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Endothelial to mesenchymal transition contributes to arsenic-trioxide-induced cardiac fibrosis

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ABSTRACT

Emerging evidence has suggested the critical role of endothelial to mesenchymal transition (EndMT) in fibrotic diseases. The present study was designed to examine whether EndMT is involved in arsenic trioxide (As2O3)-induced cardiac fibrosis and to explore the underlying mechanisms. Cardiac dysfunction was observed in rats after exposure to As2O3 for 15 days using echocardiography, and the deposition of collagen was detected by Masson’s trichrome staining and electron microscope. EndMT was indicated by the loss of endothelial cell markers (VE-cadherin and CD31) and the acquisition of mesenchymal cell markers (α-SMA and FSP1) determined by RT-PCR at the mRNA level and Western blot and immunofluorescence analysis at the protein level. In the in-vitro experiments, endothelial cells acquired a spindle-shaped morphology accompanying downregulation of the endothelial cell markers and upregulation of the mesenchymal cell markers when exposed to As2O3. As2O3 activated the AKT/GSK-3β/Snail signaling pathway, and blocking this pathway with PI3K inhibitor (LY294002) abolished EndMT in As2O3-treated endothelial cells. Our results highlight that As2O3 is an EndMT-promoting factor during cardiac fibrosis, suggesting that targeting EndMT is beneficial for preventing As2O3-induced cardiac toxicity.

No MeSH data available.


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EndMT in As2O3-induced cardiac fibrosis.(a) Representative protein bands of endothelial markers VE-cadherin (VE-cad) and CD31 and mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP1) in cardiac tissues by western blotting. (b) Analysis of western blotting results normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). (c) Quantitative real-time PCR experiments for relative mRNA levels of VE-cad, CD31, α-SMA, FSP1. No significant difference was observed between the different dosages groups. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control. Data are expressed as mean ± SEM, n = 3–5. (d) Double immunofluorescence labeling of CD31 (green) and α-SMA (red) in As2O3 treated hearts, nuclei are stained with DAPI (blue). Yellow indicates co-localization of CD31 with α-SMA expression in vessels. Scale bar = 30 μm. **p < 0.01 vs. Control, n = 3.
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f3: EndMT in As2O3-induced cardiac fibrosis.(a) Representative protein bands of endothelial markers VE-cadherin (VE-cad) and CD31 and mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP1) in cardiac tissues by western blotting. (b) Analysis of western blotting results normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). (c) Quantitative real-time PCR experiments for relative mRNA levels of VE-cad, CD31, α-SMA, FSP1. No significant difference was observed between the different dosages groups. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control. Data are expressed as mean ± SEM, n = 3–5. (d) Double immunofluorescence labeling of CD31 (green) and α-SMA (red) in As2O3 treated hearts, nuclei are stained with DAPI (blue). Yellow indicates co-localization of CD31 with α-SMA expression in vessels. Scale bar = 30 μm. **p < 0.01 vs. Control, n = 3.

Mentions: Accumulating evidence suggests that EndMT is an important contributor to cardiac fibrosis15. Therefore, we investigated whether As2O3 induced cardiac fibrosis was mediated by EndMT. As shown in Fig. 3a,b, the downregulation of endothelial–specific markers (VE-cadherin and CD31) and upregulation of mesenchymal markers (α-SMA and FSP1) were observed in hearts of As2O3 treated rats. However, these changes were only statistically significant at the high-dose group, but not at the two lower dose groups, especially for the expression of mesenchymal markers. Meanwhile, we obtained similar results using qRT-PCR analysis (Fig. 3c). Moreover, the expression of typical transcriptional factors (Snail, Twist, Slug) for EndMT was also markedly increased at the high-dose group (Supplementary Figure 2). We next performed double immunofluorescence staining of CD31 (green) and α-SMA (red) for EndMT and observed the co-localization of CD31 and α-SMA in myocardial sections of rats with the high-dose As2O3 (Fig. 3d). All of these results suggest that EndMT might be involved in As2O3-induced cardiac fibrosis.


Endothelial to mesenchymal transition contributes to arsenic-trioxide-induced cardiac fibrosis
EndMT in As2O3-induced cardiac fibrosis.(a) Representative protein bands of endothelial markers VE-cadherin (VE-cad) and CD31 and mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP1) in cardiac tissues by western blotting. (b) Analysis of western blotting results normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). (c) Quantitative real-time PCR experiments for relative mRNA levels of VE-cad, CD31, α-SMA, FSP1. No significant difference was observed between the different dosages groups. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control. Data are expressed as mean ± SEM, n = 3–5. (d) Double immunofluorescence labeling of CD31 (green) and α-SMA (red) in As2O3 treated hearts, nuclei are stained with DAPI (blue). Yellow indicates co-localization of CD31 with α-SMA expression in vessels. Scale bar = 30 μm. **p < 0.01 vs. Control, n = 3.
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f3: EndMT in As2O3-induced cardiac fibrosis.(a) Representative protein bands of endothelial markers VE-cadherin (VE-cad) and CD31 and mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP1) in cardiac tissues by western blotting. (b) Analysis of western blotting results normalized to glyceraldehyde phosphate dehydrogenase (GAPDH). (c) Quantitative real-time PCR experiments for relative mRNA levels of VE-cad, CD31, α-SMA, FSP1. No significant difference was observed between the different dosages groups. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control. Data are expressed as mean ± SEM, n = 3–5. (d) Double immunofluorescence labeling of CD31 (green) and α-SMA (red) in As2O3 treated hearts, nuclei are stained with DAPI (blue). Yellow indicates co-localization of CD31 with α-SMA expression in vessels. Scale bar = 30 μm. **p < 0.01 vs. Control, n = 3.
Mentions: Accumulating evidence suggests that EndMT is an important contributor to cardiac fibrosis15. Therefore, we investigated whether As2O3 induced cardiac fibrosis was mediated by EndMT. As shown in Fig. 3a,b, the downregulation of endothelial–specific markers (VE-cadherin and CD31) and upregulation of mesenchymal markers (α-SMA and FSP1) were observed in hearts of As2O3 treated rats. However, these changes were only statistically significant at the high-dose group, but not at the two lower dose groups, especially for the expression of mesenchymal markers. Meanwhile, we obtained similar results using qRT-PCR analysis (Fig. 3c). Moreover, the expression of typical transcriptional factors (Snail, Twist, Slug) for EndMT was also markedly increased at the high-dose group (Supplementary Figure 2). We next performed double immunofluorescence staining of CD31 (green) and α-SMA (red) for EndMT and observed the co-localization of CD31 and α-SMA in myocardial sections of rats with the high-dose As2O3 (Fig. 3d). All of these results suggest that EndMT might be involved in As2O3-induced cardiac fibrosis.

View Article: PubMed Central - PubMed

ABSTRACT

Emerging evidence has suggested the critical role of endothelial to mesenchymal transition (EndMT) in fibrotic diseases. The present study was designed to examine whether EndMT is involved in arsenic trioxide (As2O3)-induced cardiac fibrosis and to explore the underlying mechanisms. Cardiac dysfunction was observed in rats after exposure to As2O3 for 15&thinsp;days using echocardiography, and the deposition of collagen was detected by Masson&rsquo;s trichrome staining and electron microscope. EndMT was indicated by the loss of endothelial cell markers (VE-cadherin and CD31) and the acquisition of mesenchymal cell markers (&alpha;-SMA and FSP1) determined by RT-PCR at the mRNA level and Western blot and immunofluorescence analysis at the protein level. In the in-vitro experiments, endothelial cells acquired a spindle-shaped morphology accompanying downregulation of the endothelial cell markers and upregulation of the mesenchymal cell markers when exposed to As2O3. As2O3 activated the AKT/GSK-3&beta;/Snail signaling pathway, and blocking this pathway with PI3K inhibitor (LY294002) abolished EndMT in As2O3-treated endothelial cells. Our results highlight that As2O3 is an EndMT-promoting factor during cardiac fibrosis, suggesting that targeting EndMT is beneficial for preventing As2O3-induced cardiac toxicity.

No MeSH data available.


Related in: MedlinePlus