RESUMO
Extracellular vesicles (EVs) are widely studied regarding their role in cell-to-cell communication and disease, as well as for applications as biomarkers or drug delivery vehicles. EVs contain membrane and intraluminal proteins, affecting their structure and thereby likely their functioning. Here, we use atomic force microscopy for mechanical characterization of erythrocyte, or red blood cell (RBC), EVs from healthy individuals and from patients with hereditary spherocytosis (HS) due to ankyrin deficiency. While these EVs are packed with proteins, their response to indentation resembles that of fluid liposomes lacking proteins. The bending modulus of RBC EVs of healthy donors is ~15 kbT, similar to the RBC membrane. Surprisingly, whereas RBCs become more rigid in HS, patient EVs have a significantly (~40%) lower bending modulus than donor EVs. These results shed light on the mechanism and effects of EV budding and might explain the reported increase in vesiculation of RBCs in HS patients.
Assuntos
Membrana Eritrocítica/química , Eritrócitos/química , Vesículas Extracelulares/química , Esferocitose Hereditária/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Fluidez de Membrana , Microscopia de Força Atômica , Proteínas/metabolismoRESUMO
In the last few years it has become clear that, in addition to soluble molecules such as growth factors and cytokines, cells use extracellular vesicles (EVs) for intercellular communication. For example, EVs derived from cancer cells interact with endothelial cells, thereby affecting angiogenesis and metastasis, two essential processes in tumor progression. In most experiments, the interaction of EVs with target cells is investigated under static conditions. However the use of dynamic flow conditions is considered more relevant, especially when studying EV uptake by endothelial cells. Here, we describe the use of a perfusion system to investigate the interaction of (tumor) EVs with endothelial cells under dynamic flow conditions.
Assuntos
Comunicação Celular , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Fracionamento Celular/métodos , Linhagem Celular , Meios de Cultivo Condicionados , Humanos , Microfluídica/instrumentação , Microfluídica/métodosRESUMO
AIM: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). MATERIALS & METHODS: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. RESULTS: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. CONCLUSION: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior.
Assuntos
Antineoplásicos/farmacologia , Cetuximab/farmacologia , Receptores ErbB/análise , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/patologia , Neoplasias/tratamento farmacológico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Monitoramento de Medicamentos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Neoplasias/diagnóstico , Neoplasias/patologia , FosforilaçãoRESUMO
To date, cellular transplantation therapy has not yet fulfilled its high expectations for cardiac repair. A major limiting factor is lack of long-term engraftment of the transplanted cells. Interestingly, transplanted cells can positively affect their environment via secreted paracrine factors, among which are extracellular vesicles, including exosomes: small bi-lipid-layered vesicles containing proteins, mRNAs, and miRNAs. An exosome-based therapy will therefore relay a plethora of effects, without some of the limiting factors of cell therapy. Since cardiomyocyte progenitor cells (CMPC) and mesenchymal stem cells (MSC) induce vessel formation and are frequently investigated for cardiac-related therapies, the pro-angiogenic properties of CMPC and MSC-derived exosome-like vesicles are investigated. Both cell types secrete exosome-like vesicles, which are efficiently taken up by endothelial cells. Endothelial cell migration and vessel formation are stimulated by these exosomes in in vitro models, mediated via ERK/Akt-signaling. Additionally, these exosomes stimulated blood vessel formation into matrigel plugs. Analysis of pro-angiogenic factors revealed high levels of extracellular matrix metalloproteinase inducer (EMMPRIN). Knockdown of EMMPRIN on CMPCs leads to a diminished pro-angiogenic effect, both in vitro and in vivo. Therefore, CMPC and MSC exosomes have powerful pro-angiogenic effects, and this effect is largely mediated via the presence of EMMPRIN on exosomes.
Assuntos
Basigina/metabolismo , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Neovascularização Patológica/metabolismo , Células-Tronco/metabolismo , Animais , Movimento Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The identification of extracellular phospholipid vesicles as conveyors of cellular information has created excitement in the field of drug delivery. Biological therapeutics, including short interfering RNA and recombinant proteins, are prone to degradation, have limited ability to cross biological membranes, and may elicit immune responses. Therefore, delivery systems for such drugs are under intensive investigation. Exploiting extracellular vesicles as carriers for biological therapeutics is a promising strategy to overcome these issues and to achieve efficient delivery to the cytosol of target cells. Exosomes are a well studied class of extracellular vesicles known to carry proteins and nucleic acids, making them especially suitable for such strategies. However, the considerable complexity and the related high chance of off-target effects of these carriers are major barriers for translation to the clinic. Given that it is well possible that not all components of exosomes are required for their proper functioning, an alternative strategy would be to mimic these vesicles synthetically. By assembly of liposomes harboring only crucial components of natural exosomes, functional exosome mimetics may be created. The low complexity and use of well characterized components strongly increase the pharmaceutical acceptability of such systems. However, exosomal components that would be required for the assembly of functional exosome mimetics remain to be identified. This review provides insights into the composition and functional properties of exosomes, and focuses on components which could be used to enhance the drug delivery properties of exosome mimetics.
Assuntos
Materiais Biomiméticos , Sistemas de Liberação de Medicamentos , Exossomos , Animais , Materiais Biomiméticos/química , Biotecnologia , Moléculas de Adesão Celular/química , Portadores de Fármacos/química , Exossomos/química , Humanos , Lipossomos/química , Lipídeos de Membrana/química , Proteínas de Membrana/química , Nanomedicina , Nanotecnologia , RNA Interferente Pequeno/administração & dosagem , Tetraspaninas/químicaRESUMO
Cell-derived membrane vesicles (CMVs) are endogenous carriers transporting proteins and nucleic acids between cells. They appear to play an important role in many disease processes, most notably inflammation and cancer, where their efficient functional delivery of biological cargo seems to contribute to the disease progress. CMVs encompass a variety of submicron vesicular structures that include exosomes and shedding vesicles. The lipids, proteins, mRNA and microRNA (miRNA) delivered by these vesicles change the phenotype of the receiving cells. CMVs have created excitement in the drug delivery field, because they appear to have multiple advantages over current artificial drug delivery systems. Two approaches to exploit CMVs for delivery of exogenous therapeutic cargoes in vivo are currently considered. One approach is based on engineering of natural CMVs in order to target certain cell types using CMVs loaded with therapeutic compounds. In the second approach, essential characteristics of CMVs are being used to design nano-scaled drug delivery systems. Although a number of limiting factors in the clinical translation of the exciting research findings so far exist, both approaches are promising for the development of a potentially novel generation of drug carriers based on CMVs.