MicroRNA Dysregulation in Pulmonary Arteries from COPD: Relationships with Vascular Remodeling

Pulmonary vascular remodeling is an angiogenic-related process involving changes in smooth muscle cell (SMC) homeostasis, which is frequently observed in chronic obstructive pulmonary disease (COPD). MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA expression levels of many genes, leading to the manifestation of cell identity and specific cellular phenotypes. Here, we evaluate the miRNA expression profiles of pulmonary arteries (PAs) of patients with COPD and its relationship with the regulation of SMC phenotypic change. miRNA expression profiles from PAs of 12 patients with COPD, 9 smokers with normal lung function (SK), and 7 nonsmokers (NS) were analyzed using TaqMan Low-Density Arrays. In patients with COPD, expression levels of miR-98, miR-139-5p, miR-146b-5p, and miR-451 were upregulated, as compared with NS. In contrast, miR-197, miR-204, miR-485-3p, and miR-627 were downregulated. miRNA-197 expression correlated with both airflow obstruction and PA intimal enlargement. In an in vitro model of SMC differentiation, miR-197 expression was associated with an SMC contractile phenotype. miR-197 inhibition blocked the acquisition of contractile markers in SMCs and promoted a proliferative/migratory phenotype measured by both cell cycle analysis and wound-healing assay. Using luciferase assays, Western blot, and quantitative PCR, we confirmed that miR-197 targets the transcription factor E2F1. In PAs from patients with COPD, levels of E2F1 were increased as compared with NS. In PAs of patients with COPD, remodeling of the vessel wall is associated with downregulation of miR-197, which regulates SMC phenotype. The effect of miR-197 on PAs might be mediated, at least in part, by the key proproliferative factor, E2F1

Correction: Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype.

[This corrects the article DOI: 10.1371/journal.pone.0159460.]

Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype.

Previous studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling.Slug expression was decreased during both cell-to-cell contact and TGFβ1 induced SMC differentiation. Tumor necrosis factor-α (TNFα), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNFα-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGFβ1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries.Slug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases

MicroRNA Dysregulation in Pulmonary Arteries from Chronic Obstructive Pulmonary Disease. Relationships with Vascular Remodeling

Pulmonary vascular remodeling is an angiogenic-related process involving changes in smooth muscle cell (SMC) homeostasis, which is frequently observed in chronic obstructive pulmonary disease (COPD). MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA expression levels of many genes, leading to the manifestation of cell identity and specific cellular phenotypes. Here, we evaluate the miRNA expression profiles of pulmonary arteries (PAs) of patients with COPD and its relationship with the regulation of SMC phenotypic change. miRNA expression profiles from PAs of 12 patients with COPD, 9 smokers with normal lung function (SK), and 7 nonsmokers (NS) were analyzed using TaqMan Low-Density Arrays. In patients with COPD, expression levels of miR-98, miR-139-5p, miR-146b-5p, and miR-451 were upregulated, as compared with NS. In contrast, miR-197, miR-204, miR-485-3p, and miR-627 were downregulated. miRNA-197 expression correlated with both airflow obstruction and PA intimal enlargement. In an in vitro model of SMC differentiation, miR-197 expression was associated with an SMC contractile phenotype. miR-197 inhibition blocked the acquisition of contractile markers in SMCs and promoted a proliferative/migratory phenotype measured by both cell cycle analysis and wound-healing assay. Using luciferase assays, Western blot, and quantitative PCR, we confirmed that miR-197 targets the transcription factor E2F1. In PAs from patients with COPD, levels of E2F1 were increased as compared with NS. In PAs of patients with COPD, remodeling of the vessel wall is associated with downregulation of miR-197, which regulates SMC phenotype. The effect of miR-197 on PAs might be mediated, at least in part, by the key proproliferative factor, E2F1

Slug regulates genes related to proliferation and migration pathways.

<p><b>A,</b> 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>B,</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. <b>C</b>, 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.</p

Slug is upregulated in dedifferentated SMC.

<p><b>A</b>, Slug, and the related transcription factor Snail, decrease in mature SMC as determined by RT-PCR and western blot analysis. <b>B</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.</p

Analysis of Slug expression in the lungs of a mouse model of severe PAH.

<p><b>A</b>, 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>B-C</b>, Fulton index (B) and Right ventricular pressure (RVP) (C) are increased in CH and CHSU5416. <b>D</b>, 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. <b>E</b>, Correlation between Slug expression (1/dCt) with both the number of α-SMA positive vessels and <b>F</b>, the Fulton index (note that the lower the 1/dCt, the higher the Slug expression level). *p < 0.05 by Spearman analysis.</p

<i>In vitro</i> models of SMC phenotypic change.

<p><b>A</b>, 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>B</b>, Cell proliferation decreases in differentiated cells, as determined by Ki-67 expression and <b>C</b>, cell cycle analysis. <b>D</b>, 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. <b>E</b>, Gene expression of Ki-67 and <b>F</b>, 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.</p