Dataset Information

Comparison of Proangiogenic Effects of Adipose-Derived Stem Cells and Foreskin Fibroblast Exosomes on Artificial Dermis Prefabricated Flaps.

ABSTRACT: Large prefabricated flaps often suffer from necrosis or poor healing due to a lack of new blood vessels and related factors that promote angiogenesis. The innovative use of adipose-derived stem cell exosomes (ADSC-Exo) resolves the problem of vascularization of prefabricated flaps. We analyzed the differential microRNA (miRNA) expression in ADSC-Exo using next-generation sequencing (NGS) technology to explore their potential mechanisms in promoting vascularization. We observed that ADSC-Exo could significantly promote the vascularization of artificial dermis prefabricated flaps compared with human foreskin fibroblast exosomes. NGS indicated that there were some differentially expressed miRNAs in both exosomes. Bioinformatics analysis suggested that significantly upregulated hsa-miR-760 and significantly downregulated hsa-miR-423-3p in ADSC-Exo could regulate the expression of the ITGA5 and HDAC5 genes, respectively, to promote the vascularization of skin flaps. In summary, ADSC-Exo can promote skin-flap vascularization, and thereby resolve the problem of insufficient neovascularization of artificial dermis prefabricated flaps, thus expanding the application of prefabricated skin-flap transplantation.

SUBMITTER: Xiong J

PROVIDER: S-EPMC7013349 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Comparison of Proangiogenic Effects of Adipose-Derived Stem Cells and Foreskin Fibroblast Exosomes on Artificial Dermis Prefabricated Flaps.

Xiong Jiachao J Liu Zhixiao Z Wu Minliang M Sun Mengyan M Xia Yu Y Wang Yuchong Y

Stem cells international 20200131

Large prefabricated flaps often suffer from necrosis or poor healing due to a lack of new blood vessels and related factors that promote angiogenesis. The innovative use of adipose-derived stem cell exosomes (ADSC-Exo) resolves the problem of vascularization of prefabricated flaps. We analyzed the differential microRNA (miRNA) expression in ADSC-Exo using next-generation sequencing (NGS) technology to explore their potential mechanisms in promoting vascularization. We observed that ADSC-Exo coul ...[more]

PMID: 32089706

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Project description:Rationale: Mesenchymal stem cells (MSCs) play important roles in tissue repair and regeneration. However, the molecular mechanisms underlying MSCs activation remain largely unknown, thus hindering their clinical translation. Exosomes are small vesicles that act as intercellular messengers, and their potential for stem cell activation in pathological conditions has not been fully characterized yet. Here, we aim to investigate whether serum exosomes are involved in the remote activation of MSCs after myocardial infarction (MI). Methods: We established MI mouse model by ligating the left anterior descending branch of the coronary artery. Afterwards, serum exosomes were isolated from control (Con Exo) and MI mice (MI Exo) by differential centrifugation. Exosomes were characterized through transmission electron microscopy and nanoparticle tracking analysis. The cell proliferation rate was evaluated by CCK-8 and EdU incorporation assays. Exosomal miRNA and protein levels were assessed using qRT-PCR and western blotting, respectively. VEGF levels in the supernatant and serum were quantified by ELISA. Matrigel plug and tube formation assays were used to evaluate angiogenesis. To explore miR-1956 roles, overexpression and knock-down experiments were performed using mimic and inhibitor, respectively. Finally, miR-1956 target genes were confirmed using the luciferase reporter assay. Results: Both types of exosomes exhibited typical characteristics and could be internalized by adipose-derived MSCs (ADMSCs). MI Exo enhanced ADMSCs proliferation through the activation of ERK1/2. Gain- and loss-of-function studies allowed the validation of miR-1956 (enriched in MI Exo) as the functional messenger that stimulates ADMSCs-mediated angiogenesis and paracrine VEGF signaling, by downregulating Notch-1. Finally, we found that the ischemic myocardium and kidney may be the main sources that release serum exosomes after MI. Conclusions: Cardio-renal exosomes deliver miR-1956 and activate paracrine proangiogenic VEGF signaling in ADMSCs after MI; this process also involves Notch-1, which functions as the core mediator.

Project description:BACKGROUND:M2 macrophages and exosomes from adipose-derived stem cells (ASCs) are both reported to promote angiogenesis. However, the possible synergistic effects between exogenous exosomes and endogenous M2 macrophages are poorly understood. METHODS:Exosomes were isolated from conditioned medium of normoxic and hypoxic ASCs using the combined techniques of ultrafiltration and size-exclusion chromatography and were identified with nanoparticle tracking analysis and immunoblotting for exosomal markers. Macrophages were collected from the mouse peritoneal cavity. M1 and M2 macrophages were detected by immunoblotting for the intracellular markers inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) and by flow cytometry for the surface markers F4/80, CD86, and CD206. Murine models of Matrigel plug and hindlimb ischemia were employed as in vivo angiogenic assays. RESULTS:When M1 macrophages were treated with exosomes from normoxic ASCs (Nor/Exo), and particularly from hypoxic ASCs (Hyp/Exo), the expression of the M1 marker iNOS decreased, and the M2 marker Arg-1 increased in a time- and dose-dependent manner. Additionally, a decrease in the M1 surface marker CD86 and an increase in the M2 surface marker CD206 were observed, which suggested that M1 macrophages were polarized to an M2-like phenotype. Conditioned medium from these M2-like macrophages presented lower levels of proinflammatory cytokines and higher levels of proangiogenic factors and promoted endothelial cell proliferation, migration, and tube formation. Furthermore, M2 polarization and angiogenesis were induced upon the administration of exosomes in mouse Matrigel plug and hindlimb ischemia (HLI) models. Interestingly, these exosomal effects were attenuated by using a colony stimulating factor 1 receptor (CSF-1R) inhibitor, BLZ945, in vitro and in vivo. Downregulation of microRNA-21 (miR-21) in hypoxic ASCs reduced the exosomal effects on M2 polarization, Akt phosphorylation, and CSF-1 secretion. A similar reduction in exosomal activity was also observed when exosomes were administered along with BLZ945. CONCLUSION:Our findings provide evidence that exosomes from ASCs polarize macrophages toward an M2-like phenotype, which further enhances the exosomal proangiogenic effects. Exosomal delivery of miR-21 and positive feedback of secreted CSF-1 may be involved in macrophage polarization.

Project description:Tissue-engineered constructs need to become quickly vascularized in order to ensure graft take. One way of achieving this is to incorporate endothelial cells (EC) into the construct. The adipose tissue stromal vascular fraction (adipose-SVF) might provide an alternative source for endothelial cells as adipose tissue can easily be obtained by liposuction. Since adipose-EC are now gaining more interest in tissue engineering, we aimed to extensively characterize endothelial cells from adipose tissue (adipose-EC) and compare them with endothelial cells from dermis (dermal-EC). The amount of endothelial cells before purification varied between 4-16% of the total stromal population. After MACS selection for CD31 positive cells, a >99% pure population of endothelial cells was obtained within two weeks of culture. Adipose- and dermal-EC expressed the typical endothelial markers PECAM-1, ICAM-1, Endoglin, VE-cadherin and VEGFR2 to a similar extent, with 80-99% of the cell population staining positive. With the exception of CXCR4, which was expressed on 29% of endothelial cells, all other chemokine receptors (CXCR1, 2, 3, and CCR2) were expressed on less than 5% of the endothelial cell populations. Adipose-EC proliferated similar to dermal-EC, but responded less to the mitogens bFGF and VEGF. A similar migration rate was found for both adipose-EC and dermal-EC in response to bFGF. Sprouting of adipose-EC and dermal-EC was induced by bFGF and VEGF in a 3D fibrin matrix. After stimulation of adipose-EC and dermal-EC with TNF-α an increased secretion was seen for PDGF-BB, but not uPA, PAI-1 or Angiopoietin-2. Furthermore, secretion of cytokines and chemokines (IL-6, CCL2, CCL5, CCL20, CXCL1, CXCL8 and CXCL10) was also upregulated by both adipose- and dermal-EC. The similar characteristics of adipose-EC compared to their dermal-derived counterpart make them particularly interesting for skin tissue engineering. In conclusion, we show here that adipose tissue provides for an excellent source of endothelial cells for tissue engineering purposes, since they are readily available, and easily isolated and amplified.

Dataset Information

Comparison of Proangiogenic Effects of Adipose-Derived Stem Cells and Foreskin Fibroblast Exosomes on Artificial Dermis Prefabricated Flaps.

Publications

Comparison of Proangiogenic Effects of Adipose-Derived Stem Cells and Foreskin Fibroblast Exosomes on Artificial Dermis Prefabricated Flaps.

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