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Endothelial to mesenchymal transition contributes to arsenic-trioxide-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 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.

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As2O3 triggers EndMT in human aortic endothelial cells (HAECs).(a) Morphological changes of endothelial cells exposed to different concentrations of As2O3. (b) Morphological changes of endothelial cells treated with As2O3 (2 μmol/l) for 0, 24, 48 or 72 hours. (c) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in As2O3-treated HAECs. GAPDH was used as an internal control. (d) Relative expression levels of endothelial markers (VE-cad, CD31) and mesenchymal markers (α-SMA, FSP1, FN and Vimentin) were compared by qRT-PCR between control groups and As2O3-treated groups. FN, fibronectin. (e) Relative mRNA levels of fibrosis-related genes Col1a, Col3a, mmp2 and mmp9. (f) Representative confocal microscopy images showing staining of endothelial marker CD31 and mesenchymal marker α-SMA in As2O3-treated HAECs. Scale bar = 30 μm. *p < 0.05, **p < 0.01 vs. untreated condition (0 μmol/l As2O3). No significant difference was observed between the different concentrations groups. Data are represented as mean ± SEM, n = 3–5.
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f4: As2O3 triggers EndMT in human aortic endothelial cells (HAECs).(a) Morphological changes of endothelial cells exposed to different concentrations of As2O3. (b) Morphological changes of endothelial cells treated with As2O3 (2 μmol/l) for 0, 24, 48 or 72 hours. (c) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in As2O3-treated HAECs. GAPDH was used as an internal control. (d) Relative expression levels of endothelial markers (VE-cad, CD31) and mesenchymal markers (α-SMA, FSP1, FN and Vimentin) were compared by qRT-PCR between control groups and As2O3-treated groups. FN, fibronectin. (e) Relative mRNA levels of fibrosis-related genes Col1a, Col3a, mmp2 and mmp9. (f) Representative confocal microscopy images showing staining of endothelial marker CD31 and mesenchymal marker α-SMA in As2O3-treated HAECs. Scale bar = 30 μm. *p < 0.05, **p < 0.01 vs. untreated condition (0 μmol/l As2O3). No significant difference was observed between the different concentrations groups. Data are represented as mean ± SEM, n = 3–5.

Mentions: To further elucidate the relationship between As2O3 and EndMT, we used HAECs for our subsequent in vitro experiments. After treatment with varying concentrations of As2O3 for 24 h, the endothelial cells underwent a morphological transformation to fibroblast-like spindle-shaped phenotype (Fig. 4a). However, we should note that the morphological change of endothelial cells at the concentration of 2 μmol/l was not as obvious as that at 4 μmol/l and 8 μmol/l. When we prolong the treatment time to 48 h and 72 h, this change became more remarkable (Fig. 4b). Besides, As2O3 reduced the expression of endothelial cell markers (VE-cadherin and CD31) and augmented the expression of mesenchymal markers (α-SMA and FSP1) in a concentration-dependent manner as indicated by western blotting results (Fig. 4c). Similar results were obtained with qRT-PCR analysis (Fig. 4d). Additionally, we detected two other mesenchymal markers fibronectin (FN) and vimentin and found that the expression of FN and vimentin had the same trend as the expression of α-SMA and FSP1 (Fig. 4d). Meanwhile, the mRNA levels of fibrotic-related genes, including Col1a, Col3a, mmp2 and mmp9, were markedly increased (Fig. 4e). The results from double immunofluorescence staining further confirmed that endothelial cells with As2O3 treatment underwent EndMT with a decrease for the membrane staining of CD31 and increase for the cytoplasm staining of α-SMA (Fig. 4f). Taken together, we have demonstrated that As2O3 could elicit EndMT in HAECs.


Endothelial to mesenchymal transition contributes to arsenic-trioxide-induced cardiac fibrosis
As2O3 triggers EndMT in human aortic endothelial cells (HAECs).(a) Morphological changes of endothelial cells exposed to different concentrations of As2O3. (b) Morphological changes of endothelial cells treated with As2O3 (2 μmol/l) for 0, 24, 48 or 72 hours. (c) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in As2O3-treated HAECs. GAPDH was used as an internal control. (d) Relative expression levels of endothelial markers (VE-cad, CD31) and mesenchymal markers (α-SMA, FSP1, FN and Vimentin) were compared by qRT-PCR between control groups and As2O3-treated groups. FN, fibronectin. (e) Relative mRNA levels of fibrosis-related genes Col1a, Col3a, mmp2 and mmp9. (f) Representative confocal microscopy images showing staining of endothelial marker CD31 and mesenchymal marker α-SMA in As2O3-treated HAECs. Scale bar = 30 μm. *p < 0.05, **p < 0.01 vs. untreated condition (0 μmol/l As2O3). No significant difference was observed between the different concentrations groups. Data are represented as mean ± SEM, n = 3–5.
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f4: As2O3 triggers EndMT in human aortic endothelial cells (HAECs).(a) Morphological changes of endothelial cells exposed to different concentrations of As2O3. (b) Morphological changes of endothelial cells treated with As2O3 (2 μmol/l) for 0, 24, 48 or 72 hours. (c) Western blotting results for relative protein levels of VE-cad, CD31, α-SMA and FSP1 in As2O3-treated HAECs. GAPDH was used as an internal control. (d) Relative expression levels of endothelial markers (VE-cad, CD31) and mesenchymal markers (α-SMA, FSP1, FN and Vimentin) were compared by qRT-PCR between control groups and As2O3-treated groups. FN, fibronectin. (e) Relative mRNA levels of fibrosis-related genes Col1a, Col3a, mmp2 and mmp9. (f) Representative confocal microscopy images showing staining of endothelial marker CD31 and mesenchymal marker α-SMA in As2O3-treated HAECs. Scale bar = 30 μm. *p < 0.05, **p < 0.01 vs. untreated condition (0 μmol/l As2O3). No significant difference was observed between the different concentrations groups. Data are represented as mean ± SEM, n = 3–5.
Mentions: To further elucidate the relationship between As2O3 and EndMT, we used HAECs for our subsequent in vitro experiments. After treatment with varying concentrations of As2O3 for 24 h, the endothelial cells underwent a morphological transformation to fibroblast-like spindle-shaped phenotype (Fig. 4a). However, we should note that the morphological change of endothelial cells at the concentration of 2 μmol/l was not as obvious as that at 4 μmol/l and 8 μmol/l. When we prolong the treatment time to 48 h and 72 h, this change became more remarkable (Fig. 4b). Besides, As2O3 reduced the expression of endothelial cell markers (VE-cadherin and CD31) and augmented the expression of mesenchymal markers (α-SMA and FSP1) in a concentration-dependent manner as indicated by western blotting results (Fig. 4c). Similar results were obtained with qRT-PCR analysis (Fig. 4d). Additionally, we detected two other mesenchymal markers fibronectin (FN) and vimentin and found that the expression of FN and vimentin had the same trend as the expression of α-SMA and FSP1 (Fig. 4d). Meanwhile, the mRNA levels of fibrotic-related genes, including Col1a, Col3a, mmp2 and mmp9, were markedly increased (Fig. 4e). The results from double immunofluorescence staining further confirmed that endothelial cells with As2O3 treatment underwent EndMT with a decrease for the membrane staining of CD31 and increase for the cytoplasm staining of α-SMA (Fig. 4f). Taken together, we have demonstrated that As2O3 could elicit EndMT in HAECs.

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