Loss of the Serum Response Factor Cofactor, Cysteine-Rich Protein 1, Attenuates Neointima Formation in the Mouse
Objective— Cysteine-rich protein (CRP) 1 and 2 are cytoskeletal lin-11 isl-1 mec-3 (LIM)-domain proteins thought to be critical for smooth muscle differentiation. Loss of murine CRP2 does not overtly affect smooth muscle differentiation or vascular function but does exacerbate neointima formation in response to vascular injury. Because CRPs 1 and 2 are coexpressed in the vasculature, we hypothesize that CRPs 1 and 2 act redundantly in smooth muscle differentiation.
Methods and Results— We generated Csrp1 (gene name for CRP1) null mice by genetic ablation of the Csrp1 gene and found that mice lacking CRP1 are viable and fertile. Smooth muscle–containing tissues from Csrp1-null mice are morphologically indistinguishable from wild-type mice and have normal contractile properties. Mice lacking CRPs 1 and 2 are viable and fertile, ruling out functional redundancy between these 2 highly related proteins as a cause for the lack of an overt phenotype in the Csrp1-null mice. Csrp1-null mice challenged by wire-induced arterial injury display reduced neointima formation, opposite to that seen in Csrp2-null mice, whereas Csrp1/Csrp2 double-null mice produce a wild-type response.
Conclusion— Smooth muscle CRPs are not essential for normal smooth muscle differentiation during development, but may act antagonistically to modulate the smooth muscle response to pathophysiological stress.
During vascular development, smooth muscle cells are initially highly proliferative, then become quiescent and express proteins that allow the cells to assume a highly contractive phenotype. Unlike cardiac or skeletal muscle, smooth muscle cells are not terminally differentiated; instead, these cells maintain plasticity (ie, they can alternate between a contractile “quiescent” state and a highly proliferative “synthetic” state).1 This phenotypic modulation is critical for smooth muscle cells to respond to pathophysiological stresses. Thus, perturbation of the complex signaling mechanisms that direct smooth muscle phenotypic modulation may contribute to the progression of vascular disease.2
Although no single transcription factor serves as a master regulator of smooth muscle differentiation, serum response factor (SRF) has a critical role in the smooth muscle transcriptional program.3 SRF is ubiquitously expressed; therefore, tissue-specific cofactors for SRF provide target specificity. Myocardin and the related proteins myocardin-related transcription factor (MRTF)-A and MRTF-B are established SRF cofactors that are important for smooth muscle differentiation.4 The smooth muscle cysteine-rich proteins (CRPs), CRP 1 and 2, are also implicated as important SRF cofactors. Coexpression of either CRP1 or CRP2 greatly augments smooth muscle gene expression in an SRF and GATA factor-dependent manner.5 Detailed characterization of CRP2 demonstrates that it can bind both SRF and GATA, and may bridge the 2 proteins, providing a potential mechanism for CRP-directed effects on transcription. A dominant-negative CRP2 that cannot bind SRF blocks smooth muscle differentiation in vitro, strongly implicating CRP2 as a critical SRF cofactor for smooth muscle differentiation. Despite these potent effects on smooth muscle differentiation in vitro, analysis of mice deficient for CRP2 revealed that Csrp2 (gene name for CRP2) null mice are viable and fertile. However, mice lacking CRP2 display enhanced neointima formation after vascular injury, revealing a role for CRP2 in the vascular injury response.6
Our laboratory originally identified CRP1 as a smooth muscle binding partner for the actin cross-linking protein α-actinin.7 CRP1 displays 2 lin-11 isl-1 mec-3 (LIM) domains, double zinc finger structures that serve as protein binding sites,8,9 and is expressed prominently in both vascular and visceral smooth muscle cells.10–13 CRP2 is expressed primarily in vascular smooth muscle cells (VSMCs) and mesenchymal derivatives and displays some overlap with CRP1.11,14 CRP 1 and 2 are highly related at the amino acid level (88% similar), suggesting that the 2 proteins perform similar functions.
Because of the high degree of overlap in expression between CRP 1 and 2, especially in the vasculature, the lack of a phenotype in Csrp2-null mice under unchallenged physiological conditions might reflect redundancy between these 2 family members. CRP1 is the sole family member expressed in visceral smooth muscle, such as the bladder and the gastrointestinal tract. Thus, elimination of CRP1 expression would allow us to directly test whether smooth muscle differentiation can proceed in the absence of CRPs. Herein, we report the generation and characterization of mice in which CRP1 function has been eliminated by genetic ablation of the Csrp1 gene. Surprisingly, mice that lack CRP1 are viable and fertile and display normal smooth muscle contractile function. In addition, Csrp1−/−/Csrp2−/− double-null mice are also viable and fertile. Thus, the absence of an overt vascular phenotype in the Csrp1−/− or Csrp2−/− mice is not the result of redundancy between the 2 genes. Although CRP1 is not critical for normal development, the Csrp1-null mice exhibit an attenuated response to arterial injury, a response opposite to that seen in the Csrp2-null mice. Our data indicate a role for CRP family members in sensing or responding to pathological vascular stress and maintaining smooth muscle homeostasis.
Detailed methods are described in the expanded Materials and Methods in the Data Supplement (available online at: http://atvb. ahajournals.org).
Gene Targeting and Generation of Csrp1-Deficient Mice
We generated Csrp1-null mice by gene targeting. Targeted embryonic stem cells and, subsequently, mice harboring a targeted Csrp-1 allele were identified by standard Southern blot methods.
Northern and Western Blot Analysis
RNA was isolated from mouse tissues using Trizol (Invitrogen Life Technologies, Carlsbad, Calif), following manufacturer’s instructions. The Northern blot procedure was performed as described.15 Protein extract isolation and Western blot analyses were performed as previously described.16
Analysis of Smooth Muscle Contractility
Smooth muscle contractility analysis was performed as previously described.17
Cell Culture and Immunofluorescence
Primary cultures of bladder and aortic smooth muscle cells were established, in parallel, from wild-type (WT) and Csrp1-null littermates using standard procedures.
Histological Analysis of Aorta and Bladder Specimens
Tissue sections were obtained and stained for elastin or Masson trichrome (Sigma, St. Louis, Mo).
Femoral Artery Injury
Endoluminal injury to the left common femoral artery was produced by 5 passages of a 0.01-inch diameter angioplasty guide wire (Guidant) essentially as described.18
Boyden chamber migration assays were performed using standard methods.
Cell Proliferation Analysis
Cell proliferation was evaluated based on the dye incorporation/extraction assay, as previously reported.19
Apoptosis was evaluated based on the increase in cytoplasmic nucleosomes, using a commercially available kit (Cell Death Detection ELISA plus kit; Roche, Mannhein, Germany).
Transient Transfections and Luciferase Assays
Reporter assays were performed as previously described.12,20
Quantitative Reverse Transcription–Polymerase Chain Reaction Analysis
Specific smooth muscle gene transcript levels were analyzed by a commercially available system (StepOne PCR system; Applied Biosystems, Foster City, Calif), using total RNA level.
Generation of Csrp1-Deficient Mice
A targeting vector was generated by replacing Csrp1 genomic DNA that includes exons 2, 3, and 4 with a neomycin-positive selection cassette (Figure 1A). A strategy that deletes exon 2 of the murine Csrp1 gene was chosen because this exon encodes the translation initiation codon. Targeted embryonic stem cells carrying a heterozygous disruption of the Csrp1 locus (Figure 1B) were introduced into recipient C57BL/6 blastocysts to generate chimeric mice. The resulting chimeric animals were bred to C57BL/6 partners to generate heterozygous animals that carried 1 copy of the targeted Csrp1 gene. These animals were interbred to generate homozygous mutant animals. Genotypes were confirmed by Southern analysis (Figure 1C) or by polymerase chain reaction (PCR) (Figure 1D). Western and Northern blot analysis of organ samples taken from Csrp1+/+, Csrp1+/−, and Csrp1−/− animals confirmed that no CRP1 RNA or protein is detected in the homozygous mutant mice (Figure 1E and F).
Arteries and Other Smooth Muscle–Containing Organs Maintain Normal Morphology and Contractility in Csrp1-Null Mice
Genotypic analysis of the offspring derived from Csrp1+/− heterozygous parents revealed that normal mendelian ratios were obtained, indicating that zygotic expression of CRP1 is not required for embryonic development (Figure 2A). Interbreeding of Csrp1−/− animals resulted in litters of a normal size. Because CRP2 is closely related to CRP1 (90% similar), and is also expressed in vascular smooth muscle,10–14 we examined whether CRP2 expression was upregulated in the Csrp1−/− mutant animals. By Northern analysis of RNA isolated from aorta, bladder, and lung (ie, tissues that normally display robust CRP1 expression), we found no consistent evidence of increased CRP2 expression (Figure 1F).
We examined the vasculature in detail to determine if loss of CRP1 had any impact on organization of the arterial vessels. Labeling arterial cross sections with Verhoff stain did not reveal any difference in the morphology or number of smooth muscle lamellar units and elastic fibers (Figure 2B and C). Trichrome staining of aortic sections also did not indicate any gross change in organization and morphology of the arterial vessels from the Csrp1−/− mice (Figure 2D and E). Because CRP2 is also expressed in arterial smooth muscle, we also examined bladder sections, in which CRP1 is the sole family member expressed. No morphological difference was detected between Csrp1−/− bladder and WT (data not shown), indicating that loss of CRP1 does not grossly affect the development of smooth muscle–containing organs.
CRP1 has been reported to bind actin and to bundle actin filaments,21 suggesting that Csrp1−/− smooth muscle cells might be expected to exhibit an altered morphology or cytoarchitecture. We examined smooth muscle cytoskeletal organization in isolated VSMCs from WT and Csrp1−/− animals to determine if loss of CRP1 had any impact on smooth muscle cytoarchitecture. CRP1 was associated with actin filaments and adhesion sites in WT cells (Figure 2F); no CRP1 protein was detected in cells derived from the mutant animals (Figure 2F). By using smooth muscle α-actin (SMA) and vinculin, we observed that actin filament organization (Figure 2G and G’) and focal adhesions (Figure 2H and H’) appear morphologically indistinguishable in WT and Csrp1-null cells. Because VSMCs express both CRP 1 and 2, we also examined primary bladder smooth muscle cells, which normally express only CRP1. Similar to that seen for VSMCs, we did not detect any alteration in cell cytoarchitecture in bladder smooth muscle cells derived from Csrp1-null mice (data not shown).
Although smooth muscle–containing organs maintain a normal morphology in the absence of CRP1, it remained possible that smooth muscle function could still be compromised in the Csrp1−/− mice. For example, functional analysis of muscle LIM protein (MLP), the striated muscle-specific CRP isoform, revealed a critical role for MLP in cardiomyocyte contractile response.22–24 We directly tested contractility of excised smooth muscle using standard physiology methods that delineate dose-response curves and maximal force generation. Measurements of isometric force generation of aortic smooth muscle in response to potassium chloride or phenylephrine are summarized in Figure 3. Loss of CRP1 did not lead to any alterations in concentration-force relations with or without endothelium (repeated-measures ANOVA, P>0.50). Maximal force generation by either potassium chloride or phenylephrine in the endothelium-denuded condition, which solely reflects contractility of smooth muscle layers in the aorta, did not reveal a difference between WT and Csrp1−/− mutants (ANOVA, P>0.50).
Aortic smooth muscle cells express both CRP 1 and 2; therefore, we repeated the contractility analysis on bladder tissue derived from WT and Csrp1-null mice, because bladder expresses only CRP1. Force generation, elicited by either potassium chloride (Figure 3D) or carbachol (data not shown), was similar for both WT and Csrp1-null bladder samples. From these analyses, we conclude that CRP1 is not critical for normal smooth muscle contraction.
Smooth Muscle Cell Differentiation Appears Normal in Csrp1-Null Mice
CRPs have been shown to synergize with SRF and GATA factors and to potently upregulate smooth muscle gene expression.5 To directly address whether the loss of CRP1 has an impact on the steady-state expression of smooth muscle–specific proteins, we compared the expression of smooth muscle markers in tissues from WT and Csrp1-null mice. By immunoblot analysis, we did not detect any alteration in the levels of SMA or smooth muscle myosin heavy chain (SM-MHC) in aortic lysates from Csrp1- or Csrp2-null mice (Figure 4A). Vinculin, a prominent dense plaque component critical for smooth muscle cell adhesion, was also present at WT levels. Likewise, smooth muscle marker expression was not altered in mesenteric artery samples (Figure 4A), indicating that different vessel types (eg, elastic versus muscular) did not show an altered response to loss of Csrp1. We also examined smooth muscle marker expression in bladder and lung samples, 2 tissues in which CRP1 is the sole CRP family member expressed; no difference in steady-state expression of representative smooth muscle–specific proteins was observed (data not shown). Thus, despite the potent capacity of CRP1 to stimulate smooth muscle–specific gene expression in vitro, smooth muscle gene expression persists in mice lacking Csrp1.
To further assess the impact of loss of CRP1 on smooth muscle transcription, we determined the capacity of isolated VSMCs to activate an SRF-dependent reporter when stimulated with transforming growth factor (TGF) β. The SMA promoter-luciferase construct is a commonly used reporter, which has been shown to be TGF-β responsive and CArG dependent.25 Both Csrp1−/− and WT cells displayed a similar response and upregulated the SMA-luciferase reporter approximately 2-fold after TGF-β stimulation (Figure 4B). Interestingly, Csrp2−/− cells seem to be refractory to TGF-β stimulation. The ability of the WT and Csrp1−/− cells to activate the SMA reporter was SRF dependent, because an SMA reporter with a mutated CArG was not induced in either cell type by TGF-β. Consistent with our studies with the SMA reporter, a luciferase reporter containing 3 copies of the intronic CArG element from the Csrp1 gene12,26 was also upregulated by TGF-β in the WT and Csrp1−/− cells, whereas the Csrp2−/− cells were unresponsive (supplementary data, Figure S1, panel A).
To determine if the lack of activity observed in Csrp2−/− cells was the result of an inability to respond to TGF-β, we examined the induction of smooth muscle gene expression by measuring endogenous RNA. Both Csrp1−/− and Csrp2−/− cells exhibited a similar induction of SMA, smooth muscle 22α, and calponin in response to TGF-β (Figure 4C and supplementary data, Figure S1, panels B and C). These data indicate that Csrp2-null cells are not defective in TGF-β signaling per se, but have compromised SRF-dependent transcriptional activity, consistent with the normal expression of SMA and other smooth muscle proteins in the isolated tissues (Figure 4A).
Mice Lacking Both CRP 1 and 2 are Viable and Fertile
Our observation that loss of CRP1 did not overtly affect smooth muscle development or contractility, even in tissues in which it is the sole CRP expressed, strongly suggests that CRPs are not essential for smooth muscle development. To provide further support for this conclusion, we crossed the Csrp1−/− and Csrp2−/− mice to create animals that were null for both Csrp1 and Csrp2. Like the Csrp1−/− or the Csrp2−/− null animals, mice lacking both CRP 1 and 2 were viable (Figure 5A). Moreover, interbreeding of double-null Csrp1−/−/ Csrp2−/− animals resulted in normal-sized litters (approximately 8 per litter). Together, these results indicate that the smooth muscle CRPs (CRP 1 and 2) are not essential for smooth muscle development.
Mice Lacking CRP1 Show Attenuated Response to Guide Wire–Induced Arterial Injury
Although the principal function of unchallenged smooth muscle is contraction, smooth muscles also play a role in maintaining and repairing injured tissue. For example, in response to arterial injury, smooth muscle cells quickly switch from a quiescent phenotype to a proliferative and migratory phenotype, leading to neointima formation.2,27 The involvement of CRP2 in neointima formation prompted us to determine if the Csrp1−/− mice display an altered response to vascular injury. Because CRP 1 and 2 are highly related at the amino acid level, we anticipated a similar response to the wire-induced arterial injury (eg, increased neointima formation).
Surprisingly, neointima formation in Csrp1−/− mice was significantly decreased compared with WT mice (Figure 5B and C). This result was in striking contrast to the enhanced neointima formation in Csrp2−/− mice (Figure 5D and Wei et al6). Attenuated neointima formation in Csrp1−/− mice after the arterial injury led us to ask if the Csrp1−/−/Csrp2−/− double-null mice would produce a “balanced” response and, thus, show less neointima formation than Csrp2−/− mice. Indeed, double-null mice showed a statistically significant decrease in the thickness of the neointima compared with what we observed for Csrp2−/− mice (Figure 5E and F, P<0.05 by ANOVA and Bonferroni test).
The increased neointima formation seen in the Csrp2−/− mice is thought to be caused, at least in part, by increased vascular smooth muscle migration during neointima formation.6 We assessed the migration of Csrp1−/− and WT VSMCs toward the chemoattractant platelet-derived growth factor BB, and found that both cell types exhibited a similar migratory capacity (Figure 6A). Smooth muscle proliferation also contributes to neointima formation; therefore, we measured the proliferation rates of isolated Csrp1−/− and WT VSMCs during a 6-day period. Both cell types displayed similar proliferation rates during the entire time course (Figure 6B). Studies by Latonen et al28 indicate that CRP1 may have antiapoptotic properties; if loss of CRP1 resulted in increased cell death, this could account for the attenuated neointima formation seen in the Csrp1-null mice. To test this hypothesis, we determined whether the Csrp1−/− cells undergo increased apoptosis under subconfluent and confluent growth conditions. WT, Csrp1−/−, and Csrp2−/− cells all exhibit an equivalent low level of apoptosis, as detected by the accumulation of cytoplasmic nucleosomes (Figure 6C). Together, these results indicate that loss of CRP1 does not impact vascular smooth muscle proliferation, migration, or apoptosis, at least under these assay conditions.
Another aspect of phenotypic modulation is the redifferentiation of smooth muscle cells after neointima formation. We examined the redifferentiation capacity of isolated VSMCs by allowing cultures to become confluent and assaying the expression of SM-MHC. When first isolated, cultured VSMCs lose expression of SM-MHC and other smooth muscle markers but regain expression when cultures become confluent.29 In newly confluent cultures, we still detect abundant nonmuscle myosin (data not shown) and begin to see expression of SM-MHC (Figure 6D). By 2 days postconfluence, WT cells have continued to upregulate SM-MHC, but the Csrp1−/− cells do not display the same degree of SM-MHC upregulation (Figure 6D). Freshly isolated aortas from both WT and Csrp1−/− mice express similar levels of SM-MHC (Figure 4A), suggesting that the rate of re-expression of SM-MHC or maximal smooth muscle redifferentiation is compromised in the Csrp1−/− cells.
The CRPs are muscle-enriched, actin-associated proteins strongly implicated in muscle gene regulation and differentiation.5,7,13,22,30,31 Tissue culture–based experiments have shown that all 3 CRPs can greatly augment muscle gene transcription and that disruption of CRP function in vitro can block muscle differentiation. Therefore, it was unexpected that loss of the smooth muscle CRPs or striated muscle CRP (CRP3/MLP) has no overt affect on muscle development in vivo. However, these studies of CRP-deficient mice have revealed important roles for CRPs in maintaining normal muscle function. Loss of either CRP1 (present study) or CRP26 alters neointima formation in response to arterial injury, whereas MLP/Csrp3-deficient mice develop dilated cardiomyopathy postnatally.22 Collectively, these data demonstrate that CRPs are not essential for muscle development and instead have important functions in responding to pathophysiological stress and maintaining muscle homeostasis.
CRPs have the capacity to drastically potentiate smooth muscle gene expression, leading to the hypothesis that smooth muscle gene expression would be compromised in the absence of CRPs. We tested this hypothesis directly by generating single and double Csrp1/Csrp2-null mice. The resulting mice are viable and fertile, with apparently normal smooth muscle function and gene expression. However, a more detailed analysis revealed that isolated Csrp2−/− cells do not upregulate an SRF-dependent reporter in response to TGF-β (Figure 4B), indicating that loss of CRP2 can influence transcription. In addition, the expression of SM-MHC was attenuated in postconfluent Csrp1−/− cells (Figure 6D). Our results suggest that although CRPs are not absolutely essential in vivo for smooth muscle gene expression to proceed, their loss can impact smooth muscle gene expression under specific physiological or pathological conditions. Studies of smooth muscle gene regulation specifically during neointima formation in Csrp1−/− mice should help elucidate the contribution of CRP1 to smooth muscle gene expression in vivo.
The cytoskeletal role of CRPs has the potential to influence both cell morphology and gene expression. CRP 1 and 2 have been shown to bind and bundle actin filaments21,32 and, thus, may participate in actin remodeling that occurs during smooth muscle phenotypic modulation. Indeed, overexpression of CRP1 increases actin stress fiber thickness, and CRP1 translocates with actin to membrane ruffles in platelet-derived growth factor–treated fibroblasts.21 This observation is especially intriguing, given that platelet-derived growth factor is a potent humoral trigger of neointima formation in vivo.33 CRP1-mediated actin bundling could influence the G-actin to F-actin ratio, a critical regulator of SRF-dependent smooth muscle gene expression and differentiation.34–36 The identification of the mechanism by which CRPs affect actin stress fiber dynamics should help elucidate its potential contribution to smooth muscle cell behavior during vascular injury.
Several factors influence neointima formation, including smooth muscle cell apoptosis, proliferation, and migration.37 Our in vitro studies of primary VSMCs from the Csrp1-null mice did not indicate a defect in these aspects of smooth muscle cell behavior, at least under our experimental conditions. Instead, we show that cultured Csrp1−/− VSMCs express lower levels of SM-MHC under postconfluent conditions. Under these conditions, WT cells re-express late differentiation markers, such as SM-MHC, and become quiescent, essentially entering a redifferentiation program. During neointima formation, smooth muscle cells initially dedifferentiate, but as the neointima becomes established, the smooth muscle cells that invaded the intimal region redifferentiate and express smooth muscle markers.2 If smooth muscle redifferentiation is compromised, the neointima may be unstable, resulting in loss or regression of the vascular occlusion.
Alternatively, the attenuated SM-MHC expression in Csrp1−/− cells may indicate a defect in the rates of redifferentiation and dedifferentiation. Recent work by Sayers et al38 showed that loss of the focal adhesion kinase (FAK) inhibitor FRNK led to attenuated smooth muscle marker expression during postnatal growth and after vascular injury, with no overt effect on vascular function under normal physiological conditions. After vessel maturation, aortas from FRNK−/− mice expressed smooth muscle markers at normal levels, indicating that the rate of smooth muscle differentiation is compromised of loss of FRNK. These observations suggest that cellular factors can regulate the rate of smooth muscle phenotypic modulation, which may be critical for timing cellular events that lead to neointima formation.
Vascular phenotypes revealed by this study for CRP1 and by the study by Wei et al6 for CRP2 highlight intriguing features of CRP family members expressed in smooth muscle. Csrp1−/− mice show attenuated neointima formation in response to the arterial injury, whereas Csrp2−/− mice show an excessive response. These results were surprising, given that CRP 1 and 2 are highly similar (88%). However, CRP 1 and 2 also possess regions of dissimilarity that are evolutionarily conserved, suggesting the possibility of some uniqueness in the binding partner repertoire for CRP 1 and 2. Thus, the smooth muscle CRPs may have common properties (eg, actin binding) and unique protein partners in smooth muscle that allow them to perform specific functions. For example, our studies indicate that CRP 1 and 2 have distinct effects on SRF-dependent smooth muscle gene regulation. We demonstrate that Csrp2−/− VSMCs do not activate a CArG-luciferase or an SMA-luciferase reporter in response to TGF-β stimulation, whereas the Csrp1−/− cells retain this capacity (Figure 4B and Supplemental Figure 1).
The observation that animals lacking both CRP 1 and 2 have a more “balanced” response to arterial injury strongly suggests that the 2 proteins somehow antagonize each other or are involved in different cellular responses activated by arterial injury. Thus, coexpression of CRP 1 and 2 may act to fine-tune the response of the VSMC to pathological stresses, such as arterial injury. The molecular programs initiated during vascular injury are still poorly understood. Our findings highlight the importance of further characterizing the molecular functions of CRPs in normal and pathological smooth muscle, and will hopefully contribute to our understanding of the pathophysiology of vascular disease.
We thank Tess Macalma, who participated in the early stages of this study; Kirk Thomas, PhD, who provided helpful suggestions regarding the gene-targeting strategy; J. David Symons, PhD, who provided guidance in mesenteric artery dissection; and Chris Jensen and Laura Hoffman, PhD, who assisted with maintenance and backcrossing of the mouse colony.
Sources of Funding
This study was supported by grants HL076428 (Dr Lilly), HL66044 (Dr Paul), and HL60591 (Dr Beckerle) from the National Heart, Lung, and Blood Institute; the LAM Foundation (Dr Lilly); GM50877 from the National Institute of General Medicine Sciences (Dr Beckerle); the Huntsman Cancer Foundation (Dr Beckerle); and Cs-098.pp-06 from the National Health Research Institutes (Taiwan) (Dr. Yet).
Received on: January 7, 2009; final version accepted on: December 7, 2009.
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