Unknown,Transcriptomics,Genomics,Proteomics

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Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective. doi: 10.1038/nm.4109

ABSTRACT: The multiple claims about reactivation of the embryonic stem cell (ESC) pluripotency factor OCT4 in somatic cells are highly controversial due to the fact that there is no direct evidence that OCT4 has a functional role in cells other than ESCs. Herein we demonstrate that smooth muscle cell (SMC)-specific knockout of Oct4 within atherosclerotic mice resulted in increased lesion size and multiple changes consistent with decreased plaque stability. SMC-lineage tracing studies showed that lesions from SMC-specific conditional Oct4 KO mice had a reduced number of SMCs likely due to impaired SMC migration. RNA-seq analysis of lesion specimens showed that loss of Oct4 in SMCs was associated with marked activation of genes associated with inflammation and suppression of genes associated with cell migration, a number of which were shown to be activated in cultured SMCs by the combination of hypoxia and oxidized phospholipids in an OCT4-dependent manner. Activation of Oct4 within SMCs was associated with hydroxymethylation of the Oct4 promoter and was HIF1Î±- and KLF4-dependent. Results provide the first genetic evidence that OCT4 plays a functional role in somatic cells and highlight the importance of further investigation of possible OCT4 functions in somatic cells. In vivo: mRNA profiles of 18 week fed Western diet wild type (WT) and Oct4-/- mice were generated by deep sequencing, four animals per group, using Illumina HiSeq 2000. In vitro: a smooth muscle cell wild type (WT) and Oct4-/- (KO) primary aortic cell line was generated and used. mRNA profiles were generated by deep sequencing, in triplicates, using Illumina HiSeq 2000, for the following groups: WT-normoxia-vehicle; WT-normoxia-POVPC; KO-normoxia-vehicle; KO-normoxia-POVP; WT-hypoxia-vehicle; WT-hypoxia-POVPC; KO-hypoxia-vehicle; and KO-hypoxia-POVPC.

ORGANISM(S): Mus musculus

SUBMITTER: Gary Owens

PROVIDER: E-GEOD-75044 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Smooth muscle cells (SMCs) display dynamic plasticity by changing phenotype under various pathophysiological conditions. Uncovering how SMCs regulate their phenotype is a key to understanding the molecular mechanisms of a number of gastrointestinal diseases. MicroRNAs (miRNAs), generated in cells by Dicer, have been identified as regulators of the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal SMCs in a transgenic animal model. We generated SMC-specific Dicer (smDicer) null animals that express the reporter, green fluorescence protein (eGFP), in a SMC-specific manner. eGFP labeled SMCs were used for morphological, cytometric and genetic studies of smDicer null mutant and wild type control mice. The structure of the bowel wall was examined by confocal microscopy, and function was evaluated by recording spontaneous and evoked contractions. SMCs were purified with fluorescence-activated cell sorting and SM specific gene expression studies were performed with genechip arrays, PCR and quantitative PCR. Bioinformatic analyses were used to characterize the interaction between miRNAs and target genes. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs. Profiling and bioinformatic analyses showed SMC phenotype is regulated by a complex network of positive and negative feedback by SM miRNAs, serum response factor (SRF), and other transcriptional factors. SM miRNAs play an important role in growth, development and survival of SMCs. Phenotypic changes of SMCs may be regulated through multiple pathways in an interaction network of SM miRNAs, SRF, and additional transcriptional factors. We used microarrays to identify genetic changes during the development of gastric smooth muscle cells in the smDicer null mice. Mouse mRNA microarrays were performed on GeneChipÂ® Mouse Genome 430 2.0 Arrays (Affymetrix). Total RNAs isolated from the jejunal muscularis of the smDicer knockout (KO) and the wild type (WT) control mice at the ages of ~3 weeks were used for the arrays. The cDNA synthesis, hybridization, cDNA labeling, and scanning were performed. Gene expression between the smDicer KO and the WT controls were compared.

Project description:Recent smooth muscle cell (SMC) lineage tracing studies have demonstrated that SMCs undergo remarkable changes in phenotype during development of atherosclerosis, including loss of typical SMC markers such as ACTA2 and Klf4 dependent activation of multiple markers of macrophages and mesenchymal stem cells. Of major interest, we showed Klf4 in SMC is critical for overall lesion pathogenesis as SMC-specific conditional knockout (KO) of Klf4 resulted in smaller, more stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage-like and mesenchymal stem cell like cells. Herein we tested the hypothesis that Klf4 dependent SMC transitions are beneficial following myocardial infarction (MI). Utilizing SMC-specific lineage tracing mice +/- simultaneous SMC-specific conditional KO of Klf4, we demonstrate that SMCs express Klf4 and transition to a Klf4 dependent macrophage-like state as well as a Klf4 independent myofibroblast-like state. SMC specific, conditional KO of Klf4 resulted in marked exacerbation of heart failure following MI. Surprisingly, significant cardiac dilation was seen in SMC-Klf4 KO mice prior to MI. This cardiac dilation was accompanied by a reduction in peripheral resistance, as evidenced by a reduction in blood pressure, an increase in blood flow, and a larger passive diameter of mesenteric resistance arteries as measured by pressure myography. Klf4 ChIP-Seq analysis on the mesenteric resistance arteries identified potential Klf4 target genes in SMC at baseline in pathways including PDGF and FGF signaling previously shown to be important for perivascular cell investment. Interestingly, examination of microvascular tissue beds revealed gaps in lineage traced SMC coverage along the resistance arteries. Taken together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs, including being required for normal SMC coverage of resistance arteries as well as their function.

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Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective. doi: 10.1038/nm.4109

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