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. 2012 Apr 2;44(7):417-29.
doi: 10.1152/physiolgenomics.00160.2011. Epub 2012 Feb 7.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms

Matthew R Alexander  1 , Meera Murgai, Christopher W Moehle, Gary K Owens
Affiliations

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms

Matthew R Alexander et al. Physiol Genomics. .

Abstract

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

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Figures

Fig. 1.
Fig. 1.
IL-1β and PDGF-DD distinctly modulate SMC phenotype by global gene expression analysis. Venn diagrams demonstrating numbers of significantly upregulated (A) and downregulated (B) genes in smooth muscle cells (SMCs) after IL-1β or PDGF-DD treatment relative to vehicle controls.
Fig. 2.
Fig. 2.
IL-1β distinctly induces expression of multiple proinflammatory genes in SMCs. Real-time RT-PCR analysis of proinflammatory genes in SMCs 24 h after treatment with IL-1β (2.5 ng/ml) or PDGF-DD (30 ng/ml). Data represent means ± SE of mRNA levels normalized to 18s rRNA and the vehicle treatment group from 3 independent experiments (*P < 0.001 vs. vehicle by nested ANOVA; **P < 0.05 vs. vehicle by Student's t-test).
Fig. 3.
Fig. 3.
NF-κB mediates IL-1β-induced upregulation of multiple inflammatory genes in SMCs. Real-time RT-PCR analysis of VCAM1 (A), CCL20 (B), and P-selectin (C) mRNA levels 24 h after IL-1β (5 ng/ml) treatment of cultured SMCs infected with adenoviral IκBα superrepressor (Ad-IκBαSR) or control empty adenovirus (Ad-Empty). Data represent means ± SE of mRNA levels normalized to 18s rRNA and to each vehicle from 3 independent experiments performed in triplicate (*P < 0.001 and **P < 0.05 by nested ANOVA). D: representative Western blots of VCAM1 protein levels from SMC lysates after treatment with IL-1β or vehicle in cells infected with empty (Ad-E) or IκBαSR-overexpressing (AdI) adenoviruses. E: densitometry of VCAM1 band intensity relative to GAPDH by Western blot analysis as in D from 3 independent experiments. F: absorbance measures from analysis of CCL20 levels by ELISA in cell culture media 72 h after treatment with vehicle or IL-1β in cells infected with empty or IκBαSR-overexpressing adenoviruses (***P < 0.001 vs. vehicle by Student's t-test). Data represent means ± SE from 3 independent experiments.
Fig. 4.
Fig. 4.
IL-1β-induced repression of the mRNA levels of multiple SMC marker genes is NF-κB dependent. Real-time RT-PCR analysis of SM α-actin (A), SM22α (B), and calponin-1 (C) mRNA levels 24 h after IL-1β (5 ng/ml) treatment of cultured SMCs infected with adenoviral IκBα superrepressor (Ad-IκBαSR) or control empty adenovirus (Ad-Empty). Data represent means ± SE of 3 independent experiments each performed in triplicate (*P < 0.001 vs. vehicle by nested ANOVA; **P < 0.05 by Student's t-test; n = 3).
Fig. 5.
Fig. 5.
IL-1β-induced repression of smooth muscle myosin heavy chain (SMMHC) and smooth muscle (SM) α-actin protein levels are NF-κB dependent. A: representative images of Western blot analysis of multiple smooth muscle cell marker genes from cell lysates taken after 72 h of treatment with IL-1β (IL, 5 ng/ml) or vehicle (V) in cells infected with empty adenovirus (AdE) or IκBαSR-expressing adenovirus (AdIκB). GAPDH represents the loading control. Arrow, SM1 isoform of SMMHC (lower band is nonmuscle MHC). Densitometric quantitation of band intensity for SM α-actin (B), calponin-1 (C), SMMHC (D), and SM22α (E) normalized to GAPDH levels. All values were then normalized to the Ad-Empty vehicle control group. Data represent means ± SE from 3 independent experiments.
Fig. 6.
Fig. 6.
PDGF-DD does not significantly induce NF-κB activity in SMCs and PDGF-DD-induced repression of SMC marker genes is NF-κB-independent. A: luciferase activity in cell lysates from cultured SMCs transfected with 5XNF-κB-luciferase reporter constructs or control promoterless constructs and treated with IL-1β (2.5 ng/ml) or PDGF-DD (30 ng/ml) for 24 h. Data represent means ± SE of 5XNF-κB-luciferase activity normalized to promoterless control and total cellular protein followed by normalization to the vehicle group from 3 independent experiments (*P < 0.001 vs. vehicle by nested ANOVA). Real-time RT-PCR analysis of SM α-actin (B), SM22α (C), and calponin-1 (D) mRNA levels 24 h after PDGF-DD treatment (30 ng/ml) of cultured SMCs infected with empty adenovirus (Ad-Empty) or IκBαSR-expressing adenovirus (Ad-IκBαSR). Data represent means ± SE of 3 independent experiments in triplicate.
Fig. 7.
Fig. 7.
A subset of SM α-actin-negative cells within atherosclerotic plaques express an inflammatory but not proliferative phenotype marker. A: merged image from confocal microscopy of immunofluorescent staining for CCL20 (cyan), RGS17 (red), SM α-actin (green), and nuclei using DAPI (blue) of an atherosclerotic brachiocephalic artery from an Apoe−/− mouse fed a high-fat, Western-type diet for 28 wk (scale bar = 50 μm; M, media; P, plaque; L, lumen). B: single channel images from confocal microscopy at higher magnification on the boxed region in A showing the staining for each antigen separately along with the merged image on the far right (scale bars = 25 μm). Arrows indicate SM α-actin− CCL20+ RGS17− cells, boxes indicate SM α-actin+ CCL20− RGS17+ cells, circles indicate SM α-actin− CCL20− RGS17+ cells, and arrowheads indicate SM α-actin+ CCL20+ RGS17+ cells. Images are representative of brachiocephalic arteries from 3 mice.
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