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SM22 (or transgelin), an actin-binding protein abundant in vascular smooth muscle cells (VSMC), is downregulated in atherosclerosis, aneurysm and various cancers. Abolishing SM22 in apolipoprotein E knockout mice accelerates atherogenesis. However, it is unclear whether SM22 disruption independently promotes arterial inflammation.
To investigate whether SM22 disruption directly promotes inflammation upon arterial injury and to characterize the underlying mechanisms.
Using carotid denudation as an artery injury model, we showed that Sm22 knockout (Sm22−/−) mice developed enhanced inflammatory responses with higher induction of pro-inflammatory genes, including Vcam1, Icam1, Cx3cl1, Ccl2 and Ptgs2. Higher expression of these genes was confirmed in primary Sm22−/− VSMCs and in PAC1 cells after Sm22 knockdown, while SM22 recapitulation in primary Sm22−/− VSMCs decreased their expression. NF-κB pathways were prominently activated in both injured carotids of Sm22−/− mice and in PAC1 cells after Sm22 knockdown and may mediate upregulation of these pro-inflammatory genes. As a NF-κB activator, reactive oxygen species (ROS) increased in primary Sm22−/− VSMCs and in PAC1 cells after Sm22 knockdown. ROS scavengers blocked NF-κB activation and induction of pro-inflammatory genes. Furthermore, Sm22 knockdown increased Sod2 expression and activated p47phox, reflecting contributions of mitochondria and NADPH oxidase to the augmented ROS production; this may result from actin and microtubule cytoskeletal remodeling.
Our findings show that SM22 downregulation in VSMCs can independently promote arterial inflammation through activation of ROS-mediated NF-κB pathways. This study provides initial evidence linking VSMC cytoskeleton remodeling with arterial inflammation.
SM22, also known as SM22α or transgelin, is a 22 kDa protein abundant in the smooth muscle cells (SMCs) of vertebrates1. It belongs to the calponin family since it contains an N-terminal calponin homology domain and a C-terminal calponin-like domain2. The basic molecular function of SM22 is to bind actin and facilitate the formation of cytoskeletal structures such as stress fibers2.
SM22 dysregulation is observed in a variety of human diseases. For instance, expression of SM22 is decreased in several types of cancer3. Expression of SM22 is also downregulated in atherosclerotic arteries4, 5 and abdominal aortic aneurysms6. These findings suggest a correlation between decreased SM22 expression and arterial diseases. However, it is unclear whether the SM22 downregulation promotes the pathogenesis of arterial diseases or whether it is simply a passive outcome.
SM22 has been widely used as a SMC marker during embryogenesis and in adult7. Sm22 knockout (Sm22−/−) mice are viable and fertile with uncompromised vasculature development8-10. This suggests that SM22 may be either functionally redundant or compensated during vasculature development. However, the role of SM22 may not be compensated under pathological conditions. Indeed, loss of SM22 in apolipoprotein E knockout (ApoE−/−) mice led to enlarged atherosclerotic lesions with prominent macrophage infiltration, a sign of enhanced inflammation9. It is well documented that inflammation is involved in development of arterial diseases such as atherosclerosis11. However, the molecular mechanisms underlying the observed increase of inflammation in Sm22−/−ApoE−/− mice have not been characterized: it is unclear whether loss of SM22 independently promotes inflammation. To address this issue, we examined the expression of NF-κB and its target pro-inflammatory genes in Sm22−/− mice in response to carotid denudation12. We also performed this analysis in primary Sm22−/− vascular smooth muscle cells (VSMCs) as well as in PAC1 cell (a VSMC cell line) after Sm22 knockdown. Our results revealed that disruption of SM22, a cytoskeletal protein, promoted arterial inflammation through reactive oxygen species (ROS)-mediated NF-κB pathways. This study suggests that VSMC cytoskeleton integrity may be critical to maintaining artery homeostasis.
An expanded Materials and Methods section is available in the Online Supplement at http://circres.ahajournals.org.
Total RNA from mouse carotids or primary VSMCs (from aortae of Sm22−/− and Sm22+/+ mice) or PAC1 cells (a VSMC cell line from rat pulmonary artery) after transfection with Sm22 siRNA (small interfering RNA) was used for real-time RT-PCR (rtRT-PCR). The cell lysates from primary VSMCs or PAC1 cells were used for Western Blotting (WB) and electrophoresis migration shift assay (EMSA).
ROS in live primary VSMCs or PAC1 cells was detected using dihydroethidium (DHE) for superoxide and dichlorodihydrofluorescein (DCFDA) for peroxide. Immunofluorescence (IF) in PAC1 cells were performed on chamber slides.
Five Sm22−/− mice and five Sm22+/+ littermates were used in histology, immunohistochemistry (IHC) and rtRT-PCR analyses. Primary VSMCs from four Sm22−/− mice and four Sm22+/+ mice were used for rtRT-PCR analyses. Three independent Sm22 knockdown experiments were performed in PAC1 cells. Semi-quantitative analyses were performed using the Image-Pro software. Statistical analyses were performed using SPSS13.0 software.
To determine the roles of SM22 under pathological conditions, we performed carotid denudation using the Sm22−/− mice and their Sm22+/+ littermates. Before injury, the size of the media and adventitia for their carotids are similar (Online Figure IA, B). Two weeks after injury, we found that the injured carotids from Sm22−/− mice swelled significantly more than those from Sm22+/+ mice. In addition, the response was similar between Sm22+/+ and Sm22+/− littermates (Online Figure IC). These carotids adhered tightly to the surrounding tissues and were difficult to isolate. Hematoxylin and eosin (H&E) staining showed thicker and fibrotic artery walls (Fig. 1A, Online Figure IA, B) with remarkable cell infiltration in the media and adventitia of the carotids in Sm22−/− mice (Fig. 1A). IHC using F4/80 (a macrophage specific marker) (Fig. 1B) and CD3 (a T lymphocyte marker) (Fig. 1C) revealed greater macrophage and T lymphocyte infiltration in the injured carotids of Sm22−/− mice. These findings demonstrate enhanced inflammatory response of Sm22−/− mice upon artery injury.
To reveal the molecular mechanisms underlying the inflammation prone scenario in carotids of Sm22−/− mice after injury, we investigated the expression of several major pro-inflammatory molecules using rtRT-PCR in whole carotids and IHC in the VSMC-rich media of carotids. Cell adhesion molecules, including vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1), contribute to arterial inflammation via retention of inflammatory cells such as macrophages and T lymphocytes in the inflammation sites13. There was no significant difference between the two groups in mRNA expression of either adhesion molecule without injury (Fig. 2A, left panel); however, the injury induced change of Vcam1 mRNA expression was two times higher in Sm22−/− mice (Fig. 2A, right panel). IHC showed that expression of both VCAM1 (Fig. 2B) and ICAM1 (Fig. 2C) in the media was five times higher in Sm22−/− mice. Chemokine (C-X3-C motif) ligand 1 (CX3CL1) and monocyte chemotactic protein-1 (CCL2), two chemokines that potently recruit monocytes and T lymphocytes, participate in various arteriopathies13. Both the basal level and injury induced change of Cx3cl1 mRNA appears to be higher in Sm22−/− mice (Fig. 3A, left panels), while no obvious difference was observed for Ccl2 (Fig. 3A, right panels). Protein expression of CX3CL1 (Fig. 3B) in the media was three times higher in Sm22−/− mice while the difference in CCL2 (Fig. 3C) was not significant. Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cycloxygenase 2, is a characteristic inflammation marker since it mediates the synthesis of the vessel-active prostaglandin during inflammation14. Despite similar transcriptional levels in the whole carotids (Online Figure IIA), PTGS2 expression was two times higher in carotid media of Sm22−/− mice than in those of their Sm22+/+ littermates (Online Figure IIB). These findings highlight a pro-inflammatory environment in injured carotid of Sm22−/− mice where inflammatory cells were recruited, retained and activated.
Since the expression of the pro-inflammatory molecules was mainly located in the VSMC-rich artery media, we focused our efforts on analyzing VSMCs. We isolated VSMCs from aortae of both Sm22−/− and Sm22+/+ mice for primary culture. VSMCs from Sm22−/− mice expressed higher levels of Vcam1, Icam1, and Ccl2 (Fig. 4A). We also knocked down Sm22 in a VSMC line, PAC1 cells, using siRNA (Fig. 4B and 4C). As the Sm22 knockdown efficiency increased over time (Fig. 4B), the expression of the aforementioned pro-inflammatory molecules was gradually induced (Fig. 4B). Similarly, the extent of induction of Vcam1, Cx3cl1 and Ccl2 appeared to correlate with Sm22 knockdown efficiency using three different siRNAs (Online Figure IIIA). Furthermore, to investigate if recapitulation of SM22 could reverse the pro-inflammatory traits of VSMCs in the absence of Sm22, we re-introduced SM22 into primary Sm22−/− VSMCs via plasmid transfection and observed 40-60% decrease in expression of Vcam1, Icam1, Cx3cl1 and Ccl2 (Online Figure IIIB).
These observations highlight the intrinsic pro-inflammatory character of VSMCs after disruption of Sm22 expression and underscore the contributions of medial VSMCs to the enhanced arterial inflammatory responses upon injury in Sm22−/− mice.
The activation of multiple pro-inflammatory genes in injured carotids of Sm22−/− mice suggested their transcriptional coregulation. The NF-κB pathways are key pro-inflammation pathways, and the aforementioned pro-inflammatory molecules are targets of activated NF-κB. Thus, we examined whether NF-κB activation was higher in injured carotids of Sm22−/− mice than in those of their Sm22+/+ littermates. The activation of NF-κB pathways was shown by the ratio of number of RELA or NFKB2 positive nuclei to the number of all nuclei in the media from five Sm22−/− mice and Sm22+/+ littermates (Fig. 5A, 5B, the bottom panel). As a canonical NF-κB pathway activation marker, the nuclear RELA (also known as p65) appeared to be higher (about 1.5 times) in Sm22−/− mice (Fig. 5A, bottom panel). However, the nuclear NFKB2 (also known as p52), a non-canonical NF-κB pathway activation marker, was significantly higher (more than 2 times) in Sm22−/− mice (Fig. 5B, bottom panel). Consistent with this observation, we detected more nuclear NFKB2 protein in primary Sm22−/− VSMCs (Online Figure IVA) by WB using nuclear extracts, while we failed to detect nuclear RELA in either Sm22+/+ or Sm22−/− primary VSMCs (Online Figure IVA). The difference in nucleus distribution pattern between RELA and NFKB2 suggested that the non-canonical NF-κB pathway may be involved in transactivating NF-κB target genes under this condition.
To test whether NF-κB was also activated after Sm22 knockdown, we performed WB using cytoplasmic and nuclear fractions from PAC1 cells. After Sm22 knockdown, the cytoplasmic IκB level decreased, while the nuclear RELA level increased drastically; this effect was diminished by Bay-11-7082 (a NF-κB pathway inhibitor) (Fig. 6A). Since degradation of cytoplasmic IκB and nuclear translocalization of RELA reflect activation of the canonical NF-κB pathway15, we concluded that Sm22 knockdown in PAC1 cells activated the canonical NF-κB pathway. Sm22 knockdown increased NFKB2 protein level (Fig. 6B); Bay-11-7082 inhibited the processing of p100, the NFKB2 precursor, into NFKB2 and prevented NFKB2 from nuclear translocation upon Sm22 knockdown (Fig. 6B). These findings indicate that Sm22 disruption activated NF-κB pathways, consistent with the above in vivo observation (Fig. 5). We further tested alteration of nuclear NF-κB binding activity in PAC1 cells after Sm22 knockdown using a consensus NF-κB probe. The NF-κB binding activity was increased after Sm22 knockdown (Fig. 6C), and Bay-11-7082 reduced this increase (Fig. 6C).
We then investigated whether NF-κB activation participates in the induction of the aforementioned pro-inflammatory genes. As shown in Fig. 6, NF-κB inhibitor Bay-11-7082 significantly reduced the transcriptional increase of these five genes (Fig. 6D) and of VCAM1 protein in PAC1 cells after Sm22 knockdown (Fig. 6E). A similar effect was also observed using another NF-κB pathway (IKKbeta) inhibitor, IMD-0354 (Online Figure IVB).
These data demonstrate that NF-κB pathway was activated upon Sm22 disruption in PAC1 cells and promoted the transactivation of pro-inflammatory genes. However, the issue of how NF-κB activation occurs after disruption of an actin cytoskeleton protein remains to be addressed.
NF-κB is a redox-sensitive transcription factor16. We investigated ROS production based on fluorescence microscopy using DHE for superoxide and DCFDA for peroxide. Levels of both superoxide (Fig. 7A, left panel; Online Figure VA) and peroxide (Fig. 7A, left panel; Online Figure VB) were about 30% higher in Sm22−/− primary VSMCs, and 50% higher after Sm22 knockdown in PAC1 cells (Fig. 7A, right panel; Online Figure VC, VD). ROS levels seemed to correlate with Sm22 knockdown efficiency and induction of the pro-inflammatory genes over time (Online Figure VIA). To test whether the boosted ROS level contributed to NF-κB activation after Sm22 knockdown, we used Tiron (a ROS scavenger), to neutralize the ROS (Fig. 7A, right panel). Tiron decreased the expression of both nuclear RELA (Fig. 7B) and nuclear NFKB2 (Fig. 7C), indicating inhibition of NF-κB pathways. As expected, neutralization of ROS by either Tiron (Fig. 7D) or Tempol (a superoxide scavenger) (Fig. 7E) or N-acetyl-cysteine (NAC, another ROS scavenger) (Online Figure VIB) respectively repressed the upregulation of NF-κB inducible pro-inflammatory genes after Sm22 knockdown. These data indicate that ROS might act upstream of the NF-κB activation. Interestingly, it appears that superoxide rather than peroxide mediated most of these effects since Tempol lowered superoxide without significantly decreasing the elevated peroxide level and Tiron also reduced the production of superoxide more than that of peroxide (Fig. 7A, right panel).
In order to locate the sources of the elevated ROS after Sm22 disruption, we scanned mRNA expression of known ROS production related genes including SOD system, NADPH oxidase system, dual oxidase, catalase and glutathione peroxidase17. Sod2 mRNA in PAC1 after Sm22 knockdown increased about 2.5 times by rtRT-PCR (Fig. 8A); The SOD2 increase was confirmed by WB (Fig. 8A, inserted panel) and IF (Fig. 8A, lower panel). In accordance, Sod2 mRNA induction by injury was also higher in Sm22−/− mice compared to their Sm22+/+ littermates (Fig.8A); however, this difference was not statistically significant between primary VSMCs from Sm22+/+ and Sm22−/− mice (Fig. 8A). Since SOD2 is a mitochondrial matrix protein and scavenges mitochondrial superoxide, the increase of SOD2 suggested boosted mitochondrial superoxide production as a feedback. Further observation on mitochondria morphology and distribution revealed mitochondria aggregation and fusion into megamitochondria (Fig. 8B, Mito) which is associated with ROS production18. Since Sod2 is a known NF-κB target19, NF-κB activation may plausibly induce SOD2 expression. Indeed, the upregulation of SOD2 after Sm22 knockdown was repressed by Bay-11-7082 (a NF-κB pathway inhibitor) (Online Figure VIIA). Furthermore, we observed that Sm22 knockdown induced cell periphery translocation of p47phox (Fig. 8B, p47), an indication of NADPH oxidase activation20. Diphenyleneiodonium (DPI), an inhibitor of both NADPH oxidase and mitochondrial complex I, significantly blocked the upregulation of pro-inflammatory genes after Sm22 knockdown (Online Figure VIIB). Therefore, mitochondria and NADPH oxidase may both contribute to the elevated ROS.
Since SM22 is an actin associated protein and NADPH oxidase activation might be affected by change in actin cytoskeleton organization20, we examined the actin cytoskeleton and observed significantly less actin stress fiber in PAC1 cells after Sm22 knockdown (Fig. 8C, SMA). Microtubules and actin cytoskeleton cooperate functionally during a variety of cellular processes and regulate mitochondria distribution21; therefore, we examined whether SM22 disruption affect the microtubule cytoskeleton. We found that microtubules were unevenly distributed in the Sm22 knockdown cells and displayed local aggregation (Fig. 8C, TUB). These results suggest that cytoskeleton remodeling induced by disruption of SM22 in VSMCs might activate multiple ROS production machineries.
In an ongoing effort to understand the role of SM22 in SMC phenotypic modulation, we analyzed the phenotypes of Sm22−/− mice in response to arterial injury. Because of prominent injury-induced inflammation, we focused on characterizing the expression of pro-inflammatory genes in injured carotids and the underlying molecular mechanisms of inflammation using both primary VSMC and VSMC cell line systems.
Inflammation is one major event in artery injury models22. We observed macrophage and T lymphocyte infiltration in media and adventitia, excessive adventitial fibrosis, prominent thickening of denuded carotids as well as increased expression of pro-inflammatory genes VCAM1, ICAM1, CX3CL1, CCL2 and PTGS2 in arteries of Sm22−/− mice upon injury. Since expression of pro-inflammatory genes is finely regulated during inflammation13, it is not surprising that changes in some pro-inflammatory genes such as Cxcl12 (sdf-1a) and Cx3cr1(a receptor for chemokine CX3CL1) were not detected under the same condition. In our system we only observed marginal neointima formation in injured carotids: this might be due to the C57Bl/6 based mixed genetic background that may be resistant to injury-induced neointima formation23. The dominant distribution of pro-inflammatory proteins in the VSMC-rich media suggests VSMCs as the cell sources for inflammation. Consistent with this notion, primary VSMCs from Sm22−/− mice and PAC1 after Sm22 knockdown also show upregulated expression of these pro-inflammatory genes. These results imply that disruption of SM22 in VSMCs may independently establish a pro-inflammatory environment in the arteries under stressed conditions. On the other hand, adventitial cells such as fibroblasts might also play substantial roles in the arterial inflammatory responses to injury.
NF-κB was initially identified in leukocytes. Activation of NF-κB pathways in vascular cells (endothelial and smooth muscle cells) is well documented during arterial inflammation, and all five aforementioned pro-inflammatory genes are direct targets of NF-κB11, 24, 25. NF-κB pathways can be classified into canonical, non-canonical and atypical based on the different NF-κB dimers formed during activation15. Most studies thus far have focused on the activation of the canonical pathway. Surprisingly, the striking nuclear localization of NFKB2 rather than RELA in injured Sm22−/− carotids and primary Sm22−/− VSMCs indicated that non-canonical NF-κB pathways activation is predominant in our situation. However, this does not seem to fully agree with the fact that both canonical and non-canonical NF-κB pathways were activated in PAC1 cells after Sm22 knockdown. There are several possible explanations for this discrepancy. One is that the injured carotids were examined two weeks after injury, that is, outside the time window of acute inflammation, when the RELA-associated canonical pathway is activated in response to arterial injury22, 26. However, this cannot explain why the canonical pathway was activated in the Sm22 knockdown PAC1 cells, but not in the Sm22−/− primary VSMCs under the same culture condition (see Figs. Figs.6,6, ,7,7, Online Figure IV). This discrepancy may be due to different differentiation states of primary VSMCs compared to the PAC1 VSMC cell line and response variations among different systems.PAC1 cells after SM22 knockdown may more closely resemble an acute inflammation model, since our experiments were performed three days after transfection. In view of this, it would not surprise us to observe activation of NFKB2, the non-canonic pathway, in the acute phase of carotid injury. This possibility could be examined in future studies. Although it is possible that some of the NF-κB signals in injured arteries were from the infiltrated inflammatory cells, the in vitro NF-κB activation in VSMCs after Sm22 disruption lends support to the possibility of in vivo NF-κB activation in VSMCs after carotid injury.
NF-κB activation is a consequence of cell response to stress. NF-κB is a redox-sensitive transcription factor16, 27, and ROS is one key source for NF-κB activation in VSMCs in arterial diseases24, 25, 27. The increased ROS level in primary Sm22−/− VSMCs and in PAC1 after Sm22 knockdown indicated high oxidative stress in VSMCs with Sm22 disruption. Different ROS scavengers, Tiron, Tempol or NAC consistently blocked NF-κB activation and pro-inflammatory genes induction. This provides further evidence indicating that increased production of ROS may initiate NF-κB activation in PAC1 cells after Sm22 disruption. We tried to identify increased ROS production in injured carotids in vivo using both DHE and DCFDA on frozen sections. Disappointingly, high background from elastin and collagen thwarted further analysis. Although DHE and DCFDA-based assays have been used to detect ROS from live cells, ROS may not be preserved in our frozen sections. Nevertheless, we observed higher expression of Sod2 in the injured Sm22−/− carotids. Activated NF-κB perhaps induces Sod2 expression in anticipation of redox signaling. Therefore, increased expression of mitochondrial SOD2 may reflect a higher redox state in the injured carotids of Sm22−/− mice.
Mitochondria and NADPH oxidase are two important sources of ROS in VSMCs28, 29. The megamitochondria formation and mitochondria aggregation after Sm22 knockdown indicated mitochondria dysfunction associated with mitochondrial ROS production18; the upregulated SOD2 may reflect such a dysfunction and serve as a rescuing mechanism via the ROS-NF-κB feedback (Fig. 8D). NADPH oxidase, a major ROS source from VSMC membranes 28, was also activated after Sm22 knockdown. These observations suggest that disruption of Sm22 in stressed VSMCs may activate multiple ROS production mechanisms that might work together to foster a high redox environment.
How does the disruption of an actin-binding protein lead to simultaneous activation of NADPH oxidase and dysfunction of mitochondria? Activation of NADPH oxidase requires the membrane assembly of cytosolic p47phox, p67phox, p40phox and Rac220, 28. It was reported that the actin cytoskeleton and associated proteins may affect this process20. The correlation between NADPH oxidase activation and diminished stress fiber formation in PAC1 cells after Sm22 knockdown might reflect the role of the actin cytoskeleton in maintaining VSMCs phenotype. Furthermore, the actin cytoskeleton cooperates with microtubules21 in regulating organelle distribution including mitochondria21, 30. The mitochondria aggregation and formation of megamitochondria may be due to the compromised actin cytoskeleton after Sm22 knockdown or to be an outcome of subsequent disorganized microtubules (Fig. 8D). The changes in the fine structure of cytoskeleton and mitochondria after Sm22 disruption will be investigated in the future using electron microscopy.
We recently showed that actin cytoskeleton plays an important role in VSMC phenotypic modulation 31. The present study on the consequences of abolishing SM22, an actin-binding protein, offers a glimpse on how the cytoskeletal proteins could actively affect arterial pathogenesis. The SM22-associated cytoskeleton may serve as a sensor of environmental stress and participate in SMC phenotypic modulation. Consistent with this notion, the expression of SM22 is sensitive to cell shape change32, and SM22 expression is downregulated in a variety of cancers3. The finding that loss of SM22 creates a pro-inflammatory environment may also shed lights on the role of downregulation of SM22in carcinogenesis. Therefore, maintaining SM22 expression might serve as a therapeutic strategy to repress the dysregulated inflammatory responses in arterial diseases as well as in cancers. Given that carotid denudation is a simplified model for vascular injury, it is important to validate SM22's role as an anti-inflammatory agent in animal disease models such as diet-induced atherosclerosis mouse model.
In summary, based on our in vivo and in vitro results, we propose that disruption of SM22 expression in stressed VSMCs results in actin cytoskeleton and microtubules remodeling, thereby leading to a high redox state via mitochondria malfunction and NADPH oxidase activation. In turn, increased ROS production activates the NF-κB pathways required for establishing a pro-inflammatory environment (Fig. 8D). This study suggests that understanding the molecular mechanisms of cytoskeleton remodeling is critical to control inflammation in pathogenesis of vascular diseases.
The cytoskeleton plays important roles in determining vascular smooth muscle (VSMC) phenotypes. It is well known that in arterial diseases SM22 is downregulated along with several other VSMC cytoskeleton proteins. However, it is unknown whether this down-regulation is just a passive outcome or whether it actively contributes to the pathogenesis of arteriopathy. Here, we discovered that disruption of SM22 promotes arterial inflammation in SM22 knockout mice in response to arterial injury. This process is accompanied by elevated expression of inflammation markers and activation of their key inflammation regulator, NFKB, both in vitro and in vivo. Disruption of SM22 expression induces cytoskeleton remodeling, mitochondrial disorganization, and NADPH oxidase activation; these changes collectively result in increased production of reactive oxygen species that in turn activate NFKB. These observations reveal that SM22 downregulation makes VSMC pro-inflammatory: this at least partially explains why abolishing SM22 accelerates atherogenesis in hypercholesterolemic mice. The present study provides the first evidence that down-regulation of VSMC markers actively contributes to arterial inflammation. Further research should focus on whether maintaining cytoskeleton integrity may serve to prevent inflammation in arterial diseases. Also, blocking SM22 downregulation may provide a novel anti-inflammation strategy for dealing with arterial diseases.
We are grateful to Helena Kuivaniemi, Jeffrey Loeb, Giuseppe Rossi, Da-zhi Wang, and Kezhong Zhang for valuable discussion.
SOURCES OF FUNDING This work was supported by grants from the National Heart, Lung, and Blood Institute (HL058916, and HL087014 to L.L) and from the American Heart Association (0555680Z to L.L.).
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