In this study, we found that aortic SMC membrane-bound TM played a role in SMC phenotype and changes in behavior. TM affects cell behavior differently in different cell types. In epidermal epithelial A431 cells, cells expressing TM were shown to maintain compact cell colonies with an epithelial morphology, whereas TM knockdown induced the formation of cell protrusions at the edges of the colonies and increased cell migration [23]. TM expressed on monocyte works as an adhesion molecule and mediates monocytes adhesion to vascular endothelium [24]. With regards to SMCs, Tohda et al. demonstrated that exogenous recombinant TM protein treatment significantly increased the proliferation of rat vascular SMCs [10].
However, the effects of endogenous expression of TM on vascular SMCs have rarely been studied. Several studies have demonstrated that cultured vascular SMCs express TM, and this was associated with the down-regulation of contractile phenotype markers such as α-actin and tropomyosin [25, 26]. In addition, Ramachandran et al.
found that knockdown of the endogenous expression of TM in cultured human urinary bladder SMCs, a visceral SMC, did not affect bladder SMC growth but significantly attenuated PDGF-induced bladder SMC migration [27]. In the current study, we demonstrated that the TM expression on aortic SMCs was mainly associated with proliferative and proinflammatory phenotype transition, and also mildly increased aortic SMC migration. SMCs have extensive functional diversity depending on the specific demand within a given organ. Phasic and rhythmic contraction is the characteristic of visceral SMCs, while vascular SMCs in arteries usually maintain
continuous contraction to preserve vascular tone [28]. The mechanisms underlying the different effects of TM expression on different cell types, including vascular and visceral SMCs, are unclear and need further exploration.
Our in vivo experiments showed that neointima formation was more severe in the wild-type mice with TM expression compared to vascular SMC-specific TM-deficient mice. Previous studies have shown that systemic injections of exogenous recombinant TM protein or local incubation of recombinant adenoviral constructs overexpressing TM within injured arterial lumen decreased vascular injury-induced neointima formation [9, 29, 30]. The anti-thrombotic and anti-inflammatory effects of
recombinant TM protein on the endothelium are the major mechanisms underlying the anti-atherosclerosis effect of TM. Recombinant TM has been shown to decrease thrombin-induced PAR-1 activation and inflammation on the endothelium after vascular injury [31]. Recent studies have further demonstrated that recombinant TM protein has a thrombin-independent effect on the endothelium and can directly decrease the stress-induced apoptosis of endothelial cells [32, 33] and reduce leukocyte transmigration to the endothelium [34]. The accumulation of vascular SMCs in the subintima is a major feature of atherosclerosis and neointima.
Co-culturing SMCs with endothelial cells has been shown to cause a shift in SMCs to the synthetic phenotype and induced the expressions of inflammatory genes in endothelial cells [35]. Although recombinant TM has beneficial effects on the endothelium, the potential impact of vascular SMC-bound TM on endothelial cells is unknown.
Monocyte-expressed TM enhances adhesion of monocytes to endothelium and myeloid-specific TM-deficient mice had less neointima formation after carotid ligation [24]. The interplay between TM expression on monocytes, vascular SMCs and endothelial cells is unclear. The effects of TM on individual vascular cell types
are different and further investigations are needed to elucidate its role in cross talk between different cell types during atherosclerotic process. Another limitation of our study is the specificity of SM22 as a SMC-specific deletion Cre. Although commonly used to study smooth muscle specific expression, it is not the most specific marker and could express in non-muscular cells [36]. This could influence the experimental results of our study.
In conclusion, although previous studies have documented the expression of TM on neointimal SMCs and hypothesized its effect in protecting injured arteries from thrombosis, the present study showed that vascular SMC-bound TM had an important physiological effect on vascular SMC biology beyond its traditional anti-thrombotic effect.
Conflict of Interest None
Financial Support
This work was supported by grants 104-2314-B-006-083-MY2 and 106-2314-B-006-045-MY3 from the Ministry of Science and Technology, Taipei, Taiwan.
Author contributions
Wang KC, Chung HC, Tseng SY, Huang TY, and Lin YL performed the experiments and collected the data. Wang KC, Chen PS, Chao TH, and Li YH designed the study and analyzed the data. Li YH and Wang KC drafted the manuscript. Luo CY, Shi GY, and Wu HL revised the manuscript for important intellectual content.
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A.
A.
A.
Relative protein levels of p50
*
Relative protein levels of p65
***
**
**
n .s .
Figure 4
MMP-2 activity (% of siCtrl)
TNF-α
MMP-2 activity (% of siCtrl)
PDGF-BB
Figure 5
SM22-cretg/TM+/+ SM22-cretg/TMflox/flox
N/M ratio
C.
D.
SM22-cretg/TM+/+ SM22-cretg/TMflox/flox 2 week
DAPI α-SMA Ki67 Merge
SM22-cretg/TMflox/flox
Supplemental Figure 1
Figure legends
Figure 1. Phenotype changes of aortic SMCs after TM knockdown. (A) Cultured aortic SMCs expressed TM as shown by Western blot (left panel) of the cell lysates and ELISA of the cultured media (right panel). (B) Cultured aortic SMCs treated with TM-targeting siRNA (siTM) underwent morphological changes from a rhomboid (arrows) to spindle shape (arrowheads) compared with non-targeting siRNA controls (siCtrl). Scale bars, 20 μm. (C) Representative Western blot illustrating expression levels of TM, contractile and synthetic markers in aortic SMCs with (siTM) and without (siCtrl) TM knockdown. GAPDH was used as a loading control.
Quantification of protein expression (n=6) was normalized to GAPDH and expressed as mean ± SEM. *p < 0.05; **p < 0.01 and ***p < 0.001. α-SMA, α-smooth muscle actin; NM-MHC, non-muscle myosin heavy chain. (D) Representative Western blot illustrating the expressions of calponin and vimentin in aortic SMCs with the indicated treatment. GAPDH was used as a loading control.
Figure 2. Functional changes of aortic SMCs after TM knockdown. (A) Microarray results showed significant changes in cell cycle and cytokine-cytokine receptor interaction pathway in aortic SMCs with TM knockdown (siTM). *p<0.05 ,
***p<0.001 compared with controls (siCtrl). (B) Cell proliferation was measured by WST-1 cell proliferation assays. The number of cells was expressed in units of optical density (OD450). All experiments were performed at least 3 times. ***p< 0.001. (C) IL-6 mRNA expression levels in aortic SMCs and IL-6 concentrations in conditioned media were measured. Data were expressed as mean ± SEM. *p<0.05, **p<0.01,
***p<0.001, ###p<0.001, n.s. indicates not significant (n=3). (D) Expression and quantification of TM protein in pEGFPN1 vector and pEGFPN1-TM transfected
aortic SMCs with TM knockdown (n=3). IL-6 concentrations in the conditioned media from TNF-α-treated pEGFPN1 vector and TNF-α-treated pEGFPN1-TM transfected cells were determined by ELISA. Data were expressed as mean ± SEM.
*p<0.05, ***p<0.001. GAPDH was used as a loading control. Results were normalized to GAPDH and expressed as mean ± SEM.
Figure 3. TM knockdown attenuated TNF-α-induced NFκB activation in aortic SMCs. (A) Cell lysates with the indicated treatment were analyzed for TM and IκBα expressions. α-tubulin was used as a loading control. Quantification of IκBα protein expression (n=3) was shown as the bar graph. *p<0.05, **p<0.01, ***p<0.001. n.s.
indicates not significant. (B) Western blot analysis of p50 and p65 protein
expressions. Equality of nuclear sample loading was confirmed with control lamin A/C, a nuclear protein marker. Quantification of p50 and p65 protein expressions (n=3) were shown as the bar graph. Data were expressed as mean ± SEM. *p<0.05,
**p<0.01, ***p<0.001. n.s. indicates not significant.
Figure 4. Aortic SMC migration after TM knockdown. (A) Aortic SMCs received the indicated treatment and MMP-2 activity was analyzed by zymography. Data were expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, n.s. indicates not significant (n=3). (B) Migration of aortic SMCs with the indicated treatment was evaluated by Boyden chamber assay. The cells were allowed to migrate for 6 hours in response to medium with or without PDGF (20 ng/mL) in the lower chamber. The number of migrated cells was counted in 5 random fields for each well. Data were shown as mean ± SEM; n=3. *p<0.05.
Figure 5. Neointima formation in mice after carotid artery ligation. (A) Double immunofluorescent staining for α-SMA and TM in the ligated carotid arteries. Nuclei were counterstained with DAPI. TM expression was detected in the media and neointima in SM22-cretg/TM+/+ mice, but only minimal TM staining was observed in the endothelium of the ligated carotid arteries of SM22-cretg/TMflox/flox mice. Scale bar, 100 μm. (B) Hematoxylin–eosin staining of carotid arteries after carotid ligation.
The dashed lines indicated borders of neointima/media and media/adventitia. A, adventitia; L, lumen; M, media. Scale bar, 100 μm. Quantification of the neointima was calculated and shown as the bar graph. M, media; N, neointima. ***p<0.001. (C) Double immunofluorescent staining for α-SMA and Ki67 (arrows) in the carotid arteries after ligation. The dashed lines indicated borders of the neointima and media.
Scale bar, 50 μm. M, media; N, neointima. The ratio of Ki positive cells to total cells and the absolute number of Ki67 positive cells were calculated and shown as the bar graphs. *p<0.05; **p<0.01; ***p<0.001. (D) Immunohistochemical staining of SMA in the carotid arteries at 2 weeks after carotid ligation. Scale bar, 100 μm. The α-SMA positive staining intensity was quantitated and shown as the bar graph.
**p<0.01. ***p<0.001.
Supplemental Figure 1. (A) Schematic description of Cre recombination leading to TM gene inactivation in cells expressing SM22. (B) Western blotting of TM protein in SMCs isolated from SM22-cretg/TM+/+ and SM22-cretg/TMflox/flox mice. (C) The blood pressure and (D) body weight were similar between the SM22-cretg/TM+/+ and SM22-cretg/TMflox/flox mice.
106年度專題研究計畫成果彙整表
HC, Wu HL. Thrombomodulin expressed on vascular smooth muscle cell influences the behaviors of smooth muscle cell. 2018 Annual Scientific Session of the American HeartAssociation, Chicago, Illinois, USA, November 10-12, 2018.
2. Li YH, Wang KC, Chen PS, Chung HC, Wu HL. Thrombomodulin expressed on vascular smooth muscle cell influences arterial injury-induced neointima formation in mouse. 2019 Annual Scientific Session of the European Society of Cardiology, Paris, France, August 31 – September 4, 2019.
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