RESUMO
Blood-brain barrier dysfunction (BBBD) and accumulation of senescent astrocytes occur during brain aging and contribute to neuroinflammation and disease. Here, we explored the relationship between these two age-related events, hypothesizing that chronic hippocampal exposure to the blood-borne protein serum albumin could induce stress-induced premature senescence (SIPS) in astrocytes via transforming growth factor beta 1 (TGFß) signaling. We found that 1 week of albumin exposure significantly increased TGFß signaling and senescence marker expression in cultured rat hippocampal astrocytes. These changes were preventable by pharmacological inhibition of the type I TGFß receptor (TGFßR) ALK5. To study these effects in vivo, we utilized an animal model of BBBD in which albumin was continuously infused into the lateral ventricles of adult mice. Consistent with our in vitro results, 1 week of albumin infusion significantly increased TGFß signaling activation and the burden of senescent astrocytes in hippocampal tissue. Pharmacological inhibition of ALK5 TGFßR or conditional genetic knockdown of astrocytic TGFßR prior to albumin infusion was sufficient to prevent albumin-induced astrocyte senescence. Together, these results establish a link between TGFß signaling activation and astrocyte senescence and suggest that prolonged exposure to serum albumin due to BBBD can trigger these phenotypic changes.
Assuntos
Astrócitos , Barreira Hematoencefálica , Ratos , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Albumina Sérica/metabolismo , Albumina Sérica/farmacologia , Senescência CelularRESUMO
As the most abundant cell types in the brain, astrocytes form a tissue-wide signaling network that is responsible for maintaining brain homeostasis and regulating various brain activities. Here, we review some of the essential functions that astrocytes perform in supporting neurons, modulating the immune response, and regulating and maintaining the blood-brain barrier (BBB). Given their importance in brain health, it follows that astrocyte dysfunction has detrimental effects. Indeed, dysfunctional astrocytes are implicated in age-related neuropathology and participate in the onset and progression of neurodegenerative diseases. Here, we review two mechanisms by which astrocytes mediate neuropathology in the aging brain. First, age-associated blood-brain barrier dysfunction (BBBD) causes the hyperactivation of TGFß signaling in astrocytes, which elicits a pro-inflammatory and epileptogenic phenotype. Over time, BBBD-associated astrocyte dysfunction results in hippocampal and cortical neural hyperexcitability and cognitive deficits. Second, senescent astrocytes accumulate in the brain with age and exhibit a decreased functional capacity and the secretion of senescent-associated secretory phenotype (SASP) factors, which contribute to neuroinflammation and neurotoxicity. Both BBBD and senescence progressively increase during aging and are associated with increased risk of neurodegenerative disease, but the relationship between the two has not yet been established. Thus, we discuss the potential relationship between BBBD, TGFß hyperactivation, and senescence with respect to astrocytes in the context of aging and disease and identify future areas of investigation in the field.
Assuntos
Astrócitos , Doenças Neurodegenerativas , Envelhecimento/patologia , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Senescência Celular/fisiologia , Humanos , Doenças Neurodegenerativas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Aging involves a decline in neural function that contributes to cognitive impairment and disease. However, the mechanisms underlying the transition from a young-and-healthy to aged-and-dysfunctional brain are not well understood. Here, we report breakdown of the vascular blood-brain barrier (BBB) in aging humans and rodents, which begins as early as middle age and progresses to the end of the life span. Gain-of-function and loss-of-function manipulations show that this BBB dysfunction triggers hyperactivation of transforming growth factor-ß (TGFß) signaling in astrocytes, which is necessary and sufficient to cause neural dysfunction and age-related pathology in rodents. Specifically, infusion of the serum protein albumin into the young rodent brain (mimicking BBB leakiness) induced astrocytic TGFß signaling and an aged brain phenotype including aberrant electrocorticographic activity, vulnerability to seizures, and cognitive impairment. Furthermore, conditional genetic knockdown of astrocytic TGFß receptors or pharmacological inhibition of TGFß signaling reversed these symptomatic outcomes in aged mice. Last, we found that this same signaling pathway is activated in aging human subjects with BBB dysfunction. Our study identifies dysfunction in the neurovascular unit as one of the earliest triggers of neurological aging and demonstrates that the aging brain may retain considerable latent capacity, which can be revitalized by therapeutic inhibition of TGFß signaling.
Assuntos
Envelhecimento/patologia , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/fisiopatologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminas/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Doença Crônica , Disfunção Cognitiva/patologia , Disfunção Cognitiva/fisiopatologia , Técnicas de Silenciamento de Genes , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Camundongos Transgênicos , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Adulto JovemRESUMO
Immature phenotypes of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) limit the utility of these cells in clinical application and basic research. During cardiac development, postnatal cardiomyocytes experience high oxygen tension along with a concomitant downregulation of hypoxia-inducible factor 1α (HIF-1α), leading to increased fatty acid oxidation (FAO). We hypothesized that targeting HIF-1α alone or in combination with other metabolic regulators could promote the metabolic maturation of hiPSC-CMs. We examined the effect of HIF-1α inhibition on the maturation of hiPSC-CMs and investigated a multipronged approach to promote hiPSC-CM maturation by combining HIF-1α inhibition with molecules that target key pathways involved in the energy metabolism. Cardiac spheres of highly-enriched hiPSC-CMs were treated with a HIF-1α inhibitor alone or in combination with an agonist of peroxisome proliferator activated receptor α (PPARα) and three postnatal factors (triiodothyronine hormone T3, insulin-like growth factor-1 and dexamethasone). HIF-1α inhibition significantly increased FAO and basal and maximal respiration of hiPSC-CMs. Combining HIF-1α inhibition with PPARα activation and the postnatal factors further increased FAO and improved mitochondrial maturation in hiPSC-CMs. Compared with mock-treated cultures, the cultures treated with the five factors had increased mitochondrial content and contained more cells with mitochondrial distribution throughout the cells, which are features of more mature cardiomyocytes. Consistent with these observations, a number of transcriptional regulators of mitochondrial metabolic processes were upregulated in hiPSC-CMs treated with the five factors. Furthermore, these cells had significantly increased Ca2+ transient kinetics and contraction and relaxation velocities, which are functional features for more mature cardiomyocytes. Therefore, targeting HIF-1α in combination with other metabolic regulators significantly improves the metabolic maturation of hiPSC-CMs.
Assuntos
Benzamidas/farmacologia , Sinergismo Farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/fisiologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/fisiologia , PPAR alfa/agonistas , Anti-Inflamatórios/farmacologia , Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Dexametasona/farmacologia , Metabolismo Energético , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Metabolismo dos Lipídeos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Oxirredução , Transcriptoma , Tri-Iodotironina/farmacologiaRESUMO
Sensitization to prodrugs via transgenic expression of suicide genes is a leading strategy for the selective elimination of potentially tumorigenic human pluripotent stem cells (hPSCs) in regenerative medicine, but transgenic modification poses safety risks such as deleterious mutagenesis. We describe here an alternative method of delivering suicide-inducing molecules explicitly to hPSCs using virus-like particles (VLPs) and demonstrate its use in eliminating undifferentiated hPSCs in vitro. VLPs were engineered from Qß bacteriophage capsids to contain enhanced green fluorescent protein (EGFP) or cytosine deaminase (CD) and to simultaneously display multiple IgG-binding ZZ domains. After labeling with antibodies against the hPSC-specific surface glycan SSEA-5, EGFP-containing particles were shown to specifically bind undifferentiated cells in culture, and CD-containing particles were able to eliminate undifferentiated hPSCs with virtually no cytotoxicity to differentiated cells upon treatment with the prodrug 5-fluorocytosine.
Assuntos
Antimetabólitos/administração & dosagem , Proteínas do Capsídeo/química , Diferenciação Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Flucitosina/administração & dosagem , Pró-Fármacos/administração & dosagem , Vírion/química , Antimetabólitos/farmacologia , Carcinogênese/efeitos dos fármacos , Linhagem Celular , Colífagos/química , Portadores de Fármacos/química , Flucitosina/farmacologia , Proteínas de Fluorescência Verde/administração & dosagem , Humanos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Pró-Fármacos/farmacologiaRESUMO
Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.
Assuntos
Fibrose Cística/imunologia , Neutrófilos/imunologia , Animais , Compostos Azabicíclicos/farmacologia , Benzoatos/farmacologia , Células Sanguíneas , Células da Medula Óssea , Células Cultivadas , Quimiotaxia de Leucócito , Meios de Cultivo Condicionados/farmacologia , Fibrose Cística/patologia , Exocitose/efeitos dos fármacos , Citometria de Fluxo , Glicólise , Humanos , Elastase de Leucócito/metabolismo , Leucotrieno B4/farmacologia , Lipopolissacarídeos/farmacologia , Metformina/farmacologia , Camundongos , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Consumo de Oxigênio , Pinocitose , Pseudomonas aeruginosa , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Escarro/imunologia , Migração Transendotelial e Transepitelial/efeitos dos fármacosRESUMO
Understanding molecules involved in differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes and endothelial cells is important in advancing hPSCs for cell therapy and drug testing. Here, we report that LGR5, a leucine-rich repeat-containing G-protein-coupled receptor, plays a critical role in hPSC differentiation into cardiomyocytes and endothelial cells. LGR5 expression was transiently upregulated during the early stage of cardiomyocyte differentiation, and knockdown of LGR5 resulted in reduced expression of cardiomyocyte-associated markers and poor cardiac differentiation. In contrast, knockdown of LGR5 promoted differentiation of endothelial-like cells with increased expression of endothelial cell markers and appropriate functional characteristics, including the ability to form tube-like structures and to take up acetylated low-density lipoproteins. Furthermore, knockdown of LGR5 significantly reduced the proliferation of differentiated cells and increased the nuclear translocation of ß-catenin and expression of Wnt signaling-related genes. Therefore, regulation of LGR5 may facilitate efficient generation of cardiomyocytes or endothelial cells from hPSCs.
Assuntos
Diferenciação Celular/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores Acoplados a Proteínas G/genética , Padronização Corporal/genética , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Mesoderma/citologia , Mesoderma/embriologia , Via de Sinalização WntRESUMO
In recent years, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a vital cell source for in vitro modeling of genetic cardiovascular disorders, drug screening, and in vivo cardiac regeneration research. Looking forward, the ability to efficiently cryopreserve hPSC-CMs without compromising their normal biochemical and physiologic functions will dramatically facilitate their various biomedical applications. Although working protocols for freezing, storing, and thawing hPSC-CMs have been established, the question remains as to whether they are optimal. In this chapter, we discuss our current understanding of cryopreservation appertaining to hPSC-CMs, and proffer key questions regarding the mechanical, contractile, and regenerative properties of cryopreserved hPSC-CMs.
Assuntos
Criopreservação/métodos , Angina Microvascular/terapia , Isquemia Miocárdica/terapia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Criopreservação/instrumentação , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Modelos Animais de Doenças , Descoberta de Drogas , Drogas em Investigação/farmacologia , Humanos , Camundongos , Angina Microvascular/patologia , Isquemia Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/transplante , Células-Tronco Pluripotentes/fisiologia , Regeneração/fisiologiaRESUMO
Although ß-blockers can be used to eliminate stress-induced ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), this treatment is unsuccessful in â¼25% of cases. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from these patients have potential for use in investigating the phenomenon, but it remains unknown whether they can recapitulate patient-specific drug responses to ß-blockers. This study assessed whether the inadequacy of ß-blocker therapy in an individual can be observed in vitro using patient-derived CPVT iPSC-CMs. An individual with CPVT harboring a novel mutation in the type 2 cardiac ryanodine receptor (RyR2) was identified whose persistent ventricular arrhythmias during ß-blockade with nadolol were abolished during flecainide treatment. iPSC-CMs generated from this patient and two control individuals expressed comparable levels of excitation-contraction genes, but assessment of the sarcoplasmic reticulum Ca(2+) leak and load relationship revealed intracellular Ca(2+) homeostasis was altered in the CPVT iPSC-CMs. ß-adrenergic stimulation potentiated spontaneous Ca(2+) waves and unduly frequent, large and prolonged Ca(2+) sparks in CPVT compared with control iPSC-CMs, validating the disease phenotype. Pursuant to the patient's in vivo responses, nadolol treatment during ß-adrenergic stimulation achieved negligible reduction of Ca(2+) wave frequency and failed to rescue Ca(2+) spark defects in CPVT iPSC-CMs. In contrast, flecainide reduced both frequency and amplitude of Ca(2+) waves and restored the frequency, width and duration of Ca(2+) sparks to baseline levels. By recapitulating the improved response of an individual with CPVT to flecainide compared with ß-blocker therapy in vitro, these data provide new evidence that iPSC-CMs can capture basic components of patient-specific drug responses.
Assuntos
Catecolaminas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Taquicardia Ventricular/tratamento farmacológico , Taquicardia Ventricular/patologia , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/fisiopatologia , Biomarcadores/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Flecainida/farmacologia , Flecainida/uso terapêutico , Homeostase/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Linhagem , Receptores Adrenérgicos beta/metabolismo , Taquicardia Ventricular/fisiopatologiaRESUMO
Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes.
Assuntos
Simulação por Computador , Mioblastos Cardíacos/fisiologia , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Técnicas de Cultura de Órgãos , Engenharia Tecidual , Simulação de Ausência de PesoRESUMO
Cardiomyocytes derived from human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, disease modeling, and drug discovery, all of which require enriched cardiomyocytes, ideally ones with mature phenotypes. However, current methods are typically performed in 2D environments that produce immature cardiomyocytes within heterogeneous populations. Here, we generated 3D aggregates of cardiomyocytes (cardiospheres) from 2D differentiation cultures of hPSCs using microscale technology and rotary orbital suspension culture. Nearly 100% of the cardiospheres showed spontaneous contractility and synchronous intracellular calcium transients. Strikingly, from starting heterogeneous populations containing â¼10%-40% cardiomyocytes, the cell population within the generated cardiospheres featured â¼80%-100% cardiomyocytes, corresponding to an enrichment factor of up to 7-fold. Furthermore, cardiomyocytes from cardiospheres exhibited enhanced structural maturation in comparison with those from a parallel 2D culture. Thus, generation of cardiospheres represents a simple and robust method for enrichment of cardiomyocytes in microtissues that have the potential use in regenerative medicine as well as other applications.