Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail - PubMed (original) (raw)
doi: 10.1038/s41598-017-03532-z.
Jovana Serbanovic-Canic 1, Shuang Feng 1, Celine Souilhol 1, Rouyu Xing 2, Sarah Hsiao 1, Akiko Mammoto 3, Jing Chen 4, Markus Ariaans 1, Sheila E Francis 1, Kim Van der Heiden 2, Victoria Ridger 1, Paul C Evans 5
Affiliations
- PMID: 28611395
- PMCID: PMC5469771
- DOI: 10.1038/s41598-017-03532-z
Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail
Marwa M Mahmoud et al. Sci Rep. 2017.
Erratum in
- Author Correction: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail.
Mahmoud MM, Serbanovic-Canic J, Feng S, Souilhol C, Xing R, Hsiao S, Mammoto A, Chen J, Ariaans M, Francis SE, Van der Heiden K, Ridger V, Evans PC. Mahmoud MM, et al. Sci Rep. 2020 Feb 26;10(1):3870. doi: 10.1038/s41598-020-60955-x. Sci Rep. 2020. PMID: 32099026 Free PMC article.
Abstract
Blood flow influences atherosclerosis by generating wall shear stress, which alters endothelial cell (EC) physiology. Low shear stress induces dedifferentiation of EC through a process termed endothelial-to-mesenchymal transition (EndMT). The mechanisms underlying shear stress-regulation of EndMT are uncertain. Here we investigated the role of the transcription factor Snail in low shear stress-induced EndMT. Studies of cultured EC exposed to flow revealed that low shear stress induced Snail expression. Using gene silencing it was demonstrated that Snail positively regulated the expression of EndMT markers (Slug, N-cadherin, α-SMA) in EC exposed to low shear stress. Gene silencing also revealed that Snail enhanced the permeability of endothelial monolayers to macromolecules by promoting EC proliferation and migration. En face staining of the murine aorta or carotid arteries modified with flow-altering cuffs demonstrated that Snail was expressed preferentially at low shear stress sites that are predisposed to atherosclerosis. Snail was also expressed in EC overlying atherosclerotic plaques in coronary arteries from patients with ischemic heart disease implying a role in human arterial disease. We conclude that Snail is an essential driver of EndMT under low shear stress conditions and may promote early atherogenesis by enhancing vascular permeability.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Figure 1
Low shear stress induced mesenchymal genes via Snail. (a) HUVEC were exposed to low oscillatory (+/−4 dyn/cm2) or high (13 dyn/cm2) wall shear stress (WSS) using a parallel plate system. (b) PAEC were exposed to orbital flow to generate low (5 dyn/cm2) or high (15 dyn/cm2) wall shear stress. (a,b) After 72 h, levels of EndMT marker transcripts and VE-cadherin transcripts were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Data were pooled from six independent experiments using cells from different donors and mean levels +/− SEM are shown. (c–e) HUVEC were exposed to orbital flow to generate low (5 dyn/cm2) or high (15 dyn/cm2) WSS for 72 h. (c) Expression of N-cadherin (green) and VE-cadherin (red) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. The proportion of cells that expressed N-cadherin or VE-cadherin was measured. (d) The expression levels of N-cadherin (left) and VE-cadherin (right) were assessed by Western blotting using specific antibodies and anti-PDHX antibodies were used to control for total protein levels. Representative blots are shown. Bands were quantified by densitometry. (e) Expression of Snail (green) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. Fluorescence intensity was quantified in multiple cells. (f) HUVEC were transfected with siRNA targeting Snail or with scrambled sequences and exposed to orbital flow for 72 h. Cells exposed to low WSS (5 dyn/cm2) were collected and transcript levels of Slug, N cadherin and α-SMA were quantified by qRT-PCR. (c–f) Data were pooled from three independent experiments using cells from different donors and mean levels +/− SEM are shown.
Figure 2
Snail enhanced migration under low shear stress. HUVEC were treated with siRNA targeting Snail, or with scrambled non-targeting siRNA (or were untreated as a control). They were then cultured in 6 well plates prior to exposure to low or high wall shear stress (WSS) for 72 h using an orbital plate system. To assess cell migration, a scratch wound was made in the monolayer, and cells were imaged for 20 h. Representative images are shown (scale bar 100 μm). The distance migrated at multiple time points (lower left) and average velocity (lower right) was determined and mean values +/− SEM are shown. Data were pooled from four independent experiments using cells from different donors and mean levels +/− SEM are shown. Differences between means were assessed using a two-way ANOVA.
Figure 3
Snail enhanced proliferation and permeability in EC exposed to low shear stress. HUVEC were treated with siRNA targeting Snail or with scrambled (Scr) non-targeting siRNA. (a) Cells were subsequently cultured in 6 well plates prior to exposure to orbital flow to generate low or high wall shear stress (WSS) for 72 h. Cell proliferation was quantified by immunofluorescent staining using anti-Ki67 antibodies and co-staining using DAPI. Representative images are shown (Scale bar, 50 μm). The % Ki67-positive cells were calculated for multiple fields of view in four independent experiments using cells from different donors and mean levels +/− SEM are shown. Differences between means were assessed using a 2-way ANOVA. (b) Cells cultured on Transwell inserts were exposed to orbital flow (low WSS) for 72 h prior to assessment of endothelial permeability under static conditions using rhodamine (Rd)-albumin as a tracer. A schematic is shown (left panel). The concentration of Rd-albumin in the lower compartment was measured. Data were pooled from four independent experiments using cells from different donors and mean values +/− SEM are shown (right panel). Differences between means were assessed using an unpaired t-test.
Figure 4
Snail was preferentially expressed at low shear atherosusceptible sites. (a) EC were freshly-isolated from low wall shear stress (WSS; inner curvature) and high WSS (outer curvature) regions of the aorta in six pigs. Levels of Snail, Slug, N-cadherin, α-SMA and VE-cadherin mRNA were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Mean levels +/− SEM are shown. (b,c) EC at low WSS (susceptible) or high WSS (protected) regions of the aorta were studied by en face staining. (b) C57BL/6 mice (n = 5) were stained using anti-Snail antibodies (red), co-stained using anti-CD31 antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). (c) TWIST1cKO or TWIST1fl/fl mice (n = 4 each group) were stained using anti-Snail antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). Representative images (scale bar, 10 μm) and quantitation of Snail fluorescence levels (mean +/− SEM) are shown. Differences between means were assessed using a paired t-test (b) or two-way ANOVA (c).
Figure 5
Snail was induced by low oscillatory shear stress in murine carotid arteries. Flow-altering, constrictive cuffs were placed on the right carotid arteries of C57BL/6 mice. They generated anatomically distinct regions exposed to high or low oscillatory wall shear stress (WSS; as indicated). Contralateral arteries were used as an experimental control. Experimental arteries were harvested after 14 days and en face staining was performed using anti-Snail antibodies (red), anti-CD31 antibodies (green) and the nuclear counter stain TO-PRO-3 (blue). Representative images (scale bar, 10 μm) and quantitation of Snail expression (n = 3; mean +/− SEM) are shown.
Figure 6
Expression of Snail in endothelium overlying human coronary artery plaques. The expression of Snail in coronary arteries of patients with ischemic heart disease (IHD) or dilated cardiomyopathy (DCM) was studied by immunohistochemistry using specific primary antibodies and HRP-conjugated secondary antibodies (brown). Consecutive sections were stained for EC using anti-vWF antibodies (brown). Sections were counterstained using hematoxylin. Representative images are shown. The region boxed (upper panel, 10X magification) is shown at higher magnifications (centre and lower panels, 40X). Scale bar 20 μm. The proportion of EC that expressed Snail was calculated for each section. Data were pooled from six plaques and mean values +/− SEM are presented. Differences between means were assessed using an unpaired t-test.
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