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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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PI3K inhibitor LY294002 represses As2O3-induced EndMT in HAECs.(a) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in different groups. +LY294002 indicates the co-application of LY294002 and As2O3 (8 μmol/l). (b) Relative mRNA expression levels of VE-cad, CD31, α-SMA, FSP1, FN and Vimentin in each group by qRT-PCR analysis. (c) Effects of LY294002 on the mRNA expression of fibrosis-related genes Col1a, Col3a, mmp2, mmp9. *p < 0.05, **p < 0.01 vs. Control. #p < 0.05, ##p < 0.01 vs. As2O3. Data are shown as mean ± SEM, n = 3–5. (d) Immunofluorescence analysis for the expression levels of CD31 and α-SMA in Control, LY294002, As2O3 and As2O3 + LY294002 groups. Scale bar = 30 μm.
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f7: PI3K inhibitor LY294002 represses As2O3-induced EndMT in HAECs.(a) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in different groups. +LY294002 indicates the co-application of LY294002 and As2O3 (8 μmol/l). (b) Relative mRNA expression levels of VE-cad, CD31, α-SMA, FSP1, FN and Vimentin in each group by qRT-PCR analysis. (c) Effects of LY294002 on the mRNA expression of fibrosis-related genes Col1a, Col3a, mmp2, mmp9. *p < 0.05, **p < 0.01 vs. Control. #p < 0.05, ##p < 0.01 vs. As2O3. Data are shown as mean ± SEM, n = 3–5. (d) Immunofluorescence analysis for the expression levels of CD31 and α-SMA in Control, LY294002, As2O3 and As2O3 + LY294002 groups. Scale bar = 30 μm.

Mentions: We also detected EndMT-associated markers in the condition of AKT pathway inhibition. Western blotting analysis showed that LY294002 effectively prevented the downregulation of endothelial markers and upregulation of mesenchymal markers by As2O3 (Fig. 7a). Consistently, the qRT-PCR results revealed that the As2O3-induced reduction of endothelial markers and augmentation of mesenchymal markers and fibrosis-related gene expression were attenuated by pre-treatment with LY294002 (Fig. 7b,c). Moreover, the immunostaining assay confirmed the inhibitory effects of the PI3K inhibitor (LY294002) on EndMT (Fig. 7d).


Endothelial to mesenchymal transition contributes to arsenic-trioxide-induced cardiac fibrosis
PI3K inhibitor LY294002 represses As2O3-induced EndMT in HAECs.(a) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in different groups. +LY294002 indicates the co-application of LY294002 and As2O3 (8 μmol/l). (b) Relative mRNA expression levels of VE-cad, CD31, α-SMA, FSP1, FN and Vimentin in each group by qRT-PCR analysis. (c) Effects of LY294002 on the mRNA expression of fibrosis-related genes Col1a, Col3a, mmp2, mmp9. *p < 0.05, **p < 0.01 vs. Control. #p < 0.05, ##p < 0.01 vs. As2O3. Data are shown as mean ± SEM, n = 3–5. (d) Immunofluorescence analysis for the expression levels of CD31 and α-SMA in Control, LY294002, As2O3 and As2O3 + LY294002 groups. Scale bar = 30 μm.
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f7: PI3K inhibitor LY294002 represses As2O3-induced EndMT in HAECs.(a) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in different groups. +LY294002 indicates the co-application of LY294002 and As2O3 (8 μmol/l). (b) Relative mRNA expression levels of VE-cad, CD31, α-SMA, FSP1, FN and Vimentin in each group by qRT-PCR analysis. (c) Effects of LY294002 on the mRNA expression of fibrosis-related genes Col1a, Col3a, mmp2, mmp9. *p < 0.05, **p < 0.01 vs. Control. #p < 0.05, ##p < 0.01 vs. As2O3. Data are shown as mean ± SEM, n = 3–5. (d) Immunofluorescence analysis for the expression levels of CD31 and α-SMA in Control, LY294002, As2O3 and As2O3 + LY294002 groups. Scale bar = 30 μm.
Mentions: We also detected EndMT-associated markers in the condition of AKT pathway inhibition. Western blotting analysis showed that LY294002 effectively prevented the downregulation of endothelial markers and upregulation of mesenchymal markers by As2O3 (Fig. 7a). Consistently, the qRT-PCR results revealed that the As2O3-induced reduction of endothelial markers and augmentation of mesenchymal markers and fibrosis-related gene expression were attenuated by pre-treatment with LY294002 (Fig. 7b,c). Moreover, the immunostaining assay confirmed the inhibitory effects of the PI3K inhibitor (LY294002) on EndMT (Fig. 7d).

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