AoSMC response to FGF2: Difference between revisions
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|TCOverview=Vascular smooth muscle cells (SMCs) are key components in our blood vessels, and show remarkable plasticity. SMCs are normally growth-quiescent in the normal adult vessels, but are activated by injury, or exposure to growth factors, such as fibroblast growth factor-2 (FGF-2) and pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). These cues are sensed by these cells through changes in immediate-early gene expression, and can lead to increased proliferation and migration. These responses are associated with the initiation and progression of a range of vascular diseases including atherosclerosis, post-angioplasty restenosis and bypass graft stenosis. | |TCOverview=Vascular smooth muscle cells (SMCs) are key components in our blood vessels, and show remarkable plasticity. SMCs are normally growth-quiescent in the normal adult vessels, but are activated by injury, or exposure to growth factors, such as fibroblast growth factor-2 (FGF-2) and pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). These cues are sensed by these cells through changes in immediate-early gene expression, and can lead to increased proliferation and migration. These responses are associated with the initiation and progression of a range of vascular diseases including atherosclerosis, post-angioplasty restenosis and bypass graft stenosis. | ||
|TCQuality_control=Early growth response-1 (Egr-1) is an immediate-early gene (encoding a zinc finger transcription factor) that is poorly expressed in growth-quiescent cells and serves as a marker of cell activation or stress. Total RNA provided to the RIKEN Yokohama Institute was first analysed for Egr-1 expression by qRT-PCR. This demonstrated peak inducible expression after 30 min by FGF-2 and after 60 min by IL-1beta (Fig 1). Transient expression of this biomarker within 30-60 min is supported by the literature (e.g. Zhu et al. 2007). <br><html><img src="/resource_browser/images/TC_qc/500px-MSC_Fig1.jpg" /></html><br>Figure 1 (qRT-PCR analysis, EGR-1) <br><br><br>CAGE analysis on these samples revealed that Egr-1 underwent transient induction within 30-60 min in response to the growth factor or cytokine (Fig 2). In contrast, CAGE analysis revealed no change in expression of alpha-actin 2 (ACTA2) in response to FGF-2 or IL-1beta within the 6h time frame (Fig 2). <br><br><html><img src=" | |TCQuality_control=Early growth response-1 (Egr-1) is an immediate-early gene (encoding a zinc finger transcription factor) that is poorly expressed in growth-quiescent cells and serves as a marker of cell activation or stress. Total RNA provided to the RIKEN Yokohama Institute was first analysed for Egr-1 expression by qRT-PCR. This demonstrated peak inducible expression after 30 min by FGF-2 and after 60 min by IL-1beta (Fig 1). Transient expression of this biomarker within 30-60 min is supported by the literature (e.g. Zhu et al. 2007). <br><html><img src="/resource_browser/images/TC_qc/500px-MSC_Fig1.jpg" /></html><br>Figure 1 (qRT-PCR analysis, EGR-1) <br><br><br>CAGE analysis on these samples revealed that Egr-1 underwent transient induction within 30-60 min in response to the growth factor or cytokine (Fig 2). In contrast, CAGE analysis revealed no change in expression of alpha-actin 2 (ACTA2) in response to FGF-2 or IL-1beta within the 6h time frame (Fig 2). <br><br><html><img src="/resource_browser/images/TC_qc/500px-MSC_Fig2.jpg" /></html><br>Figure 2 (CAGE analysis, EGR-1 and ACTA2) <br><br><br>CAGE analysis (Fig 3) and subsequent qRT-PCR analysis using separate samples in which Egr-1 was induced (Fig 4) also revealed dynamic changes in the expression of two other prototypic immediate-early genes, c-FOS and FOSB, in response to FGF-2 or IL-1beta (Fig 5). <br><html><img src="/resource_browser/images/TC_qc/500px-MSC_Fig3.jpg" /></html><br>Figure 3 (CAGE analysis, c-FOS and FOSB) <br><br><br><html><img src="/resource_browser/images/TC_qc/500px-MSC_Fig4.jpg" /></html><br>Figure 4 (qRT-PCR analysis, EGR-1) <br><br><br><html><img src="/resource_browser/images/TC_qc/500px-MSC_Fig5.jpg" /></html><br>Figure 5 (qRT-PCR analysis, c-FOS and FOSB) <br> | ||
|TCSample_description=We provided total RNA (in triplicate) from growth arrested human aortic SMCs (Cell Applications) treated with IL-1beta or FGF-2 for periods of up to 6 hours. SMCs (pool of 3 donors) were grown in 100 mm petri dishes in Waymouth’s medium, pH 7.4, supplemented with 1 mM L-glutamine, 10 units/ml penicillin, 10 mcg/ml streptomycin and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO2. The cells were rendered growth-quiescent at 80-90% confluency by incubation in serum-free medium for 24h. The SMCs were then exposed to IL-1beta (10ng/ml) or FGF-2 (50ng/ml) for various times up to 6 hours (0, 15, 30, 45, 60, 120, 180, 240, 300 and 360 min). 0 min samples represent growth arrested and unstimulated cells. RNA was harvested using TRIzol reagent, quantitated using a Nanodrop spectrophometer and validated for the transient induction of Egr-1 mRNA (by quantitative real-time PCR) prior to shipment to the Omics Science Center, RIKEN Yokohama Institute (Japan) for CAGE analysis. | |TCSample_description=We provided total RNA (in triplicate) from growth arrested human aortic SMCs (Cell Applications) treated with IL-1beta or FGF-2 for periods of up to 6 hours. SMCs (pool of 3 donors) were grown in 100 mm petri dishes in Waymouth’s medium, pH 7.4, supplemented with 1 mM L-glutamine, 10 units/ml penicillin, 10 mcg/ml streptomycin and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO2. The cells were rendered growth-quiescent at 80-90% confluency by incubation in serum-free medium for 24h. The SMCs were then exposed to IL-1beta (10ng/ml) or FGF-2 (50ng/ml) for various times up to 6 hours (0, 15, 30, 45, 60, 120, 180, 240, 300 and 360 min). 0 min samples represent growth arrested and unstimulated cells. RNA was harvested using TRIzol reagent, quantitated using a Nanodrop spectrophometer and validated for the transient induction of Egr-1 mRNA (by quantitative real-time PCR) prior to shipment to the Omics Science Center, RIKEN Yokohama Institute (Japan) for CAGE analysis. | ||
|Time_Course= | |Time_Course= | ||
Revision as of 20:08, 12 February 2015
| Series: | IN_VITRO DIFFERENTIATION SERIES |
|---|---|
| Species: | Human (Homo sapiens) |
| Genomic View: | Zenbu |
| Expression table: | FILE |
| Link to TET: | TET |
| Sample providers : | Levon Khachigian |
| Germ layer: | mesoderm |
| Primary cells or cell line: | primary cells |
| Time span: | 6 hours |
| Number of time points: | 9 |
| Overview |
|---|
|
Vascular smooth muscle cells (SMCs) are key components in our blood vessels, and show remarkable plasticity. SMCs are normally growth-quiescent in the normal adult vessels, but are activated by injury, or exposure to growth factors, such as fibroblast growth factor-2 (FGF-2) and pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). These cues are sensed by these cells through changes in immediate-early gene expression, and can lead to increased proliferation and migration. These responses are associated with the initiation and progression of a range of vascular diseases including atherosclerosis, post-angioplasty restenosis and bypass graft stenosis. |
| Sample description |
|---|
|
We provided total RNA (in triplicate) from growth arrested human aortic SMCs (Cell Applications) treated with IL-1beta or FGF-2 for periods of up to 6 hours. SMCs (pool of 3 donors) were grown in 100 mm petri dishes in Waymouth’s medium, pH 7.4, supplemented with 1 mM L-glutamine, 10 units/ml penicillin, 10 mcg/ml streptomycin and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO2. The cells were rendered growth-quiescent at 80-90% confluency by incubation in serum-free medium for 24h. The SMCs were then exposed to IL-1beta (10ng/ml) or FGF-2 (50ng/ml) for various times up to 6 hours (0, 15, 30, 45, 60, 120, 180, 240, 300 and 360 min). 0 min samples represent growth arrested and unstimulated cells. RNA was harvested using TRIzol reagent, quantitated using a Nanodrop spectrophometer and validated for the transient induction of Egr-1 mRNA (by quantitative real-time PCR) prior to shipment to the Omics Science Center, RIKEN Yokohama Institute (Japan) for CAGE analysis. |
| Quality control |
|---|
|
Early growth response-1 (Egr-1) is an immediate-early gene (encoding a zinc finger transcription factor) that is poorly expressed in growth-quiescent cells and serves as a marker of cell activation or stress. Total RNA provided to the RIKEN Yokohama Institute was first analysed for Egr-1 expression by qRT-PCR. This demonstrated peak inducible expression after 30 min by FGF-2 and after 60 min by IL-1beta (Fig 1). Transient expression of this biomarker within 30-60 min is supported by the literature (e.g. Zhu et al. 2007). |
Profiled time course samples
Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples
| 12642-134G5 | Aortic smooth muscle cell response to FGF2 | 00hr00min | biol_rep1 |
| 12643-134G6 | Aortic smooth muscle cell response to FGF2 | 00hr15min | biol_rep1 |
| 12644-134G7 | Aortic smooth muscle cell response to FGF2 | 00hr30min | biol_rep1 |
| 12645-134G8 | Aortic smooth muscle cell response to FGF2 | 00hr45min | biol_rep1 |
| 12646-134G9 | Aortic smooth muscle cell response to FGF2 | 01hr | biol_rep1 |
| 12647-134H1 | Aortic smooth muscle cell response to FGF2 | 02hr | biol_rep1 |
| 12648-134H2 | Aortic smooth muscle cell response to FGF2 | 03hr | biol_rep1 |
| 12650-134H4 | Aortic smooth muscle cell response to FGF2 | 05hr | biol_rep1 |
| 12651-134H5 | Aortic smooth muscle cell response to FGF2 | 06hr | biol_rep1 |
| 12740-135I4 | Aortic smooth muscle cell response to FGF2 | 00hr00min | biol_rep2 |
| 12741-135I5 | Aortic smooth muscle cell response to FGF2 | 00hr15min | biol_rep2 |
| 12742-135I6 | Aortic smooth muscle cell response to FGF2 | 00hr30min | biol_rep2 |
| 12743-135I7 | Aortic smooth muscle cell response to FGF2 | 00hr45min | biol_rep2 |
| 12745-135I9 | Aortic smooth muscle cell response to FGF2 | 02hr | biol_rep2 |
| 12746-136A1 | Aortic smooth muscle cell response to FGF2 | 03hr | biol_rep2 |
| 12748-136A3 | Aortic smooth muscle cell response to FGF2 | 05hr | biol_rep2 |
| 12749-136A4 | Aortic smooth muscle cell response to FGF2 | 06hr | biol_rep2 |
| 12839-137B4 | Aortic smooth muscle cell response to FGF2 | 00hr15min | biol_rep3 |
| 12840-137B5 | Aortic smooth muscle cell response to FGF2 | 00hr30min | biol_rep3 |
| 12841-137B6 | Aortic smooth muscle cell response to FGF2 | 00hr45min | biol_rep3 |
| 12842-137B7 | Aortic smooth muscle cell response to FGF2 | 01hr | biol_rep3 |
| 12843-137B8 | Aortic smooth muscle cell response to FGF2 | 02hr | biol_rep3 |
| 12844-137B9 | Aortic smooth muscle cell response to FGF2 | 03hr | biol_rep3 |
| 12846-137C2 | Aortic smooth muscle cell response to FGF2 | 05hr | biol_rep3 |
| 12847-137C3 | Aortic smooth muscle cell response to FGF2 | 06hr | biol_rep3 |
