calpain inhibition. Moreover, mRNA levels of p53-dependent genes (serving as a readout for transcriptional activity of p53) were increased after 12 h of cyclic stretching compared to quiescent cells. Administration of the calpain inhibitors calpeptin or PD150606 further augmented the stretch-induced expression of p21Cip1- and Mdm2-mRNA, indicating that the augmented protein expression of p53 results in an augmentation of transcriptional activity as well.
To examine the posttranscriptional regulation of p53, VSMCs were exposed to cyclic stretching for 8 h to induce p53 expression. After the administration of cycloheximide (10 µg/ml) to prevent de novo protein synthesis, the kinetics of p53 degradation was quantified by Western blot and densitometric analysis of the resulting bands. Addition of the calpain inhibitor calpeptin resulted in a significant twofold increase of the half-life of p53 protein from 41 ± 6 to 84 ± 9 min (n=3; P<0.05), indicating that calpain regulates p53 protein levels via a post-translational mechanism.
To further evaluate calpain’s involvement in p53 regulation, we examined whether p53 is a proteolytic substrate for calpain-dependent degradation in an in vitro assay. Recombinant full-length human p53 was exposed to human calpain I, which resulted in a rapid degradation of p53 (Fig. 4C
). In the absence of calpain I, hardly any p53 degradation was detected. Furthermore, prevention of calpain activation by the calcium chelator EGTA or inhibition by calpeptin prevented p53 cleavage in vitro. Degradation was limited, since a number of proteolytic products (with a predominant product occurring at 40 kDa) accumulated in the reaction mixture, with no further degradation seen after 60 min. This finding is in accordance with the fact that calpains usually degrade their substrates to a limited extent (29)
. The degradation of isolated recombinant human p53 by purified human calpain I in vitro provides conclusive evidence that p53 is a substrate for calpains. Notably, this in vitro assay indicates that there is no dependence on additional cytoplasmic cofactors for p53 degradation by calpain I.
Mechanical strain-induced apoptosis is dependent on p53 functional activity
The previous results indicate that activated calpain I not only regulates expression and functional activity of p53 but also prevents excessive apoptosis of VSMCs exposed to cyclic stretching. We next examined the causal relation between p53 expression and increased apoptosis seen after blockade of calpain. VSMCs were exposed to cyclic stretching for 24 h, and the apoptotic index was determined (Fig. 5
A). Inhibition of p53 function by the highly specific p53-inhibitor pifithrin prevented the stretch-induced apoptosis. Furthermore, pifithrin was able to prevent the augmented stretch-induced apoptosis seen after administration of calpain inhibitors calpeptin or PD150606 as well (Fig. 5A
). However, p53 inhibition did not decrease the fraction of apoptotic cells not exposed to cyclic stretching (data not shown). Verification of these results was provided by transfection of VSMCs with p53 antisense ODNs 24 h before exposure to cyclic stretching: p53 antisense ODNs effectively prevented stretch-induced p53 expression (Fig. 5B
) and significantly reduced stretch-induced apoptosis of VSMCs, whereas treatment with reverse or scrambled ODNs had no effect (Fig. 5B, C
). These results provide evidence that stretch-induced apoptosis of VSMCs is mainly mediated in a p53-dependent manner. Furthermore, the increased apoptosis seen after inhibition of calpain activity is due to the enhanced expression of functionally active p53. These data indicate that activated calpain counteracts an excessive apoptotic response of VSMCs during exposure to mechanical stress through active regulation of p53 function by controlling the p53 protein level.
Inhibition of calpain increases medial apoptosis after balloon injury in the rat carotid artery in a p53-dependent manner
To examine the role of calpain in mechanical stress-induced apoptosis in vivo, rat carotid arteries were injured by balloon dilatation in the absence or presence of the specific calpain inhibitor calpeptin (250 µg/kg body weight). Calpeptin efficiently prevented calpain activation in the vessel wall, as determined 30 min after dilatation by Ac-LLY-AFC assay (Fig. 6
A). Furthermore, a significant increase of apoptotic nuclei within the media was observed 24 h after dilatation (12.3±2.2 vs. 0.2±0.2%; n=6; P<0.05; Fig. 6B, C
). Administration of the specific calpain inhibitor calpeptin before the injury further increased the number of apoptotic nuclei compared to dilated arteries treated with vehicle only (20.2±3.3 vs. 12.3±2.2%; n=6; P<0.05). However, no effect of calpeptin treatment vs. vehicle treatment on apoptosis was noted in noninjured arteries (0.2±0.4 vs. 0.2±0.2%; n=6; P<0.05).
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To determine whether p53 is involved in the angioplasty-induced apoptotic response and whether this is modulated by calpain in vivo as well, we examined the effect of calpeptin after deleting p53 using p53 antisense ODNs: p53 was found to be elevated 12 h after dilatation of the carotid artery, and p53 antisense ODNs effectively prevented angioplasty-induced p53 expression (Fig. 7
A). p53 antisense ODNs also significantly reduced apoptotic rate determined within the media 24 h after angioplasty, whereas treatment with scrambled ODNs had no effect (Fig. 7B
). Calpeptin treatment of the animals further augmented p53 expression in vivo, which correlated with a significantly increased number of apoptotic VSMCs 24 h after dilatation (Fig. 7A, B
). Additional p53 antisense ODN treatment reduced the number of apoptotic VSMCs to the levels in animals that had been treated with p53 antisense ODNs alone, suggesting that the augmented apoptotic response seen after calpeptin treatment is due to increased p53 expression (n=4 animals per group; *P<0.01).
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These results indicate that calpain I also acts as an endogenous regulator of mechanical stress-induced apoptosis in vivo, preventing excessive apoptosis after mechanical injury of the vessel wall.
Initial augmentation of apoptosis induced by calpain inhibition results in increased neointima formation
To elucidate whether an initially increased apoptotic response affects neointima lesion development and restenosis, we quantified neointimal lesion size 2 wk after balloon injury of the carotid artery. Single administration of the calpain inhibitor calpeptin at the day of injury resulted in significantly increased neointima formation after 2 wk (neointima to media ratio: 1.5±0.3 vs. 0.9±0.24; n=6; *P<0.01; Fig. 8
). Luminal area was reduced as well (0.17±0.03 vs. 0.26±0.05 mm2; n=6; *P<0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Postnatally, an altered balance between apoptosis and proliferation not only allows vessel remodeling but also promotes cardiovascular disease development (1)
. Hemodynamic and mechanical forces have been demonstrated to contribute to these physiological and pathological processes. For example, VSMCs exposed to mechanical stress undergo apoptosis in vitro (27
, 30)
, and a rapid wave of apoptotic VSMC death contributes to decreased cellularity in the media within hours of traumatic balloon injury in vivo (6
, 9)
. Although this initial medial VSMC apoptosis may provoke wound healing responses resulting in exacerbated neointimal lesion formation, apoptosis at later time points seems confined to VSMCs of the developing neointima and, therefore, may limit lesion growth (2
, 31)
. Indeed, an inhibitory effect of a locally delivered caspase inhibitor on neointima formation in a balloon injury model has been demonstrated (32)
. On the other hand, induction of apoptosis through inhibition of Bcl-X expression has also been shown to result in regression of vascular disease (33)
, an effect that may be explained by Bcl-X’s preferential expression in neointimal VSMCs. Our present study not only supports the concept that early arterial trauma-mediated apoptosis of medial VSMCs causes neointima formation. We further define a regulatory pathway controlling vessel architecture in disease by providing evidence that the force-induced activation of the calcium-dependent cysteine protease calpain plays an important role in limiting excess medial apoptosis through degradation and inhibition of p53.
Cell viability is governed at the molecular level by a balance between proapoptotic and antiapoptotic signals mediated by a number of gene families, the most prominent being the Bcl-2 family. The protective protein Bcl-X is abundantly expressed in normal medial VSMCs but is down-regulated after balloon injury (6)
. Similarly, in our in vitro model, mechanical stress of VSMCs resulted in a shift from the antiapoptotic Bcl-2 to the proapoptotic Bax, which was markedly pronounced when calpain activation was prevented by pharmacologic or antisense ODN inhibition. The transcription factor involved in positive Bax and negative Bcl-2 expression is p53 (21)
. p53 has not only been reported to accumulate in human atherosclerotic lesions (17)
, it has also been described to be a key mediator of mechanical stress-induced apoptosis of vascular smooth muscle cells in vitro and in vivo (27
, 28)
. In these studies, Mayr et al. provide compelling evidence for the involvement of p38, a subfamily of mitogen-activated protein kinases, in mechanical stress-induced activation of p53. We were able to prevent stretch-induced apoptosis in vitro by pretreatment of VSMCs with p53 antisense ODNs or the pharmacologic p53 inhibitor pifithrin, thereby supporting the central role of p53 in mechanosensitive VSMC apoptosis. However, simultaneous calpain activation limits excess p53 expression and thereby represents a potent counterbalance to all p53-mediated proapoptotic stimuli during mechanical stress.
In nonvascular cells, a role of calpains in degradation of p53, besides the ubiquitin-proteasome pathway, has been suggested (34)
, and in vivo activation of calpain or expression of its inhibitor, calpastatin, has been shown to modulate p53 levels (14)
. Most important, recent studies suggest a link between the role of calpain in degradation of wild-type p53 and consecutive prevention of p53-dependent apoptosis (14
, 35)
. The present study demonstrates that in VSMCs in vitro and in the vasculature in vivo, calpain is rapidly activated by mechanical stress in a calcium-dependent manner, modulating stress-induced p53 levels.
Although our study suggests that calpain mediates its antiapoptotic properties within the vasculature predominantly through p53 degradation, a certain effect of the protease on other apoptosis-related proteins has not been ruled out. Caspases themselves have been shown to be a target of calpains (36)
. On the other hand, calpains have been reported to cleave procaspase-12 to generate an active caspase and to cleave the loop region of Bcl-Xl to change an antiapoptotic molecule into a proapoptotic molecule (37)
. This way, calpains may act as negative and as positive regulators of apoptosis, resulting in a balanced effect in our models (13)
.
Mechanical stress not only induces apoptosis, it also triggers cell cycle entry of quiescent VSMCs (38)
. Vascular remodeling processes as a response to changes of mechanical force seem to be a result of a delicate balance between positive and negative regulators of cell death and cell proliferation. p53 itself is well known to function in a dual way. In addition to its proapoptotic effect, p53 negatively regulates cell proliferation through its ability to induce the cyclin-dependent kinase inhibitor p21Cip1 (22)
. Overexpression of p53 induces VSMC apoptosis in vitro and inhibits neointima formation in vivo (18
, 19)
. Consistently, p53 deficiency exacerbates atherosclerotic lesion expansion in ApoE-deficient mice fed a high-fat diet (39)
. However, this lack of p53 had no effect on the frequency of vascular cell apoptosis within the lesions; the increase in lesion size appeared to result from increased vascular cell proliferation. Similarly, calpain proves to be a multifunctional modulator of cell death and cell growth. Besides limiting excess p53-mediated apoptosis in mechanically stressed VSMCs, the requirement for calpain activity in serum-stimulated VSMC proliferation has been suggested as well (40)
. Since a recent study (41)
demonstrated an unexpected up-regulation of p53 after serum-stimulated VSMCs, thereby minimizing inappropriate accumulation of cells, an interesting question arises as to whether calpain is involved in the modulation of VSMC response to serum mitogens and contact inhibition as well.
In summary, our study supports the concept that early trauma-induced apoptosis results in increased late neointima formation and that p53 is the critical factor involved in this process. We have identified the calcium-dependent cystein protease calpain as the key modulator of trauma-induced p53 expression levels. Calpain activation counteracts mechanically induced excessive VSMC apoptosis through its p53-degrading properties, which identifies calpain as a key regulator of mechanosensitive remodeling processes of the vascular wall. Our study helps to further define regulatory pathways controlling vessel architecture and function in development and in degenerative vascular diseases.
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ACKNOWLEDGMENTS |
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Received for publication April 23, 2007. Accepted for publication August 9, 2007.
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