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BACKGROUND: Integrative multiomics can elucidate pulmonary arterial hypertension (PAH) pathobiology, but procuring human PAH lung samples is rare. METHODS: We leveraged transcriptomic profiling and deep phenotyping of the largest multicenter PAH lung biobank to date (96 disease and 52 control) by integration with clinicopathologic data, genome-wide association studies, Bayesian regulatory networks, single-cell transcriptomics, and pharmacotranscriptomics. RESULTS: We identified 2 potentially protective gene network modules associated with vascular cells, and we validated ASPN, coding for asporin, as a key hub gene that is upregulated as a compensatory response to counteract PAH. We found that asporin is upregulated in lungs and plasma of multiple independent PAH cohorts and correlates with reduced PAH severity. We show that asporin inhibits proliferation and transforming growth factor-ß/phosphorylated SMAD2/3 signaling in pulmonary artery smooth muscle cells from PAH lungs. We demonstrate in Sugen-hypoxia rats that ASPN knockdown exacerbated PAH and recombinant asporin attenuated PAH. CONCLUSIONS: Our integrative systems biology approach to dissect the PAH lung transcriptome uncovered asporin as a novel protective target with therapeutic potential in PAH.
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Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer.
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Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Lesões Pré-Cancerosas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma de Pulmão/induzido quimicamente , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Microdissecção e Captura a Laser/métodos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Reação em Cadeia da Polimerase/métodos , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Sequenciamento Completo do Genoma/métodosRESUMO
Mechanisms driving adaptive developmental responses to chronic high-altitude (HA) exposure are incompletely known. We developed a novel rat model mimicking the human condition of cardiopulmonary adaptation to HA starting at conception and spanning the in utero and postnatal timeframe. We assessed lung growth and cardiopulmonary structure and function and performed transcriptome analyses to identify mechanisms facilitating developmental adaptations to chronic hypoxia. To generate the model, breeding pairs of Sprague-Dawley rats were exposed to hypobaric hypoxia (equivalent to 9,000 ft elevation). Mating, pregnancy, and delivery occurred in hypoxic conditions. Six weeks postpartum, structural and functional data were collected in the offspring. RNA-Seq was performed on right ventricle (RV) and lung tissue. Age-matched breeding pairs and offspring under room air (RA) conditions served as controls. Hypoxic rats exhibited significantly lower body weights and higher hematocrit levels, alveolar volumes, pulmonary diffusion capacities, RV mass, and RV systolic pressure, as well as increased pulmonary artery remodeling. RNA-Seq analyses revealed multiple differentially expressed genes in lungs and RVs from hypoxic rats. Although there was considerable similarity between hypoxic lungs and RVs compared with RA controls, several upstream regulators unique to lung or RV were identified. We noted a pattern of immune downregulation and regulation patterns of immune and hormonal mediators similar to the genome from patients with pulmonary arterial hypertension. In summary, we developed a novel murine model of chronic hypoxia exposure that demonstrates functional and structural phenotypes similar to human adaptation. We identified transcriptomic alterations that suggest potential mechanisms for adaptation to chronic HA.
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Adaptação Fisiológica/fisiologia , Altitude , Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Transcriptoma/fisiologia , Animais , Modelos Animais de Doenças , Pulmão/fisiopatologia , Ratos Sprague-Dawley , Remodelação Vascular/fisiologiaRESUMO
Inflammation and vascular smooth muscle cell (VSMC) phenotypic switching are causally linked to pulmonary arterial hypertension (PAH) pathogenesis. Carbonic anhydrase inhibition induces mild metabolic acidosis and exerts protective effects in hypoxic pulmonary hypertension. Carbonic anhydrases and metabolic acidosis are further known to modulate immune cell activation. To evaluate if carbonic anhydrase inhibition modulates macrophage activation, inflammation, and VSMC phenotypic switching in severe experimental pulmonary hypertension, pulmonary hypertension was assessed in Sugen 5416/hypoxia (SU/Hx) rats after treatment with acetazolamide or ammonium chloride (NH4Cl). We evaluated pulmonary and systemic inflammation and characterized the effect of carbonic anhydrase inhibition and metabolic acidosis in alveolar macrophages and bone marrow-derived macrophages (BMDMs). We further evaluated the treatment effects on VSMC phenotypic switching in pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs) and corroborated some of our findings in lungs and pulmonary arteries of patients with PAH. Both patients with idiopathic PAH and SU/Hx rats had increased expression of lung inflammatory markers and signs of PASMC dedifferentiation in pulmonary arteries. Acetazolamide and NH4Cl ameliorated SU/Hx-induced pulmonary hypertension and blunted pulmonary and systemic inflammation. Expression of carbonic anhydrase isoform 2 was increased in alveolar macrophages from SU/Hx animals, classically (M1) and alternatively (M2) activated BMDMs, and lungs of patients with PAH. Carbonic anhydrase inhibition and acidosis had distinct effects on M1 and M2 markers in BMDMs. Inflammatory cytokines drove PASMC dedifferentiation, and this was inhibited by acetazolamide and acidosis. The protective antiinflammatory effect of acetazolamide in pulmonary hypertension is mediated by a dual mechanism of macrophage carbonic anhydrase inhibition and systemic metabolic acidosis.
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Acetazolamida/uso terapêutico , Cloreto de Amônio/uso terapêutico , Inibidores da Anidrase Carbônica/uso terapêutico , Anidrases Carbônicas/fisiologia , Hipertensão Pulmonar/tratamento farmacológico , Acidose/induzido quimicamente , Acidose/complicações , Acidose/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proteínas Contráteis/biossíntese , Proteínas Contráteis/genética , Avaliação Pré-Clínica de Medicamentos , Humanos , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Inflamação , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/enzimologia , Masculino , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Isoformas de Proteínas/antagonistas & inibidores , Artéria Pulmonar/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary artery pressure and vascular resistance, typically leading to right heart failure and death. Current therapies improve quality of life of the patients but have a modest effect on long-term survival. A detailed transcriptomics and systems biology view of the PAH lung is expected to provide new testable hypotheses for exploring novel treatments. We completed transcriptomics analysis of PAH and control lung tissue to develop disease-specific and clinical data/tissue pathology gene expression classifiers from expression datasets. Gene expression data were integrated into pathway analyses. Gene expression microarray data were collected from 58 PAH and 25 control lung tissues. The strength of the dataset and its derived disease classifier was validated using multiple approaches. Pathways and upstream regulators analyses was completed with standard and novel graphical approaches. The PAH lung dataset identified expression patterns specific to PAH subtypes, clinical parameters, and lung pathology variables. Pathway analyses indicate the important global role of TNF and transforming growth factor signaling pathways. In addition, novel upstream regulators and insight into the cellular and innate immune responses driving PAH were identified. Finally, WNT-signaling pathways may be a major determinant underlying the observed sex differences in PAH. This study provides a transcriptional framework for the PAH-diseased lung, supported by previously reported findings, and will be a valuable resource to the PAH research community. Our investigation revealed novel potential targets and pathways amenable to further study in a variety of experimental systems.
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Pulmão/metabolismo , Pulmão/patologia , Hipertensão Arterial Pulmonar/genética , Análise de Sistemas , Transcriptoma/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Hipertensão Arterial Pulmonar/patologia , Caracteres Sexuais , Transdução de Sinais/genética , Adulto JovemRESUMO
The endothelial glycocalyx is a heparan sulfate (HS)-rich endovascular structure critical to endothelial function. Accordingly, endothelial glycocalyx degradation during sepsis contributes to tissue edema and organ injury. We determined the endogenous mechanisms governing pulmonary endothelial glycocalyx reconstitution, and if these reparative mechanisms are impaired during sepsis. We performed intravital microscopy of wild-type and transgenic mice to determine the rapidity of pulmonary endothelial glycocalyx reconstitution after nonseptic (heparinase-III mediated) or septic (cecal ligation and puncture mediated) endothelial glycocalyx degradation. We used mass spectrometry, surface plasmon resonance, and in vitro studies of human and mouse samples to determine the structure of HS fragments released during glycocalyx degradation and their impact on fibroblast growth factor receptor (FGFR) 1 signaling, a mediator of endothelial repair. Homeostatic pulmonary endothelial glycocalyx reconstitution occurred rapidly after nonseptic degradation and was associated with induction of the HS biosynthetic enzyme, exostosin (EXT)-1. In contrast, sepsis was characterized by loss of pulmonary EXT1 expression and delayed glycocalyx reconstitution. Rapid glycocalyx recovery after nonseptic degradation was dependent upon induction of FGFR1 expression and was augmented by FGF-promoting effects of circulating HS fragments released during glycocalyx degradation. Although sepsis-released HS fragments maintained this ability to activate FGFR1, sepsis was associated with the downstream absence of reparative pulmonary endothelial FGFR1 induction. Sepsis may cause vascular injury not only via glycocalyx degradation, but also by impairing FGFR1/EXT1-mediated glycocalyx reconstitution.
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Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glicocálix/metabolismo , Pulmão/metabolismo , Transdução de Sinais , Animais , Ceco/patologia , Heparitina Sulfato/metabolismo , Homeostase , Ligadura , Masculino , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeo-Liases/metabolismo , Punções , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Sepse/patologiaRESUMO
Epigenetic mechanisms, including DNA methylation and histone acetylation, regulate gene expression in idiopathic pulmonary arterial hypertension (IPAH). These mechanisms can modulate expression of extracellular superoxide dismutase (SOD3 or EC-SOD), a key vascular antioxidant enzyme, and loss of vascular SOD3 worsens outcomes in animal models of pulmonary arterial hypertension. We hypothesized that SOD3 gene expression is decreased in patients with IPAH due to aberrant DNA methylation and/or histone deacetylation. We used lung tissue and pulmonary artery smooth muscle cells (PASMC) from subjects with IPAH at transplantation and from failed donors (FD). Lung SOD3 mRNA expression and activity was decreased in IPAH vs. FD. In contrast, mitochondrial SOD (Mn-SOD or SOD2) protein expression was unchanged and intracellular SOD activity was unchanged. Using bisulfite sequencing in genomic lung or PASMC DNA, we found the methylation status of the SOD3 promoter was similar between FD and IPAH. Furthermore, treatment with 5-aza-2'-deoxycytidine did not increase PASMC SOD3 mRNA, suggesting DNA methylation was not responsible for PASMC SOD3 expression. Though total histone deacetylase (HDAC) activity, histone acetyltransferase (HAT) activity, acetylated histones, and acetylated SP1 were similar between IPAH and FD, treatment with two selective class I HDAC inhibitors increased SOD3 only in IPAH PASMC. Class I HDAC3 siRNA also increased SOD3 expression. Trichostatin A, a pan-HDAC inhibitor, decreased proliferation in IPAH, but not in FD PASMC. These data indicate that histone deacetylation, specifically via class I HDAC3, decreases SOD3 expression in PASMC and HDAC inhibitors may protect IPAH in part by increasing PASMC SOD3 expression.
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Histonas/metabolismo , Hipertensão Pulmonar/enzimologia , Processamento de Proteína Pós-Traducional , Superóxido Dismutase/metabolismo , Acetilação , Adulto , Animais , Células Cultivadas , Repressão Enzimática , Feminino , Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Regiões Promotoras Genéticas , Ratos , Superóxido Dismutase/genética , Adulto JovemRESUMO
UNLABELLED: Blood transcriptional signatures are promising for tuberculosis (TB) diagnosis but have not been evaluated among U.S. PATIENTS: To be used clinically, transcriptional classifiers need reproducible accuracy in diverse populations that vary in genetic composition, disease spectrum and severity, and comorbidities. In a prospective case-control study, we identified novel transcriptional classifiers for active TB among U.S. patients and systematically compared their accuracy to classifiers from published studies. Blood samples from HIV-uninfected U.S. adults with active TB, pneumonia, or latent TB infection underwent whole-transcriptome microarray. We used support vector machines to classify disease state based on transcriptional patterns. We externally validated our classifiers using data from sub-Saharan African cohorts and evaluated previously published transcriptional classifiers in our population. Our classifier distinguishing active TB from pneumonia had an area under the concentration-time curve (AUC) of 96.5% (95.4% to 97.6%) among U.S. patients, but the AUC was lower (90.6% [89.6% to 91.7%]) in HIV-uninfected Sub-Saharan Africans. Previously published comparable classifiers had AUC values of 90.0% (87.7% to 92.3%) and 82.9% (80.8% to 85.1%) when tested in U.S. PATIENTS: Our classifier distinguishing active TB from latent TB had AUC values of 95.9% (95.2% to 96.6%) among U.S. patients and 95.3% (94.7% to 96.0%) among Sub-Saharan Africans. Previously published comparable classifiers had AUC values of 98.0% (97.4% to 98.7%) and 94.8% (92.9% to 96.8%) when tested in U.S. PATIENTS: Blood transcriptional classifiers accurately detected active TB among U.S. adults. The accuracy of classifiers for active TB versus that of other diseases decreased when tested in new populations with different disease controls, suggesting additional studies are required to enhance generalizability. Classifiers that distinguish active TB from latent TB are accurate and generalizable across populations and can be explored as screening assays.
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Biomarcadores , Mycobacterium tuberculosis , Transcriptoma , Tuberculose/diagnóstico , Tuberculose/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Humanos , Tuberculose Latente , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/fisiologia , Pneumonia/sangue , Pneumonia/diagnóstico , Pneumonia/genética , Curva ROC , Tuberculose/sangue , Tuberculose/epidemiologia , Estados Unidos/epidemiologia , Estados Unidos/etnologia , Adulto JovemAssuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Síndrome de Hermanski-Pudlak/genética , Proteínas de Membrana/genética , Fibrose Pulmonar/genética , Mutação da Fase de Leitura , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Haplótipos , Síndrome de Hermanski-Pudlak/complicações , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismoRESUMO
RATIONALE: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary artery pressure, vascular remodeling, and ultimately right ventricular heart failure. PAH can have a genetic component (heritable PAH), most often through mutations of bone morphogenetic protein receptor 2, and idiopathic and associated forms. Heritable PAH is not completely penetrant within families, with approximately 20% concurrence of inactivating bone morphogenetic protein receptor 2 mutations and delayed onset of PAH disease. Because one of the treatment options is using prostacyclin analogs, we hypothesized that prostacyclin synthase promoter sequence variants associated with increased mRNA expression may play a protective role in the bone morphogenetic protein receptor 2 unaffected carriers. OBJECTIVES: To characterize the range of prostacyclin synthase promoter variants and assess their transcriptional activities in PAH-relevant cell types. To determine the distribution of prostacyclin synthase promoter variants in PAH, unaffected carriers in heritable PAH families, and control populations. METHODS: Polymerase chain reaction approaches were used to genotype prostacyclin synthase promoter variants in more than 300 individuals. Prostacyclin synthase promoter haplotypes' transcriptional activities were determined with luciferase reporter assays. MEASUREMENTS AND MAIN RESULTS: We identified a comprehensive set of prostacyclin synthase promoter variants and tested their transcriptional activities in PAH-relevant cell types. We demonstrated differences of prostacyclin synthase promoter activities dependent on their haplotype. CONCLUSIONS: Prostacyclin synthase promoter sequence variants exhibit a range of transcriptional activities. We discovered a significant bias for more active prostacyclin synthase promoter variants in unaffected carriers as compared with affected patients with PAH.
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Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Sistema Enzimático do Citocromo P-450/genética , Heterozigoto , Hipertensão Pulmonar/genética , Oxirredutases Intramoleculares/genética , Polimorfismo Genético , Estudos de Casos e Controles , Sistema Enzimático do Citocromo P-450/fisiologia , Progressão da Doença , Hipertensão Pulmonar Primária Familiar , Feminino , Haplótipos , Humanos , Oxirredutases Intramoleculares/fisiologia , Masculino , Mutação , Reação em Cadeia da PolimeraseRESUMO
Pulmonary hypertension (PH) is associated with significant morbidity and mortality. RASA3 is a GTPase activating protein integral to angiogenesis and endothelial barrier function. In this study, we explore the association of RASA3 genetic variation with PH risk in patients with sickle cell disease (SCD)-associated PH and pulmonary arterial hypertension (PAH). Cis-expression quantitative trait loci (eQTL) were queried for RASA3 using whole genome genotype arrays and gene expression profiles derived from peripheral blood mononuclear cells (PBMC) of three SCD cohorts. Genome-wide single nucleotide polymorphisms (SNPs) near or in the RASA3 gene that may associate with lung RASA3 expression were identified, reduced to 9 tagging SNPs for RASA3 and associated with markers of PH. Associations between the top RASA3 SNP and PAH severity were corroborated using data from the PAH Biobank and analyzed based on European or African ancestry (EA, AA). We found that PBMC RASA3 expression was lower in patients with SCD-associated PH as defined by echocardiography and right heart catheterization and was associated with higher mortality. One eQTL for RASA3 (rs9525228) was identified, with the risk allele correlating with PH risk, higher tricuspid regurgitant jet velocity and higher pulmonary vascular resistance in patients with SCD-associated PH. rs9525228 associated with markers of precapillary PH and decreased survival in individuals of EA but not AA. In conclusion, RASA3 is a novel candidate gene in SCD-associated PH and PAH, with RASA3 expression appearing to be protective. Further studies are ongoing to delineate the role of RASA3 in PH.
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Pulmonary arterial hypertension (PAH) remains an incurable and often fatal disease despite currently available therapies. Multiomics systems biology analysis can shed new light on PAH pathobiology and inform translational research efforts. Using RNA sequencing on the largest PAH lung biobank to date (96 disease and 52 control), we aim to identify gene co-expression network modules associated with PAH and potential therapeutic targets. Co-expression network analysis was performed to identify modules of co-expressed genes which were then assessed for and prioritized by importance in PAH, regulatory role, and therapeutic potential via integration with clinicopathologic data, human genome-wide association studies (GWAS) of PAH, lung Bayesian regulatory networks, single-cell RNA-sequencing data, and pharmacotranscriptomic profiles. We identified a co-expression module of 266 genes, called the pink module, which may be a response to the underlying disease process to counteract disease progression in PAH. This module was associated not only with PAH severity such as increased PVR and intimal thickness, but also with compensated PAH such as lower number of hospitalizations, WHO functional class and NT-proBNP. GWAS integration demonstrated the pink module is enriched for PAH-associated genetic variation in multiple cohorts. Regulatory network analysis revealed that BMPR2 regulates the main target of FDA-approved riociguat, GUCY1A2, in the pink module. Analysis of pathway enrichment and pink hub genes (i.e. ANTXR1 and SFRP4) suggests the pink module inhibits Wnt signaling and epithelial-mesenchymal transition. Cell type deconvolution showed the pink module correlates with higher vascular cell fractions (i.e. myofibroblasts). A pharmacotranscriptomic screen discovered ubiquitin-specific peptidases (USPs) as potential therapeutic targets to mimic the pink module signature. Our multiomics integrative study uncovered a novel gene subnetwork associated with clinicopathologic severity, genetic risk, specific vascular cell types, and new therapeutic targets in PAH. Future studies are warranted to investigate the role and therapeutic potential of the pink module and targeting USPs in PAH.
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Most patients with familial pulmonary arterial hypertension (FPAH) carry mutations in the bone morphogenic protein receptor 2 gene (BMPR2). Yet carriers have only a 20% risk of disease, suggesting that other factors influence penetrance. Thrombospondin-1 (TSP1) regulates activation of TGF-ß and inhibits endothelial and smooth muscle cell proliferation, pathways coincidentally altered in pulmonary arterial hypertension (PAH). To determine whether a subset of FPAH patients also have mutations in the TSP1 gene (THBS1) we resequenced the type I repeats of THBS1 encoding the TGF-ß regulation and cell growth inhibition domains in 60 FPAH probands, 70 nonfamilial PAH subjects, and in large control groups. We identified THBS1 mutations in three families: a novel missense mutation in two (Asp362Asn), and an intronic mutation in a third (IVS8+255 G/A). Neither mutation was detected in population controls. Mutant 362Asn TSP1 had less than half of the ability of wild-type TSP1 to activate TGF-ß. Mutant 362Asn TSP1 also lost the ability to inhibit growth of pulmonary arterial smooth muscle cells and was over threefold less effective at inhibiting endothelial cell growth. The IVS8+255 G/A mutation decreased and/or eliminated local binding of the transcription factors SP1 and MAZ but did not affect RNA splicing. These novel mutations implicate THBS1 as a modifier gene in FPAH. These THBS1 mutations have implications in the genetic evaluation of FPAH patients. However, since FPAH is rare, these data are most relevant as evidence for the importance of TSP1 in pulmonary vascular homeostasis. Further examination of THBS1 in the pathogenesis of PAH is warranted.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Mutação de Sentido Incorreto , Sítios de Ligação , Processos de Crescimento Celular/genética , Células Cultivadas , Estudos de Coortes , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Hipertensão Pulmonar Primária Familiar , Feminino , Humanos , Íntrons , Masculino , Miócitos de Músculo Liso/metabolismo , Polimorfismo Genético , Ligação Proteica , Artéria Pulmonar/metabolismo , Splicing de RNA/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
The aberrant proliferation of pulmonary artery smooth muscle (PASMCs) cells is a defining characteristic of pulmonary arterial hypertension (PAH) and leads to increased vascular resistance, elevated pulmonary pressure, and right heart failure. The sphingosine kinase 1 (SPHK1)/sphingosine-1 phosphate/sphingosine-1 phosphate receptor 2 pathway promotes vascular remodeling and induces PAH. The aim of this study was to identify genes and cellular processes that are modulated by over-expression of SPHK1 in human PASMCs (hPASMCs). RNA was purified and submitted for RNA sequencing to identify differentially expressed genes. Using a corrected p-value threshold of <0.05, there were 294 genes significantly up-regulated while 179 were significantly down-regulated. Predicted effects of these differentially expressed genes were evaluated using the freeware tool Enrichr to assess general gene set over-representation (enrichment) and ingenuity pathway analysis (IPA™) for upstream regulator predictions. We found a strong change in genes that regulated the cellular immune response. IL6, STAT1, and PARP9 were elevated in response to SPHK1 over-expression in hPASMCs. The gene set enrichment mapped to a few immune-modulatory signaling networks, including IFNG. Furthermore, PARP9 and STAT1 protein were elevated in primary hPASMCs isolated from PAH patients. In conclusion, these data suggest a role of Sphk1 regulates pulmonary vascular immune response in PAH.
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Fosfotransferases (Aceptor do Grupo Álcool)RESUMO
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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BACKGROUND: Primary graft dysfunction (PGD) is the leading cause of early mortality after lung transplantation. Anti-collagen type-V (col(V)) immunity has been observed in animal models of ischemia-reperfusion injury (IRI) and in PGD. We hypothesized that collagen type-V is an innate danger signal contributing to PGD pathogenesis. METHODS: Anti-col(V) antibody production was detected by flow cytometric assay following cultures of murine CD19+ splenic cells with col.(V). Responding murine B cells were phenotyped using surface markers. RNA-Seq analysis was performed on murine CD19+ cells. Levels of anti-col(V) antibodies were measured in 188 recipients from the Lung Transplant Outcomes Group (LTOG) after transplantation. RESULTS: Col(V) induced rapid production of anti-col(V) antibodies from murine CD19+ B cells. Subtype analysis demonstrated innate B-1 B cells bound col.(V). Col(V) induced a specific transcriptional signature in CD19+ B cells with similarities to, yet distinct from, B cell receptor (BCR) stimulation. Rapid de novo production of anti-col(V) Abs was associated with an increased incidence of clinical PGD after lung transplant. CONCLUSIONS: This study demonstrated that col.(V) is an rapidly recognized by B cells and has specific transcriptional signature. In lung transplants recipients the rapid seroconversion to anti-col(V) Ab is linked to increased risk of grade 3 PGD.
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Subpopulações de Linfócitos B/fisiologia , Colágeno Tipo V/imunologia , Rejeição de Enxerto/imunologia , Transplante de Pulmão , Adulto , Idoso , Animais , Formação de Anticorpos , Antígenos CD19/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Imunidade Inata , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , TranscriptomaRESUMO
Pharmacological activators of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibit growth of non-small-cell lung cancer (NSCLC) cell lines in vitro and in xenograft models. Because these agents engage off-target pathways, we have assessed the effects of PPARgamma by overexpressing the protein in NSCLC cells. We reported previously that increased PPARgamma inhibits transformed growth and invasiveness and promotes epithelial differentiation in a panel of NSCLC expressing oncogenic K-Ras. These cells express high levels of cyclooxygenase-2 (COX-2) and produce high levels of prostaglandin E(2) (PGE(2)). The goal of these studies was to identify the molecular mechanisms whereby PPARgamma inhibits tumorigenesis. Increased PPARgamma inhibited expression of COX-2 protein and promoter activity, resulting in decreased PGE(2) production. Suppression of COX-2 was mediated through increased activity of the tumor suppressor phosphatase and tensin homolog, leading to decreased levels of phospho-Akt and inhibition of nuclear factor-kappaB activity. Pharmacological inhibition of PGE(2) production mimicked the effects of PPARgamma on epithelial differentiation in three-dimensional culture, and exogenous PGE(2) reversed the effects of increased PPARgamma activity. Transgenic mice overexpressing PPARgamma under the control of the surfactant protein C promoter had reduced expression of COX-2 in type II cells and were protected against developing lung tumors in a chemical carcinogenesis model. These data indicate that high levels of PGE(2) as a result of elevated COX-2 expression are critical for promoting lung tumorigenesis and that the antitumorigenic effects of PPARgamma are mediated in part through blocking this pathway.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo-Oxigenase 2/fisiologia , Neoplasias Pulmonares/metabolismo , NF-kappa B/antagonistas & inibidores , PPAR gama/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Meios de Cultura , Dinoprostona/análise , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Luciferases/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Técnicas de Cultura de Órgãos , PPAR gama/uso terapêutico , PTEN Fosfo-Hidrolase/análise , PTEN Fosfo-Hidrolase/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sulindaco/análogos & derivados , Sulindaco/farmacologia , Transfecção , TransgenesRESUMO
The importance of the arachidonic acid pathway has been established in colon and lung cancers, as well as in inflammatory diseases. In these diseases, prostacyclin I(2) (PGI2) and prostaglandin E(2) (PGE2) are thought to have antagonistic activities, with PGI2 exerting anti-inflammatory and antiproliferative activities, whereas PGE2 is proinflammatory and antiapoptotic. In human lung cancer, prostacyclin synthase (PGIS) and PGI2 are down-regulated, whereas PGE2 synthase (PGES) and PGE2 are up-regulated. Murine carcinogenesis models of human lung cancer reciprocate the relationship between PGIS and PGES expression. PGIS-overexpressing transgenic mice are protected from carcinogen- and tobacco smoke-induced lung tumor formation, suggesting that PGI2 may play a role in chemoprevention. We investigated several potential mechanisms for the down-regulation of PGIS in human lung cancer. Using transcription reporter assays, we show that single nucleotide polymorphisms in the PGIS promoter can affect transcriptional activity. In addition, PGIS expression in several human lung cancer cell lines is silenced by CpG methylation, and we have mapped these sites across the variable number of tandem repeats (VNTR) sequence in the promoter, as well as CpGs within exon 1 and the first intron. Finally, using fluorescence in situ hybridization, we show that human lung cancer cell lines and lung cancer tissues do not have a loss of the PGIS genomic region but multiple copies. These results show that an individual's PGIS promoter haplotype can play an important role in the predisposition for lung cancer and CpG methylation provides an epigenetic mechanism for the down-regulated PGIS expression.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Oxirredutases Intramoleculares/genética , Neoplasias Pulmonares/genética , Sequência de Bases , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Regulação para Baixo , Genes Reporter , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/enzimologia , Repetições Minissatélites , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análiseRESUMO
Renal cell carcinoma is associated with mutation of the von Hippel-Lindau (VHL) tumor suppressor gene. Cell lines derived from these tumors cannot exit the cell cycle when deprived of growth factors, and the ability to exit the cell cycle can be restored by the reintroduction of wild-type protein VHL (pVHL). Here, we report that cyclin D1 is overexpressed and remains inappropriately high in during contact inhibition in pVHL-deficient cell lines. In addition, hypoxia increased the expression of cyclin D1 specifically in pVHL-negative cell lines into which pVHL expression was restored. Hypoxic-induction of cyclin D1 was not observed in other pVHL-positive cell lines. This suggests a model whereby in some kidney cell types, pVHL may regulate a proliferative response to hypoxia, whereas the loss of pVHL leads to constitutively elevated cyclin D1 and abnormal proliferation under normal growth conditions.
Assuntos
Carcinoma de Células Renais/metabolismo , Ciclina D1/biossíntese , Neoplasias Renais/metabolismo , Ligases/fisiologia , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Hipóxia Celular/fisiologia , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Ligases/genética , Transfecção , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
BACKGROUND: Hypoplastic left heart syndrome (HLHS) is a heterogeneous, lethal combination of congenital malformations characterized by severe underdevelopment of left heart structures, resulting in a univentricular circulation. The genetic determinants of this disorder are largely unknown. Evidence of copy number variants (CNVs) contributing to the genetic etiology of HLHS and other congenital heart defects has been mounting. However, the functional effects of such CNVs have not been examined, particularly in cases where the variant of interest is found in only a single patient. METHODS AND RESULTS: Whole-genome SNP microarrays were employed to detect CNVs in two patient cohorts (N = 70 total) predominantly diagnosed with some form of nonsyndromic HLHS. We discovered 16 rare or private variants adjacent to or overlapping 20 genes associated with cardiovascular or premature lethality phenotypes in mouse knockout models. We evaluated the impact of selected variants on the expression of nine of these genes through quantitative PCR on cDNA derived from patient heart tissue. Four genes displayed significantly altered expression in patients with an overlapping or proximal CNV verses patients without such CNVs. CONCLUSION: Rare and private genomic imbalances perturb transcription of genes that potentially affect cardiogenesis in a subset of nonsyndromic HLHS patients.