RESUMO
Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.
Assuntos
Vesículas Extracelulares , Microscopia/métodos , Animais , Corantes/química , Epitopos , Vesículas Extracelulares/química , Vesículas Extracelulares/patologia , Vesículas Extracelulares/fisiologia , Corantes Fluorescentes/química , HumanosRESUMO
Langerhans cell histiocytosis (LCH) is a potentially life-threatening inflammatory myeloid neoplasia linked to pediatric neurodegeneration, whereby transformed LCH cells form agglomerated lesions in various organs. Although MAP-kinase pathway mutations have been identified in LCH cells, the functional consequences of these mutations and the mechanisms that cause the pathogenic behavior of LCH cells are not well understood. In our study, we used an in vitro differentiation system and RNA-sequencing to compare monocyte-derived dendritic cells from LCH patients to those derived from healthy controls or patients with Crohn's disease, a non-histiocytic inflammatory disease. We observed that interferon-γ treatment exacerbated intrinsic differences between LCH patient and control cells, including strikingly increased endo- and exocytosis gene activity in LCH patients. We validated these transcriptional patterns in lesions and functionally confirmed that LCH cells exhibited increased endo- and exocytosis. Furthermore, RNA-sequencing of extracellular vesicles revealed the enrichment of pathological transcripts involved in cell adhesion, MAP-kinase pathway, vesicle trafficking and T-cell activation in LCH patients. Thus, we tested the effect of the LCH secretome on lymphocyte activity and found significant activation of NK cells. These findings implicate extracellular vesicles in the pathology of LCH for the first time, in line with their established roles in the formation of various other tumor niches. Thus, we describe novel traits of LCH patient cells and suggest a pathogenic mechanism of potential therapeutic and diagnostic importance.
Assuntos
Histiocitose de Células de Langerhans , Neoplasias , Humanos , Criança , Secretoma , Histiocitose de Células de Langerhans/genética , Histiocitose de Células de Langerhans/tratamento farmacológico , Histiocitose de Células de Langerhans/patologia , Células Mieloides/metabolismo , Células Matadoras Naturais/metabolismoRESUMO
The International Society for Cell & Gene Therapy Scientific Signature Series event "Therapeutic Advances With Native and Engineered Human EVs" took place as part of the International Society for Cell & Gene Therapy 2022 Annual Meeting, held from May 4 to 7, 2022, in San Francisco, California, USA. This was the first signature series event on extracellular vesicles (EVs) and a timely reflection of the growing interest in EVs, including both native and engineered human EVs, for therapeutic applications. The event successfully gathered academic and industrial key opinion leaders to discuss the current state of the art in developing and understanding native and engineered EVs and applying our knowledge toward advancing EV therapeutics. Latest advancements in understanding the mechanisms by which native and engineered EVs exert their therapeutic effects against different diseases in animal models were presented, with some diseases such as psoriasis and osteoarthritis already reaching clinical testing of EVs. The discussion also covered various aspects relevant to advancing the clinical translation of EV therapies, including EV preparation, manufacturing, consistency, site(s) of action, route(s) of administration, and luminal cargo delivery of RNA and other compounds.
Assuntos
Vesículas Extracelulares , Animais , Humanos , Terapia Baseada em Transplante de Células e Tecidos , Terapia GenéticaRESUMO
mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy.
Assuntos
COVID-19 , Vacinas Anticâncer , Nanopartículas , Animais , Imunização/métodos , Imunoterapia , RNA Mensageiro/metabolismo , SARS-CoV-2/genética , Baço , Distribuição Tecidual , Vacinação/métodosRESUMO
The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.
Assuntos
Progressão da Doença , Distrofia Muscular de Duchenne/genética , Mutação/genética , Proteômica , Animais , Diferenciação Celular , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Reprodutibilidade dos Testes , Regulação para CimaRESUMO
Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
Assuntos
Neoplasias do Colo/tratamento farmacológico , DNA Glicosilases/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Poli(ADP-Ribose) Polimerase-1/imunologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Dano ao DNA , DNA Glicosilases/antagonistas & inibidores , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.
Assuntos
Vesículas Extracelulares , Biofísica , Comunicação Celular , Criança , Humanos , Microscopia de Fluorescência , TetraspaninasRESUMO
Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Vesículas Extracelulares/metabolismo , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Exossomos/metabolismo , Citometria de Fluxo/métodos , HumanosRESUMO
Cerebrospinal fluid (CSF) extracellular vesicles (EVs) show promise as a source of neurological disease biomarkers, although their precise origin is poorly understood. Current extraction techniques produce disappointing yield and purity. This study describes the application of ultrafiltration LC (UFLC) to CSF-EVs, compared with ultracentrifugation (UC), and explores CSF-EV origin. EVs are extracted from human CSF by UC and UFLC and characterized using nanoparticle tracking analysis, electron microscopy, and immunoblotting. EV and CSF proteomes are analyzed by LC-MS/MS. UFLC-isolated particles have size, morphology, and marker expression characteristic of EVs. UFLC provides greater EV yield (UFLC 7.90 × 108 ± SD 1.31 × 108 EVs mL-1 CSF, UC 1.06 × 108 ± 0.57 × 108 p < 0.001). UFLC enhances purity, proteomic depth (UFLC 622 ± 49, UC 298 ± 50, p = 0.001), and consistency of quantification (CV 17% vs 23%). EVs contain more intracellular proteins (Odds ratio [OR] 2.63 p < 0.001) and fewer plasma proteins than CSF (OR 0.60, p < 0.001). CSF and EV-enriched proteomes show overrepresentation of brain-specific proteins (EV OR 3.18, p < 0.001; CSF OR 3.37, p < 0.001). Overrepresentation of cerebral white matter (OR 1.99, p = 0.015) and choroid plexus proteins (OR 1.87, p<0.001) is observed in EVs. UFLC improves yield and purity of CSF-EVs. The EV-enriched proteome better reflects the intracellular and white matter proteome than whole CSF.
Assuntos
Biomarcadores/líquido cefalorraquidiano , Cromatografia Líquida/métodos , Vesículas Extracelulares/metabolismo , Doenças do Sistema Nervoso/diagnóstico , Proteoma/metabolismo , Ultrafiltração/métodos , Humanos , Doenças do Sistema Nervoso/líquido cefalorraquidianoRESUMO
MyomiRs are muscle-specific microRNAs (miRNAs) that regulate myoblast proliferation and differentiation. Extracellular myomiRs (ex-myomiRs) are highly enriched in the serum of Duchenne Muscular Dystrophy (DMD) patients and dystrophic mouse models and consequently have potential as disease biomarkers. The biological significance of miRNAs present in the extracellular space is not currently well understood. Here we demonstrate that ex-myomiR levels are elevated in perinatal muscle development, during the regenerative phase that follows exercise-induced myoinjury, and concomitant with myoblast differentiation in culture. Whereas ex-myomiRs are progressively and specifically released by differentiating human primary myoblasts and C2C12 cultures, chemical induction of apoptosis in C2C12 cells results in indiscriminate miRNA release. The selective release of myomiRs as a consequence of cellular differentiation argues against the idea that they are solely waste products of muscle breakdown, and suggests they may serve a biological function in specific physiological contexts. Ex-myomiRs in culture supernatant and serum are predominantly non-vesicular, and their release is independent of ceramide-mediated vesicle secretion. Furthermore, ex-myomiRs levels are reduced in aged dystrophic mice, likely as a consequence of chronic muscle wasting. In conclusion, we show that myomiR release accompanies periods of myogenic differentiation in cell culture and in vivo. Serum myomiR abundance is therefore a function of the regenerative/degenerative status of the muscle, overall muscle mass, and tissue expression levels. These findings have implications for the use of ex-myomiRs as biomarkers for DMD disease progression and monitoring response to therapy.
Assuntos
MicroRNAs/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Espaço Extracelular/genética , Humanos , Camundongos , MicroRNAs/sangue , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Especificidade de Órgãos , Cultura Primária de CélulasRESUMO
A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Transporte Biológico , Proteína C9orf72 , Células COS , Linhagem Celular , Chlorocebus aethiops , Expansão das Repetições de DNA , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Íntrons , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oligonucleotídeos Antissenso/farmacologia , Linhagem , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/patologia , Proteínas/genética , Proteínas rab de Ligação ao GTP , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules, including proteins and RNA, between cells. As a result of their suggested natural capability to functionally deliver RNA, EVs may be harnessed as therapeutic RNA carriers. One major limitation for their translation to therapeutic use is the lack of an efficient, robust, and scalable method to load EVs with RNA molecules of interest. Here, we evaluated and optimized methods to load EVs with cholesterol-conjugated small interfering RNAs (cc-siRNAs) by systematic evaluation of the influence of key parameters, including incubation time, volume, temperature, and EV:cc-siRNA ratio. EV loading under conditions that resulted in the highest siRNA retention percentage, incubating 15 molecules of cc-siRNA per EV at 37°C for 1 hr in 100 µL, facilitated concentration-dependent silencing of human antigen R (HuR), a therapeutic target in cancer, in EV-treated cells. These results may accelerate the development of EV-based therapeutics.
Assuntos
Colesterol/química , Sistemas de Liberação de Medicamentos , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Vesículas Extracelulares/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , RNA Interferente Pequeno/metabolismo , Animais , Sequência de Bases , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Colesterol/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Vesículas Extracelulares/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Permeabilidade , RNA Interferente Pequeno/genética , TemperaturaRESUMO
Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.
Assuntos
Peptídeos Penetradores de Células/genética , Distrofina/biossíntese , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/genética , Animais , Peptídeos Penetradores de Células/administração & dosagem , Modelos Animais de Doenças , Distrofina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/administração & dosagemRESUMO
Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle.
Assuntos
Distrofina/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Terapia Genética , Masculino , Camundongos , Camundongos Endogâmicos mdx , MicroRNAs/metabolismo , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Distrofia Muscular de Duchenne/terapia , Mutação , Proteômica , RNA Mensageiro/metabolismoRESUMO
Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.
Assuntos
Exossomos/genética , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/genética , Neovascularização Fisiológica/genética , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Comunicação Parácrina/genética , Proteoma/genética , Transdução de Sinais , Adulto JovemRESUMO
AIMS/HYPOTHESIS: The crosstalk between skeletal muscle (SkM) and beta cells plays a role in diabetes aetiology. In this study, we have investigated whether SkM-released exosome-like vesicles (ELVs) can be taken up by pancreatic beta cells and can deliver functional cargoes. METHODS: Mice were fed for 16 weeks with standard chow diet (SCD) or with standard diet enriched with 20% palmitate (HPD) and ELVs were purified from quadriceps muscle. Fluorescent ELVs from HPD or SCD quadriceps were injected i.v. or intramuscularly (i.m.) into mice to determine their biodistributions. Micro (mi)RNA quantification in ELVs was determined using quantitative real-time RT-PCR (qRT-PCR)-based TaqMan low-density arrays. Microarray analyses were performed to determine whether standard diet ELVs (SD-ELVs) and high palmitate diet ELVs (HPD-ELVs) induced specific transcriptional signatures in MIN6B1 cells. RESULTS: In vivo, muscle ELVs were taken up by pancreas, 24 h post-injection. In vitro, both SD-ELVs and HPD-ELVs transferred proteins and miRNAs to MIN6B1 cells and modulated gene expressions whereas only HPD-ELVs induced proliferation of MIN6B1 cells and isolated islets. Bioinformatic analyses suggested that transferred HPD-ELV miRNAs may participate in these effects. To validate this, we demonstrated that miR-16, which is overexpressed in HPD-ELVs, was transferred to MIN6B1 cells and regulated Ptch1, involved in pancreas development. In vivo, islets from HPD mice showed increased size and altered expression of genes involved in development, including Ptch1, suggesting that the effect of palm oil on islet size in vivo was reproduced in vitro by treating beta cells with HPD-ELVs. CONCLUSIONS/INTERPRETATION: Our data suggest that muscle ELVs might have an endocrine effect and could participate in adaptations in beta cell mass during insulin resistance.
Assuntos
Exossomos/metabolismo , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Músculo Esquelético/metabolismo , Animais , Linhagem Celular , Masculino , Camundongos , MicroRNAs/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMO
Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.
Assuntos
Nanopartículas/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Receptores Depuradores Classe A/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Éxons , Terapia Genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Micelas , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Receptores Depuradores Classe A/genéticaRESUMO
X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.
Assuntos
Agamaglobulinemia/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Tirosina Quinase da Agamaglobulinemia , Processamento Alternativo , Animais , Humanos , Mutação , Proteínas Tirosina Quinases/genética , RNA Mensageiro/genética , Transdução de SinaisRESUMO
BACKGROUND: Acute respiratory distress syndrome (ARDS) is a devastating disorder. Despite enormous efforts in clinical research, effective treatment options are lacking, and mortality rates remain unacceptably high. OBJECTIVES: A male patient with severe ARDS showed no clinical improvement with conventional therapies. Hence, an emergent experimental intervention was performed. METHODS: We performed intratracheal administration of autologous peripheral blood-derived mononuclear cells (PBMCs) and erythropoietin (EPO). RESULTS: We found that after 2 days of initial PBMC/EPO application, lung function improved and extracorporeal membrane oxygenation (ECMO) support was reduced. Bronchoscopy and serum inflammatory markers revealed reduced inflammation. Additionally, serum concentration of miR-449a, b, c and miR-34a, a transient upregulation of E-cadherin and associated chromatin marks in PBMCs indicated airway epithelial differentiation. Extracellular vesicles from PBMCs demonstrated anti-inflammatory capacity in a TNF-α-mediated nuclear factor-x03BA;B in vitro assay. Despite improving respiratory function, the patient died of multisystem organ failure on day 38 of ECMO treatment. CONCLUSIONS: This case report provides initial encouraging evidence to use locally instilled PBMC/EPO for treatment of severe refractory ARDS. The observed clinical improvement may partially be due to the anti-inflammatory effects of PBMC/EPO to promote tissue regeneration. Further studies are needed for more in-depth understanding of the underlying mechanisms of in vivo regeneration.
Assuntos
Leucócitos Mononucleares/transplante , Síndrome do Desconforto Respiratório/terapia , Caderinas/sangue , Citocinas/sangue , Regulação para Baixo , Eritropoetina/administração & dosagem , Oxigenação por Membrana Extracorpórea , Evolução Fatal , Humanos , Masculino , MicroRNAs/sangue , Insuficiência de Múltiplos Órgãos/etiologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/sangue , Transplante Autólogo , Regulação para Cima , Adulto JovemRESUMO
Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.