Smooth muscle protein 22α-Cre recombination in resting cardiac fibroblasts and hematopoietic precursors.
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ABSTRACT:
SUBMITTER: Ikeda S
PROVIDER: S-EPMC9263136 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During development of the embryonic mouse lung, the pulmonary mesenchyme differentiates into smooth muscle that wraps around the airway epithelium. Inhibiting smooth muscle differentiation leads to cystic airways, while enhancing it stunts epithelial branching. These findings support a conceptual model wherein the differentiation of smooth muscle sculpts the growing epithelium into branches at precise positions and with stereotyped morphologies. Unfortunately, most approaches to manipulate the differentiation of airway smooth muscle rely on pharmacological or physical perturbations that are conducted ex vivo. Here, we explored the use of diphtheria toxin-based genetic ablation strategies to eliminate airway smooth muscle in the embryonic mouse lung. Surprisingly, neither airway smooth muscle wrapping nor epithelial branching were affected in embryos in which the expression of diphtheria toxin or its receptor were driven by several different smooth muscle-specific Cre lines. Close examination of spatial patterns of Cre activity in the embryonic lung revealed that none of these commonly used Cre lines target embryonic airway smooth muscle robustly or specifically. Our findings demonstrate the need for airway smooth muscle-specific Cre lines that are active in the embryonic lung, and serve as a resource for researchers contemplating the use of these commonly used Cre lines for studying embryonic airway smooth muscle.
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Project description:Mutations in the LMNA gene (encoding lamin A/C proteins) cause several human cardiac diseases, including dilated cardiomyopathies (LMNA-DCM). The main clinical risks in LMNA-DCM patients are sudden cardiac death and progressive left ventricular ejection fraction deterioration, and therefore most human and animal studies have sought to define the mechanisms through which LMNA mutations provoke cardiac alterations, with a particular focus on cardiomyocytes. To investigate if LMNA mutations also cause vascular alterations that might contribute to the etiopathogenesis of LMNA-DCM, we generated and characterized Lmnaflox/floxSM22αCre mice, which constitutively lack lamin A/C in vascular smooth muscle cells (VSMCs), cardiac fibroblasts, and cardiomyocytes. Like mice with whole body or cardiomyocyte-specific lamin A/C ablation, Lmnaflox/floxSM22αCre mice recapitulated the main hallmarks of human LMNA-DCM, including ventricular systolic dysfunction, cardiac conduction defects, cardiac fibrosis, and premature death. These alterations were associated with elevated expression of total and phosphorylated (active) Smad3 and cleaved (active) caspase 3 in the heart. Lmnaflox/floxSM22αCre mice also exhibited perivascular fibrosis in the coronary arteries and a switch of aortic VSMCs from the 'contractile' to the 'synthetic' phenotype. Ex vivo wire myography in isolated aortic rings revealed impaired maximum contraction capacity and an altered response to vasoconstrictor and vasodilator agents in Lmnaflox/floxSM22αCre mice. To our knowledge, our results provide the first evidence of phenotypic alterations in VSMCs that might contribute significantly to the pathophysiology of some forms of LMNA-DCM. Future work addressing the mechanisms underlying vascular defects in LMNA-DCM may open new therapeutic avenues for these diseases.
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Project description:Adult bone marrow-derived cells can participate in muscle regeneration after bone marrow transplantation. In recent studies a single hematopoietic stem cell (HSC) was shown to give rise to cells that not only reconstituted all of the lineages of the blood, but also contributed to mature muscle fibers. However, the relevant HSC derivative with this potential has not yet been definitively identified. Here we use fluorescence-activated cell sorter-based protocols to test distinct hematopoietic fractions and show that only fractions containing c-kit(+) immature myelomonocytic precursors are capable of contributing to muscle fibers after i.m. injection. Although these cells belong to the myeloid lineage, they do not include mature CD11b(+) myelomonocytic cells, such as macrophages. Of the four sources of mature macrophages tested that were derived either from monocytic culture, bone marrow, peripheral blood after granulocyte colony-stimulating factor mobilization, or injured muscle, none contributed to muscle. In addition, after transplantation of bone marrow isolated from CD11b-Cre-transgenic mice into the Cre-reporter strain (Z/EG), no GFP myofibers were detected, demonstrating that macrophages expressing CD11b do not fuse with myofibers. Irrespective of the underlying mechanisms, these data suggest that the HSC derivatives that integrate into regenerating muscle fibers exist in the pool of hematopoietic cells known as myelomonocytic progenitors.
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Project description:Mechanical cues activate cardiac fibroblasts and induce differentiation into myofibroblasts, which are key steps for development of cardiac fibrosis. In vitro, the high stiffness of plastic culturing conditions will also induce these changes. It is therefore challenging to study resting cardiac fibroblasts and their activation in vitro. Here we investigate the extent to which disrupting mechanotransduction by culturing cardiac fibroblasts on soft hydrogels or in the presence of biochemical inhibitors can be used to maintain resting cardiac fibroblasts in vitro. Primary cardiac fibroblasts were isolated from adult mice and cultured on plastic or soft (4.5 kPa) polyacrylamide hydrogels. Myofibroblast marker gene expression and smooth muscle α-actin (SMA) fibers were quantified by real-time PCR and immunostaining, respectively. Myofibroblast differentiation was prevented on soft hydrogels for 9 days, but had occurred after 15 days on hydrogels. Transferring myofibroblasts to soft hydrogels reduced expression of myofibroblast-associated genes, albeit SMA fibers remained present. Inhibitors of transforming growth factor β receptor I (TGFβRI) and Rho-associated protein kinase (ROCK) were effective in preventing and reversing myofibroblast gene expression. SMA fibers were also reduced by blocker treatment although cell morphology did not change. Reversed cardiac fibroblasts maintained the ability to re-differentiate after the removal of blockers, suggesting that these are functionally similar to resting cardiac fibroblasts. However, actin alpha 2 smooth muscle (Acta2), lysyl oxidase (Lox) and periostin (Postn) were no longer sensitive to substrate stiffness, suggesting that transient treatment with mechanotransduction inhibitors changes the mechanosensitivity of some fibrosis-related genes. In summary, our results bring novel insight regarding the relative importance of specific mechanical signaling pathways in regulating different myofibroblast-associated genes. Furthermore, combining blocker treatment with the use of soft hydrogels has not been tested previously and revealed that only some genes remain mechano-sensitive after phenotypic reversion. This is important information for researchers using inhibitors to maintain a "resting" cardiac fibroblast phenotype in vitro as well as for our current understanding of mechanosensitive gene regulation.
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Project description:The molecular events that control cell fate determination in cardiac and smooth muscle lineages remain elusive. Myocardin is an important transcription cofactor that regulates cell proliferation, differentiation, and development of the cardiovascular system. Here, we describe the construction and analysis of a dual Cre and enhanced green fluorescent protein (EGFP) knock-in mouse line in the Myocardin locus (Myocd(KI)). We report that the Myocd(KI) allele expresses the Cre enzyme and the EGFP in a manner that recapitulates endogenous Myocardin expression patterns. We show that Myocardin expression marks the earliest cardiac and smooth muscle lineages. Furthermore, this genetic model allows for the identification of a cardiac cell population, which maintains both Myocardin and Isl-1 expression, in E7.75-E8.0 embryos, highlighting the contribution and merging of the first and second heart fields during cardiogenesis. Therefore, the Myocd(KI) allele is a unique tool for studying cardiovascular development and lineage-specific gene manipulation.
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Project description:Excessive collagen deposition by myofibroblasts during adverse cardiac remodeling leads to myocardial fibrosis that can compromise cardiac function. Unraveling the mechanisms underlying collagen gene expression in cardiac myofibroblasts is therefore an important clinical goal. The collagen receptors, discoidin domain receptor 2 (DDR2), a collagen-specific receptor tyrosine kinase, and integrin-β1, are reported to mediate tissue fibrosis. Here, we probed the role of DDR2-integrin-β1 cross-talk in the regulation of collagen α1(I) gene expression in angiotensin II (Ang II)-stimulated cardiac fibroblasts. Results from gene silencing/overexpression approaches, electrophoretic mobility shift assays, and ChIP revealed that DDR2 acts via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK)-dependent transforming growth factor-β1 (TGF-β1) signaling to activate activator protein-1 (AP-1) that in turn transcriptionally enhances the expression of collagen-binding integrin-β1 in Ang II-stimulated cardiac fibroblasts. The DDR2-integrin-β1 link was also evident in spontaneously hypertensive rats and DDR2-knockout mice. Further, DDR2 acted via integrin-β1 to regulate α-smooth muscle actin (α-SMA) and collagen type I expression in Ang II-exposed cardiac fibroblasts. Downstream of the DDR2-integrin-β1 axis, α-SMA was found to regulate collagen α1(I) gene expression via the Ca2+ channel, transient receptor potential cation channel subfamily C member 6 (TRPC6), and the profibrotic transcription factor, Yes-associated protein (YAP). This finding indicated that fibroblast-to-myofibroblast conversion is mechanistically coupled to collagen expression. The observation that collagen receptor cross-talk underlies α-SMA-dependent collagen type I expression in cardiac fibroblasts expands our understanding of the complex mechanisms involved in collagen gene expression in the heart and may be relevant to cardiac fibrogenesis.
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Project description:Smooth muscle alpha-actin gene activity appears in promyocardial cells well before cardiac myocyte differentiation and is down-regulated during the onset of rhythmic contractility and cardiac morphogenesis. The levels of LIM-only CRP2 correlated well with smooth muscle gene activity. Cardiomyocyte-specific expression of CRP2 in transgenic mice showed robust expression of smooth muscle cell-specific transcripts and protein filaments in the adult heart. Protein transduction of a recombinant CRP2 protein, fused to the protein transduction domain of HIV, into neonatal heart cells induced de novo synthesis of smooth muscle cell-specific transcripts and proteins. The LIM zinc fingers in CRP2 were found to collaborate with Brg1 of the SNF/SWI complexes, recruited serum response factor, and remodeled smooth muscle target gene chromatin through histone acetylation. CRP2 may have a cytoskeletal role, but as a nuclear protein, CRP2 acted as a potent transcription coadaptor that remodeled silent cardiac myocyte chromatin and directed serum response factor-dependent smooth muscle gene activity.
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Project description:BackgroundA critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (α-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating α-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in α-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of α-SMA expression.MethodsThe association of MeCP2 with α-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor β1 (TGF-β1). The regulation of α-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on α-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of α-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing.ResultsThe human lung fibroblasts with increased α-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced α-SMA upregulation by TGF-β1. The 5'-Aza-treated cells have decreased α-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of α-SMA after being treated by TGF-β1 or 5'-aza.ConclusionOur data indicate that the methyl-binding protein MeCP2 is critical for α-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of α-SMA is not a determinative factor for its inducible expression.
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Project description: Not available
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