Fig 1.
In vitro models of SMC phenotypic change.
A, RT-PCR and immunofluorescence of the SMC differentiation markers (myoCD, sm22α, calponin, caldesmon, GATA6, α-SMA and SM-MHC, and the transcription factor KLF4 at D0, D2 and D6 states show the acquisition of a mature phenotype in SMC during differentiation. B, Cell proliferation decreases in differentiated cells, as determined by Ki-67 expression and C, cell cycle analysis. D, RT-PCR of SMC markers and KLF4 and immunofluorescence of α-SMA, calponin and SM-MHC in TNFα treated SMC showing the induction of SMC dedifferentiation by this cytokine. E, Gene expression of Ki-67 and F, cell cycle analysis increase significantly in TNFα treated SMC when compared with controls, indicating greater proliferation. Data are expressed as the mean ± SEM of five independent experiments performed in duplicate. *p < 0.05 by one-way ANOVA.
Fig 2.
Slug is upregulated in dedifferentated SMC.
A, Slug, and the related transcription factor Snail, decrease in mature SMC as determined by RT-PCR and western blot analysis. B, RT-PCR and western blot analyses show increased Slug expression in contractile SMC after 48 h of TNFα treatment. Data are expressed as the mean ± SEM of five independent experiments performed in duplicate. *p < 0.05 by one-way ANOVA.
Fig 3.
Slug regulates SMC proliferation and migration rate.
A, RT-PCR and inmunofluorescence analysis showed that Slug inhibition promoted downregulation of Ki-67 expression, consistent with decreased S phase of cell cycle analyzed by flow cytometry (B). C, Slug overexpression increases SMC proliferation analyzed by Ki67 expression. F and G, Cell migration was measured by wound-healing assay and expressed as the percentage (%) of SMC migrating/time. Slug knockdown cells display lower migration rates than control cells (F), while Slug-overexpressing SMC exhibit higher migration rate than control cells (G). Data are expressed as the mean ± SEM of four independent experiments performed in duplicate. *p < 0.05 by paired t-test.
Fig 4.
Slug regulates both TNFα-induced dedifferentiation and TGFβ1-induced differentiation.
A, The increase of KLF4 and the decrease of SMC marker genes induced by TNFα treatment is prevented by Slug knockdown measured by RT-PCR B-C, TGFβ1-induced increase of SMC markers is dampened in Slug overexpressing cells as observed by RT-PCR (B) and immunofluorescence (C). Data are expressed as mean ± SEM of three independent experiments performed in duplicate. *p < 0.05 by paired t-test.
Fig 5.
Slug regulates genes related to proliferation and migration pathways.
A, Scatter plot of the per-gene log2-fold changes following Slug knockdown (x-axis). Differentially expressed genes in Slug knockdown cells respect to control are marked in red. Validated genes are marked in blue. The vertical dashed lines mark the two fold changes. B, Enrichment plot of the GSEA cell cycle. GSEA gave a normalized enrichment score of -1.621 and an FDR of 0.0, indicating a significant enrichment of downregulated cell cycle-associated genes. C, Validation of Slug, Snail and other genes array performed by RT-PCR show the downregulation of HBEGF, CCNA2, and the upregulation of CLDN1, RARRES3 and KRT19. Data are expressed as the mean ± SEM of five independent experiments performed in duplicate. *p < 0.05 by one-way ANOVA.
Fig 6.
Analysis of Slug expression in the lungs of a mouse model of severe PAH.
A, Partially (within 25–75% visible muscularization) and totally muscularized (within 75–100% visible muscularization) intrapulmonary vessels with a diameter <50 μm analyzed in CTL: control group (n = 6), CH: animals exposed to chronic hypoxia (n = 5), CH+SU5416: animals exposed to chronic hypoxia plus Sugen 5416 (n = 5) show positive immunostaining for α-SMA in CH and CH+SU5416 animals. *p<0.05 by one way ANOVA. B-C, Fulton index (B) and Right ventricular pressure (RVP) (C) are increased in CH and CHSU5416. D, RT-PCR in lung homogenates showed increased Slug, but not Snail expression, in the group exposed to CH and CHSU5416 analyzed by one-way ANOVA. E, Correlation between Slug expression (1/dCt) with both the number of α-SMA positive vessels and F, the Fulton index (note that the lower the 1/dCt, the higher the Slug expression level). *p < 0.05 by Spearman analysis.
Fig 7.
Analysis of Slug expression in human pulmonary arteries.
A, Samples were classified according to the degree of vascular remodeling: low (R1), mild (R2) and high (R3). Slug but not Snail expression was significantly increased in R3 arteries compared with R1. *p<0.05 by one way ANOVA. B, Slug expression (1/dCt) displays a positive correlation with the intimal enlargement of pulmonary arteries measured by % of intimal thickness of the pulmonary artery (r = 0.44, p<0.05 by Pearson test). Note that the lower the 1/dCt, the higher the expression level. C, Representative images of Slug immunostaining; Slug expression is significantly increased in dedifferentiated intimal pulmonary SMC and EC of highly remodeled pulmonary arteries (right image) compared with less remodeled (left image). Negative control is shown in the first picture. Slug positive areas were quantified in highly remodeled (n = 3) and less remodeled (n = 4) arteries and normalized by the total wall area. Highly remodeled pulmonary arteries display a significant increase in Slug positive cells compared with less remodeled arteries. *p<0.05 by t-test. D, Slug (red), α-SMA (green) and merged (yellow) immunofluorescence images. Slug expression is predominantly found in the intima layer of remodeled pulmonary arteries.
Fig 8.
Slug induces a proliferative phenotype of SMC.
A, Schematic figure showing the contribution of both Slug and Snail to the phenotypic switch of EC and SMC. Under an inflammatory/hypoxic environment the expression of Slug and Snail increase in EC and promotes EnMT, while only Slug increases in SMC to induce a proliferative SMC phenotype. B, Schematic figure representing the most prominent molecular players involved in the phenotypic switch of SMC. In contractile cells, MYCD-SRF/MRTF-SRF complexes bind to CArG boxes, while bHLH transcription factors, like USF bind to E-boxes to activate and maintain expression of SMC specific markers. In this stage, Slug is minimally expressed. In consequence, expression of its known targets, like CDN1 and KRT19, as well as the tumor suppressor RARRES3, are high and migration is repressed. Expression of the cell cycle related genes, CCNA2 and HBEGF, are highly expressed and the rate of proliferation is very low. Under specific stimuli, such as inflammation and/or hypoxia, the expression of Slug is triggered. Expression of its target genes CDN1 and KRT19 are blocked and cell migration is activated. Moreover, expression of CCNA2 and HBEGF decrease and cells become proliferative. In this condition, the expression of the stem cell factor KLF4 is upregulated. Expression of MYCD and its binding to CArG boxes is also repressed resulting in decreased expression of SMC specific markers. MYCD, myocardin; SRF, serum response factor; MRTF, myocardin-related transcription factors; KLF4, krüppel like factor 4; CDN1, claudin1; KRT19, keratin 19; RARRES3, retinoic acid receptor related gene 3; CCNA2, cyclinA2; HBEGF, Heparin-Binding EGF-Like Growth Factor.