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AoSMC response to IL1b

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Series:IN_VITRO DIFFERENTIATION SERIES
Species:Human (Homo sapiens)
Genomic View:Zenbu
Expression table:TET
Link to TET:FILE
Sample providers :Levon Khachigian
Germ layer:mesoderm
Primary cells or cell line:primary cells
Time span:6 hours
Number of time points:10

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




Sample Name Time point Donor/Replica
12652-134H6 Aortic smooth muscle cell response to IL1b 00hr00min biol_rep1 (LK31)
12653-134H7 Aortic smooth muscle cell response to IL1b 00hr15min biol_rep1 (LK34)
12654-134H8 Aortic smooth muscle cell response to IL1b 00hr30min biol_rep1 (LK37)
12655-134H9 Aortic smooth muscle cell response to IL1b 00hr45min biol_rep1 (LK40)
12656-134I1 Aortic smooth muscle cell response to IL1b 01hr biol_rep1 (LK43)
12658-134I3 Aortic smooth muscle cell response to IL1b 03hr biol_rep1 (LK49)
12659-134I4 Aortic smooth muscle cell response to IL1b 04hr biol_rep1 (LK52)
12660-134I5 Aortic smooth muscle cell response to IL1b 05hr biol_rep1 (LK55)
12661-134I6 Aortic smooth muscle cell response to IL1b 06hr biol_rep1 (LK58)
12750-136A5 Aortic smooth muscle cell response to IL1b 00hr00min biol_rep2 (LK32)
12751-136A6 Aortic smooth muscle cell response to IL1b 00hr15min biol_rep2 (LK35)
12752-136A7 Aortic smooth muscle cell response to IL1b 00hr30min biol_rep2 (LK38)
12753-136A8 Aortic smooth muscle cell response to IL1b 00hr45min biol_rep2 (LK41)
12754-136A9 Aortic smooth muscle cell response to IL1b 01hr biol_rep2 (LK44)
12755-136B1 Aortic smooth muscle cell response to IL1b 02hr biol_rep2 (LK47)
12756-136B2 Aortic smooth muscle cell response to IL1b 03hr biol_rep2 (LK50)
12757-136B3 Aortic smooth muscle cell response to IL1b 04hr biol_rep2 (LK53)
12758-136B4 Aortic smooth muscle cell response to IL1b 05hr biol_rep2 (LK56)
12759-136B5 Aortic smooth muscle cell response to IL1b 06hr biol_rep2 (LK59)
12848-137C4 Aortic smooth muscle cell response to IL1b 00hr00min biol_rep3 (LK33)
12849-137C5 Aortic smooth muscle cell response to IL1b 00hr15min biol_rep3 (LK36)
12850-137C6 Aortic smooth muscle cell response to IL1b 00hr30min biol_rep3 (LK39)
12851-137C7 Aortic smooth muscle cell response to IL1b 00hr45min biol_rep3 (LK42)
12853-137C9 Aortic smooth muscle cell response to IL1b 02hr biol_rep3 (LK48)
12855-137D2 Aortic smooth muscle cell response to IL1b 04hr biol_rep3 (LK54)
12857-137D4 Aortic smooth muscle cell response to IL1b 06hr biol_rep3 (LK60)