RESUMO
BACKGROUND: Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points. METHODS: In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes from FUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings. FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems. RESULTS: We observed a dysregulation of astrocyte homeostasis, which resulted in a FUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/ß-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs. CONCLUSIONS: Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular , Proteína FUS de Ligação a RNA/fisiologiaRESUMO
During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKAiEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.
Assuntos
Vasos Sanguíneos , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico , Células Endoteliais , Fígado , Albuminas , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiologia , AMP Cíclico , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Homeostase , Hipoalbuminemia , Hipoglicemia , Fígado/metabolismo , Fígado/fisiologia , Camundongos , RecombinasesRESUMO
In the present work, an insoluble polymer, i.e., ethyl cellulose (EC), was combined with the water-soluble polyvinylpyrrolidone (PVP) as a carrier system for the formulation of amorphous solid dispersions. The rationale was that by conjoining these two different types of carriers a more gradual drug release could be created with less risk for precipitation. Our initial hypothesis was that upon contact with the dissolution medium, PVP would be released, creating a porous EC matrix through which the model drug indomethacin could diffuse. On the basis of observations of EC as a coating material, the effect of the molecular weight of PVP, and the ratio of EC/PVP on the miscibility of the polymer blend, the solid state of the solid dispersion and the drug release from these solid dispersions were investigated. X-ray powder diffraction, modulated differential scanning calorimetry, and solid-state nuclear magnetic resonance were used to unravel the miscibility and solid-state properties of these blends and solid dispersions. Solid-state nuclear magnetic resonance appeared to be a crucial technique for this aspect as modulated differential scanning calorimetry was not sufficient to grasp the complex phase behavior of these systems. Both EC/PVP K12 and EC/PVP K25 blends were miscible over the entire composition range, and addition of indomethacin did not alter this. Concerning the drug release, it was initially thought that more PVP would lead to faster drug release with a higher probability that all of the drug molecules would be able to diffuse out of the EC network as more pores would be created. However, this view on the release mechanism appeared to be too simplistic as an optimum was observed for both blends. On the basis of this work, it could be concluded that drug release from this complex ternary system was affected not only by the ratio of EC/PVP and the molecular weight of PVP but also by interactions between the three components, the wettability of the formulations, and the viscosity layer that was created around the particles.
Assuntos
Excipientes , Povidona , Varredura Diferencial de Calorimetria , Celulose/análogos & derivados , Indometacina/química , Polímeros/química , Porosidade , Povidona/química , Solubilidade , Difração de Raios XRESUMO
Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cistina/metabolismo , Cistinose/patologia , Modelos Animais de Doenças , Rim/patologia , Mutação , Proteínas de Peixe-Zebra/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Cistinose/etiologia , Humanos , Rim/metabolismo , Fenótipo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies.
Assuntos
Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células da Medula Óssea/citologia , Movimento Celular/fisiologia , Células Cultivadas , Camundongos , Nicho de Células-Tronco/fisiologia , Transplante de Células-Tronco/métodosRESUMO
Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.
Assuntos
Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Dispositivos Lab-On-A-Chip , Mutação , Junção Neuromuscular/genética , Junção Neuromuscular/fisiopatologia , Proteína FUS de Ligação a RNA/genética , Agrina/metabolismo , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Imunofluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/metabolismo , Técnicas Analíticas Microfluídicas , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacosRESUMO
To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.
Assuntos
Células Endoteliais/metabolismo , Fígado/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Animais , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/citologia , Humanos , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Pluripotentes/citologiaRESUMO
BACKGROUND: Chronic pressure overload predisposes to heart failure, but the pathogenic role of microvascular endothelial cells (MiVEC) remains unknown. We characterized transcriptional, metabolic, and functional adaptation of cardiac MiVEC to pressure overload in mice and patients with aortic stenosis (AS). METHODS: In Tie2-Gfp mice subjected to transverse aortic constriction or sham surgery, we performed RNA sequencing of isolated cardiac Gfp+-MiVEC and validated the signature in freshly isolated MiVEC from left ventricle outflow tract and right atrium of patients with AS. We next compared their angiogenic and metabolic profiles and finally correlated molecular and pathological signatures with clinical phenotypes of 42 patients with AS (50% women). RESULTS: In mice, transverse aortic constriction induced progressive systolic dysfunction, fibrosis, and reduced microvascular density. After 10 weeks, 25 genes predominantly involved in matrix-regulation were >2-fold upregulated in isolated MiVEC. Increased transcript levels of Cartilage Intermediate Layer Protein (Cilp), Thrombospondin-4, Adamtsl-2, and Collagen1a1 were confirmed by quantitative reverse transcription polymerase chain reaction and recapitulated in left ventricle outflow tract-derived MiVEC of AS (P<0.05 versus right atrium-MiVEC). Fatty acid oxidation increased >2-fold in left ventricle outflow tract-MiVEC, proline content by 130% (median, IQR, 58%-474%; P=0.008) and procollagen secretion by 85% (mean [95% CI, 16%-154%]; P<0.05 versus right atrium-MiVEC for all). The altered transcriptome in left ventricle outflow tract-MiVEC was associated with impaired 2-dimensional-vascular network formation and 3-dimensional-spheroid sprouting (P<0.05 versus right atrium-MiVEC), profibrotic ultrastructural changes, and impaired diastolic left ventricle function, capillary density and functional status, especially in female AS. CONCLUSIONS: Pressure overload induces major transcriptional and metabolic adaptations in cardiac MiVEC resulting in excess interstitial fibrosis and impaired angiogenesis. Molecular rewiring of MiVEC is worse in women, compromises functional status, and identifies novel targets for intervention.
Assuntos
Estenose da Valva Aórtica/genética , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Microvasos/metabolismo , Proteínas ADAMTS/genética , Idoso , Animais , Aorta , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/cirurgia , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Constrição Patológica , Vasos Coronários/patologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Proteínas da Matriz Extracelular/genética , Ácidos Graxos/metabolismo , Feminino , Perfilação da Expressão Gênica , Átrios do Coração/patologia , Implante de Prótese de Valva Cardíaca , Ventrículos do Coração/patologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Densidade Microvascular , Microvasos/patologia , Pró-Colágeno/metabolismo , Prolina/metabolismo , Pirofosfatases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Trombospondinas/genéticaRESUMO
OBJECTIVE: Although recent advances in pulmonary valve replacement have enabled excellent hemodynamics, infective endocarditis remains a serious complication, particularly for implanted bovine jugular vein (BJV) conduits. METHODS: We investigated contributions by platelets and plasma fibrinogen to endocarditis initiation on various grafts used for valve replacement. Thus, adherence of Staphylococcus aureus and platelets to 5 graft tissues was studied quantitatively in perfusion chambers, assisted by microscopic analysis. We also evaluated standard antiplatelet therapy to prevent onset of S aureus endocarditis. RESULTS: Of all tissues, bovine pericardium (BP) showed the greatest fibrinogen binding. Perfusion of all plasma-precoated tissues identified BP and BJVwall with the greatest affinity for S aureus. Perfusions of anticoagulated human blood over all tissues also triggered more platelet adhesion to BP and BJVwall as single platelets. Several controls confirmed that both S aureus and platelets were recruited on immobilized fibrinogen. In addition, perfusions (and controls) over plasma-coated tissues with whole blood, spiked with S aureus, revealed that bacteria exclusively bound to adhered platelets. Both the platelet adhesion and platelet-mediated S aureus recruitment required platelet αIIbß3 and coated or soluble fibrinogen, respectively, interactions abrogated by the αIIbß3-antagonist eptifibatide. Also, standard antiplatelet therapy (aspirin/ticagrelor) reduced the adherence of S aureus in blood to BJV 3-fold. CONCLUSIONS: Binding of plasma fibrinogen to especially BJV grafts enables adhesion of single platelets via αIIbß3. S aureus then attaches from blood to (activated) bound platelet αIIbß3 via plasma fibrinogen. Dual antiplatelet therapy appears a realistic approach to prevent endocarditis and its associated mortality.
Assuntos
Bioprótese , Endocardite Bacteriana , Valvas Cardíacas , Inibidores da Agregação Plaquetária , Infecções Estafilocócicas , Animais , Aderência Bacteriana , Plaquetas/fisiologia , Bovinos , Fibrinogênio , Valvas Cardíacas/microbiologia , Valvas Cardíacas/fisiopatologia , Ligação Proteica , Staphylococcus aureusRESUMO
The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.
Assuntos
Mecanotransdução Celular/fisiologia , Análise de Célula Única , Pele/citologia , Pele/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Hidrogéis/administração & dosagem , Hidrogéis/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , RNA Mensageiro/genética , RNA-Seq , Pele/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum chaperone, with a central role in maintaining protein homeostasis. Recently, an alternative role for GRP78 under stress conditions has been proposed, with stress-induced extracellular secretion and translocation of GRP78 to the cell surface where it acts as a multifunctional signaling receptor. Here we demonstrate translocation of GRP78 to the surface of human EndoC-ßH1 cells and primary human islets upon cytokine exposure, in analogy to observations in rodent INS-1E and MIN6 beta cell lines. We show that GRP78 is shuttled via the anterograde secretory pathway, through the Golgi complex and secretory granules, and identify the DNAJ homolog subfamily C member 3 (DNAJC3) as a GRP78-interacting protein that facilitates its membrane translocation. Evaluation of downstream signaling pathways, using N- and C-terminal anti-GRP78 blocking antibodies, demonstrates that both GRP78 signaling domains initiate pro-apoptotic signaling cascades in beta cells. Extracellular GRP78 itself is identified as a ligand for cell surface GRP78 (sGRP78), increasing caspase 3/7 activity and cell death upon binding, which is accompanied by enhanced Chop and Bax mRNA expression. These results suggest that inflammatory cytokines induce a self-destructive pro-apoptotic feedback loop through the secretion and membrane translocation of GRP78. This proapoptotic function distinguishes the role of sGRP78 in beta cells from its reported anti-apoptotic and proliferative role in cancer cells, opening the road for the use of compounds that block sGRP78 as potential beta cell-preserving therapies in type 1 diabetes.
Assuntos
Apoptose/efeitos dos fármacos , Membrana Celular/metabolismo , Citocinas/farmacologia , Proteínas de Choque Térmico/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Citocinas/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Retroalimentação Fisiológica/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/imunologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Chaperonas Moleculares/metabolismo , RatosRESUMO
Despite the many advantages of small interfering RNA (siRNA) inhalation therapy and a growing prevalence of respiratory pathologies, its clinical translation is severely hampered by inefficient intracellular delivery. To this end, we previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). Interestingly, the PS shell was shown to markedly improve particle stability as well as intracellular siRNA delivery in vitro and in vivo. The major aim of this work was to identify the key molecular components of PS responsible for the enhanced siRNA delivery and evaluate how the complexity of the PS coat could be reduced. We identified surfactant protein B (SP-B) as a potent siRNA delivery enhancer when reconstituted in proteolipid coated hydrogel nanocomposites. Improved cytosolic siRNA delivery was achieved by inserting SP-B into a simplified phospholipid mixture prior to nanogel coating. This effect was observed both in vitro (lung epithelial cell line) and in vivo (murine acute lung injury model), albeit that distinct phospholipids were required to achieve these results. Importantly, the developed nanocomposites have a low in vivo toxicity and are efficiently taken up by resident alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Our results demonstrate the potential of the endogenous protein SP-B as an intracellular siRNA delivery enhancer, paving the way for future design of nanoformulations for siRNA inhalation therapy. STATEMENT OF SIGNIFICANCE: Despite the therapeutic potential of small interfering RNA (siRNA) and a growing prevalence of lung diseases for which innovative therapies are needed, a safe and effective siRNA inhalation therapy remains non-existing due to a lack of suitable formulations. We identified surfactant protein B (SP-B) as a potent enhancer of siRNA delivery by proteolipid coated nanogel formulations in vitro in a lung epithelial cell line. The developed nanocomposites have a low in vivo toxicity and show a high uptake by alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Importantly, in vivo SP-B is also critical for the developed formulation to obtain a significant silencing of TNFα in a murine LPS-induced acute lung injury model.
Assuntos
Técnicas de Transferência de Genes , Polietilenoglicóis/química , Polietilenoimina/química , Proteolipídeos/química , Proteína B Associada a Surfactante Pulmonar/química , RNA Interferente Pequeno/administração & dosagem , Terapia Respiratória , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/terapia , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Inativação Gênica , Humanos , Lipopolissacarídeos , Camundongos Endogâmicos BALB C , Nanogéis , Fosfolipídeos/química , Polietilenoglicóis/toxicidade , Polietilenoimina/toxicidade , Proteolipídeos/toxicidade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.
Assuntos
Esclerose Lateral Amiotrófica/genética , Transporte Axonal , Desacetilase 6 de Histona/metabolismo , Neurônios Motores/metabolismo , Proteína FUS de Ligação a RNA/genética , Adolescente , Adulto , Idoso , Sistemas CRISPR-Cas , Feminino , Desacetilase 6 de Histona/antagonistas & inibidores , Humanos , Ácidos Hidroxâmicos , Indóis , Células-Tronco Pluripotentes Induzidas , Masculino , Mutação Puntual , Cultura Primária de Células , PirimidinasRESUMO
BACKGROUND: Cytochrome oxidase (COX) IV complex regulates energy production in mitochondria. Impaired COX gene expression is related to obesity and type 2 diabetes mellitus, but whether it is directly related to the incidence of cardiovascular events is unknown. We investigated whether COX gene expression in monocytes is predictive for cardiovascular events in coronary artery disease patients. To avoid monocyte isolation from fresh blood, we then aimed to validate our findings in monocyte-derived microvesicles isolated from plasma. METHODS AND RESULTS: We enrolled 142 consecutive patients undergoing diagnostic coronary angiography between June 2010 and January 2011 and followed 67 patients with stable coronary artery disease prospectively for at least 3 years. Twenty-two patients experienced a new cardiovascular event (32.8%). Circulating CD14+ monocytes and microvesicles were isolated with magnetic beads, and COX mRNA levels were measured with quantitative polymerase chain reaction, after normalization with 5 validated house-keeping genes. Patients in the lowest tertile of mitochondrial cytochrome oxidase, subunit I (MT-COI) in monocytes at baseline had a higher risk for developing a new event after adjusting for age, sex, (ex)smoking, body mass index, blood pressure, diabetes mellitus, low-density lipoprotein- and high-density lipoprotein-cholesterol, triglycerides, high-sensitivity C-reactive protein, interleukin-6, and number of diseased vessels (harzard ratio [HR], 3.95; 95% CI, 1.63-9.57). Patients in the lowest tertile of MT-COI in monocyte-specific microvesicles had also a higher risk of developing a new event (adjusted HR, 5.00; 95% CI, 1.77-14). CONCLUSIONS: In the current blinded study, low MT-COI in monocytes of coronary artery disease patients identifies a population at risk for new cardiovascular events. For the first time, we show that signatures in monocyte-specific microvesicles in plasma have similar predictive properties.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Doença da Artéria Coronariana/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Monócitos/metabolismo , Doenças Cardiovasculares/mortalidade , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Feminino , Fator C1 de Célula Hospedeira , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Isquemia Miocárdica/epidemiologia , Isquemia Miocárdica/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/metabolismoRESUMO
γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-2/análise , Complexo 1 de Proteínas Adaptadoras/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Endossomos/química , Humanos , Lisossomos/química , Camundongos , Presenilina-1/análise , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratos , Especificidade por SubstratoRESUMO
Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis.
Assuntos
Exossomos/química , Análise Espectral Raman/métodos , Algoritmos , Ouro/química , Nanopartículas Metálicas/químicaRESUMO
Focal amplifications of chromosome 3p13-3p14 occur in about 10% of melanomas and are associated with a poor prognosis. The melanoma-specific oncogene MITF resides at the epicentre of this amplicon. However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA (lncRNA) gene SAMMSON is consistently co-gained with MITF. In addition, SAMMSON is a target of the lineage-specific transcription factor SOX10 and its expression is detectable in more than 90% of human melanomas. Whereas exogenous SAMMSON increases the clonogenic potential in trans, SAMMSON knockdown drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state and BRAF, NRAS or TP53 mutational status. Moreover, SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics both in vitro and in patient-derived xenograft models. Mechanistically, SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that silencing of the lineage addiction oncogene SAMMSON disrupts vital mitochondrial functions in a cancer-cell-specific manner; this silencing is therefore expected to deliver highly effective and tissue-restricted anti-melanoma therapeutic responses.
Assuntos
Melanoma/genética , Melanoma/patologia , Oncogenes/genética , RNA Longo não Codificante/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Proteínas de Transporte , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Cromossomos Humanos Par 3/genética , Células Clonais/metabolismo , Células Clonais/patologia , Feminino , Amplificação de Genes/genética , Técnicas de Silenciamento de Genes , Humanos , Melanoma/terapia , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Terapia de Alvo Molecular , RNA Longo não Codificante/uso terapêutico , Fatores de Transcrição SOXE/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacteriemia/tratamento farmacológico , Proteínas de Bactérias/metabolismo , Agregados Proteicos/efeitos dos fármacos , Animais , Antibacterianos/biossíntese , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Desenho de Fármacos , Feminino , Células HCT116 , Células HEK293 , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Sepse/terapia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
Sox9 is a transcription factor expressed in most solid tumors. However, the molecular mechanisms underlying Sox9 function during tumorigenesis remain unclear. Here, using a genetic mouse model of basal cell carcinoma (BCC), the most frequent cancer in humans, we show that Sox9 is expressed from the earliest step of tumor formation in a Wnt/ß-catenin-dependent manner. Deletion of Sox9 together with the constitutive activation of Hedgehog signaling completely prevents BCC formation and leads to a progressive loss of oncogene-expressing cells. Transcriptional profiling of oncogene-expressing cells with Sox9 deletion, combined with in vivo ChIP sequencing, uncovers a cancer-specific gene network regulated by Sox9 that promotes stemness, extracellular matrix deposition, and cytoskeleton remodeling while repressing epidermal differentiation. Our study identifies the molecular mechanisms regulated by Sox9 that link tumor initiation and invasion.
Assuntos
Autorrenovação Celular/fisiologia , Transformação Celular Neoplásica/genética , Células-Tronco Neoplásicas/fisiologia , Oncogenes , Fatores de Transcrição SOX9/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Carcinogênese , Carcinoma Basocelular/etiologia , Carcinoma Basocelular/genética , Carcinoma Basocelular/fisiopatologia , Adesão Celular , Autorrenovação Celular/genética , Matriz Extracelular/fisiologia , Feminino , Deleção de Genes , Proteínas Hedgehog/fisiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Mutação , Invasividade Neoplásica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/fisiopatologia , Receptor SmoothenedRESUMO
Recently, a number of aggregation disease polypeptides have been shown to spread from cell to cell, thereby displaying prionoid behavior. Studying aggregate internalization, however, is often hampered by the complex kinetics of the aggregation process, resulting in the concomitant uptake of aggregates of different sizes by competing mechanisms, which makes it difficult to isolate pathway-specific responses to aggregates. We designed synthetic aggregating peptides bearing different aggregation propensities with the aim of producing modes of uptake that are sufficiently distinct to differentially analyze the cellular response to internalization. We found that small acidic aggregates (≤500 nm in diameter) were taken up by nonspecific endocytosis as part of the fluid phase and traveled through the endosomal compartment to lysosomes. By contrast, bigger basic aggregates (>1 µm) were taken up through a mechanism dependent on cytoskeletal reorganization and membrane remodeling with the morphological hallmarks of phagocytosis. Importantly, the properties of these aggregates determined not only the mechanism of internalization but also the involvement of the proteostatic machinery (the assembly of interconnected networks that control the biogenesis, folding, trafficking, and degradation of proteins) in the process; whereas the internalization of small acidic aggregates is HSF1-independent, the uptake of larger basic aggregates was HSF1-dependent, requiring Hsp70. Our results show that the biophysical properties of aggregates determine both their mechanism of internalization and proteostatic response. It remains to be seen whether these differences in cellular response contribute to the particular role of specific aggregated proteins in disease.