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Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization.

Hirasawa M, Noda K, Noda S, Suzuki M, Ozawa Y, Shinoda K, Inoue M, Ogawa Y, Tsubota K, Ishida S - Mol. Vis. (2011)

Bottom Line: Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina.Furthermore, TGF-β induced relocalization of Snail to the nucleus in RPE cells.The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retinal Cell Biology, Keio University School of Medicine, Tokyo, Japan

ABSTRACT

Purpose: To investigate the transcriptional factors associated with epithelial-mesenchymal transition (EMT) in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD).

Methods: Paraffin sections of CNV obtained from patients with AMD (n = 12) were stained for transcriptional factors related to EMT, i.e., Snail, Slug, SIP1, and Twist. As a control, postmortem sections of ocular normal tissue were used. Furthermore, using a human retinal pigment epithelial (RPE) cell line (ARPE-19), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence microscopy were performed to explore the cellular localization and expression levels of EMT-associated transcriptional factors upon cytokine stimulation.

Results: Of 12 specimens, 11 CNV tissues (91.6%) showed staining for Snail localized in cellular nuclei, particularly in those of RPE cells. Snail was strongly co-localized with α-smooth muscle antigen (SMA) in RPE cells. In contrast, postmortem human retina showed no Snail staining in RPE cells. Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina. In ARPE-19 cells, RT-PCR and immunofluorescence microscopy showed that Snail mRNA was upregulated by transforming growth factor (TGF)-β and VEGF stimulation. Furthermore, TGF-β induced relocalization of Snail to the nucleus in RPE cells.

Conclusions: The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV.

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Related in: MedlinePlus

Snail expression after cytokine stimulation in ARPE-19 cells. A: RT–PCR amplification of Snail mRNAs from ARPE-19 cells after cytokine stimulation. ARPE-19 cells were incubated with PBS or recombinant human TGF-β, TNF-α, VEGF, CTGF, bFGF, and IGF-1. After 4 h, RNA was extracted from these cells. Snail mRNA expression was measured by reverse-transcription PCR. B: Gel densitometric analysis. Values are mean±SEM (n=6 in each group). *p<0.05. C: Immunofluorescence for Snail after cytokine stimulation. Left-Control. Middle-8 h after TGF-β stimulation (10 ng/ml). Right-8 h after VEGF stimulation (10 ng/ml). Scale bar=25 µm.
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f5: Snail expression after cytokine stimulation in ARPE-19 cells. A: RT–PCR amplification of Snail mRNAs from ARPE-19 cells after cytokine stimulation. ARPE-19 cells were incubated with PBS or recombinant human TGF-β, TNF-α, VEGF, CTGF, bFGF, and IGF-1. After 4 h, RNA was extracted from these cells. Snail mRNA expression was measured by reverse-transcription PCR. B: Gel densitometric analysis. Values are mean±SEM (n=6 in each group). *p<0.05. C: Immunofluorescence for Snail after cytokine stimulation. Left-Control. Middle-8 h after TGF-β stimulation (10 ng/ml). Right-8 h after VEGF stimulation (10 ng/ml). Scale bar=25 µm.

Mentions: Next, at 7 days after passage we stimulated ARPE-19 cells with cytokines, TGF-β, TNF-α, VEGF, CTGF, bFGF, and IGF-1, previously found in human CNV samples. Among them, TGF-β and VEGF significantly upregulated Snail mRNA (Figure 5AB). However, the mRNA level of Snail was not changed with stimulation of the other cytokines. Furthermore, fluorescence microscopy depicted the enhanced staining of Snail in the nucleus and cytoplasm of ARPE-19 cells stimulated with TGF-β at 7 days after passage (Figure 5C), indicating a role for TGF-β in the upregulation and nuclear relocalization of Snail in RPE cells. By contrast, VEGF enhanced immunoreactivity of Snail mainly in the cytoplasm, but not in the nucleus (Figure 5C).


Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization.

Hirasawa M, Noda K, Noda S, Suzuki M, Ozawa Y, Shinoda K, Inoue M, Ogawa Y, Tsubota K, Ishida S - Mol. Vis. (2011)

Snail expression after cytokine stimulation in ARPE-19 cells. A: RT–PCR amplification of Snail mRNAs from ARPE-19 cells after cytokine stimulation. ARPE-19 cells were incubated with PBS or recombinant human TGF-β, TNF-α, VEGF, CTGF, bFGF, and IGF-1. After 4 h, RNA was extracted from these cells. Snail mRNA expression was measured by reverse-transcription PCR. B: Gel densitometric analysis. Values are mean±SEM (n=6 in each group). *p<0.05. C: Immunofluorescence for Snail after cytokine stimulation. Left-Control. Middle-8 h after TGF-β stimulation (10 ng/ml). Right-8 h after VEGF stimulation (10 ng/ml). Scale bar=25 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102030&req=5

f5: Snail expression after cytokine stimulation in ARPE-19 cells. A: RT–PCR amplification of Snail mRNAs from ARPE-19 cells after cytokine stimulation. ARPE-19 cells were incubated with PBS or recombinant human TGF-β, TNF-α, VEGF, CTGF, bFGF, and IGF-1. After 4 h, RNA was extracted from these cells. Snail mRNA expression was measured by reverse-transcription PCR. B: Gel densitometric analysis. Values are mean±SEM (n=6 in each group). *p<0.05. C: Immunofluorescence for Snail after cytokine stimulation. Left-Control. Middle-8 h after TGF-β stimulation (10 ng/ml). Right-8 h after VEGF stimulation (10 ng/ml). Scale bar=25 µm.
Mentions: Next, at 7 days after passage we stimulated ARPE-19 cells with cytokines, TGF-β, TNF-α, VEGF, CTGF, bFGF, and IGF-1, previously found in human CNV samples. Among them, TGF-β and VEGF significantly upregulated Snail mRNA (Figure 5AB). However, the mRNA level of Snail was not changed with stimulation of the other cytokines. Furthermore, fluorescence microscopy depicted the enhanced staining of Snail in the nucleus and cytoplasm of ARPE-19 cells stimulated with TGF-β at 7 days after passage (Figure 5C), indicating a role for TGF-β in the upregulation and nuclear relocalization of Snail in RPE cells. By contrast, VEGF enhanced immunoreactivity of Snail mainly in the cytoplasm, but not in the nucleus (Figure 5C).

Bottom Line: Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina.Furthermore, TGF-β induced relocalization of Snail to the nucleus in RPE cells.The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retinal Cell Biology, Keio University School of Medicine, Tokyo, Japan

ABSTRACT

Purpose: To investigate the transcriptional factors associated with epithelial-mesenchymal transition (EMT) in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD).

Methods: Paraffin sections of CNV obtained from patients with AMD (n = 12) were stained for transcriptional factors related to EMT, i.e., Snail, Slug, SIP1, and Twist. As a control, postmortem sections of ocular normal tissue were used. Furthermore, using a human retinal pigment epithelial (RPE) cell line (ARPE-19), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence microscopy were performed to explore the cellular localization and expression levels of EMT-associated transcriptional factors upon cytokine stimulation.

Results: Of 12 specimens, 11 CNV tissues (91.6%) showed staining for Snail localized in cellular nuclei, particularly in those of RPE cells. Snail was strongly co-localized with α-smooth muscle antigen (SMA) in RPE cells. In contrast, postmortem human retina showed no Snail staining in RPE cells. Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina. In ARPE-19 cells, RT-PCR and immunofluorescence microscopy showed that Snail mRNA was upregulated by transforming growth factor (TGF)-β and VEGF stimulation. Furthermore, TGF-β induced relocalization of Snail to the nucleus in RPE cells.

Conclusions: The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV.

Show MeSH
Related in: MedlinePlus