RESUMEN
Neutrophils can contribute to inflammatory disease propagation via innate mechanisms intended for inflammation resolution. For example, neutrophil extracellular traps (NETs) are necessary for trapping pathogens but can contribute to clot formation and blood flow restriction, that is, ischemia. Currently, no therapeutics in the clinic directly target NETs despite the known involvement of NETs contributing to mortality and increased disease severity. Vascular-deployed particle-based therapeutics are a novel and robust alternative to traditional small-molecule drugs by enhancing drug delivery to cells of interest. This work designs a high-throughput assay to investigate the immunomodulatory behavior and functionality of salicylic acid-based polymer-based particle therapeutics against NETosis in human neutrophils. Briefly, this work finds that polymeric composition plays a role, and particle size can also influence rates of NETosis. Salicylate-based polymeric (Poly-SA) particles are found to functionally inhibit NETosis depending on the particle size and concentration exposed to neutrophils. This work demonstrates the high throughput method can help fast-track particle-based therapeutic optimization and design, more efficiently preparing this innovative therapeutics for the clinic.
RESUMEN
The combination of inflammation and thrombosis is a hallmark of many cardiovascular diseases. Under such conditions, platelets are recruited to an area of inflammation by forming platelet-leukocyte aggregates via interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can bind to the endothelium. While particulate drug carriers have been utilized to passively redirect leukocytes from areas of inflammation, the downstream impact of these carriers on platelet accumulation in thromboinflammatory conditions has yet to be studied. Here, we explore the ability of polymeric particles to divert platelets away from inflamed blood vessels both in vitro and in vivo. We find that untargeted and targeted micron-sized polymeric particles can successfully reduce platelet adhesion to an inflamed endothelial monolayer in vitro in blood flow systems and in vivo in a lipopolysaccharide-induced, systemic inflammation murine model. Our data represent initial work in developing cargo-free, anti-platelet therapeutics specifically for conditions of thromboinflammation.
Asunto(s)
Neutrófilos , Trombosis , Humanos , Animales , Ratones , Neutrófilos/metabolismo , Inflamación/metabolismo , Tromboinflamación , Trombosis/metabolismo , Plaquetas/metabolismo , Leucocitos/metabolismo , Selectina-P/metabolismoRESUMEN
Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Replicación del ADN/genética , ADN Ribosómico/genética , Recombinación Genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Ratones , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMEN
Aging leads to hypothalamic inflammation, but does so more slowly in mice whose lifespan has been extended by mutations that affect GH/IGF-1 signals. Early-life exposure to GH by injection, or to nutrient restriction in the first 3 weeks of life, also modulate both lifespan and the pace of hypothalamic inflammation. Three drugs extend lifespan of UM-HET3 mice in a sex-specific way: acarbose (ACA), 17-α-estradiol (17αE2), and nordihydroguaiaretic acid (NDGA), with more dramatic longevity increases in males in each case. In this study, we examined the effect of these anti-aging drugs on neuro-inflammation in hypothalamus and hippocampus. We found that age-associated hypothalamic inflammation is reduced in males but not in females at 12 months of age by ACA and 17αE2 and at 22 months of age in NDGA-treated mice. The three drugs blocked indices of hypothalamic reactive gliosis associated with aging, such as Iba-1-positive microglia and GFAP-positive astrocytes, as well as age-associated overproduction of TNF-α. This effect was not observed in drug-treated female mice or in the hippocampus of the drug-treated animals. On the other hand, caloric restriction (CR; an intervention that extends the lifespan in both sexes) significantly reduced hypothalamic microglia and TNF-α in both sexes at 12 months of age. Together, these results suggest that the extent of drug-induced changes in hypothalamic inflammatory processes is sexually dimorphic in a pattern that parallels the effects of these agents on mouse longevity and that mimics the changes seen, in both sexes, of long-lived nutrient restricted or mutant mice.
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Acarbosa/farmacología , Restricción Calórica , Estradiol/farmacología , Hipotálamo/efectos de los fármacos , Longevidad/efectos de los fármacos , Masoprocol/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Hipotálamo/metabolismo , Hipotálamo/patología , Inflamación/prevención & control , Longevidad/genética , Masculino , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Factores Sexuales , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: The GH/IGF-1 axis has important roles in growth and metabolism. GH and GH receptor (GHR) are active in the central nervous system (CNS) and are crucial in regulating several aspects of metabolism. In the hypothalamus, there is a high abundance of GH-responsive cells, but the role of GH signaling in hypothalamic neurons is unknown. Previous work has demonstrated that the Ghr gene is highly expressed in LepRb neurons. Given that leptin is a key regulator of energy balance by acting on leptin receptor (LepRb)-expressing neurons, we tested the hypothesis that LepRb neurons represent an important site for GHR signaling to control body homeostasis. METHODS: To determine the importance of GHR signaling in LepRb neurons, we utilized Cre/loxP technology to ablate GHR expression in LepRb neurons (LeprEYFPΔGHR). The mice were generated by crossing the Leprcre on the cre-inducible ROSA26-EYFP mice to GHRL/L mice. Parameters of body composition and glucose homeostasis were evaluated. RESULTS: Our results demonstrate that the sites with GHR and LepRb co-expression include ARH, DMH, and LHA neurons. Leptin action was not altered in LeprEYFPΔGHR mice; however, GH-induced pStat5-IR in LepRb neurons was significantly reduced in these mice. Serum IGF-1 and GH levels were unaltered, and we found no evidence that GHR signaling regulates food intake and body weight in LepRb neurons. In contrast, diminished GHR signaling in LepRb neurons impaired hepatic insulin sensitivity and peripheral lipid metabolism. This was paralleled with a failure to suppress expression of the gluconeogenic genes and impaired hepatic insulin signaling in LeprEYFPΔGHR mice. CONCLUSION: These findings suggest the existence of GHR-leptin neurocircuitry that plays an important role in the GHR-mediated regulation of glucose metabolism irrespective of feeding.
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Glucosa/metabolismo , Hipotálamo/metabolismo , Hígado/metabolismo , Neuronas/metabolismo , Receptores de Leptina/metabolismo , Receptores de Somatotropina/metabolismo , Animales , Hipotálamo/citología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Mice in which the genes for growth hormone (GH) or GH receptor (GHR(-/-) ) are disrupted from conception are dwarfs, possess low levels of IGF-1 and insulin, have low rates of cancer and diabetes, and are extremely long-lived. Median longevity is also increased in mice with deletion of hypothalamic GH-releasing hormone (GHRH), which leads to isolated GH deficiency. The remarkable extension of longevity in hypopituitary Ames dwarf mice can be reversed by a 6-week course of GH injections started at the age of 2 weeks. Here, we demonstrate that mutations that interfere with GH production or response, in the Snell dwarf, Ames dwarf, or GHR(-/-) mice lead to reduced formation of both orexigenic agouti-related peptide (AgRP) and anorexigenic proopiomelanocortin (POMC) projections to the main hypothalamic projection areas: the arcuate nucleus (ARH), paraventricular nucleus (PVH), and dorsomedial nucleus (DMH). These mutations also reduce hypothalamic inflammation in 18-month-old mice. GH injections, between 2 and 8 weeks of age, reversed both effects in Ames dwarf mice. Disruption of GHR specifically in liver (LiGHRKO), a mutation that reduces circulating IGF-1 but does not lead to lifespan extension, had no effect on hypothalamic projections or inflammation, suggesting an effect of GH, rather than peripheral IGF-1, on hypothalamic development. Hypothalamic leptin signaling, as monitored by induction of pStat3, is not impaired by GHR deficiency. Together, these results suggest that early-life disruption of GH signaling produces long-term hypothalamic changes that may contribute to the longevity of GH-deficient and GH-resistant mice.
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Proteínas Portadoras/genética , Enanismo Hipofisario/genética , Hormona del Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Longevidad/genética , Proteína Relacionada con Agouti/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Enanismo Hipofisario/metabolismo , Hormona del Crecimiento/genética , Inflamación/patología , Leptina/metabolismo , Hígado/metabolismo , Masculino , Núcleo Talámico Mediodorsal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Núcleo Hipotalámico Paraventricular/metabolismo , Proopiomelanocortina/metabolismo , Factor de Transcripción STAT3/biosíntesisRESUMEN
Transient nutrient restriction in the 3 weeks between birth and weaning (producing "crowded litter" or CL mice) leads to a significant increase in lifespan and is associated with permanent changes in energy homeostasis, leptin, and insulin sensitivity. Here, we show this brief period of early food restriction leads to permanent modulation of the arcuate nucleus of the hypothalamus (ARH), markedly increasing formation of both orexigenic agouti-related peptide (AgRP) and anorexigenic proopiomelanocortin (POMC) projections to the paraventricular nucleus of the hypothalamus (PVH). An additional 4 weeks of caloric restriction, after weaning, does not further intensify the formation of AgRP and POMC projections. Acute leptin stimulation of 12-month-old mice leads to a stronger increase in the levels of hypothalamic pStat3 and cFos activity in CL mice than in controls, suggesting that preweaning food restriction leads to long-lasting enhancement of leptin signaling. In contrast, FoxO1 nuclear exclusion in response to insulin is equivalent in young adult CL and control mice, suggesting that hypothalamic insulin signaling is not modulated by the crowded litter intervention. Markers of hypothalamic reactive gliosis associated with aging, such as Iba1-positive microglia and GFAP-positive astrocytes, are significantly reduced in CL mice as compared to controls at 12 and 22 months of age. Lastly, age-associated overproduction of TNF-α in microglial cells is reduced in CL mice than in age-matched controls. Together, these results suggest that transient early life nutrient deprivation leads to long-term hypothalamic changes which may contribute to the longevity of CL mice.
RESUMEN
The action of nutrients on early postnatal growth can influence mammalian aging and longevity. Recent work has demonstrated that limiting nutrient availability in the first 3 wk of life [by increasing the number of pups in the crowded-litter (CL) model] leads to extension of mean and maximal lifespan in genetically normal mice. In this study, we aimed to characterize the impact of early-life nutrient intervention on glucose metabolism and energy homeostasis in CL mice. In our study, we used mice from litters supplemented to 12 or 15 pups and compared those to control litters limited to eight pups. At weaning and then throughout adult life, CL mice are significantly leaner and consume more oxygen relative to control mice. At 6 mo of age, CL mice had low fasting leptin concentrations, and low-dose leptin injections reduced body weight and food intake more in CL female mice than in controls. At 22 mo, CL female mice also have smaller adipocytes compared with controls. Glucose and insulin tolerance tests show an increase in insulin sensitivity in 6 mo old CL male mice, and females become more insulin sensitive later in life. Furthermore, ß-cell mass was significantly reduced in the CL male mice and was associated with reduction in ß-cell proliferation rate in these mice. Together, these data show that early-life nutrient intervention has a significant lifelong effect on metabolic characteristics that may contribute to the increased lifespan of CL mice.
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Aglomeración/psicología , Metabolismo Energético/fisiología , Homeostasis/fisiología , Resistencia a la Insulina/fisiología , Tejido Adiposo Blanco/anatomía & histología , Tejido Adiposo Blanco/metabolismo , Envejecimiento/fisiología , Animales , Proliferación Celular , Femenino , Prueba de Tolerancia a la Glucosa , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/anatomía & histología , Islotes Pancreáticos/fisiología , Leptina/fisiología , Masculino , Ratones , Estado Nutricional , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Estrés Psicológico/metabolismo , DesteteRESUMEN
Insufficient cell proliferation has been suggested as a potential cause of age-related tissue dysgenesis in mammals. However, genetic manipulation of cell cycle regulators in the germ lines of mice results in changes in animal size but not progeroid phenotypes. Here we increase levels of the cyclin-dependent kinase inhibitor Cdkn1b (p27kip1) in adult mice through doxycycline-inducible expression and show this results in reduced cell proliferation in multiple tissues. The mice undergo changes resembling ageing even in the absence of an elevated DNA damage response or evidence of senescent cells, suggesting an altered balance between genetic and tissue ageing. In contrast, suppressing cell proliferation by doxycycline treatment of neonates retards growth, but the onset of degenerative changes is delayed during the period of reduced body mass. These results support the hypothesis that many of the most recognizable features of mammalian ageing can result from an imbalance between cell production and the mass of tissue that must be maintained.