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. , , 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−/−
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 (). 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 (). 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 (). This study suggests that understanding the molecular mechanisms of cytoskeleton remodeling is critical to control inflammation in pathogenesis of vascular diseases.
Novelty and Significance
What is known?
- SM22 is an actin-binding cytoskeleton protein.
- SM22 expression is down-regulated along with other VSMC markers in atherosclerosis and aneurysm in mice and humans.
- Inflammation is broadly involved in pathogenesis of arterial diseases.
What is New?
- Disruption of SM22 induces proinflammatory VSMC.
- SM22 down-regulation compromises cytoskeleton formation, increases the redox status, and activates NF-κB pathways in VSMCs.
- This is the first evidence indicating that disruption of VSMC cytoskeleton actively participates in the pathogenesis of arterial 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.