RESUMEN
Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Edema Macular , Animales , Ratones , Humanos , Edema Macular/tratamiento farmacológico , Edema Macular/etiología , Retinopatía Diabética/tratamiento farmacológico , Inhibidores de la Angiogénesis/uso terapéutico , Células Endoteliales , Senoterapéuticos , Senescencia CelularRESUMEN
Blood cell production is a complex process, partly genetically determined and influenced by acquired factors. However, there is a paucity of data on how these factors interplay in the context of aging, which is associated with a myeloid proliferation bias, clonal hematopoiesis (CH), and an increased incidence of myeloid cancers. We investigated hereditary and acquired factors underlying blood cell trait variability in a cohort of 2996 related and unrelated women from Quebec aged from 55 to 101 years. We performed a genome-wide association study, evaluated the impact of chronic diseases, and performed targeted deep sequencing of CH driver genes and X-chromosome inactivation (XCI)-based clonality analyses. Multivariable analyses were conducted using generalized linear mixed models. We document that aging is associated with increasing neutrophil and monocyte counts and decreasing lymphocyte counts. Neutrophil counts were influenced by the variants in the region of GSDMA and PSMD3-CSF3, but this association decreased with age; in parallel, older individuals with cardiometabolic comorbidities exhibited significantly higher neutrophil counts (4.1 × 109/L vs 3.83 × 109/L; P < .001) than younger individuals. These age-related diseases were also associated with an increase in other myeloid-derived cells. Neither CH nor XCI clonality correlated with neutrophil counts. In conclusion, we show that neutrophil counts are genetically influenced, but as individuals age, this contribution decreases in favor of acquired factors. Aging is associated with a myeloid proliferation bias which is greater in the presence of cardiometabolic comorbidities but not of CH. These findings support that cell-extrinsic factors may contribute to the myeloid shift possibly through low-grade inflammation.
Asunto(s)
Enfermedades Cardiovasculares , Neutrófilos , Humanos , Femenino , Anciano , Estudio de Asociación del Genoma Completo , Recuento de Leucocitos , Envejecimiento/genética , Proteínas Citotóxicas Formadoras de PorosRESUMEN
Pathological neovascularization in age-related macular degeneration (nvAMD) drives the principal cause of blindness in the elderly. While there is a robust genetic association between genes of innate immunity and AMD, genome-to-phenome relationships are low, suggesting a critical contribution of environmental triggers of disease. Possible insight comes from the observation that a past history of infection with pathogens such as Chlamydia pneumoniae, or other systemic inflammation, can predispose to nvAMD in later life. Using a mouse model of nvAMD with prior C. pneumoniae infection, endotoxin exposure, and genetic ablation of distinct immune cell populations, we demonstrated that peripheral infections elicited epigenetic reprogramming that led to a persistent memory state in retinal CX3CR1+ mononuclear phagocytes (MNPs). The immune imprinting persisted long after the initial inflammation had subsided and ultimately exacerbated choroidal neovascularization in a model of nvAMD. Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) identified activating transcription factor 3 (ATF3) as a central mediator of retina-resident MNP reprogramming following peripheral inflammation. ATF3 polarized MNPs toward a reparative phenotype biased toward production of proangiogenic factors in response to subsequent injury. Therefore, a past history of bacterial endotoxin-induced inflammation can lead to immunological reprograming within CNS-resident MNPs and aggravate pathological angiogenesis in the aging retina.
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Neovascularización Coroidal , Degeneración Macular , Humanos , Microglía/patología , Retina/patología , Neovascularización Coroidal/genética , Degeneración Macular/genética , Degeneración Macular/patología , Inflamación/patologíaRESUMEN
Individuals with age-related clonal hematopoiesis (CH) are at greater risk for hematologic malignancies and cardiovascular diseases. However, predictive preclinical animal models to recapitulate the spectrum of human CH are lacking. Through error-corrected sequencing of 56 human CH/myeloid malignancy genes, we identified natural CH driver mutations in aged rhesus macaques matching genes somatically mutated in human CH, with DNMT3A mutations being the most frequent. A CH model in young adult macaques was generated via autologous transplantation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene-edited hematopoietic stem and progenitor cells (HSPCs), targeting the top human CH genes with loss-of-function (LOF) mutations. Long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared with minimal expansion of clones bearing other mutations. Although the blood counts of these CH macaques were normal, their bone marrows were hypercellular and myeloid-predominant. TET2-disrupted myeloid colony-forming units isolated from these animals showed a distinct hyperinflammatory gene expression profile compared with wild type. In addition, mature macrophages purified from the CH macaques showed elevated NLRP3 inflammasome activity and increased interleukin-1ß (IL-1ß) and IL-6 production. The model was used to test the impact of IL-6 blockage by tocilizumab, documenting a slowing of TET2-mutated expansion, suggesting that interruption of the IL-6 axis may remove the selective advantage of mutant HSPCs. These findings provide a model for examining the pathophysiology of CH and give insights into potential therapeutic interventions.
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Hematopoyesis Clonal , Dioxigenasas , Humanos , Adulto Joven , Animales , Anciano , Hematopoyesis Clonal/genética , Hematopoyesis/genética , Interleucina-1beta/genética , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Macaca mulatta , Proteína 9 Asociada a CRISPR , Interleucina-6/genética , Células Clonales , Proteínas de Unión al ADN/genética , Dioxigenasas/genéticaRESUMEN
The beneficial effects of brown adipose tissue (BAT) on obesity and associated metabolic diseases are mediated through its capacity to dissipate energy as heat. While immune cells, such as tissue-resident macrophages, are known to influence adipose tissue homeostasis, relatively little is known about their contribution to BAT function. Here we report that neuropilin-1 (NRP1), a multiligand single-pass transmembrane receptor, is highly expressed in BAT-resident macrophages. During diet-induced obesity (DIO), myeloid-resident NRP1 influences interscapular BAT mass, and consequently vascular morphology, innervation density and ultimately core body temperature during cold exposure. Thus, NRP1-expressing myeloid cells contribute to the BAT homeostasis and potentially its thermogenic function in DIO.
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Tejido Adiposo Pardo/fisiología , Homeostasis , Células Mieloides/metabolismo , Neuropilina-1/fisiología , Obesidad/prevención & control , Termogénesis , Animales , Dieta/efectos adversos , Metabolismo Energético , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
Age-related macular degeneration (AMD) in its various forms is a leading cause of blindness in industrialized countries. Here, we provide evidence that ligands for neuropilin-1 (NRP1), such as Semaphorin 3A and VEGF-A, are elevated in the vitreous of patients with AMD at times of active choroidal neovascularization (CNV). We further demonstrate that NRP1-expressing myeloid cells promote and maintain CNV. Expression of NRP1 on cells of myeloid lineage is critical for mitigating production of inflammatory factors such as IL6 and IL1ß. Therapeutically trapping ligands of NRP1 with an NRP1-derived trap reduces CNV. Collectively, our findings identify a role for NRP1-expressing myeloid cells in promoting pathological angiogenesis during CNV and introduce a therapeutic approach to counter neovascular AMD.
Asunto(s)
Neovascularización Coroidal , Degeneración Macular Húmeda , Inhibidores de la Angiogénesis , Humanos , Inflamación , Neuropilina-1/genética , Factor A de Crecimiento Endotelial Vascular , Agudeza VisualRESUMEN
Neovascularization contributes to multiple visual disorders including age-related macular degeneration (AMD) and retinopathy of prematurity. Current therapies for treating ocular angiogenesis are centered on the inhibition of vascular endothelial growth factor (VEGF). While clinically effective, some AMD patients are refractory or develop resistance to anti-VEGF therapies and concerns of increased risks of developing geographic atrophy following long-term treatment have been raised. Identification of alternative pathways to inhibit pathological angiogenesis is thus important. We have identified a novel inhibitor of angiogenesis, COCO, a member of the Cerberus-related DAN protein family. We demonstrate that COCO inhibits sprouting, migration and cellular proliferation of cultured endothelial cells. Intravitreal injections of COCO inhibited retinal vascularization during development and in models of retinopathy of prematurity. COCO equally abrogated angiogenesis in models of choroidal neovascularization. Mechanistically, COCO inhibited TGFß and BMP pathways and altered energy metabolism and redox balance of endothelial cells. Together, these data show that COCO is an inhibitor of retinal and choroidal angiogenesis, possibly representing a therapeutic option for the treatment of neovascular ocular diseases.
Asunto(s)
Neovascularización Coroidal , Cocos , Neovascularización Coroidal/tratamiento farmacológico , Células Endoteliales , Humanos , Péptidos y Proteínas de Señalización Intercelular , Retina , Factor A de Crecimiento Endotelial VascularRESUMEN
Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.
Asunto(s)
Senescencia Celular , Enfermedades de la Retina/patología , Proteína bcl-X/metabolismo , Animales , Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Flavonoles/química , Flavonoles/farmacología , Flavonoles/uso terapéutico , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Patológica , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/metabolismo , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
In developed countries, the leading causes of blindness such as diabetic retinopathy are characterized by disorganized vasculature that can become fibrotic. Although many such pathological vessels often naturally regress and spare sight-threatening complications, the underlying mechanisms remain unknown. Here, we used orthogonal approaches in human patients with proliferative diabetic retinopathy and a mouse model of ischemic retinopathies to identify an unconventional role for neutrophils in vascular remodeling during late-stage sterile inflammation. Senescent vasculature released a secretome that attracted neutrophils and triggered the production of neutrophil extracellular traps (NETs). NETs ultimately cleared diseased endothelial cells and remodeled unhealthy vessels. Genetic or pharmacological inhibition of NETosis prevented the regression of senescent vessels and prolonged disease. Thus, clearance of senescent retinal blood vessels leads to reparative vascular remodeling.
Asunto(s)
Envejecimiento/patología , Retinopatía Diabética/patología , Trampas Extracelulares/inmunología , Vasos Retinianos/patología , Animales , Senescencia Celular , Retinopatía Diabética/inmunología , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Células Endoteliales/patología , Humanos , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Vasos Retinianos/inmunologíaRESUMEN
Clonal hematopoiesis of indeterminate potential (CHIP) is predictive of hematological cancers and cardiovascular diseases, but the etiology of CHIP initiation and clonal expansion is unknown. Several lines of evidence suggest that proinflammatory cytokines may favor mutated hematopoietic stem cell expansion. To investigate the potential link between inflammation and CHIP, we performed targeted deep sequencing of 11 genes previously implicated in CHIP in 1887 subjects aged >70 years from the Montreal Heart Institute Biobank, of which 1359 had prior coronary artery disease (CAD), and 528 controls did not. We assessed association of CHIP with log transformed high-sensitivity C-reactive protein (hs-CRP), a validated biomarker of inflammation. CHIP was identified in 427 of the 1887 subjects (22.6%). CHIP mutations were more frequently identified in DNMT3A (11.6%) and TET2 (6.1%), with a higher proportion of TET2 mutations occurring in controls than in patients with CAD (9.0% vs 4.9%, P < .001). CHIP carriers had 21% higher hs-CRP levels compared with their noncarrier counterparts (eß = 1.21, 95% confidence interval [CI]: 1.08 to 1.36; P = .001). A similar effect was observed in the subgroup of patients with known CAD (eß = 1.22, 95% CI: 1.06 to 1.41; P = .005). These findings confirm the association between inflammation and CHIP. This association may open investigational avenues aimed at documenting mechanisms linking inflammation to clonal progression and ultimately supports prevention interventions to attenuate CHIP's impact on cardiovascular disease and cancer.
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Proteína C-Reactiva , Hematopoyesis Clonal , Hematopoyesis , Intervención Coronaria Percutánea , Anciano , Antagonistas de Receptores de Angiotensina , Inhibidores de la Enzima Convertidora de Angiotensina , Hematopoyesis/genética , Células Madre Hematopoyéticas , Humanos , MasculinoRESUMEN
The history of clonal hematopoiesis (CH) research is punctuated by several seminal discoveries that have forged our understanding of cancer development. The clever application of the principle of random X-chromosome inactivation (XCI) in females led to the development of the first test to identify clonal derivation of cells. Initially limited by a low level of informativeness, the applicability of these assays expanded with differential methylation-based assays at highly polymorphic genes such as the human androgen receptor (HUMARA). Twenty years ago, the observation that skewing of XCI ratios increases as women age was the first clue that led to the identification of mutations in the TET2 gene in hematologically normal aging individuals. In 2014, large-scale genomic approaches of three cohorts allowed definition of CH, which was reported to increase the risk of developing hematologic cancers and cardiovascular diseases. These observations created a fertile field of investigation aimed at investigating the etiology and consequences of CH. The most frequently mutated genes in CH are DNMT3A, TET2, and ASXL1, which have a role in hematopoietic stem cell (HSC) development and self-renewal. These mutations confer a competitive advantage to the CH clones. However, the penetrance of CH is age dependent but incomplete, suggesting the influence of extrinsic factors. Recent data attribute a modest role to genetic predisposition, but several observations point to the impact of a pro-inflammatory milieu that advantages the mutated clones. CH may be a barometer of nonhealthy aging, and interventions devised at curbing its initiation or progression should be a research priority.
Asunto(s)
Envejecimiento/genética , Investigación Biomédica/historia , Cromosomas Humanos X/genética , Hematopoyesis/genética , Mutación , Inactivación del Cromosoma X , Adulto , Proteínas de Unión al ADN/genética , Dioxigenasas , Femenino , Células Madre Hematopoyéticas , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Masculino , Proteínas Proto-Oncogénicas/genética , Receptores Androgénicos/genética , Proteínas Represoras/genéticaRESUMEN
Diabetic macular edema is a major complication of diabetes resulting in loss of central vision. Although heightened vessel leakiness has been linked to glial and neuronal-derived factors, relatively little is known on the mechanisms by which mature endothelial cells exit from a quiescent state and compromise barrier function. Here we report that endothelial NOTCH1 signaling in mature diabetic retinas contributes to increased vascular permeability. By providing both human and mouse data, we show that NOTCH1 ligands JAGGED1 and DELTA LIKE-4 are up-regulated secondary to hyperglycemia and activate both canonical and rapid noncanonical NOTCH1 pathways that ultimately disrupt endothelial adherens junctions in diabetic retinas by causing dissociation of vascular endothelial-cadherin from ß-catenin. We further demonstrate that neutralization of NOTCH1 ligands prevents diabetes-induced retinal edema. Collectively, these results identify a fundamental process in diabetes-mediated vascular permeability and provide translational rational for targeting the NOTCH pathway (primarily JAGGED1) in conditions characterized by compromised vascular barrier function.
Asunto(s)
Permeabilidad Capilar , Retinopatía Diabética/patología , Receptor Notch1/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Activación Enzimática , Hiperglucemia/metabolismo , Proteína Jagged-1/biosíntesis , Ratones , Óxido Nítrico/biosíntesis , Vasos Retinianos/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
The recent characterization of clonal hematopoiesis in a large segment of the aging population has raised tremendous interest and concern alike. Mutations have been documented in genes associated with hematological cancers and in non-driver candidates. These mutations are present at low frequency in the majority of individuals after middle-age, and principally affect the epigenetic modifiers DNMT3A and TET2. In 10%-40% of cases, the clone will progress to meet the diagnostic criteria for Clonal Hematopoiesis of Indeterminate Potential, which is associated with an increased risk of hematological cancer and cardiovascular mortality. Blood cell parameters appear unmodified in these individuals, but a minority of them will develop a hematologic malignancy. At this time, the factors put forward as potentially influencing the risk of cancer development are clone size, specific gene, specific mutation, and the number of mutations. Specific stress on hematopoiesis also gives rise to clonal expansion. Genotoxic exposure (such as chemotherapy), or immune attack (as in aplastic anemia) selects/provides a fitness advantage to clones with a context-specific signature. Clonal hematopoiesis offers a new opportunity to understand the biology and adaptation mechanisms of aging hematopoiesis and provides insight into the mechanisms underlying malignant transformation. Furthermore, it might shed light on common denominators of age-associated medical conditions and help devise global strategies that will impact the prevention of hematologic cancers and promote healthy aging. Stem Cells 2018;36:1287-1294.
Asunto(s)
Transformación Celular Neoplásica/genética , Hematopoyesis/genética , Células Madre/metabolismo , Factores de Edad , HumanosRESUMEN
We analyzed DNA from polymorphonuclear (PMN) cells, monocytes, B cells, and T cells of 107 individuals with clonal hematopoiesis of indeterminate potential (CHIP) to perform lineage restriction analysis of different gene mutations. Three lineage categories were defined: myeloid (PMN with or without monocytes), myelolympho-B (myeloid and B cells), and multipotent (myeloid, B and T cells). Six individuals with aberrant patterns were excluded from analysis. Ninety-four had a single mutation (56 in DNMT3A, 24 in TET2, 7 in other genes [JAK2, ASXL1, CBL or TP53]). Fourteen had multiple mutations. The lineage restriction patterns of single DNMT3A- or TET2-mutated individuals were different. The proportion of myeloid restricted mutations was higher for TET2 (54.2%, 13 of 24) than for DNMT3A (23.2%, 13 of 56) (P < .05). It was similar for myelolympho-B category but with a 1.5 fold greater proportion of myeloid cells for TET2 individuals (P < .05). Importantly, 0% (0 of 24) of the individuals with TET2 mutation in the multipotent category in contrast to 35.7% (20 of 56) for DNMT3A (P < .01). The clone size predicted multipotent pattern for DNMT3A suggesting a time delay for extensive lineage clonal dominance. These distinctive features may be important in deciphering the transformation mechanisms of these frequent mutations.
Asunto(s)
Linaje de la Célula/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Unión al ADN/genética , Células Madre Multipotentes/metabolismo , Proteínas Proto-Oncogénicas/genética , Anciano , Anciano de 80 o más Años , Biomarcadores , Diferenciación Celular , Inmunoprecipitación de Cromatina , Evolución Clonal/genética , ADN Metiltransferasa 3A , Dioxigenasas , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , MutaciónRESUMEN
Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2-/- mice8,9 and humans with TET2 mutations1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.
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Enfermedades Asintomáticas , Fenómenos Fisiológicos Bacterianos , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Leucemia/microbiología , Leucemia/patología , Proteínas Proto-Oncogénicas/deficiencia , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Fenómenos Fisiológicos Bacterianos/inmunología , Proteínas de Unión al ADN/genética , Dioxigenasas , Femenino , Vida Libre de Gérmenes , Inflamación/microbiología , Interleucina-6/inmunología , Mucosa Intestinal/metabolismo , Lactobacillus/química , Lactobacillus/citología , Lactobacillus/inmunología , Masculino , Ratones , Penetrancia , Permeabilidad , Proteínas Proto-Oncogénicas/genética , Receptor Toll-Like 2/agonistasRESUMEN
Age-associated clonal hematopoiesis caused by acquired mutations in myeloid cancer-associated genes is highly prevalent in the normal population. Its etiology, biological impact on hematopoiesis, and oncogenic risk is poorly defined at this time. To gain insight into this phenomenon, we analyzed a cohort of 2530 related and unrelated hematologically normal individuals (ages 55 to 101 years). We used a sensitive gene-targeted deep sequencing approach to gain precision on the exact prevalence of driver mutations and the proportions of affected genes. Mutational status was correlated with biological parameters. We report a higher overall prevalence of driver mutations (13.7%), which occurred mostly (93%) in DNMT3A or TET2 and were highly age-correlated. Mutation in these 2 genes had some distinctive effects on end points. TET2 mutations were more age-dependent, associated with a modest neutropenic effect (9%, P = .012), demonstrated familial aggregation, and associated with chronic obstructive pulmonary disease. Mutations in DNMT3A had no impact on blood counts or indices. Mutational burden of both genes correlated with X-inactivation skewing but no significant association with age-adjusted telomere length reduction was documented. The discordance between the high prevalence of mutations in these 2 genes and their limited biological impact raise the question of the potential role of dysregulated epigenetic modifiers in normal aging hematopoiesis, which may include support to failing hematopoiesis.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Unión al ADN/genética , Hematopoyesis , Mutación , Proteínas Proto-Oncogénicas/genética , Factores de Edad , Anciano , Anciano de 80 o más Años , Envejecimiento , Recuento de Células Sanguíneas , Células Clonales , Estudios de Cohortes , ADN Metiltransferasa 3A , Dioxigenasas , Femenino , Predisposición Genética a la Enfermedad , Neoplasias Hematológicas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Homeostasis del Telómero , Inactivación del Cromosoma XRESUMEN
Epigenetic alteration may play a role in age-associated dysfunction of stem cells and predispose to the development of hematological cancers. We analyzed global levels of hematopoietic 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in a cross-sectional study comprising 198 unrelated individuals from four age categories (neonates, 25-30, 70-75, and >90 years old) by liquid chromatography-electrospray ionization-tandem mass spectrometry with multiple reaction monitoring. X-chromosome inactivation (XCI) ratios and telomere length (TL) were measured in all individuals by polymerase chain reaction. Sequencing of epigenetic regulator genes (including TET2, DNMT3A, ASXL1, IDH1, IDH2, and WT1) was performed in the two older subcohorts. We found that global 5hmC levels declined with age in human blood cells (27.5% reduction from birth to old age, p < 0.0005). The levels of 5mC underwent a more modest reduction (2.4% drop) between newborns and the elderly (p < 0.0005). Low 5hmC was associated with increased skewing of XCI (age-adjusted p = 0.0304) and reduced TL (age-adjusted p = 0.0354), both surrogate markers of clonal dominance. Of the 100 individuals over the age of 70, 16 had somatic mutations in TET2, 14 in DNMT3A, and none in IDH1, IDH2, or WT1. Individuals with TET2 mutations had significantly lower 5hmC (relative to unmutated individuals), whereas DNMT3A-mutated subjects did not. However, mutations in TET2 cannot account solely for the decline in 5hmC levels observed with aging because unmutated older individuals also had lower 5hmC levels compared with younger individuals. This suggests that the age-associated decline in 5hmC is multifactorial. Larger prospective studies are needed to determine whether 5hmC reduction is a biomarker of hematological cancer development.
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5-Metilcitosina/análogos & derivados , Células Sanguíneas/metabolismo , Proteínas de Unión al ADN/genética , Mutación , Proteínas Proto-Oncogénicas/genética , 5-Metilcitosina/metabolismo , Factores de Edad , Anciano , Anciano de 80 o más Años , Biomarcadores , Cromatografía Liquida , Dioxigenasas , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Espectrometría de Masas , Metabolómica/métodosRESUMEN
In mammals, combinatorial assembly of alternative families of subunits confers functional specificity to adenosine triphosphate (ATP)-dependent SWI/SNF-like Brg/Brm-associated factor (BAF) chromatin remodeling complexes by creating distinct polymorphic surfaces for interaction with regulatory elements and DNA-binding factors. Although redundant in terms of biochemical activity, the core ATPase subunits, BRG/SMARCA4 and BRM/SMARCA2, are functionally distinct and may contribute to complex specificity. Here we show using quantitative proteomics that BAF complexes expressed in leukemia are specifically assembled around the BRG ATPase. Moreover, using a mouse model of acute myeloid leukemia, we demonstrate that BRG is essential for leukemia maintenance, as leukemic cells lacking BRG rapidly undergo cell-cycle arrest and apoptosis. Most importantly, we show that BRG is dispensable for the maintenance of immunophenotypic long-term repopulating hematopoietic stem cells, suggesting that adroit targeting of BRG in leukemia may have potent and specific therapeutic effects.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , ADN Helicasas/fisiología , Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Animales , Southern Blotting , Cromatografía Liquida , ADN Helicasas/genética , Modelos Animales de Enfermedad , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Factores de Transcripción/genéticaRESUMEN
ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes are emerging as key regulators of embryonic and adult stem cell function. Particularly intriguing are the findings that specialized assemblies of BAF complexes are required for establishing and maintaining pluripotent and multipotent states in cells. However, little is known on the importance of these complexes in normal and leukemic hemopoiesis. Here we provide the first evidence that the actin-related protein BAF53a, a subunit of BAF complexes preferentially expressed in long-term repopulating stem cells, is essential for adult hemopoiesis. Conditional deletion of BAF53a resulted in multilineage BM failure, aplastic anemia, and rapid lethality. These severe hemopoietic defects originate from a proliferative impairment of BM HSCs and progenitors and decreased progenitor survival. Using hemopoietic chimeras, we show that the impaired function of BAF53a-deficient HSCs is cell-autonomous and independent of the BM microenvironment. Altogether, our studies highlight an unsuspected role for BAF chromatin remodeling complexes in the maintenance of HSC and progenitor cell properties.
Asunto(s)
Actinas/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Células Madre Hematopoyéticas/metabolismo , Sistema Hematopoyético/metabolismo , Transcriptoma , Actinas/metabolismo , Anemia Aplásica/genética , Anemia Aplásica/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Proteínas de Ciclo Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Feto , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/fisiología , Sistema Hematopoyético/crecimiento & desarrollo , Hígado , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Transcriptional co-repressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate the expression of a variety of genes and are involved in numerous developmental processes in both invertebrate and vertebrate species. More specifically, Gro/TLE1 participates in mechanisms that inhibit/delay the differentiation of cerebral cortex neural progenitor cells into neurons during mammalian forebrain development. The anti-neurogenic function of Gro/TLE1 depends on the formation of protein complexes with specific DNA-binding transcription factors that engage Gro/TLE1 through WRP(W/Y) sequences. Interaction with those transcription partners results in Gro/TLE1 recruitment to selected DNA sites and causes increased Gro/TLE1 phosphorylation. The physiological significance of the latter event, termed "cofactor-activated phosphorylation," had not been determined. Therefore, this study aimed at clarifying the role of cofactor-activated phosphorylation in the anti-neurogenic function of Gro/TLE1. METHODS AND PRINCIPAL FINDINGS: A combination of site-directed mutagenesis, mass spectrometry, biochemistry, primary cell culture, and immunocytochemical assays was utilized to characterize point mutations of Ser-286, a residue that is phosphorylated in vivo and is located within the serine/proline-rich (SP) domain of Gro/TLE1. Mutation of Ser-286 to alanine or glutamic acid does not perturb the interaction of Gro/TLE1 with DNA-binding partners, including the basic helix-loop-helix transcription factor Hes1, a prototypical anti-neurogenic WRP(W/Y) motif protein. Ser-286 mutations do not prevent the recruitment of Gro/TLE1 to DNA, but they impair cofactor-activated phosphorylation and weaken the interaction of Gro/TLE1 with chromatin. These effects are correlated with an impairment of the anti-neurogenic activity of Gro/TLE1. Similar results were obtained when mutations of Ser-289 and Ser-298, which are also located within the SP domain of Gro/TLE1, were analyzed. CONCLUSION: Based on the positive correlation between Gro/TLE1 cofactor-activated phosphorylation and ability to inhibit cortical neuron differentiation, we propose that hyperphosphorylation induced by cofactor binding plays a positive role in the regulation of Gro/TLE1 anti-neurogenic activity.