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{{TimeCourse
 
{{TimeCourse
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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.
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|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>
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|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=
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|category_treatment=Differentiation
 
|collaborators=Levon Khachigian
 
|collaborators=Levon Khachigian
 
|description=human_Aortic_smooth_muscle_cell_FGF2
 
|description=human_Aortic_smooth_muscle_cell_FGF2
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|germ_layer=mesoderm
 
|libraryids=CNhs13339,CNhs13340,CNhs13341,CNhs13343,CNhs12741,CNhs13344,CNhs13345,CNhs13347,CNhs13348,CNhs13358,CNhs13359,CNhs13360,CNhs13361,CNhs13363,CNhs13364,CNhs13367,CNhs13368,CNhs13568,CNhs13569,CNhs13571,CNhs13683,CNhs13572,CNhs13573,CNhs13575,CNhs13576
 
|libraryids=CNhs13339,CNhs13340,CNhs13341,CNhs13343,CNhs12741,CNhs13344,CNhs13345,CNhs13347,CNhs13348,CNhs13358,CNhs13359,CNhs13360,CNhs13361,CNhs13363,CNhs13364,CNhs13367,CNhs13368,CNhs13568,CNhs13569,CNhs13571,CNhs13683,CNhs13572,CNhs13573,CNhs13575,CNhs13576
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|number_time_points=9
 
|page_name=human_Aortic_smooth_muscle_cell_FGF2
 
|page_name=human_Aortic_smooth_muscle_cell_FGF2
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|primary_cells=primary cells
 
|series=IN_VITRO DIFFERENTIATION SERIES
 
|series=IN_VITRO DIFFERENTIATION SERIES
 
|species=Human (Homo sapiens)
 
|species=Human (Homo sapiens)
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=tS4-Tkr9BZ8h1ceHaImYgB
+
|tet_config=http://fantom.gsc.riken.jp/5/suppl/tet/Aortic_smooth_muscle_cell_FGF2.tsv.gz
 +
|tet_file=http://fantom.gsc.riken.jp/5/tet#!/search/?filename=hg19.cage_peak_phase1and2combined_tpm_ann_decoded.osc.txt.gz&file=1&c=1&c=110&c=111&c=113&c=114&c=115&c=116&c=117&c=118&c=119&c=120&c=121&c=122&c=124&c=125&c=126&c=127&c=128&c=129&c=130&c=134&c=135&c=136&c=137&c=138&c=139
 +
|time_points=0hr
 +
|time_span=6 hours
 +
|timepoint_design=Early focus
 +
|tissue_cell_type=Aortic smooth muscle
 +
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=_UNEnOckJFPiYiMWXz39eC
 
}}
 
}}

Revision as of 07:49, 10 March 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).

Figure 1 (qRT-PCR analysis, EGR-1)


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).


Figure 2 (CAGE analysis, EGR-1 and ACTA2)


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).

Figure 3 (CAGE analysis, c-FOS and FOSB)



Figure 4 (qRT-PCR analysis, EGR-1)



Figure 5 (qRT-PCR analysis, c-FOS and FOSB)

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



12642-134G5Aortic smooth muscle cell response to FGF200hr00minbiol_rep1
12643-134G6Aortic smooth muscle cell response to FGF200hr15minbiol_rep1
12644-134G7Aortic smooth muscle cell response to FGF200hr30minbiol_rep1
12645-134G8Aortic smooth muscle cell response to FGF200hr45minbiol_rep1
12646-134G9Aortic smooth muscle cell response to FGF201hrbiol_rep1
12647-134H1Aortic smooth muscle cell response to FGF202hrbiol_rep1
12648-134H2Aortic smooth muscle cell response to FGF203hrbiol_rep1
12650-134H4Aortic smooth muscle cell response to FGF205hrbiol_rep1
12651-134H5Aortic smooth muscle cell response to FGF206hrbiol_rep1
12740-135I4Aortic smooth muscle cell response to FGF200hr00minbiol_rep2
12741-135I5Aortic smooth muscle cell response to FGF200hr15minbiol_rep2
12742-135I6Aortic smooth muscle cell response to FGF200hr30minbiol_rep2
12743-135I7Aortic smooth muscle cell response to FGF200hr45minbiol_rep2
12745-135I9Aortic smooth muscle cell response to FGF202hrbiol_rep2
12746-136A1Aortic smooth muscle cell response to FGF203hrbiol_rep2
12748-136A3Aortic smooth muscle cell response to FGF205hrbiol_rep2
12749-136A4Aortic smooth muscle cell response to FGF206hrbiol_rep2
12839-137B4Aortic smooth muscle cell response to FGF200hr15minbiol_rep3
12840-137B5Aortic smooth muscle cell response to FGF200hr30minbiol_rep3
12841-137B6Aortic smooth muscle cell response to FGF200hr45minbiol_rep3
12842-137B7Aortic smooth muscle cell response to FGF201hrbiol_rep3
12843-137B8Aortic smooth muscle cell response to FGF202hrbiol_rep3
12844-137B9Aortic smooth muscle cell response to FGF203hrbiol_rep3
12846-137C2Aortic smooth muscle cell response to FGF205hrbiol_rep3
12847-137C3Aortic smooth muscle cell response to FGF206hrbiol_rep3