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1.
Stem Cell Res Ther ; 7(1): 115, 2016 08 17.
Article in English | MEDLINE | ID: mdl-27530160

ABSTRACT

BACKGROUND: The Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune system and of bone formation. Inappropriate activation of these pathways, as in conditions of congenital heterotopic ossification, are thought to activate an osteogenic program in endothelial cells. However, if and how this occurs in human endothelial cells remains unclear. METHODS: We used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP), a congenital disease of heterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy allowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of transgenic expression. These cells were challenged with BMP or Activin A ligand, and tested for their ability to activate osteogenesis, extracellular matrix production, and differential downstream signaling in the BMP/Activin A pathways. RESULTS: We found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally permissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining, suggesting formation of immature osteoblasts, but failed to show mature osteoblastic features. However, FOP iECs expressed more fibroblastic genes and Collagen 1/2 compared to control iECs, suggesting a mechanism for the tissue fibrosis seen in early heterotopic lesions. Finally, FOP iECs showed increased SMAD1/5/8 signaling upon BMP4 stimulation. Contrary to FOP hiPSCs, FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon Activin A stimulation, suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition, we found that the expression of ACVR1 and type II receptors were different in hiPSCs and iECs, which could explain the cell type-specific SMAD signaling. CONCLUSIONS: Our results suggest that the ACVR1 R206H mutation may not directly increase the formation of mature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell dysfunction, increase expression of fibrogenic matrix proteins, and cause differential downstream signaling of the ACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute to the pathogenesis of heterotopic ossification.


Subject(s)
Activin Receptors, Type I/genetics , Bone Morphogenetic Protein 4/metabolism , Collagen/metabolism , Endothelial Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Myositis Ossificans/genetics , Smad Proteins/metabolism , Activins/metabolism , Cell Differentiation/physiology , Cell Line , Chondrogenesis/genetics , Chondrogenesis/physiology , Endothelial Cells/physiology , Extracellular Matrix/metabolism , Humans , Induced Pluripotent Stem Cells/physiology , Ligands , Mutation , Myositis Ossificans/metabolism , Myositis Ossificans/pathology , Ossification, Heterotopic/genetics , Ossification, Heterotopic/metabolism , Ossification, Heterotopic/pathology , Osteoblasts/metabolism , Osteoblasts/physiology , Osteogenesis/physiology , Phosphorylation/genetics , Phosphorylation/physiology , Signal Transduction/physiology
2.
J Mol Cell Cardiol ; 84: 13-23, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25871831

ABSTRACT

Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on human aortic valve endothelium. mRNA-sequencing of primary human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, matrix gla protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.


Subject(s)
Aortic Valve Stenosis/genetics , Aortic Valve/pathology , Calcinosis/genetics , Calcium-Binding Proteins/metabolism , Endothelium, Vascular/metabolism , Extracellular Matrix Proteins/metabolism , Gene Regulatory Networks , Receptor, Notch1/metabolism , Aortic Valve Stenosis/pathology , Calcinosis/pathology , Chromatin Immunoprecipitation , Cluster Analysis , DNA/metabolism , Endothelial Cells/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Genome, Human , Humans , Protein Binding , Rheology , Sequence Analysis, RNA , Signal Transduction/genetics , Stress, Mechanical , Matrix Gla Protein
3.
Cell ; 160(6): 1072-86, 2015 Mar 12.
Article in English | MEDLINE | ID: mdl-25768904

ABSTRACT

The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. Here, we utilized human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Hemodynamic shear stress, which protects valves from calcification in vivo, activated anti-osteogenic and anti-inflammatory networks in NOTCH1(+/+), but not NOTCH1(+/-), iPSC-derived ECs. NOTCH1 haploinsufficiency altered H3K27ac at NOTCH1-bound enhancers, dysregulating downstream transcription of more than 1,000 genes involved in osteogenesis, inflammation, and oxidative stress. Computational predictions of the disrupted NOTCH1-dependent gene network revealed regulatory nodes that, when modulated, restored the network toward the NOTCH1(+/+) state. Our results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention.


Subject(s)
Epigenesis, Genetic , Gene Regulatory Networks , Receptor, Notch1/genetics , Endothelial Cells/metabolism , Female , Haploinsufficiency , Histone Code , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation/metabolism , Male , Osteogenesis , Pedigree , Receptor, Notch1/metabolism , Stress, Mechanical , Transcription, Genetic
4.
Stem Cells ; 31(1): 92-103, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23079999

ABSTRACT

Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized, and defined growth factors were used to generate KDR(+) EC progenitors. Magnetic purification of a KDR(+) progenitor subpopulation resulted in an expanding, homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders.


Subject(s)
Embryonic Stem Cells/metabolism , Endothelial Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Cell Differentiation/genetics , Cell Line , Cell Lineage , Gene Expression , Gene Expression Profiling , Gene Expression Regulation, Developmental , Genetic Variation , Humans , Mesoderm/embryology , Mesoderm/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Transcriptome/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
5.
Circ Res ; 111(11): 1421-33, 2012 Nov 09.
Article in English | MEDLINE | ID: mdl-22955733

ABSTRACT

RATIONALE: Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood. OBJECTIVE: To identify microRNAs that regulate angiogenesis. METHODS AND RESULTS: We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating proangiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth in the trunk of zebrafish larvae, whereas overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells. We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters vascular endothelial growth factor, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in human umbilical venous endothelial cells inhibited the angiogenic behavior of endothelial cells largely by antagonizing vascular endothelial growth factor receptor 2 signaling. CONCLUSIONS: Our study provides insights into how FLT1 and vascular endothelial growth factor receptor 2 signaling is titrated in a microRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.


Subject(s)
Endothelial Cells/metabolism , MicroRNAs/genetics , Neovascularization, Physiologic/genetics , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-2/genetics , Animals , Animals, Genetically Modified , Base Sequence , Cell Proliferation/drug effects , Cells, Cultured , Female , Gene Knockdown Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunoblotting , Larva/genetics , Larva/metabolism , Male , Mice , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Signal Transduction/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Zebrafish
6.
Cell Stem Cell ; 11(1): 91-9, 2012 Jul 06.
Article in English | MEDLINE | ID: mdl-22770243

ABSTRACT

Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.


Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/metabolism , X Chromosome Inactivation/genetics , Cell Differentiation/genetics , Cell Line , Chromosomes, Human, X/genetics , Feeder Cells/cytology , Feeder Cells/metabolism , Female , Gene Expression Regulation , Genes, X-Linked , Humans , Induced Pluripotent Stem Cells/cytology , Sequence Analysis, DNA
7.
Development ; 137(24): 4307-16, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21098571

ABSTRACT

Neural crest cells (NCCs) are a subset of multipotent, migratory stem cells that populate a large number of tissues during development and are important for craniofacial and cardiac morphogenesis. Although microRNAs (miRNAs) have emerged as important regulators of development and disease, little is known about their role in NCC development. Here, we show that loss of miRNA biogenesis by NCC-specific disruption of murine Dicer results in embryos lacking craniofacial cartilaginous structures, cardiac outflow tract septation and thymic and dorsal root ganglia development. Dicer mutant embryos had reduced expression of Dlx2, a transcriptional regulator of pharyngeal arch development, in the first pharyngeal arch (PA1). miR-452 was enriched in NCCs, was sufficient to rescue Dlx2 expression in Dicer mutant pharyngeal arches, and regulated non-cell-autonomous signaling involving Wnt5a, Shh and Fgf8 that converged on Dlx2 regulation in PA1. Correspondingly, knockdown of miR-452 in vivo decreased Dlx2 expression in the mandibular component of PA1, leading to craniofacial defects. These results suggest that post-transcriptional regulation by miRNAs is required for differentiation of NCC-derived tissues and that miR-452 is involved in epithelial-mesenchymal signaling in the pharyngeal arch.


Subject(s)
Branchial Region/embryology , MicroRNAs/metabolism , Neural Crest/embryology , Animals , Cell Line , DEAD-box RNA Helicases/genetics , Endoribonucleases/genetics , Fibroblast Growth Factor 8/genetics , Hedgehog Proteins/genetics , Homeodomain Proteins/genetics , In Situ Hybridization , Mice , Mice, Mutant Strains , MicroRNAs/genetics , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease III , Signal Transduction/genetics , Signal Transduction/physiology , Transcription Factors/genetics , Wnt Proteins/genetics , Wnt-5a Protein
8.
Nature ; 460(7256): 705-10, 2009 Aug 06.
Article in English | MEDLINE | ID: mdl-19578358

ABSTRACT

MicroRNAs (miRNAs) are regulators of myriad cellular events, but evidence for a single miRNA that can efficiently differentiate multipotent stem cells into a specific lineage or regulate direct reprogramming of cells into an alternative cell fate has been elusive. Here we show that miR-145 and miR-143 are co-transcribed in multipotent murine cardiac progenitors before becoming localized to smooth muscle cells, including neural crest stem-cell-derived vascular smooth muscle cells. miR-145 and miR-143 were direct transcriptional targets of serum response factor, myocardin and Nkx2-5 (NK2 transcription factor related, locus 5) and were downregulated in injured or atherosclerotic vessels containing proliferating, less differentiated smooth muscle cells. miR-145 was necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. Furthermore, miR-145 and miR-143 cooperatively targeted a network of transcription factors, including Klf4 (Kruppel-like factor 4), myocardin and Elk-1 (ELK1, member of ETS oncogene family), to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells.


Subject(s)
Cell Lineage , MicroRNAs/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Animals , Cell Differentiation , Cell Proliferation , Female , Gene Expression Regulation , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/metabolism , Kruppel-Like Factor 4 , Male , Mice , Mice, Transgenic , MicroRNAs/genetics , Models, Biological , Myocardium/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Vascular Diseases/metabolism , ets-Domain Protein Elk-4/metabolism
9.
Exp Gerontol ; 43(10): 882-91, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18755260

ABSTRACT

The observation that long-lived and relatively healthy animals can be obtained by simple genetic manipulation prompts the search for chemical compounds that have similar effects. Since aging is the most important risk factor for many socially and economically important diseases, the discovery of a wide range of chemical modulators of aging in model organisms could prompt new strategies for attacking age-related disease such as diabetes, cancer and neurodegenerative disorders [Collins, J.J., Evason, K., Kornfeld, K., 2006. Pharmacology of delayed aging and extended lifespan of Caenorhabditis elegans. Exp. Gerontol.; Floyd, R.A., 2006. Nitrones as therapeutics in age-related diseases. Aging Cell 5, 51-57; Gill, M.S., 2006. Endocrine targets for pharmacological intervention in aging in Caenorhabditis elegans. Aging Cell 5, 23-30; Hefti, F.F., Bales, R., 2006. Regulatory issues in aging pharmacology. Aging Cell 5, 3-8]. Resistance to multiple types of stress is a common trait in long-lived genetic variants of a number of species; therefore, we have tested compounds that act as stress response mimetics. We have focused on compounds with antioxidant properties and identified those that confer thermal stress resistance in the nematode Caenorhabditis elegans. Some of these compounds (lipoic acid, propyl gallate, trolox and taxifolin) also extend the normal lifespan of this simple invertebrate, consistent with the general model that enhanced stress resistance slows aging.


Subject(s)
Antioxidants/pharmacology , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Endocrine System/metabolism , Longevity/drug effects , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Genes, Helminth/drug effects , Genes, Helminth/physiology , Longevity/genetics , Models, Biological , Oxidative Stress/drug effects , Oxidative Stress/genetics
10.
Aging Cell ; 5(4): 283-91, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16913876

ABSTRACT

Population density, temperature and food availability all regulate the formation of the Caenorhabditis elegans dauer larva by modulating endocrine signaling pathways. The orphan nuclear receptor DAF-12 is pivotal for the decision to form a dauer or to undergo normal reproductive development. The DAF-12 ligand has been predicted to be a sterol that is metabolized by DAF-9, a cytochrome P450. Here we chemically characterize purified lipophilic nematode extracts and show that the ligand for DAF-12 contains a carboxyl moiety and is likely to be derived from a sterol. Using a candidate ligand approach we find that the C27 bile acid cholestenoic acid (5-cholesten-3beta-ol-(25S)-carboxylic acid) promotes reproductive growth in dauer-constitutive mutants in a daf-9- and daf-12-dependent manner. Furthermore, we find that cholestenoic acid can act as a DAF-12 ligand by activating DAF-12 in a cell-based transcription assay. Analysis of dauer-rescuing lipophilic extracts from nematodes by gas chromatography-mass spectrometry indicates the presence of several regioisomers of cholestenoic acid that are distinct from Delta(5)-cholestenoic acid and are not present in extracts from daf-9 mutants. These data suggest that carboxylated sterols may be key determinants of life history.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/growth & development , Larva/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Cell Line , Cholestenes/pharmacology , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Gene Expression Regulation, Developmental , Humans , Larva/metabolism , Ligands , Mutation , Phenotype , Receptors, Cytoplasmic and Nuclear/genetics , Transfection
11.
Article in English | MEDLINE | ID: mdl-20483265

ABSTRACT

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.

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