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1.
Ann Anat ; 245: 152006, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36183939

RESUMO

BACKGROUND: Cell-cell communication through extracellular vesicles (EVs) including exosomes, microvesicles and apoptotic bodies has been shown to be important in physiological homoeostasis as well as pathological processes such as atherosclerosis. However, while the cellular machinery controlling EV formation and composition has been studied during the past decade, less is known about the morphological process of their formation and release. METHODS: Using different electron microscopic approaches including transmission-, scanning-, immun-, and serial block face electron microscopy we studied the morphogenetic events of EV formation and release. We analysed the different steps of EV formation and release in cultured myocardial endothelial (MyEnd) and aortic endothelial (AoEnd) cell lines under serum starvation and under inflammatory conditions. RESULTS: We show that in a narrow time frame, the number of active cells and microvesicle (MV) producing cells increased in dependence of time spent in cultivation and additional stimulation by TNF-α. However, MV secretion was a highly heterogeneous process which couldn´t be seen in all cells cultivated under the same conditions. Release of MVs could be observed all over the cells' surface with no preferred release site. While no single specific microscopic approach applied was sufficient to provide a comprehensive analysis of EV biogenesis, we show that the limitations of one technique could be compensated by the qualities of the respective other applied techniques, thus enabling us to provide a detailed ultrastructural analysis of MV and exosome biogenesis. Surprisingly, exosome release in endothelial cells occurred via a yet undescribed process indicating that MVBs were incorporated into a novel distinct cellular compartment covered by fenestrated endothelium before exosome release. Lastly, we could show that TNF-α stimulation of AoEnd cells leads not only to the upregulation of CD44 in parental cells, but also to incorporation of CD44 into the membranes of generated MVs and exosomes. CONCLUSIONS: Taken together, our data contribute to a better understanding of biogenesis and release of EVs. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells at distant sites including vessel wall cells and this could be a mechanism by which MVs may change the and thus contribute to the development and progression of atherosclerotic lesions.


Assuntos
Aterosclerose , Exossomos , Vesículas Extracelulares , Humanos , Células Endoteliais , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Vesículas Extracelulares/metabolismo , Exossomos/química , Exossomos/metabolismo , Exossomos/ultraestrutura , Endotélio
2.
Methods Mol Biol ; 2582: 39-57, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370343

RESUMO

Cellular Communication Network (CCN) proteins are growth factors that play key roles in many pathophysiological events, including bone formation, wound healing, and cancer. CCN factors and fragments generated by metalloproteinases-dependent cleavage are often associated with extracellular matrix (ECM) or small extracellular vesicles (sEVs) such as exosomes or matrix-coated vesicles. We provide reliable methods and protocols for Western blotting to analyze CCN factors and fragments in cells, sEVs, and vesicle-free fractions.


Assuntos
Exossomos , Vesículas Extracelulares , Exossomos/metabolismo , Proteínas de Sinalização Intercelular CCN/metabolismo , Comunicação Celular , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Western Blotting
3.
Methods Mol Biol ; 2582: 59-76, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370344

RESUMO

Cellular Communication Network (CCN) proteins are secretory growth factors often associated with extracellular matrix (ECM) and extracellular vesicles (EVs) such as exosomes or matrix-coated vesicles. CCN factors and fragments loaded on/in EVs may play key roles in cell communication networks in cancer biology, bone and cartilage metabolism, wound healing, and tissue regeneration. CCN proteins and EVs/exosomes are found in body fluids, such as blood, urine, milk, and supernatants of the two-dimensionally (2D) cultured cells and three-dimensionally (3D) cultured tissues, such as spheroids or organoids. More than ten methods to isolate exosomes or EVs have been developed with different properties. Here, we introduce comprehensive protocols for polymer-based precipitation, affinity purification, ultracentrifugation methods combined with the ultrafiltration method for isolating CCN-loaded exosomes/EVs from 2D and 3D cultured tissues, and proteome analysis using mass spectrometry for comprehensive analysis of CCN proteins.


Assuntos
Exossomos , Vesículas Extracelulares , Exossomos/metabolismo , Proteoma/metabolismo , Ultracentrifugação/métodos , Vesículas Extracelulares/metabolismo , Comunicação Celular
4.
Clin. transl. oncol. (Print) ; 24(11): 2074-2080, noviembre 2022.
Artigo em Inglês | IBECS | ID: ibc-210136

RESUMO

Colorectal cancer (CRC) is one of the most common cancers worldwide and one of the main causes of cancer-associated mortality. At the period of diagnosis, metastases to other tissues will be present in around 30% of CRC individuals. Individuals with CRC continue to have a poor prognosis despite advances in medication. There is a growing body of literature that CRC develops as a result of the aggregation of various mutations in tumor oncogenes or suppressor genes and that diagnosing cancer in its initial phases may assist in increasing the overall lifespan of individuals with the illness. On the other hand, tumor cells may discharge exosomes in response to oncogenic mutations. By Inhibiting signaling pathways, including the Kirsten rat sarcoma virus (KRAS) mechanism, which is important in a variety of cell activities, exosomes have been shown to cause colorectal cancer in animal studies. The purpose of this review was to summarize the latest discoveries on the modulation of KRAS signaling by exosomes extracted from colorectal cancer. (AU)


Assuntos
Humanos , Neoplasias Colorretais/patologia , Exossomos/metabolismo , Vírus do Sarcoma Murino de Kirsten/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Mutação , Transdução de Sinais
5.
J Exp Clin Cancer Res ; 41(1): 319, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36324182

RESUMO

In the last two decades, clinical oncology has been revolutionized by the advent of targeted drugs. However, the efficacy of these therapies is significantly limited by primary and acquired resistance, that relies not only on cell-autonomous mechanisms but also on tumor microenvironment cues. Cancer-associated fibroblasts (CAFs) are extremely plastic cells of the tumor microenvironment. They not only produce extracellular matrix components that build up the structure of tumor stroma, but they also release growth factors, chemokines, exosomes, and metabolites that affect all tumor properties, including response to drug treatment. The contribution of CAFs to tumor progression has been deeply investigated and reviewed in several works. However, their role in resistance to anticancer therapies, and in particular to molecular therapies, has been largely overlooked. This review specifically dissects the role of CAFs in driving resistance to targeted therapies and discusses novel CAF targeted therapeutic strategies to improve patient survival.


Assuntos
Fibroblastos Associados a Câncer , Exossomos , Neoplasias , Humanos , Fibroblastos Associados a Câncer/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Microambiente Tumoral , Exossomos/metabolismo
6.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 44(5): 785-793, 2022 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-36325775

RESUMO

Objective To explore the role and mechanism of microRNA-204(miR-204) carried by the exosomes of human umbilical cord-derived mesenchymal stem cells(hUC-MSC) in regulating the polarization of macrophages in a mouse model of myocardial ischemia-reperfusion(I/R) injury. Methods After the hUC-MSCs were isolated,cultured,and identified,their adipogenic and osteogenic differentiation capabilities were determined.The exosomes of hUC-MSCs were separated by ultracentrifugation,and the expression of CD81,CD63,tumor susceptibility gene 101(Tsg101),and calnexin in the exosomes was determined by Nanoparticle Tracking Analysis software,transmission electron microscopy,and Western blotting.Three groups(hUC-MSC,miR-204 mimic,and negative control) were designed for the determination of the expression of miR-204 in the cells and their exosomes by qRT-PCR.The C57BL/6J mice were randomly assigned into a sham operation group,an I/R group,a hUC-MSC exosomes group,a negative control group,and a miR-204 mimic group.Except the sham operation group,the I/R model was established by ligating the left anterior descending artery.The echocardiography system was employed to detect the heart function of mice.HE staining was employed to observe the pathological changes of mouse myocardium.ELISA was employed to determine the levels of interleukin-1ß(IL-1ß),tumor necrosis factor-α(TNF-α),arginase 1(Arg-1),and IL-10 in the myocardial tissue.After the macrophages of mouse myocardial tissue were isolated,flow cytometry was employed to determine the expression of CD11c and CD206,and ELISA to measure the levels of IL-1ß,TNF-α,Arg-1,and IL-10 in the macrophages. Results hUC-MSCs had adipogenic and osteogenic differentiation capabilities,and the exosomes were successfully identified.Compared with the negative control group,the miR-204 mimic group showed up-regulated expression of miR-204 in hUC-MSCs and their exosomes(P<0.001,P<0.001).Compared with the sham operation group,the modeling of I/R increased the left ventricular end-diastolic diameter(LVEDD)(P<0.001),left ventricular end-systolic diameter(LVESD)(P<0.001),myocardial injury score(P<0.001),and the levels of IL-1ß(P<0.001),TNF-α(P<0.001),and CD11c(P<0.001).Meanwhile,it lowered the left ventricular ejection fraction(LVEF)(P<0.001),left ventricular fractional shortening(LVFS)(P<0.001),Arg-1(P<0.001),IL-10(P<0.001),and CD206(P<0.001).Compared with those in the I/R group,the LVEDD(P<0.001),LVESD(P<0.001),myocardial injury score(P<0.001),and the levels of IL-1ß(P<0.001),TNF-α(P=0.010),and CD11c(P<0.001) reduced,while LVEF(P<0.001),LVFS(P<0.001),and the levels of Arg-1(P<0.001),IL-10(P=0.028),and CD206(P=0.022) increased in the hUC-MSC exosomes group.Compared with those in the negative control group,the LVEDD(P<0.001),LVESD(P<0.001),myocardial injury score(P=0.001),and the levels of IL-1ß(P=0.048),TNF-α(P<0.001),and CD11c(P=0.007) reduced,while the LVEF(P<0.001),LVFS(P<0.001),and the levels of Arg-1(P<0.001),IL-10(P=0.001),and CD206(P=0.001) increased in the miR-204 mimic group. Conclusion The hUC-MSC exosomes overexpressing miR-204 can inhibit the polarization of macrophages in the I/R mouse model to M1-type and promote the polarization to M2-type.


Assuntos
Exossomos , Células-Tronco Mesenquimais , MicroRNAs , Traumatismo por Reperfusão Miocárdica , Animais , Humanos , Camundongos , Modelos Animais de Doenças , Exossomos/metabolismo , Exossomos/patologia , Interleucina-10/metabolismo , Macrófagos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Osteogênese , Volume Sistólico , Fator de Necrose Tumoral alfa/metabolismo , Cordão Umbilical/metabolismo , Cordão Umbilical/patologia , Função Ventricular Esquerda
7.
Arch Biochem Biophys ; 731: 109430, 2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36326546

RESUMO

Diabetic cardiovascular complication is a common systemic disease with high morbidity and mortality worldwide. We hypothesise that exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs-exos) can rescue these disorders and alleviate vascular remodeling in diabetes. Morphological, non-targeted metabolomics and 4D label-free proteomics techniques were used to analyze the aortas of db/m mice as normal control group (NCA), saline treated db/db mice (DMA), and hUCMSCs-exos treated db/db mice (DMTA), and to clarify the molecular mechanism of the protection of hUCMSCs-exos in vascular remodeling from a new point of view. The results showed that 74 metabolites were changed significantly in diabetic aortas, of which 15 were almost restored by hUCMSCs-exos. In proteomics, 30 potential targets such as Stromal cell-derived factor 2-like protein 1, Leukemia inhibitory factor receptor, Peroxisomal membrane protein and E3 ubiquitin-protein ligase MYCBP2 were detected. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based analysis showed that Central carbon metabolism in cancer and Galactose metabolism pathway were up-regulated to near normal by hUCMSCs-exos in metabolomics, with janus associated kinase-signal transducer and activator of transcription (JAK-STAT) pathway displayed in proteomics. According to bioinformatics and integrated analysis, these targeted molecules of hUCMSCs-exos to attenuate the vascular remodeling were mainly associated with regulation of energy metabolism, oxidative stress, inflammation, and cellular communications. This study provided a reference for the therapy of diabetes-induced cardiovascular complications.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Exossomos/metabolismo , Cordão Umbilical , Proteômica , Remodelação Vascular , Células-Tronco Mesenquimais/metabolismo , Aorta
8.
J Adv Res ; 41: 113-128, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36328741

RESUMO

INTRODUCTION: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. OBJECTIVES: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. METHODS: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. RESULTS: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-ß, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-ß, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. CONCLUSION: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines.


Assuntos
Exossomos , Exossomos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteômica , Interleucina-6/metabolismo , Imunidade , Fator de Crescimento Transformador beta/metabolismo
9.
Drug Deliv ; 29(1): 3270-3280, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36330597

RESUMO

Liver fibrosis is a global life-threatening disorder with no approved treatment. It leads to serious hepatic complications when progressive, such as cirrhosis and carcinoma. Luteolin (LUT) is a plant flavonoid possessing a promising therapeutic potential in various liver diseases particularly, liver fibrosis. It was reported to have potent anti-inflammatory and antioxidant properties. It also suppresses the proliferation of activated hepatic stellate cells (HSC) and induces their apoptosis. However, its poor aqueous solubility and exposure to metabolism hinder its clinical use. Mesenchymal stem cells (MSCs)-derived exosomes, nano-sized extracellular vesicles, have recently emerged as natural biocompatible drug delivery vehicles permitting efficient drug delivery. Accordingly, the present study aimed for the first time to investigate the potential of bone marrow MSCs-derived exosomes to improve LUTs antifibrotic effectiveness. LUT-loaded exosomes (LUT-Ex) were successfully developed, optimized and subjected to both in vitro and in vivo characterization. The elaborated LUT-Ex presented good colloidal properties (size; 150 nm, PDI; 0.3 and ζ-potential; -28 mV), typical vesicular shape, reasonable drug entrapment efficiency (40%) with sustained drug release over 72 h. Additionally, the cellular uptake study of coumarin-6-loaded exosomes in HEP-G2 revealed a significant enhancement in their uptake by 78.4% versus free coumarin-6 solution (p ≤ 0.001). Following a single intraperitoneal injection, LUT-Ex revealed a superior antifibrotic activity compared with either LUT-suspension or blank exosomes as evidenced by the results of biochemical and histopathological evaluation. In conclusion, the elaborated LUT-Ex could be addressed as a promising nanocarrier for effective treatment of liver fibrosis.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Humanos , Exossomos/metabolismo , Luteolina/farmacologia , Luteolina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Cumarínicos
10.
Int J Mol Sci ; 23(21)2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36362078

RESUMO

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.


Assuntos
Exossomos , Neoplasias Ovarianas , Fosfolipase D , Feminino , Humanos , Apoptose , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Exossomos/metabolismo , Isoenzimas , Neoplasias Ovarianas/genética , Fosfolipase D/genética , Fosfolipase D/metabolismo
11.
J Interv Cardiol ; 2022: 5451947, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36419957

RESUMO

Pulmonary hypertension (PH) is a type of clinical pathophysiological syndrome characterized by a progressive increase in pulmonary vascular resistance and subsequent progressive failure of the right heart function, and is a common complication of many diseases. Mesenchymal stem cells (MSCs) autonomously home to sites damaged by disease, repair damaged tissues, and participate in the regulation of systemic inflammation and immune responses, which have good clinical application prospects. Extracellular vesicles (EVs), such as exosomes and microvesicles, participate in various biological activities by regulating intercellular communication. Exosomes secreted into the extracellular environment also affect the host immune system. MSC-derived extracellular vesicles (MSC-EVs), as a mediator in the paracrine processes of MSCs, carry biologically active substances such as proteins, lipids, mRNA, and micro-RNA. MSC-EVs therapies, safer than cell-based treatments, have been shown to be effective in modulating macrophages to support anti-inflammatory phenotypes, which are strongly related to histological and functional benefits in preclinical models of pulmonary hypertension. The main effects of active substances and their potential medical value have attracted wide attention from researchers. This article reviews the role and relevant mechanisms of MSC-EVs in the treatment of pulmonary hypertension in recent studies and provides a basis for their future clinical applications.


Assuntos
Exossomos , Vesículas Extracelulares , Hipertensão Pulmonar , Células-Tronco Mesenquimais , MicroRNAs , Humanos , Hipertensão Pulmonar/terapia , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Exossomos/metabolismo , MicroRNAs/metabolismo
12.
Front Immunol ; 13: 1005307, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36420273

RESUMO

Diabetes is the most prevalent metabolic disease in the world today. In addition to elevated blood glucose, it also causes serious complications, which has a significant effect on the quality of life of patients. Diabetic trauma is one of complications as a result of the interaction of diabetic neuropathy, peripheral vascular disease, infection, trauma, and other factors. Diabetic trauma usually leads to poor healing of the trauma and even to severe foot ulcers, wound gangrene, and even amputation, causing serious psychological, physical, and financial burdens to diabetic patients. Non-coding RNAs (ncRNAs) carried by exosomes have been demonstrated to be relevant to the development and treatment of diabetes and its complications. Exosomes act as vehicle, which contain nucleic acids such as mRNA and microRNA (miRNA), and play a role in the intercellular communication and the exchange of substances between cells. Because exosomes are derived from cells, there are several advantages over synthetic nanoparticle including good biocompatibility and low immunogenicity. Exosomal ncRNAs could serve as markers for the clinical diagnosis of diabetes and could also be employed to accelerate diabetic wound healing via the regulation of the immune response and modulation of cell function. ncRNAs in exosomes can be employed to promote diabetic wound healing by regulating inflammation and accelerating re-vascularization, re-epithelialization, and extracellular matrix remodeling. Herein, exosomes in terms of ncRNA (miRNA, lncRNA, and circRNA) to accelerate diabetic wounds healing were summarized, and we discussed the challenge of the loading strategy of ncRNA into exosomes.


Assuntos
Diabetes Mellitus , Exossomos , MicroRNAs , Humanos , Qualidade de Vida , Cicatrização/genética , Diabetes Mellitus/metabolismo , Exossomos/genética , Exossomos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo
13.
Proc Natl Acad Sci U S A ; 119(48): e2210532119, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36409902

RESUMO

A hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, or c9ALS/FTD. The RNA transcribed from the expansion, r(G4C2)exp, causes various pathologies, including intron retention, aberrant translation that produces toxic dipeptide repeat proteins (DPRs), and sequestration of RNA-binding proteins (RBPs) in RNA foci. Here, we describe a small molecule that potently and selectively interacts with r(G4C2)exp and mitigates disease pathologies in spinal neurons differentiated from c9ALS patient-derived induced pluripotent stem cells (iPSCs) and in two c9ALS/FTD mouse models. These studies reveal a mode of action whereby a small molecule diminishes intron retention caused by the r(G4C2)exp and allows the liberated intron to be eliminated by the nuclear RNA exosome, a multi-subunit degradation complex. Our findings highlight the complexity of mechanisms available to RNA-binding small molecules to alleviate disease pathologies and establishes a pipeline for the design of brain penetrant small molecules targeting RNA with novel modes of action in vivo.


Assuntos
Exossomos , Demência Frontotemporal , Animais , Camundongos , Demência Frontotemporal/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , RNA/genética , Exossomos/metabolismo , Barreira Hematoencefálica/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Nuclear
14.
ACS Appl Mater Interfaces ; 14(45): 50626-50636, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36342824

RESUMO

Exosomes serve as a promising therapeutic nanoplatform. However, the exosomes produced by donor cells are a heterogeneous group, with only a small portion having high therapeutic efficacy. Specific isolation of the subpopulation with high efficacy is important for lowering the dose and minimizing toxicity. In this study, we loaded target mRNA and displayed specific Flag in engineered exosomes simultaneously. Briefly, the donor cells were transfected with plasmid expressing a fusion protein Flag-TCS-PTGFRN-CTSL-MCP, namely, exosome sorter. During biogenesis, the RNA-binding motif MCP can specifically bind with MS2-containing RNA and sort the target RNA into the lumen of exosomes. Anti-Flag magnetic beads can capture and thus purify the engineered exosomes via recognition of the Flag on the surface of exosomes. After purification, the Flag could be cleaved by thrombin treatment while MCP can be separated from the fusion protein by CTSL autocleavage upon exosome acidification, minimizing the side effects and augmenting the therapeutic effects. By the proof-of-concept experiment, the exosome sorter-based "all-in-one" strategy was confirmed effective in both the encapsulation of therapeutic mRNA (Ldlr-MS2) into exosomes and the subsequent purification. The purified Ldlr-MS2-containing exosomes had much higher efficacy in alleviating atherosclerosis, in comparison with the bulk exosomes, confirming the advantage of the proposed "all-in-one" strategy.


Assuntos
Exossomos , Hiperlipoproteinemia Tipo II , Humanos , Exossomos/metabolismo , Hiperlipoproteinemia Tipo II/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo
15.
Oxid Med Cell Longev ; 2022: 9042345, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36388171

RESUMO

Both hair follicle stem cells (HFSC) and dermal papilla cells (DPC) are essential for hair follicle growth and proliferation. In this study, HFSCs and DPCs that made signature proteins like KRT14, KRT15, KRT19, α-SMA, and Versican were obtained. Cell coculture systems between HFSCs and DPCs were used to measure the increased PCNA protein content in HFSCs. Additionally, exosomes from dermal papilla cells (DPC-Exos), the overexpression and silencing of Wnt3a, could regulate the Wnt/ß-catenin signaling pathway downstream genes. After collecting DPC-ExosOE-Wnt3a, the treatment of HFSC with DPC-ExosOE-Wnt3a showed that DPC-ExosOE-Wnt3a could upregulate the mRNA expression of downstream genes in the Wnt/ß-catenin signaling pathway and that DPC-ExosOE-Wnt3a enhanced the proliferation of HFSCs while inhibiting their apoptosis. These findings suggest that DPC-Exos could regulate HFSC cell proliferation via the Wnt3a/ß-catenin signaling pathway. This research offers novel concepts for the molecular breeding and efficient production of Angora rabbits, as well as for the treatment of human hair problems.


Assuntos
Exossomos , beta Catenina , Animais , Humanos , Coelhos , beta Catenina/metabolismo , Folículo Piloso , Exossomos/metabolismo , Via de Sinalização Wnt , Proliferação de Células , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo
16.
Sci Rep ; 12(1): 19786, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396670

RESUMO

Extracellular matrix (ECM) secretion, deposition and assembly are part of a whole complex biological process influencing the microenvironment and other cellular behaviors. Emerging evidence is attributing a significant role to extracellular vesicles (EVs) and exosomes in a plethora of ECM-associated functions, but the role of dermal fibroblast-derived EVs in paracrine signalling is yet unclear. Herein, we investigated the effect of exosomes isolated from stimulated human dermal fibroblasts. We report that tridimensional (3D) cell culture of dermal fibroblasts promotes secretion of exosomes carrying a large quantity of proteins involved in the formation, organisation and remodelling of the ECM. In our 3D model, gene expression was highly modulated and linked to ECM, cellular migration and proliferation, as well as inflammatory response. Mass spectrometry analysis of exosomal proteins, isolated from 3D cultured fibroblast-conditioned media, revealed ECM protein enrichment, of which many were associated with the matrisome. We also show that the cytokine interleukin 6 (IL-6) is predicted to be central to the signalling pathways related to ECM formation and contributing to cell migration and proliferation. Overall, our data suggest that dermal fibroblast-derived EVs participate in many steps of the establishment of dermis's ECM.


Assuntos
Exossomos , Humanos , Exossomos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Matriz Extracelular/metabolismo
17.
Cell Transplant ; 31: 9636897221133345, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36324284

RESUMO

Human umbilical cord mesenchymal stem cell (HUMSC)-exosome gel played a significant role in promoting thin endometrial receptivity and improving the pregnancy rate by inhibiting endometrial fibrosis and accelerating subendometrial microangiogenesis. High-quality HUMSC-exosome were obtained by pretreating HUMSC with transforming growth factor-ß1 (TGF-ß1). Exosome gel mixture has good biocompatibility and physical rheological properties, stabilizing the structure of exosomes and prolonging the action of exosomes in the uterine cavity. HUMSC or HUMSC-derived exosomes were used to treat rat model of thin endometrium. In animal experiments, four groups, including the HUMSC, HUMSC-exosome, model (negative control), and sham operation groups, were designed. The therapeutic effects were evaluated by the thickness of the endometrium, the number of glands, the subendometrial vessel density, the markers of endometrial receptivity, and the pregnancy rate. In an in vivo study, three groups, involving HUMSC-coculture, HUMSC-exosome, and the control, were explored. The proliferation and migration of the human endometrial stromal cells (HESCs) were further determined by cell scratch and 5-ethynyl-2'-deoxyuridine (EdU) assays. The protein expression of the TGF-ß1/smad2/3 signaling pathway was determined by Western blot. After treatment, the thickness of the endometrium, the number of glands, and the subendometrial microangiogenesis were obviously increased, and the level of inhibition of endometrial fibrosis, molecular markers of endometrial receptivity, and the pregnancy rate were also significantly improved. HUMSC-exosome and HUMSC significantly promoted the migration and proliferation of HESCs. And it was confirmed that HUMSC-exosome were superior to HUMSC in inhibiting HESCs fibrosis through TGF-ß1/smad2/3 signaling pathway at the protein expression level.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Gravidez , Feminino , Ratos , Humanos , Animais , Exossomos/metabolismo , Taxa de Gravidez , Cordão Umbilical , Endométrio/metabolismo , Fibrose
18.
Cell Commun Signal ; 20(1): 171, 2022 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-36316715

RESUMO

Presently, more than half of cancer patients receive radiotherapy to cure localized cancer, palliate symptoms, or control the progression of cancer. However, radioresistance and radiation-induced bystander effects (RIBEs) are still challenging problems in cancer treatment. Exosomes, as a kind of extracellular vesicle, have a significant function in mediating and regulating intercellular signaling pathways. An increasing number of studies have shown that radiotherapy can increase exosome secretion and alter exosome cargo. Furthermore, radiation-induced exosomes are involved in the mechanism of radioresistance and RIBEs. Therefore, exosomes hold great promise for clinical application in radiotherapy. In this review, we not only focus on the influence of radiation on exosome biogenesis, secretion and cargoes but also on the mechanism of radiation-induced exosomes in radioresistance and RIBEs, which may expand our insight into the cooperative function of exosomes in radiotherapy. Video abstract.


Assuntos
Exossomos , Vesículas Extracelulares , Neoplasias , Humanos , Exossomos/metabolismo , Efeito Espectador , Comunicação Celular , Transdução de Sinais , Vesículas Extracelulares/metabolismo , Neoplasias/radioterapia , Neoplasias/metabolismo
19.
Cells ; 11(21)2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36359787

RESUMO

Epithelial ovarian cancer (EOC) patients frequently develop peritoneal metastasis, especially in the human omentum. However, the mechanism underlying this propensity remains unknown. A previous study found that human omental adipose-derived mesenchymal stem cells are potentially involved in ovarian cancer growth and metastasis, but the results were inconsistent and even contradictory. In addition, the underlying mechanisms of visceral adipose metastasis remain poorly understood. Here, our goal is to clarify the role and mechanism of human omental adipose-derived mesenchymal stem cells (HO-ADSCs) in EOC cancer growth and metastasis. We first found that human omental tissue conditioned medium (HO-CM) enhances EOC cell function. Subsequent coculture studies indicated that HO-ADSCs increase the growth, migratory and invasive capabilities of ovarian cancer cells. Then, we demonstrated that exosomes secreted by HO-ADSCs (HO-ADSC exosomes) enhanced ovarian cancer cell function, and further mechanistic studies showed that the FOXM1, Cyclin F, KIF20A, and MAPK signaling pathways were involved in this process. In addition, subcutaneous tumorigenesis and peritoneal metastatic xenograft experiments provided evidence that HO-ADSC exosomes promote ovarian cancer growth and metastasis in vivo. Finally, our clinical studies provided evidence that ascites from ovarian cancer patients enhance EOC cell line proliferation, migration, and invasion in vitro. The present study indicated that HO-ADSC exosomes are secreted into ascites and exert a tumor-promoting effect on EOC growth and metastasis, providing a new perspective and method to develop future novel therapeutic strategies for the treatment of ovarian cancer.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Neoplasias Ovarianas , Neoplasias Peritoneais , Humanos , Feminino , Carcinoma Epitelial do Ovário/patologia , Exossomos/metabolismo , Omento/metabolismo , Omento/patologia , Ascite/patologia , Tecido Adiposo/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neoplasias Ovarianas/patologia , Processos Neoplásicos
20.
Cells ; 11(21)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36359776

RESUMO

The cancer burden continues to grow globally, and drug resistance remains a substantial challenge in cancer therapy. It is well established that cancerous cells with clonal dysplasia generate the same carcinogenic lesions. Tumor cells pass on genetic templates to subsequent generations in evolutionary terms and exhibit drug resistance simply by accumulating genetic alterations. However, recent evidence has implied that tumor cells accumulate genetic alterations by progressively adapting. As a result, intratumor heterogeneity (ITH) is generated due to genetically distinct subclonal populations of cells coexisting. The genetic adaptive mechanisms of action of ITH include activating "cellular plasticity", through which tumor cells create a tumor-supportive microenvironment in which they can proliferate and cause increased damage. These highly plastic cells are located in the tumor microenvironment (TME) and undergo extreme changes to resist therapeutic drugs. Accordingly, the underlying mechanisms involved in drug resistance have been re-evaluated. Herein, we will reveal new themes emerging from initial studies of drug resistance and outline the findings regarding drug resistance from the perspective of the TME; the themes include exosomes, metabolic reprogramming, protein glycosylation and autophagy, and the relates studies aim to provide new targets and strategies for reversing drug resistance in cancers.


Assuntos
Bullying , Exossomos , Neoplasias , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Microambiente Tumoral/genética , Exossomos/metabolismo
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