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1.
Arterioscler Thromb Vasc Biol ; 44(6): 1330-1345, 2024 06.
Artículo en Inglés | MEDLINE | ID: mdl-38602103

RESUMEN

BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.


Asunto(s)
Proteína Similar al Receptor de Calcitonina , Enfermedad de la Arteria Coronaria , Células Endoteliales , Elementos de Facilitación Genéticos , Polimorfismo de Nucleótido Simple , Estrés Mecánico , Humanos , Células Endoteliales/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Proteína Similar al Receptor de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina/metabolismo , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Mecanotransducción Celular , Células Cultivadas , Regulación de la Expresión Génica , Unión Proteica , Predisposición Genética a la Enfermedad , Sitios de Unión
2.
Elife ; 122024 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-38578680

RESUMEN

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.


Asunto(s)
Aterosclerosis , Enfermedad de la Arteria Coronaria , Humanos , Aterosclerosis/metabolismo , Cromatina/metabolismo , Enfermedad de la Arteria Coronaria/genética , Células Endoteliales/metabolismo , Estudio de Asociación del Genoma Completo
3.
J Cell Biol ; 223(3)2024 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-38231044

RESUMEN

Vascular homeostasis and pathophysiology are tightly regulated by mechanical forces generated by hemodynamics. Vascular disorders such as atherosclerotic diseases largely occur at curvatures and bifurcations where disturbed blood flow activates endothelial cells while unidirectional flow at the straight part of vessels promotes endothelial health. Integrated analysis of the endothelial transcriptome, the 3D epigenome, and human genetics systematically identified the SNP-enriched cistrome in vascular endothelium subjected to well-defined atherosclerosis-prone disturbed flow or atherosclerosis-protective unidirectional flow. Our results characterized the endothelial typical- and super-enhancers and underscored the critical regulatory role of flow-sensitive endothelial super-enhancers. CRISPR interference and activation validated the function of a previously unrecognized unidirectional flow-induced super-enhancer that upregulates antioxidant genes NQO1, CYB5B, and WWP2, and a disturbed flow-induced super-enhancer in endothelium which drives prothrombotic genes EDN1 and HIVEP in vascular endothelium. Our results employing multiomics identify the cis-regulatory architecture of the flow-sensitive endothelial epigenome related to atherosclerosis and highlight the regulatory role of super-enhancers in mechanotransduction mechanisms.


Asunto(s)
Aterosclerosis , Células Endoteliales , Mecanotransducción Celular , Humanos , Aterosclerosis/genética , Endotelio Vascular
4.
bioRxiv ; 2024 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-37066416

RESUMEN

Objective: Endothelial cells (ECs), macrophages, and vascular smooth muscle cells (VSMCs) are major cell types in atherosclerosis progression, and heterogeneity in EC sub-phenotypes are becoming increasingly appreciated. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Approach and Results: To create an in vitro dataset to study human EC heterogeneity, multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs). To model pro-inflammatory and activating environments characteristic of the atherosclerotic microenvironment in vitro, HAECs from at least three donors were exposed to three distinct perturbations with their respective controls: transforming growth factor beta-2 (TGFB2), interleukin-1 beta (IL1B), and siRNA-mediated knock-down of the endothelial transcription factor ERG (siERG). To form a comprehensive in vivo/ex vivo dataset of human atherosclerotic cell types, meta-analysis of single cell transcriptomes across 17 human arterial specimens was performed. Two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and in vivo cell profiles. HAEC cultures were reproducibly populated by 4 major clusters with distinct pathway enrichment profiles: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Exposure to siERG, IL1B or TGFB2 elicited mostly distinct transcriptional and accessible chromatin responses. EC1 and EC2, the most canonically 'healthy' EC populations, were affected predominantly by siERG; the activated cluster EC3 was most responsive to IL1B; and the mesenchymal population EC4 was most affected by TGFB2. Quantitative comparisons between in vitro and in vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated SNPs from GWAS, suggesting these cell phenotypes harbor CAD-modulating mechanisms. Conclusion: Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here, which have been reported by others to be involved in the pathogenesis of atherosclerosis as well as induce endothelial-to-mesenchymal transition (EndMT), only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and in vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

5.
Arterioscler Thromb Vasc Biol ; 44(1): 238-253, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38031841

RESUMEN

BACKGROUND: Biological sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (ECs) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC. METHODS: We used human and mouse aortic EC as well as EC-lineage tracing (Cdh5-CreERT2 Rosa-YFP [yellow fluorescence protein]) atherosclerotic Apoe-/- mice to investigate the biological sexual dimorphism of the EC functions in vitro and in vivo. Bioinformatics analyses were performed on male and female mouse aortic EC and human lung and aortic EC. RESULTS: In vitro, female human and mouse aortic ECs showed more apoptosis and higher cellular reactive oxygen species levels than male EC. In addition, female mouse aortic EC had lower mitochondrial membrane potential (ΔΨm), lower TFAM (mitochondrial transcription factor A) levels, and decreased angiogenic potential (tube formation, cell viability, and proliferation) compared with male mouse aortic EC. In vivo, female mice had significantly higher lipid accumulation within the aortas, impaired glucose tolerance, and lower endothelial-mediated vasorelaxation than males. Using the EC-lineage tracing approach, we found that female lesions had significantly lower rates of intraplaque neovascularization and endothelial-to-mesenchymal transition within advanced atherosclerotic lesions but higher incidents of missing EC lumen coverage and higher levels of oxidative products and apoptosis. RNA-seq analyses revealed that both mouse and human female EC had higher expression of genes associated with inflammation and apoptosis and lower expression of genes related to angiogenesis and oxidative phosphorylation than male EC. CONCLUSIONS: Our study delineates critical sex-specific differences in EC relevant to proinflammatory, pro-oxidant, and angiogenic characteristics, which are entirely consistent with a vulnerable phenotype in females. Our results provide a biological basis for sex-specific proatherosclerotic mechanisms.


Asunto(s)
Enfermedades de la Aorta , Aterosclerosis , Femenino , Masculino , Humanos , Ratones , Animales , Células Endoteliales/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/patología , Aorta/patología , Células Cultivadas , Especies Reactivas de Oxígeno/metabolismo , Inflamación/metabolismo , Ratones Endogámicos C57BL
6.
Proc Natl Acad Sci U S A ; 120(38): e2218150120, 2023 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-37695914

RESUMEN

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.


Asunto(s)
Proteasa La , Factor de Necrosis Tumoral alfa , Humanos , Factor de Necrosis Tumoral alfa/farmacología , Acetilcoenzima A , Células Endoteliales , Histonas , Citocinas
7.
J Lipid Res ; 64(8): 100411, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37437844

RESUMEN

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs.


Asunto(s)
Citocinas , Células Endoteliales , Humanos , Activación Transcripcional , Células Endoteliales/metabolismo , Citocinas/metabolismo , Colesterol/metabolismo , Factores de Transcripción/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Factor 6 Similar a Kruppel/genética , Factor 6 Similar a Kruppel/metabolismo
8.
Elife ; 122023 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-36971339

RESUMEN

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA sequencing and protein expression by liquid chromatography-mass spectrometry directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.


Asunto(s)
Células Endoteliales , Perfilación de la Expresión Génica , Humanos , Células Endoteliales/metabolismo , Endotelio , Células Cultivadas , Técnicas de Cocultivo
10.
Genome Res ; 32(3): 409-424, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35193936

RESUMEN

Functional consequences of genetic variation in the noncoding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of functional noncoding SNPs with effects in human endothelial cells (ECs), we utilized our previous molecular quantitative trait locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional noncoding SNPs. Together with information from genome-wide association studies (GWASs) for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5711 variants validated, whose enriched attributes included: (1) mutations to TF binding motifs for ETS or AP-1 that are regulators of the EC state; (2) location in accessible and H3K27ac-marked EC chromatin; and (3) molQTL associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of noncoding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlighted evidence suggesting mechanisms by which noncoding SNPs at two loci affect risk for pulse pressure/large artery stroke and abdominal aortic aneurysm through respective effects on transcriptional regulation of POU4F1 and LDAH Together, we highlight the attributes and context dependence of functional noncoding SNPs and provide new mechanisms underlying vascular disease risk.


Asunto(s)
Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , Alelos , Células Endoteliales , Predisposición Genética a la Enfermedad , Humanos , Sitios de Carácter Cuantitativo
11.
Elife ; 112022 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-35049498

RESUMEN

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPß binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPß at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.


Asunto(s)
Regulación de la Expresión Génica , Genómica/métodos , Factores de Transcripción/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Humanos , Células K562 , Masculino , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética
12.
Nat Commun ; 12(1): 7104, 2021 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-34876579

RESUMEN

Idiopathic pulmonary arterial hypertension (IPAH) is a rare but fatal disease diagnosed by right heart catheterisation and the exclusion of other forms of pulmonary arterial hypertension, producing a heterogeneous population with varied treatment response. Here we show unsupervised machine learning identification of three major patient subgroups that account for 92% of the cohort, each with unique whole blood transcriptomic and clinical feature signatures. These subgroups are associated with poor, moderate, and good prognosis. The poor prognosis subgroup is associated with upregulation of the ALAS2 and downregulation of several immunoglobulin genes, while the good prognosis subgroup is defined by upregulation of the bone morphogenetic protein signalling regulator NOG, and the C/C variant of HLA-DPA1/DPB1 (independently associated with survival). These findings independently validated provide evidence for the existence of 3 major subgroups (endophenotypes) within the IPAH classification, could improve risk stratification and provide molecular insights into the pathogenesis of IPAH.


Asunto(s)
Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/metabolismo , Perfilación de la Expresión Génica , Transcriptoma , 5-Aminolevulinato Sintetasa , Regulación hacia Abajo , Cadenas beta de HLA-DP , Humanos , Hipertensión Arterial Pulmonar
13.
Nat Metab ; 3(11): 1552-1568, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34697471

RESUMEN

We have previously suggested a central role for mitochondria in the observed sex differences in metabolic traits. However, the mechanisms by which sex differences affect adipose mitochondrial function and metabolic syndrome are unclear. Here we show that in both mice and humans, adipose mitochondrial functions are elevated in females and are strongly associated with adiposity, insulin resistance and plasma lipids. Using a panel of diverse inbred strains of mice, we identify a genetic locus on mouse chromosome 17 that controls mitochondrial mass and function in adipose tissue in a sex- and tissue-specific manner. This locus contains Ndufv2 and regulates the expression of at least 89 mitochondrial genes in females, including oxidative phosphorylation genes and those related to mitochondrial DNA content. Overexpression studies indicate that Ndufv2 mediates these effects by regulating supercomplex assembly and elevating mitochondrial reactive oxygen species production, which generates a signal that increases mitochondrial biogenesis.


Asunto(s)
Tejido Adiposo/metabolismo , Biomarcadores , Regulación de la Expresión Génica , Síndrome Metabólico/etiología , Síndrome Metabólico/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , NADH Deshidrogenasa/genética , Adiposidad/genética , Animales , Respiración de la Célula/genética , Cromosomas Humanos Par 17 , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Perfilación de la Expresión Génica , Estudios de Asociación Genética , Humanos , Masculino , Síndrome Metabólico/diagnóstico , Ratones , NADH Deshidrogenasa/metabolismo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Carácter Cuantitativo Heredable , Especies Reactivas de Oxígeno/metabolismo , Factores Sexuales
14.
Cell Rep ; 35(13): 109293, 2021 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-34192535

RESUMEN

While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.


Asunto(s)
Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Fenómenos Biomecánicos , Médula Ósea/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Mecanotransducción Celular , Invasividad Neoplásica , Microambiente Tumoral
15.
Immunometabolism ; 3(3)2021.
Artículo en Inglés | MEDLINE | ID: mdl-34178388

RESUMEN

BACKGROUND: Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity in atherosclerotic plaques, and among other approaches, has been modeled in vitro by cholesterol loading. METHODS: Meta-analysis of scRNA-seq data from VSMC lineage traced cells across five experiments of murine atherosclerosis was performed. In vivo expression profiles were compared to three in vitro datasets of VSMCs loaded with cholesterol and three datasets of polarized macrophages. RESULTS: We identified 24 cell clusters in the meta-analysis of single cells from mouse atherosclerotic lesions with notable heterogeneity across studies, especially for macrophage populations. Trajectory analysis of VSMC lineage positive cells revealed several possible paths of state transitions with one traversing from contractile VSMC to macrophages by way of a proliferative cell cluster. Transcriptome comparisons between in vivo and in vitro states underscored that data from three in vitro cholesterol-treated VSMC experiments did not mirror cell state transitions observed in vivo. However, all in vitro macrophage profiles analyzed (M1, M2, and oxLDL) were more similar to in vivo profiles of macrophages than in vitro VSMCs were to in vivo profiles of VSMCs. oxLDL loaded macrophages showed the most similarity to in vivo states. In contrast to the in vitro data, comparison between mouse and human in vivo data showed many similarities. CONCLUSIONS: Identification of the sources of variation across single cell datasets in atherosclerosis will be an important step towards understanding VSMC fate transitions in vivo. Also, we conclude that cholesterol-loading in vitro is insufficient to model the VSMC cell state transitions observed in vivo, which underscores the need to develop better cell models. Mouse models, however, appear to reproduce a number of the features of VSMCs in human plaques.

17.
Am J Hum Genet ; 108(3): 411-430, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33626337

RESUMEN

Genetic factors underlying coronary artery disease (CAD) have been widely studied using genome-wide association studies (GWASs). However, the functional understanding of the CAD loci has been limited by the fact that a majority of GWAS variants are located within non-coding regions with no functional role. High cholesterol and dysregulation of the liver metabolism such as non-alcoholic fatty liver disease confer an increased risk of CAD. Here, we studied the function of non-coding single-nucleotide polymorphisms in CAD GWAS loci located within liver-specific enhancer elements by identifying their potential target genes using liver cis-eQTL analysis and promoter Capture Hi-C in HepG2 cells. Altogether, 734 target genes were identified of which 121 exhibited correlations to liver-related traits. To identify potentially causal regulatory SNPs, the allele-specific enhancer activity was analyzed by (1) sequence-based computational predictions, (2) quantification of allele-specific transcription factor binding, and (3) STARR-seq massively parallel reporter assay. Altogether, our analysis identified 1,277 unique SNPs that display allele-specific regulatory activity. Among these, susceptibility enhancers near important cholesterol homeostasis genes (APOB, APOC1, APOE, and LIPA) were identified, suggesting that altered gene regulatory activity could represent another way by which genetic variation regulates serum lipoprotein levels. Using CRISPR-based perturbation, we demonstrate how the deletion/activation of a single enhancer leads to changes in the expression of many target genes located in a shared chromatin interaction domain. Our integrative genomics approach represents a comprehensive effort in identifying putative causal regulatory regions and target genes that could predispose to clinical manifestation of CAD by affecting liver function.


Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Elementos de Facilitación Genéticos/genética , Predisposición Genética a la Enfermedad , Sitios de Carácter Cuantitativo/genética , Alelos , Cromatina/genética , Enfermedad de la Arteria Coronaria/patología , Femenino , Estudio de Asociación del Genoma Completo/métodos , Genómica , Humanos , Hígado/metabolismo , Masculino , Anotación de Secuencia Molecular , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Factores de Riesgo
18.
Eur Heart J ; 42(9): 919-933, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33532862

RESUMEN

AIMS: While most patients with myocardial infarction (MI) have underlying coronary atherosclerosis, not all patients with coronary artery disease (CAD) develop MI. We sought to address the hypothesis that some of the genetic factors which establish atherosclerosis may be distinct from those that predispose to vulnerable plaques and thrombus formation. METHODS AND RESULTS: We carried out a genome-wide association study for MI in the UK Biobank (n∼472 000), followed by a meta-analysis with summary statistics from the CARDIoGRAMplusC4D Consortium (n∼167 000). Multiple independent replication analyses and functional approaches were used to prioritize loci and evaluate positional candidate genes. Eight novel regions were identified for MI at the genome wide significance level, of which effect sizes at six loci were more robust for MI than for CAD without the presence of MI. Confirmatory evidence for association of a locus on chromosome 1p21.3 harbouring choline-like transporter 3 (SLC44A3) with MI in the context of CAD, but not with coronary atherosclerosis itself, was obtained in Biobank Japan (n∼165 000) and 16 independent angiography-based cohorts (n∼27 000). Follow-up analyses did not reveal association of the SLC44A3 locus with CAD risk factors, biomarkers of coagulation, other thrombotic diseases, or plasma levels of a broad array of metabolites, including choline, trimethylamine N-oxide, and betaine. However, aortic expression of SLC44A3 was increased in carriers of the MI risk allele at chromosome 1p21.3, increased in ischaemic (vs. non-diseased) coronary arteries, up-regulated in human aortic endothelial cells treated with interleukin-1ß (vs. vehicle), and associated with smooth muscle cell migration in vitro. CONCLUSIONS: A large-scale analysis comprising ∼831 000 subjects revealed novel genetic determinants of MI and implicated SLC44A3 in the pathophysiology of vulnerable plaques.


Asunto(s)
Enfermedad de la Arteria Coronaria , Infarto del Miocardio , Enfermedad de la Arteria Coronaria/genética , Células Endoteliales , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Humanos , Japón , Infarto del Miocardio/genética , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo
19.
Am J Respir Crit Care Med ; 203(11): 1410-1418, 2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-33326355

RESUMEN

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4ß1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.


Asunto(s)
Integrina alfa4beta1/metabolismo , Mycoplasma pneumoniae , Neumonía por Mycoplasma/prevención & control , Uteroglobina/metabolismo , Animales , Adhesión Celular , Modelos Animales de Enfermedad , Ratones , Infiltración Neutrófila/fisiología , Neumonía por Mycoplasma/metabolismo , Unión Proteica
20.
Pulm Circ ; 10(4): 2045894020968531, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33343881

RESUMEN

Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of pulmonary arterial hypertension cases versus controls, highlighting potentially novel candidate genes involved in pulmonary arterial hypertension development.

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