RESUMEN
Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (â¼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).
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Trasplante de Células Madre Hematopoyéticas , Leucodistrofia de Células Globoides , Perros , Animales , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Galactosilceramidasa/genética , Psicosina , Trasplante de Células Madre Hematopoyéticas/métodos , Terapia Genética/métodos , Modelos Animales de EnfermedadRESUMEN
The small airway epithelium (SAE), the first site of smoking-induced lung pathology, exhibits genome-wide changes in gene expression in response to cigarette smoking. Based on the increasing evidence that the epigenome can respond to external stimuli in a rapid manner, we assessed the SAE of smokers for genome-wide DNA methylation changes compared with nonsmokers, and whether changes in SAE DNA methylation were linked to the transcriptional output of these cells. Using genome-wide methylation analysis of SAE DNA of nonsmokers and smokers, the data identified 204 unique genes differentially methylated in SAE DNA of smokers compared with nonsmokers, with 67% of the regions with differential methylation occurring within 2 kb of the transcriptional start site. Among the genes with differential methylation were those related to metabolism, transcription, signal transduction and transport. For the differentially methylated genes, 35 exhibited a correlation with gene expression, 54% with an inverse correlation of DNA methylation with gene expression and 46% a direct correlation. These observations provide evidence that cigarette smoking alters the DNA methylation patterning of the SAE and that, for some genes, these changes are associated with the smoking-related changes in gene expression.
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Epigénesis Genética , Epitelio/metabolismo , Mucosa Respiratoria/metabolismo , Fumar/genética , Adulto , Estudios de Casos y Controles , Metilación de ADN/efectos de los fármacos , Epitelio/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Mucosa Respiratoria/efectos de los fármacos , Fumar/efectos adversos , Fumar/metabolismo , Sitio de Iniciación de la Transcripción , Adulto JovenRESUMEN
MOTIVATION: Identification of expression Quantitative Trait Loci (eQTL), the genetic loci that contribute to heritable variation in gene expression, can be obstructed by factors that produce variation in expression profiles if these factors are unmeasured or hidden from direct analysis. METHODS: We have developed a method for Hidden Expression Factor analysis (HEFT) that identifies individual and pleiotropic effects of eQTL in the presence of hidden factors. The HEFT model is a combined multivariate regression and factor analysis, where the complete likelihood of the model is used to derive a ridge estimator for simultaneous factor learning and detection of eQTL. HEFT requires no pre-estimation of hidden factor effects; it provides P-values and is extremely fast, requiring just a few hours to complete an eQTL analysis of thousands of expression variables when analyzing hundreds of thousands of single nucleotide polymorphisms on a standard 8 core 2.6 G desktop. RESULTS: By analyzing simulated data, we demonstrate that HEFT can correct for an unknown number of hidden factors and significantly outperforms all related hidden factor methods for eQTL analysis when there are eQTL with univariate and multivariate (pleiotropic) effects. To demonstrate a real-world application, we applied HEFT to identify eQTL affecting gene expression in the human lung for a study that included presumptive hidden factors. HEFT identified all of the cis-eQTL found by other hidden factor methods and 91 additional cis-eQTL. HEFT also identified a number of eQTLs with direct relevance to lung disease that could not be found without a hidden factor analysis, including cis-eQTL for GTF2H1 and MTRR, genes that have been independently associated with lung cancer. AVAILABILITY: Software is available at http://mezeylab.cb.bscb.cornell.edu/Software.aspx. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Perfilación de la Expresión Génica/métodos , Sitios de Carácter Cuantitativo , Expresión Génica , Humanos , Pulmón/metabolismo , Polimorfismo de Nucleótido Simple , Análisis de Regresión , Programas InformáticosRESUMEN
Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared with 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Premarital genetic screening in Qatar tests for only four out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance.
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Cromosomas Humanos/genética , Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Variación Genética , Análisis de Secuencia de ADN , Bases de Datos Genéticas , Exoma , Femenino , Frecuencia de los Genes , Enfermedades Genéticas Congénitas/epidemiología , Humanos , Masculino , Prevalencia , Qatar/epidemiologíaRESUMEN
Activation of the human embryonic stem cell (hESC) signature genes has been observed in various epithelial cancers. In this study, we found that the hESC signature is selectively induced in the airway basal stem/progenitor cell population of healthy smokers (BC-S), with a pattern similar to that activated in all major types of human lung cancer. We further identified a subset of 6 BC-S hESC genes, whose coherent overexpression in lung adenocarcinoma (AdCa) was associated with reduced lung function, poorer differentiation grade, more advanced tumor stage, remarkably shorter survival, and higher frequency of TP53 mutations. BC-S shared with hESC and a considerable subset of lung carcinomas a common TP53 inactivation molecular pattern which strongly correlated with the BC-S hESC gene expression. These data provide transcriptome-based evidence that smoking-induced reprogramming of airway BC toward the hESC-like phenotype might represent a common early molecular event in the development of aggressive lung carcinomas in humans.
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Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Pulmón/patología , Fumar/genética , Fumar/patología , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Epitelio/metabolismo , Epitelio/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Pulmón/metabolismo , Ratones , Análisis Multivariante , Fenotipo , Modelos de Riesgos Proporcionales , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking. METHODS: Using the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function. RESULTS: Analysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers. CONCLUSION: These data provide biologic evidence that smoking accelerates aging of the small airway epithelium.
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Senescencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Humo/efectos adversos , Fumar/efectos adversos , Adulto , Estudios de Casos y Controles , Senescencia Celular/genética , Células Epiteliales/química , Células Epiteliales/patología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Mucosa Respiratoria/química , Mucosa Respiratoria/patología , Fumar/genética , Fumar/patología , Telómero/genética , Acortamiento del TelómeroRESUMEN
People of the Qatar peninsula represent a relatively recent founding by a small number of families from three tribes of the Arabian Peninsula, Persia, and Oman, with indications of African admixture. To assess the roles of both this founding effect and the customary first-cousin marriages among the ancestral Islamic populations in Qatar's population genetic structure, we obtained and genotyped with Affymetrix 500k SNP arrays DNA samples from 168 self-reported Qatari nationals sampled from Doha, Qatar. Principal components analysis was performed along with samples from the Human Genetic Diversity Project data set, revealing three clear clusters of genotypes whose proximity to other human population samples is consistent with Arabian origin, a more eastern or Persian origin, and individuals with African admixture. The extent of linkage disequilibrium (LD) is greater than that of African populations, and runs of homozygosity in some individuals reflect substantial consanguinity. However, the variance in runs of homozygosity is exceptionally high, and the degree of identity-by-descent sharing generally appears to be lower than expected for a population in which nearly half of marriages are between first cousins. Despite the fact that the SNPs of the Affymetrix 500k chip were ascertained with a bias toward SNPs common in Europeans, the data strongly support the notion that the Qatari population could provide a valuable resource for the mapping of genes associated with complex disorders and that tests of pairwise interactions are particularly empowered by populations with elevated LD like the Qatari.
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Árabes/genética , Pueblo Asiatico/genética , Población Negra/genética , Consanguinidad , Femenino , Genética de Población , Homocigoto , Humanos , Desequilibrio de Ligamiento , Masculino , Nombres , Polimorfismo de Nucleótido Simple , Análisis de Componente Principal , QatarRESUMEN
MOTIVATION: Computational inference methods that make use of graphical models to extract regulatory networks from gene expression data can have difficulty reconstructing dense regions of a network, a consequence of both computational complexity and unreliable parameter estimation when sample size is small. As a result, identification of hub genes is of special difficulty for these methods. METHODS: We present a new algorithm, Empirical Light Mutual Min (ELMM), for large network reconstruction that has properties well suited for recovery of graphs with high-degree nodes. ELMM reconstructs the undirected graph of a regulatory network using empirical Bayes conditional independence testing with a heuristic relaxation of independence constraints in dense areas of the graph. This relaxation allows only one gene of a pair with a putative relation to be aware of the network connection, an approach that is aimed at easing multiple testing problems associated with recovering densely connected structures. RESULTS: Using in silico data, we show that ELMM has better performance than commonly used network inference algorithms including GeneNet, ARACNE, FOCI, GENIE3 and GLASSO. We also apply ELMM to reconstruct a network among 5492 genes expressed in human lung airway epithelium of healthy non-smokers, healthy smokers and individuals with chronic obstructive pulmonary disease assayed using microarrays. The analysis identifies dense sub-networks that are consistent with known regulatory relationships in the lung airway and also suggests novel hub regulatory relationships among a number of genes that play roles in oxidative stress and secretion. AVAILABILITY AND IMPLEMENTATION: Software for running ELMM is made available at http://mezeylab.cb.bscb.cornell.edu/Software.aspx. CONTACT: ramimahdi@yahoo.com or jgm45@cornell.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Algoritmos , Teorema de Bayes , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Biología Computacional/métodos , Simulación por Computador , Humanos , Mucosa Respiratoria/metabolismo , Programas InformáticosRESUMEN
BACKGROUND: Ciliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development. METHODS: To test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes. RESULTS: Basal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3. CONCLUSION: FOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.
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Cilios/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/ultraestructura , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Células Cultivadas , Cilios/ultraestructura , Regulación de la Expresión Génica/fisiología , Humanos , Factores de Transcripción del Factor Regulador XRESUMEN
The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors, 'hemangiocytes,' constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+ VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2-/- Csf3-/-), profound impairment in neovascularization was detected in sKitL-deficient Mmp9-/- as well as thrombocytopenic Thpo-/- and TPO receptor-deficient (Mpl-/-) mice. SDF-1-mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo-/-, Mpl-/- and Mmp9-/- mice. Transplantation of CXCR4+ VEGFR1+ hemangiocytes into Mmp9-/- mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A-mediated mobilization of CXCR4+ VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+ VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies.
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Quimiocinas CXC/metabolismo , Citocinas/sangre , Neovascularización Fisiológica , Receptores CXCR4/metabolismo , Regeneración , Células Madre/fisiología , Animales , Plaquetas/metabolismo , Quimiocina CXCL12 , Quimiocinas CXC/genética , Humanos , Isquemia/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR4/genética , Factor de Células Madre/metabolismo , Trombocitopenia/metabolismo , Trombopoyetina/sangre , Trombopoyetina/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The human airway epithelium is a pseudostratified heterogenous layer comprised of ciliated, secretory, intermediate, and basal cells. As the stem/progenitor population of the airway epithelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the three major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2-dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell-secreted VEGFA activated endothelium to express mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA-mediated cross-talk between airway basal cells and endothelium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.
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Células Endoteliales/fisiología , Comunicación Paracrina/fisiología , Receptor Cross-Talk/fisiología , Mucosa Respiratoria/citología , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Cartilla de ADN/genética , Células Endoteliales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Isoformas de Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Mucosa Respiratoria/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Based on the observation that humans have variable responses of gene expression with the same dose of an adeno-associated vector, we hypothesized that there are deleterious variants in genes coding for processes required for adeno-associated virus (AAV)-mediated gene transfer/expression that may hamper or enhance the effectiveness of AAV-mediated gene therapy. To assess this hypothesis, we evaluated 69,442 whole genome sequences from three populations (European, African/African American, and Qatari) for predicted deleterious variants in 62 genes known to play a role in AAV-mediated gene transfer/expression. The analysis identified 5,564 potentially deleterious mutations of which 27 were classified as common based on an allele frequency ≥1% in at least one population studied. Many of these deleterious variants are predicated to prevent while others enhance effective AAV gene transfer/expression, and several are linked to known hereditary disorders. The data support the hypothesis that, like other drugs, human genetic variability contributes to the person-to-person effectiveness of AAV gene therapy and the screening for genetic variability should be considered as part of future clinical trials.
RESUMEN
BACKGROUND: The small airway epithelium (SAE), the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq). RESULTS: The data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype. CONCLUSIONS: These observations provide insights into the unique biology of human SAE by providing quantitative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.
Asunto(s)
Perfilación de la Expresión Génica , Mucosa Respiratoria/metabolismo , Transcriptoma , Empalme Alternativo , Humanos , Masculino , Anotación de Secuencia Molecular , Familia de Multigenes , Especificidad de Órganos/genética , Análisis de Secuencia de ARN , Fumar/efectos adversos , Ubiquitinación/genéticaRESUMEN
BACKGROUND: Populations of the Arabian Peninsula have a complex genetic structure that reflects waves of migrations including the earliest human migrations from Africa and eastern Asia, migrations along ancient civilization trading routes and colonization history of recent centuries. RESULTS: Here, we present a study of genome-wide admixture in this region, using 156 genotyped individuals from Qatar, a country located at the crossroads of these migration patterns. Since haplotypes of these individuals could have originated from many different populations across the world, we have developed a machine learning method "SupportMix" to infer loci-specific genomic ancestry when simultaneously analyzing many possible ancestral populations. Simulations show that SupportMix is not only more accurate than other popular admixture discovery tools but is the first admixture inference method that can efficiently scale for simultaneous analysis of 50-100 putative ancestral populations while being independent of prior demographic information. CONCLUSIONS: By simultaneously using the 55 world populations from the Human Genome Diversity Panel, SupportMix was able to extract the fine-scale ancestry of the Qatar population, providing many new observations concerning the ancestry of the region. For example, as well as recapitulating the three major sub-populations in Qatar, composed of mainly Arabic, Persian, and African ancestry, SupportMix additionally identifies the specific ancestry of the Persian group to populations sampled in Greater Persia rather than from China and the ancestry of the African group to sub-Saharan origin and not Southern African Bantu origin as previously thought.
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Genética de Población , Genoma Humano , Bases de Datos Factuales , Genotipo , Haplotipos , Migración Humana , Humanos , Desequilibrio de Ligamiento , Cadenas de Markov , Qatar , Máquina de Vectores de SoporteRESUMEN
Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1(-)E3(-) adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine.
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Cocaína/análogos & derivados , Cocaína/inmunología , Trastornos Relacionados con Sustancias , Vacunas , Adenoviridae/genética , Animales , Anticuerpos/sangre , Cocaína/metabolismo , Virus Defectuosos/genética , Virus Defectuosos/inmunología , Virus Defectuosos/metabolismo , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Locomoción/efectos de los fármacos , Locomoción/inmunología , Ratones , Ratones Endogámicos BALB C , Actividad Motora/efectos de los fármacos , Actividad Motora/inmunología , Trastornos Relacionados con Sustancias/inmunología , Trastornos Relacionados con Sustancias/prevención & control , Vacunas/administración & dosificación , Vacunas/inmunologíaRESUMEN
The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1) is expressed on human CD34(+) and mouse Lin(-)Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1, but not VEGFR2, blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Placental growth factor (PlGF), which signals through VEGFR1, restored early and late phases of hematopoiesis following BM suppression. PlGF enhanced early phases of BM recovery directly through rapid chemotaxis of VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9, mediating the release of soluble Kit ligand. Thus, PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative BM microenvironment, favoring differentiation, mobilization and reconstitution of hematopoiesis.
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Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteínas Gestacionales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Antimetabolitos Antineoplásicos/farmacología , Separación Celular , Trasplante de Células , Quimiotaxis , Femenino , Fluorouracilo/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos , Factor de Crecimiento Placentario , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Quimera por Trasplante , Trasplante Heterólogo , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.
Asunto(s)
Médula Ósea/irrigación sanguínea , Quimiocinas/fisiología , Células Madre Hematopoyéticas/citología , Trombopoyesis/fisiología , Animales , Antígenos CD , Cadherinas/fisiología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Megacariocitos/citología , Ratones , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/fisiología , Receptores CXCR4/fisiología , Receptores de Citocinas/genética , Receptores de Citocinas/fisiología , Receptores de Trombopoyetina , Trombopoyetina/genética , Trombopoyetina/fisiologíaRESUMEN
Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.
Asunto(s)
Adenoviridae/genética , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Células Endoteliales/citología , Neovascularización Fisiológica/fisiología , Animales , Células de la Médula Ósea/citología , Carcinoma Embrionario , Supervivencia Celular/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Células Endoteliales/fisiología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células HL-60 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Venas Umbilicales/citologíaRESUMEN
BACKGROUND: The production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. Based on studies suggesting a role of the airway epithelial GABAergic system in asthma-related mucus overproduction, we hypothesized that cigarette smoking, another disorder associated with increased mucus production, may modulate GABAergic system-related gene expression levels in the airway epithelium. METHODS: We assessed expression of the GABAergic system in human airway epithelium obtained using bronchoscopy to sample the epithelium and microarrays to evaluate gene expression. RT-PCR was used to confirm gene expression of GABAergic system gene in large and small airway epithelium from heathy nonsmokers and healthy smokers. The differences in the GABAergic system gene was further confirmed by TaqMan, immunohistochemistry and Western analysis. RESULTS: The data demonstrate there is a complete GABAergic system expressed in the large and small human airway epithelium, including glutamate decarboxylase, GABA receptors, transporters and catabolism enzymes. Interestingly, of the entire GABAergic system, smoking modified only the expression of GAD67, with marked up-regulation of GAD67 gene expression in both large (4.1-fold increase, p < 0.01) and small airway epithelium of healthy smokers (6.3-fold increase, p < 0.01). At the protein level, Western analysis confirmed the increased expression of GAD67 in airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05). There was a significant positive correlation between GAD67 and MUC5AC gene expression in both large and small airway epithelium (p < 0.01), implying a link between GAD67 and mucin overproduction in association with smoking. CONCLUSIONS: In the context that GAD67 is the rate limiting enzyme in GABA synthesis, the correlation of GAD67 gene expression with MUC5AC expressions suggests that the up-regulation of airway epithelium expression of GAD67 may contribute to the increase in mucus production observed in association with cigarette smoking. TRIAL REGISTRATION: NCT00224198; NCT00224185.
Asunto(s)
Glutamato Descarboxilasa/metabolismo , Pulmón/metabolismo , Mucina 5AC/metabolismo , Fumar/metabolismo , Adulto , Femenino , Humanos , Masculino , Mucosa Respiratoria , Distribución Tisular , Regulación hacia Arriba/fisiologíaRESUMEN
RATIONALE: The airway epithelium of smokers is subject to a variety of mechanisms of injury with consequent modulation of epithelial regeneration and disordered differentiation. Several signaling pathways, including the Notch pathway, control epithelial differentiation in lung morphogenesis, but little is known about the role of these pathways in adults. OBJECTIVES: We tested the hypotheses that Notch-related genes are expressed in the normal nonsmoker small airway epithelium of human adults, and that Notch-related gene expression is down-regulated in healthy smokers and smokers with chronic obstructive pulmonary disease (COPD). METHODS: We used microarray technology to evaluate the expression of 55 Notch-related genes in the small airway epithelium of nonsmokers. We used TaqMan quantitative polymerase chain reaction (PCR) to confirm the expression of key genes and we used immunohistochemistry to assess the expression of Notch-related proteins in the airway epithelium. Changes in expression of Notch genes in healthy smokers and smokers with COPD compared with nonsmokers were evaluated by PCR. MEASUREMENTS AND MAIN RESULTS: Microarray analysis demonstrated that 45 of 55 Notch-related genes are expressed in the small airway epithelium of adults. TaqMan PCR confirmed the expression of key genes with highest expression of the ligand DLL1, the receptor NOTCH2, and the downstream effector HES1. Immunohistochemistry demonstrated the expression of Jag1, Notch2, Hes1, and Hes5 in airway epithelium. Several Notch ligands, receptors, and downstream effector genes were down-regulated in smokers, with more genes down-regulated in smokers with COPD than in healthy smokers. CONCLUSIONS: These observations are consistent with the hypothesis that the Notch pathway likely plays a role in the human adult airway epithelium, with down-regulation of Notch pathway gene expression in association with smoking and COPD.