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Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts.
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Drosophila/citología , Drosophila/metabolismo , Gotas Lipídicas/metabolismo , Nicho de Células Madre/efectos de los fármacos , Animales , Antioxidantes/farmacología , Proliferación Celular , Drosophila/crecimiento & desarrollo , Ácidos Grasos Insaturados/farmacología , Larva/citología , Larva/crecimiento & desarrollo , Larva/metabolismo , Neuroglía/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C(+) cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface.
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Células Asesinas Naturales/inmunología , Receptores KIR/química , Receptores KIR/metabolismo , Zinc/farmacología , Células Cultivadas , Células HEK293 , Antígenos HLA/metabolismo , Humanos , Sinapsis Inmunológicas/metabolismo , Polimerizacion , Receptores KIR/genética , Transducción de SeñalRESUMEN
BACKGROUND: The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age. Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart. Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the effect of exercise on the generation of new cardiomyocytes in the aged heart. METHODS: Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15N-thymidine incorporation and multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts. RESULTS: Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged hearts on the basis of the detection of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes. No mononucleated/diploid 15N-thymidine-labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further. CONCLUSIONS: Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.
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Miocitos Cardíacos , Condicionamiento Físico Animal , Animales , Calcineurina/metabolismo , Humanos , Lactante , Ratones , Miocitos Cardíacos/citología , Timidina/metabolismoRESUMEN
A major challenge in managing depression is that antidepressant drugs take a long time to exert their therapeutic effects. For the development of faster-acting treatments, it is crucial to get an improved understanding of the molecular mechanisms underlying antidepressant mode of action. Here, we used a novel mass spectrometry-based workflow to investigate how antidepressant treatment affects brain protein turnover at single-cell and subcellular resolution. We combined stable isotope metabolic labeling, quantitative Tandem Mass Spectrometry (qTMS) and Multi-isotope Imaging Mass Spectrometry (MIMS) to simultaneously quantify and image protein synthesis and turnover in hippocampi of mice treated with the antidepressant paroxetine. We identified changes in turnover of individual hippocampal proteins that reveal altered metabolism-mitochondrial processes and report on subregion-specific turnover patterns upon paroxetine treatment. This workflow can be used to investigate brain protein turnover changes in vivo upon pharmacological interventions at a resolution and precision that has not been possible with other methods to date. Our results reveal acute paroxetine effects on brain protein turnover and shed light on antidepressant mode of action.
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Antidepresivos , Paroxetina , Animales , Antidepresivos/metabolismo , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Hipocampo/metabolismo , Marcaje Isotópico/métodos , Isótopos/metabolismo , Isótopos/farmacología , Ratones , Paroxetina/metabolismo , Paroxetina/farmacología , Espectrometría de Masas en TándemRESUMEN
Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the 'immortal strand hypothesis', which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with (15)N-thymidine from gestation until post-natal week 8, we find no (15)N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered (15)N-thymidine pulse-chase, we find that proliferating crypt cells dilute the (15)N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.
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División Celular , Espectrometría de Masas/métodos , Células Madre/citología , Células Madre/metabolismo , Animales , Animales Recién Nacidos , ADN/biosíntesis , ADN/genética , ADN/metabolismo , Drosophila melanogaster/citología , Enterocitos/citología , Fibroblastos/citología , Humanos , Intestino Delgado/citología , Marcaje Isotópico , Isótopos , Leucocitos/citología , Metabolismo de los Lípidos , Linfopoyesis , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Células Madre/patología , Moldes Genéticos , Timidina/metabolismoRESUMEN
In the field of secondary ion mass spectrometry at nanometer scale (NanoSIMS), configuration of parallel detectors to routinely measure isotope ratios in sub-100 nm domains brings classical stable isotope tracer studies from the whole tissue level down to the suborganelle level. Over the past decade, the marriage of stable isotope tracers with NanoSIMS has been applied to a range of fundamental biological questions that were largely inaccessible by other means. Although multiplexed measurement of different stable isotope tracers is feasible, in practice there remains a gap in the current analytical capacity to efficiently measure stable isotopes commonly utilized in tracer studies. One such example is the measurement of deuterated tracers. The most obvious approach to measuring deuterium/hydrogen isotope ratios is at mass 2/1. However, the radius of the magnetic sector limits concomitant measurement of other masses critical to multiplexed exploration of biological samples. Here we determine the experimental parameters to measure deuterated tracers in biological samples using the C2H- polyatomic ion species (C2D-/C2H-) while operating the NanoSIMS at a reduced Mass Resolving Power of 14,000. Through control of the sputtering parameters, we demonstrate that there is an analytical window during which the C2D-/C2H- isotope ratio can be measured with sufficient precision for biological studies where the degree of D-labeling is typically well above natural abundance. We provide validation of this method by comparing the C2D measurement of D-water labeling in the murine small intestine relative to measurements of native D/H conducted in the same analytical fields. Additional proof-of-concept demonstrations include measurement of D-water, D-glucose, and D-thymidine in biological specimens. Therefore, this study provides a practical template for deuterium-based tracer studies in biological systems.
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Combinatorial signaling by proinflammatory cytokines synergizes to exacerbate toxicity to cells and tissue injury during acute infections. To explore synergism at the gene-regulatory level, we investigated the dynamics of transcription and chromatin signaling in response to dual cytokines by integrating nascent RNA imaging mass spectrometry, RNA sequencing, amplification-independent mRNA quantification, assay for transposase-accessible chromatin using sequencing (ATAC-seq), and transcription factor profiling. Costimulation with interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) synergistically induced a small subset of genes, including the chemokines CXCL9, -10, and -11. Gene induction coincided with increased chromatin accessibility at non-coding regions enriched for p65 and STAT1 binding sites. To discover coactivator dependencies, we conducted a targeted chemogenomic screen of transcriptional inhibitors followed by modeling of inhibitor dose-response curves. These results identified high efficacy of either p300/CREB-binding protein (CBP) or bromodomain and extra-terminal (BET) bromodomain inhibitors to disrupt induction of synergy genes. Combination p300/CBP and BET bromodomain inhibition at half-maximal inhibitory concentrations (subIC50) synergistically abrogated IFNγ/TNFα-induced chemokine gene and protein levels.
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Myofibroblast differentiation, essential for driving extracellular matrix synthesis in pulmonary fibrosis, requires increased glycolysis. While glycolytic cells must export lactate, the contributions of lactate transporters to myofibroblast differentiation are unknown. In this study, we investigated how MCT1 and MCT4, key lactate transporters, influence myofibroblast differentiation and experimental pulmonary fibrosis. Our findings reveal that inhibiting MCT1 or MCT4 reduces TGFß-stimulated pulmonary myofibroblast differentiation in vitro and decreases bleomycin-induced pulmonary fibrosis in vivo. Through comprehensive metabolic analyses, including bioenergetics, stable isotope tracing, metabolomics, and imaging mass spectrometry in both cells and mice, we demonstrate that inhibiting lactate transport enhances oxidative phosphorylation, reduces reactive oxygen species production, and diminishes glucose metabolite incorporation into fibrotic lung regions. Furthermore, we introduce VB253, a novel MCT4 inhibitor, which ameliorates pulmonary fibrosis in both young and aged mice, with comparable efficacy to established antifibrotic therapies. These results underscore the necessity of lactate transport for myofibroblast differentiation, identify MCT1 and MCT4 as promising pharmacologic targets in pulmonary fibrosis, and support further evaluation of lactate transport inhibitors for patients for whom limited therapeutic options currently exist.
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Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular fibroblasts rewire their metabolism to sustain collagen biosynthesis remains unknown. Here, we found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAFs). Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions for treating cardiopulmonary vascular disease.
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Glutamina , Serina , Rigidez Vascular , Animales , Glutamina/metabolismo , Serina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fibroblastos/metabolismo , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Humanos , Colágeno/metabolismo , RatasRESUMEN
In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-Seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all aa and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest that elevated glucose and proline avidity underlie increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.
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Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Humanos , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , Hipertensión Pulmonar/metabolismo , Células Endoteliales/metabolismo , Biomasa , Arteria Pulmonar/patologíaRESUMEN
The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues.
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Adipose tissue (AT) expands by a combination of two fundamental cellular mechanisms: hypertrophic growth of existing adipocytes or through generation of new adipocytes, also known as hyperplastic growth. Multiple lines of evidence suggest a limited capacity for hyperplastic growth of AT in adulthood and that adipocyte number is relatively stable, even with fluctuations in AT mass. If the adipocyte number is stable in adulthood, despite well-documented birth and death of adipocytes, then this would suggest that birth may be coupled to death in a regenerative cycle. To test this hypothesis, we examined the dynamics of birth of new fat cells in relationship to adipocyte death by using high-fidelity stable isotope tracer methods in C57Bl6 mice. We discovered birth of new adipocytes at higher frequency in histological proximity to dead adipocytes. In diet-induced obesity, adipogenesis surged after an adipocyte death peak beyond 8 weeks of high-fat feeding. Through transcriptional analyses of AT and fractionated adipocytes, we found that the dominant cell death signals were inflammasome related. Proinflammatory signals were particularly evident in hypertrophied adipocytes or with deletion of a constitutive oxygen sensor and inhibitor of hypoxia-inducible factor, Egln1. We leveraged the potential role for the inflammasome in adipocyte death to test the adipocyte death-birth hypothesis, finding that caspase 1 loss of function attenuated adipocyte death and birth in murine visceral AT. These data collectively point to a regenerative cycle of adipocyte death and birth as a driver of adipogenesis in adult murine AT.
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Adipocitos/fisiología , Adipogénesis/fisiología , Muerte Celular , Inflamación/fisiopatología , Grasa Intraabdominal/fisiopatología , Obesidad/fisiopatología , Células 3T3-L1 , Animales , Caspasa 1/genética , Caspasa 1/fisiología , Dieta Alta en Grasa , Hipertrofia , Inflamasomas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiologíaRESUMEN
Cancer and antimicrobial resistance to antibiotics are two of the most worrying healthcare concerns that humanity is facing nowadays. Some of the most promising solutions for these healthcare problems may come from nanomedicine. While the traditional synthesis of nanomaterials is often accompanied by drawbacks such as high cost or the production of toxic by-products, green nanotechnology has been presented as a suitable solution to overcome such challenges. In this work, an approach for the synthesis of tellurium (Te) nanostructures in aqueous media has been developed using aloe vera (AV) extracts as a unique reducing and capping agent. Te-based nanoparticles (AV-TeNPs), with sizes between 20 and 60 nm, were characterized in terms of physicochemical properties and tested for potential biomedical applications. A significant decay in bacterial growth after 24 h was achieved for both Methicillin-resistant Staphylococcus aureus and multidrug-resistant Escherichia coli at a relative low concentration of 5 µg/mL, while there was no cytotoxicity towards human dermal fibroblasts after 3 days of treatment. AV-TeNPs also showed anticancer properties up to 72 h within a range of concentrations between 5 and 100 µg/mL. Consequently, here, we present a novel and green approach to produce Te-based nanostructures with potential biomedical applications, especially for antibacterial and anticancer applications.
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Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aß) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aß accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aß. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper's transparent peer review process is included in the Supplemental Information.
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Enfermedad de Alzheimer/genética , Terminales Presinápticos/metabolismo , Proteómica/métodos , Animales , Modelos Animales de Enfermedad , Ratones , Ratones TransgénicosRESUMEN
The helium ion microscope (HIM) is a focussed ion beam instrument with unprecedented spatial resolution for secondary electron imaging but has traditionally lacked microanalytical capabilities. With the addition of the secondary ion mass spectrometry (SIMS) attachment, the capabilities of the instrument have expanded to microanalysis of isotopes from Li up to hundreds of atomic mass units, effectively opening up the analysis of all natural and geological systems. However, the instrument has thus far been underutilised by the geosciences community, due in no small part to a lack of a thorough understanding of the quantitative capabilities of the instrument. Li represents an ideal element for an exploration of the instrument as a tool for geological samples, due to its importance for economic geology and a green economy, and the difficult nature of observing Li with traditional microanalytical techniques. Also Li represents a "best-case" scenario for isotopic measurements. Here we present details of sample preparation, instrument sensitivity, theoretical, and measured detection limits for both elemental and isotopic analysis as well as practicalities for geological sample analyses of Li alongside a discussion of potential geological use cases of the HIM-SIMS instrument.
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Quantification of stable isotope tracers after metabolic labeling provides a snapshot of the dynamic state of living cells and tissue. A form of imaging mass spectrometry quantifies isotope ratios with a lateral resolution <50 nm, using a methodology that we refer to as multi-isotope imaging mass spectrometry (MIMS). Despite lateral resolution exceeding diffraction-limited light microscopy, lack of contrast has largely limited use of MIMS to large or specialized subcellular structures, such as the nucleus and stereocilia. In this study, we repurpose the engineered peroxidase APEX2 as the first genetically encoded marker for MIMS. Coupling APEX2 labeling of lysosomes and metabolic labeling of protein, we identify that individual lysosomes exhibit substantial heterogeneity in protein age, which is lost in iPSC-derived neurons lacking the lysosomal protein progranulin. This study expands the practical use of MIMS for cell biology by enabling measurements of metabolic function from stable isotope labeling within individual organelles in situ.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Marcaje Isotópico/métodos , Lisosomas/metabolismo , Espectrometría de Masas/métodos , Neuronas/metabolismo , Orgánulos/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , ProteolisisRESUMEN
Malignant tumors exhibit high degrees of genomic heterogeneity at the cellular level, leading to the view that subpopulations of tumor cells drive growth and treatment resistance. To examine the degree to which tumors also exhibit metabolic heterogeneity at the level of individual cells, we employed multi-isotope imaging mass spectrometry (MIMS) to quantify utilization of stable isotopes of glucose and glutamine along with a label for cell division. Mouse models of melanoma and malignant peripheral nerve sheath tumors (MPNSTs) exhibited striking heterogeneity of substrate utilization, evident in both proliferating and non-proliferating cells. We identified a correlation between metabolic heterogeneity, proliferation, and therapeutic resistance. Heterogeneity in metabolic substrate usage as revealed by incorporation of glucose and glutamine tracers is thus a marker for tumor proliferation. Collectively, our data demonstrate that MIMS provides a powerful tool with which to dissect metabolic functions of individual cells within the native tumor environment.
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Atherosclerotic plaques feature local proliferation of leukocytes and vascular smooth muscle cells (VSMCs) and changes in cellular metabolism. Yet the relationship between glucose utilization and proliferation has been technically impossible to study directly in cells of atherosclerotic plaques in vivo. We used multi-isotope imaging mass spectrometry (MIMS), a quantitative imaging platform, to measure coincident cell division and glucose utilization at suborganelle resolution in atherosclerotic plaques. In established plaques, 65% of intimal foam cells and only 4% of medial VSMCs were labeled with 15N-thymidine after 1 week of isotope treatment. Dividing cells demonstrated heightened glucose labeling. MIMS detected 2H-glucose label in multiple subcellular compartments within foam cells, including lipid droplets, the cytosol, and chromatin. Unexpectedly, we identified an intensely focal region of 2H-label in VSMCs underlying plaques. This signal diminished in regions of aorta without atherosclerosis. In advanced plaques, 15N-thymidine and 2H-glucose labeling in foam cells and VSMCs significantly decreased. These data demonstrate marked heterogeneity in VSMC glucose metabolism that was dependent on both proliferative status and proximity of VSMCs to plaques. Furthermore, these results reveal how quantitative mass spectrometry coupled with isotope imaging can complement other methods used to study cell biology directly in the growing atherosclerotic plaque in vivo.
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Aterosclerosis/diagnóstico por imagen , Proliferación Celular/fisiología , Espectrometría de Masas/métodos , Imagen Molecular/métodos , Animales , Aorta/citología , Aorta/diagnóstico por imagen , Aorta/metabolismo , Aterosclerosis/metabolismo , Modelos Animales de Enfermedad , Células Espumosas/metabolismo , Leucocitos/citología , Leucocitos/metabolismo , Masculino , Ratones , Músculo Liso Vascular/diagnóstico por imagen , Músculo Liso Vascular/metabolismo , Isótopos de Nitrógeno/química , Isótopos de Nitrógeno/metabolismo , Timidina/química , Timidina/metabolismoRESUMEN
One million patients with congenital heart disease (CHD) live in the United States. They have a lifelong risk of developing heart failure. Current concepts do not sufficiently address mechanisms of heart failure development specifically for these patients. Here, analysis of heart tissue from an infant with tetralogy of Fallot with pulmonary stenosis (ToF/PS) labeled with isotope-tagged thymidine demonstrated that cardiomyocyte cytokinesis failure is increased in this common form of CHD. We used single-cell transcriptional profiling to discover that the underlying mechanism of cytokinesis failure is repression of the cytokinesis gene ECT2, downstream of ß-adrenergic receptors (ß-ARs). Inactivation of the ß-AR genes and administration of the ß-blocker propranolol increased cardiomyocyte division in neonatal mice, which increased the number of cardiomyocytes (endowment) and conferred benefit after myocardial infarction in adults. Propranolol enabled the division of ToF/PS cardiomyocytes in vitro. These results suggest that ß-blockers could be evaluated for increasing cardiomyocyte division in patients with ToF/PS and other types of CHD.
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Citocinesis/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Antagonistas Adrenérgicos beta/farmacología , Animales , Animales Recién Nacidos , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Miocitos Cardíacos/efectos de los fármacos , Propranolol/farmacología , Proteínas Proto-Oncogénicas/metabolismo , RatasRESUMEN
Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency.