Project description:Cardiovascular disease is the leading cause of death in developed countries and there is compelling evidence that the majority of these fatalities are secondary to rupture of unstable atherosclerotic plaques. However, the mechanisms that control plaque stability are poorly understood, although it is widely believed that plaques having a decreased ratio of cells positive for smooth muscle cell (SMC) markers such as ACTA2 relative to macrophage markers are more likely to rupture. Herein we employMyh11-CreERT2 ROSA floxed STOP eYFP Apoe-/- mice to trace SMC lineage and show that traditional methods for detecting SMCs based on immunostaining for SMC markers like ACTA2 are unable to detect 82% of SMC-derived cells within advanced atherosclerotic lesions. Moreover, we show that these SMC-derived cells within advanced lesions exhibit multiple phenotypes including activation of multiple markers of macrophages, mesenchymal stem cells (MSC), and myofibroblasts (MF). Importantly, we show that SMC-derived macrophage like cells are phagocytic based on electron microscope YFP immunolabeling. In addition, results of single cell epigenetic assays developed in our lab shows that nearly 20% of macrophage-like cells within human lesions are of SMC not myeloid origin. These phenotypic transitions appear to be critical in lesion pathogenesis, in that we show that SMC-specific conditional knockout (KO) of the pluripotency factor, Krüppel-like factor 4 (KLF4), resulted in marked reductions in lesion size, increases in multiple indices of plaque stability, and reduced numbers of SMC-derived MSC- and macrophage-like cells. Finally, we show that cholesterol loading of cultured SMC is associated with activation of multiple markers of macrophages and MSC, secretion of pro-inflammatory cytokines, and increased phagocytosis, all of which are Klf4 dependent. Taken together, results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that loss of Klf4 within SMC result in major changes in SMC phenotype and function that play a major role in lesion pathogenesis. Examination of KLF4 transcription factor in an atherosclerosis setting

Project description:Cardiovascular disease is the leading cause of death in developed countries and there is compelling evidence that the majority of these fatalities are secondary to rupture of unstable atherosclerotic plaques. However, the mechanisms that control plaque stability are poorly understood, although it is widely believed that plaques having a decreased ratio of cells positive for smooth muscle cell (SMC) markers such as ACTA2 relative to macrophage markers are more likely to rupture. Herein we employMyh11-CreERT2 ROSA floxed STOP eYFP Apoe-/- mice to trace SMC lineage and show that traditional methods for detecting SMCs based on immunostaining for SMC markers like ACTA2 are unable to detect 82% of SMC-derived cells within advanced atherosclerotic lesions. Moreover, we show that these SMC-derived cells within advanced lesions exhibit multiple phenotypes including activation of multiple markers of macrophages, mesenchymal stem cells (MSC), and myofibroblasts (MF). Importantly, we show that SMC-derived macrophage like cells are phagocytic based on electron microscope YFP immunolabeling. In addition, results of single cell epigenetic assays developed in our lab shows that nearly 20% of macrophage-like cells within human lesions are of SMC not myeloid origin. These phenotypic transitions appear to be critical in lesion pathogenesis, in that we show that SMC-specific conditional knockout (KO) of the pluripotency factor, Krüppel-like factor 4 (KLF4), resulted in marked reductions in lesion size, increases in multiple indices of plaque stability, and reduced numbers of SMC-derived MSC- and macrophage-like cells. Finally, we show that cholesterol loading of cultured SMC is associated with activation of multiple markers of macrophages and MSC, secretion of pro-inflammatory cytokines, and increased phagocytosis, all of which are Klf4 dependent. Taken together, results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that loss of Klf4 within SMC result in major changes in SMC phenotype and function that play a major role in lesion pathogenesis.

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:SMCs express plasminogen activator inhibitor-1 (PAI-1), which regulates SMC function and vascular remodeling. However, whether PAI-1 controls SMC cytoskeletal dynamics and stiffness is unknown, and the causal role of PAI-1 in arterial stiffening is undefined. SMCs from human coronary arteries and aortae of wild-type vs. PAI-1-deficient mice were cultured with or without PAI-039, a specific PAI-1 inhibitor, after which cell stiffness was measured by atomic force microscopy, filamentous actin structures were assessed by confocal microscopy, and the activities cofilin, LIM domain kinase 1 (LIMK), slingshot homolog 1 (SSH), and AMP-activated protein kinase (AMPK) were measured. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. Effects of PAI-039 on aortic stiffness were assessed by pulse wave velocity. PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by significant decreases in cytoplasmic actin filaments. Similar effects were observed in wild-type, but not in PAI-1-deficient SMCs. Mechanistically, PAI-039 significantly increased the activity of cofilin, an actin depolymerase, in SMCs expressing PAI-1, but not in PAI-1-deficient cells. PAI-039 had no significant effects on LIMK or SSH activity. RNA-sequencing analysis suggested that PAI-039 up-regulates AMPK signaling in SMCs, which was confirmed by western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness without significantly altering peri-aortic fibrosis. PAI-039 decreases intrinsic SMC stiffness by reducing cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacologic inhibition of PAI-1 has potential to treat cardiovascular diseases mediated by accelerated arterial stiffening.

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Klf4 has an unexpected protective role in perivascular cells within the microvasculature

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