Published online before print March 25, 2004, doi: 10.1161/01.RES.0000126417.38728.F6
© 2004 American Heart Association, Inc.
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SM22
Modulates Vascular Smooth Muscle Cell Phenotype During Atherogenesis
From the Institut für Pharmakologie und Toxikologie, Technische Universität, München, Germany.
Correspondence to Robert Feil, Institut für Pharmakologie und Toxikologie, Technische Universität, Biedersteiner Str. 29, 80802 München, Germany. E-mail feil{at}ipt.med.tu-muenchen.de
Abstract
The function of cytoskeletal proteins in the modulation of vascular smooth muscle cell (SMC) phenotype during vascular disease is poorly understood. In this report, we used a combination of gene targeting and Cre/lox-mediated cell fate mapping in mice to investigate the role of SM22
, an SMC-specific cytoskeletal protein of unknown function, in the development of atherosclerosis. In hypercholesterolemic ApoE-deficient mice, genetic ablation of SM22 resulted in increased atherosclerotic lesion area and a higher proportion of proliferating SMC-derived plaque cells. These results identify a role for SM22 in the regulation of SMC phenotype during atherogenesis.
Key Words: mouse • vascular remodeling • phenotypic modulation • Cre recombinase • fate mapping
SM22 (also known as transgelin, WS3-10, or p27) is a 22-kDa protein that is considered a marker of contractile smooth muscle cells (SMCs) and is exclusively and abundantly expressed in SMCs of adult animals. It is structurally related to the actin-binding protein calponin and has been localized within the cytoskeletal apparatus (see review1). The analysis of SM22-deficient mice showed that the protein is not required for the development and basal homeostatic functions of SMCs,2 suggesting that its function can be compensated by homologous proteins like calponin and SM22ß.3 The role of SM22 under pathophysiological settings such as atherosclerosis was not studied yet. A critical step in atherogenesis is the phenotypic modulation of vascular SMCs from contractile to synthetic/proliferative cells,4 a process which may involve the cytoskeleton.5 In this study, we have investigated the function of SM22 in a mouse model of atherosclerosis by using gene targeting technology combined with temporally controlled Cre/lox-mediated activation of a reporter gene to visualize the fate of SMCs during plaque development.6,7
Materials and Methods
Western blot analysis, immunohistochemistry, X-Gal and Oil Red O staining, measurement of plasma lipids, and quantification of atherosclerotic lesion area were performed as described previously7,8 and in the online data supplement at http://circres.ahajournals.org.
Results and Discussion
The generation of mice carrying a SM22 knock-in allele, also designated SM-CreERT2(ki), has been described.8 The structures of the SM22 wild-type (+) and knock-in (–) allele are depicted inFigure 1A. The knock-in allele expresses the tamoxifen-activated CreERT2 recombinase instead of the endogenous SM22 gene. Western blot analysis demonstrated that the SM22 protein was expressed in SMC-containing tissues of SM22+/+ and SM22+/– mice, but not in SM22–/– mice (Figure 1B). Confirming previous results with another SM22-deficient mouse line,2 the SM22–/– mice generated in the present study were viable and fertile, and exhibited no obvious phenotype of SMC-containing tissues under basal conditions (data not shown). As measured by the tail-cuff method, heart rate and mean blood pressure of SM22–/– mice (644±19 bpm and 87±5 mm Hg) was not different from wild-type mice (620±24 bpm and 88±5 mm Hg).
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To study the role of SM22 in the modulation of SMC phenotype during atherogenesis, control (CTR) and SM22 knockout (KO) mice were generated on an ApoE-deficient9 C57BL/6 genetic background (genotype: SM22+/–; ApoE–/– and SM22–/–; ApoE–/–, respectively) and fed an atherogenic diet (20% fat, 1.5% cholesterol) for 8 or 16 weeks. The presence of the knock-in allele in both CTR and KO mice allowed us to control for potential unspecific effects related to CreERT2 expression as well as to label SMCs during atherogenesis (see later). SM22 was expressed in aortic SMCs of CTR mice and appeared to be downregulated in atherosclerotic lesions (Figure 1C), in agreement with published data.10 Body weight and plasma lipid levels were not significantly different between CTR and KO mice (online Table 1 in the online data supplement).
The mean atherosclerotic lesion area, as determined by Oil Red O staining of the aorta, was significantly elevated in KO mice compared with CTR mice (Figures 2A through 2D). In KO mice, the lesion area in the aortic arch (Figures 2A and 2C) after 8 and 16 weeks on the atherogenic diet, respectively, was increased by 
39% and 27% in female animals, and by 60% and 28% in male animals. Similarly, the lesion area of KO mice in the brachiocephalic artery, left carotid artery, and left subclavian artery (Figures 2B and 2D) after 8 and 16 weeks on the atherogenic diet, respectively, was increased by 82% and 48% in female animals, and by 65% and 23% in male animals. These results indicated that SM22 inhibits plaque growth at various stages and sites of atherogenesis in both female and male mice. The relative increase in lesion area of KO versus CTR mice was higher after 8 weeks than 16 weeks, suggesting a role for SM22 in the initiation of SMC phenotypic modulation.
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To determine the effect of SM22 deficiency on the properties of plaque SMCs in vivo, the fate of SMCs during the development of atherosclerotic lesions was followed in CTR and KO mice that carried the ROSA26 Cre reporter (R26R) allele11 (genotype: SM22+/–; ApoE–/–; R26R+/– and SM22–/–; ApoE–/–; R26R+/–, respectively). The R26R allele encodes a nonfunctionallacZ gene, which is activated by Cre-mediated recombination. The activated R26R allele is inherited to all progeny cells, which allows one to follow the fate of the originally labeled cells by staining tissues with X-Gal for ß-galactosidase activity. Both CTR and KO mice expressed the tamoxifen-dependent CreERT2 recombinase from the endogenous SM22 promoter (Figure 1A). Thus, CTR and KO mice were treated with tamoxifen to label SM22-expressing and SM22-deficient SMCs, respectively. After 16 weeks on the atherogenic diet, plaques were stained with X-Gal. Blue-stained cells in the lesions of CTR and KO mice should have been derived from SM22-expressing and SM22-deficient medial SMCs, respectively. We did not detect positive X-Gal staining in the bone marrow of tamoxifen-treated mice. Thus, it is unlikely that bone marrow–derived cells contributed to the stained lesional cells. In sections of atherosclerotic plaques, blue-stained cells were detected in the media and inside the lesion. The stained plaque area was strongly increased in KO mice compared with CTR mice (Figure 2E). Quantitative analysis of SMC-derived cells (X-Gal positive) and of proliferating cells (positive for proliferating cell nuclear antigen, PCNA) revealed significant differences in plaque composition between CTR and KO mice (Figure 2E; online Table 2. In plaques from KO mice, the fraction of SMC-derived cells and of proliferating cells was increased. Proliferating SMC-derived cells accounted for 6±1% and 15±1% of total plaque cells in CTR and KO mice, respectively. Thus, the increased lesion area in KO mice can be attributed, at least in part, to a higher number of proliferative SMC-derived plaque cells. Interestingly, some of the blue-stained cells were also positive for the macrophage marker, MAC-2 (Figure 2E), indicating that these cells were derived by transdifferentiation of SMCs to a macrophage-like state.12
These results suggest that SM22 restricts plaque growth by inhibiting the phenotypic modulation of SMCs from contractile to synthetic/proliferative cells. Although the phenotypic modulation of SMCs has been considered a key pathogenic factor in atherogenesis, it is increasingly recognized that intimal SMCs play an important role in synthesizing and maintaining the protective fibrous cap of an advanced lesion.13,14 Failure of this repair response leads to weakening of the cap and plaque rupture, with potentially fatal consequences. Thus, SM22 might restrict both plaque growth and stability making it difficult to judge whether the overall effect of this protein in atherogenesis is protective or deleterious.
Taken together, the analysis of SM22-deficient mice supports the concept that cytoskeleton-associated proteins play an important functional role in the phenotypic modulation of SMCs during vascular remodeling. The present study focused on the role of SM22 in the development and proliferation of SMC-derived plaque cells. However, SM22 may also regulate SMC apoptosis as well as factors that are secreted by SMCs and affect the recruitment and proliferation of other cells in the lesion. The molecular mechanism behind the effect of SM22 on SMC phenotype is presently unknown, but may involve the reorganization of the actin cytoskeleton.2,15,16
Acknowledgments
This work was supported by the VolkswagenStiftung and the Deutsche Forschungsgemeinschaft. We thank Sabine Brummer, Anna-Maria Knorn, Wiebke Wolfsgruber, and Carsten Wotjak for help and Mario Gimona for the antiserum to SM22.
Footnotes
Original received October 15, 2003; first resubmission received January 12, 2004; second resubmission received February 12, 2004; revised resubmission received March 4, 2004; accepted March 11, 2004.
References
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