SMC timecourses

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Primary Aortic Smooth Muscle Cells (AoSMC) Response to Interleukin-1beta (IL1-beta) and Fibroblast Growth Factor-2 (FGF-2)

Time course ID: human_Aortic_smooth_muscle_cell_FGF2 and human_Aortic_smooth_muscle_cell_IL1b
Sample provider:  Levon Khachigian, Margaret Patrikakis, Ahmad M.N. Alhendi

Introduction

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.

Samples

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).

MSC Fig1.jpg
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).

MSC Fig2.jpg
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).

MSC Fig3.jpg
Figure 3 (CAGE analysis, c-FOS and FOSB)


MSC Fig4.jpg
Figure 4 (qRT-PCR analysis, EGR-1)


MSC Fig5.jpg
Figure 5 (qRT-PCR analysis, c-FOS and FOSB)

References

[1] Zhu et al. Cardiovasc Res. 2007;76(1):141-8
[2] Silverman et al. Am J Pathol. 1999;155(4):1311-7






















































































Beginning of non-public section

Key Research Questions

1. Dynamics of gene expression: 1a. What are the top 100 most responsive genes (based on fold increase at any time point) in the SMC timecourse set? 1b. Which 100 genes are most differentially responsive to FGF-2 and IL-1beta in SMCs? For example, VCAM1 is induced 15 fold by IL-1beta at 2h but only a few fold by FGF-2. 1c. Can I correctly assume that FGFR1 is more basally expressed than IL1R based on the expression data? 1d. In relation to cluster analysis 1d(i). Which genes have an inducible profile (similar to Egr-1) in FGF-2-treated samples, IL-1beta-treated samples, or both? For example, Fos. 1d(ii). Which genes have an inducible profile like VCAM1 (post IEG induction), in FGF-2-treated samples, IL-1beta-treated samples, or both?

2. ISMARA and Motif analysis: 2a. Is ISMARA analysis available for the FGF-2-treated samples, IL-1beta-treated samples, or both? What are the similarities and differences? Specifically, which motifs are preferentially used (or used then no longer) used over time?

3. Chromatin remodelling: 3a. Does the IEG response impact on chromatin remodelling in the promoters/enhancers of these IEGs or other genes?

4. Promoter switching: 4a. To what extent does promoter switching occur in FGF-2-treated samples, IL-1beta-treated samples, or both? What are the similarities and differences? Is there a timecourse relationship? What about promoter switching in IEGs?

5. miRNA dynamics: 5a. Are there similarities and differences in miRNA expression in FGF-2-treated samples and IL-1beta-treated samples over time?

Please contact L.Khachigian@unsw.edu.au if you can help with any of these, or can pose or analyse other questions.


Zenbu configurations and status (COMPLETED)


Expression profiles


MARA based network results


Self organising maps


Related samples

AoSMC were exposed to either IL-1beta or FGF-2


References

Zhu et al. Cardiovasc Res. 2007;76(1):141-8
Silverman et al. Am J Pathol. 1999;155(4):1311-7


Quality control

Marker gene expression


ISMARA analysis results

Response to IL-1beta

All samples: http://ismara.unibas.ch/timecourses/IL1b/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/IL1b-avgd/averaged_report/index.html


Response to FGF-2

All samples: http://ismara.unibas.ch/timecourses/FGF2/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/FGF2-avgd/averaged_report/index.html

For more information, see ISMARA.

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