Extracellular Vesicle-Derived microRNAs of Human Wharton’s Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis
Abstract
:1. Introduction
2. Results
2.1. Isolation of MSCs and Immunophenotype Analysis
2.2. Quantitative Evaluation of the Secretome of Fetal Dermal MSCs, WJ- and UC-MSCs
2.3. In Vitro Angiogenesis Induced by Secretome and EVs of WJ-MSCs
2.4. Characterization of WJ-MSC EVs by Nanoparticle Tracking Analysis (NTA) and Western Blot
2.5. Differential Expression of EV-Derived miRNAs and Their Association with GO Biological Processes Related to Angiogenesis and Tissue Repair
2.6. Effect of the WJ-MSC Secretome and EVs on the Migration and Proliferation of HUVECs
2.7. Validation of miRNA Putative Target Genes, VEGF-A and THBS1 by Luminex and Western Blot
2.8. Cellular Uptake of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE)-Labeled EVs
3. Discussion
4. Materials and Methods
4.1. Tissue Procurement, Cell Isolation and Culture
4.2. Immunophenotype Analysis by Flow Cytometry
4.3. Harvesting of Secretome and Quantification of Soluble Factors by Luminex
4.4. In Vitro Angiogenesis Induced by Secretome and EVs of WJ-MSCs
4.5. Isolation of EVs and Physical Characterization by NTA
4.6. Quantification of Total Protein Amounts and Western Blot Analysis
4.7. EV miRNA Expression Profile with TaqMan Low-Density Array (TLDA)
4.8. Gene Ontology (GO) Enrichment Analysis of EV miRNAs and Biological Validation of Putative Target Genes
4.9. In Vitro Migration and Proliferation of HUVECs Induced by Secretome or EVs of WJ-MSCs
4.10. EV Labeling with Fluorescent Dye and Cellular Uptake Assay
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
WJ | Wharton’s jelly |
UC | Umbilical cord |
EVs | Extracellular vesicles |
GO | Gene Ontology |
THBS1 | Thrombospondin 1 |
NTA | Nanoparticle Tracking Analysis |
TLDA | TaqMan low-density array |
RTCA | Real-time cell analyzer |
CI | Cell index |
RIU | Relative Intensity Unit |
CFSE | carboxyfluorescein diacetate succinimidyl ester |
DAPI | 4′,6-diamidino-2-phenylindole |
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Soluble Factors (pg/mL/106 Cells/24 h) | Fetal Dermal MSC Secretome | WJ-MSC Secretome | UC-MSC Secretome | WJ-MSC EVs | UC-MSC EVs |
---|---|---|---|---|---|
VEGF-A | 5600 ± 150 | 170 ± 24 *** | 180 ± 18 *** | 100 ± 29 *** | 150 ± 30 *** |
SDF-1 alpha | 3120 ± 900 | 4190 ± 920 | 5725 ± 900 * | 5880 ± 120 * | 5040 ± 280 |
Selected GO Terms Associated with Angiogenesis (VEGF-A Target Gene) | log10 p-Value |
---|---|
lymph vessel morphogenesis | 0.029070346 |
coronary vein morphogenesis | 0.029070346 |
VEGF-activated neuropilin signaling pathway | 0.029070346 |
positive regulation of cell proliferation by VEGF-activated platelet derived growth factor receptor signaling pathway | 0.029070346 |
positive regulation of lymphangiogenesis | 0.029070346 |
positive regulation of endothelial cell chemotaxis by VEGF-activated vascular endothelial growth factor receptor signaling pathway | 0.029070346 |
coronary artery morphogenesis | 0.035697463 |
vascular endothelial growth factor signaling pathway | 0.035697463 |
positive regulation of vascular permeability | 0.035697463 |
endothelial cell chemotaxis | 0.035697463 |
positive regulation of cell migration involved in sprouting angiogenesis | 0.035697463 |
tube formation | 0.036090227 |
lung vasculature development | 0.037567858 |
positive regulation of vascular endothelial growth factor receptor signaling pathway | 0.042118988 |
induction of positive chemotaxis | 0.043066423 |
cell migration involved in sprouting angiogenesis | 0.043946017 |
positive regulation of blood vessel endothelial cell migration | 0.044267247 |
cellular response to vascular endothelial growth factor stimulus | 0.047669382 |
MiRNA Name | Role in Angiogenesis | References |
---|---|---|
hsa-let-7b-5p | it targets VEGF gene; validated role in angiogenesis | [39,40] |
hsa-let-7e-5p | validated role in angiogenesis | [40] |
hsa-miR-17-5p | angiogenesis promoter; it targets VEGF gene | [19,39,41] |
hsa-miR-21-5p | angiogenesis promoter | [41] |
hsa-miR-24-3p | validated role in angiogenesis; highly expressed by endothelial cells | [40,42] |
hsa-miR-31-5p | angiogenesis promoter | [43] |
hsa-miR-92a-3p | validated role in angiogenesis | [40,41] |
hsa-miR-99a-5p | highly expressed by endothelial cells | [42] |
hsa-miR-99b-3p | angiogenesis promoter | [44] |
hsa-miR-100-5p | angiogenesis promoter | [45] |
hsa-miR-106a-5p | it targets VEGF gene | [39] |
hsa-miR-125b-5p | angiogenesis promoter; tube formation of HUVECs | [46] |
hsa-miR-145-5p | angiogenesis promoter | [47] |
hsa-miR-146a-3p | it targets VEGF gene | [48] |
hsa-miR-191-5p | it may regulate the angiogenic actions of VEGF | [40] |
hsa-miR-193b-3p | it targets VEGF gene | [39] |
hsa-miR-199a-3p | it targets VEGF gene | [39] |
hsa-miR-214-3p | it targets VEGF gene | [39] |
hsa-miR-221-3p | validated role in angiogenesis | [40] |
hsa-miR-222-3p | angiogenesis in wound healing; validated role in angiogenesis | [40,49] |
hsa-miR-320a | it targets VEGF gene; validated role in angiogenesis | [39,40] |
hsa-miR-484 | it targets VEGF gene | [50] |
Selected GO Terms Associated with Angiogenesis and Tissue Repair (THBS1 Target Gene) | log10 p-Value |
---|---|
positive regulation of transforming growth factor beta1 production | 0.002834 |
positive regulation of transforming growth factor beta production | 0.002834 |
positive regulation of fibroblast migration | 0.002834 |
positive regulation of endothelial cell apoptotic process | 0.002834 |
positive regulation of chemotaxis | 0.002834 |
negative regulation of focal adhesion assembly | 0.002834 |
negative regulation of cell-matrix adhesion | 0.002905399 |
negative regulation of fibroblast growth factor receptor signaling pathway | 0.002971086 |
negative regulation of blood vessel endothelial cell migration | 0.003082249 |
positive regulation of blood vessel endothelial cell migration | 0.003082249 |
negative regulation of endothelial cell migration | 0.003082249 |
positive regulation of transforming growth factor beta receptor signaling pathway | 0.003884392 |
negative regulation of endothelial cell proliferation | 0.004108779 |
sprouting angiogenesis | 0.00471601 |
positive regulation of endothelial cell migration | 0.00471601 |
blood vessel morphogenesis | 0.00471601 |
positive regulation of cell-substrate adhesion | 0.00471601 |
negative regulation of extrinsic apoptotic signaling pathway | 0.004811209 |
cellular response to growth factor stimulus | 0.005970634 |
negative regulation of angiogenesis | 0.006063442 |
response to mechanical stimulus | 0.006624871 |
activation of MAPK activity | 0.007836227 |
positive regulation of angiogenesis | 0.009069898 |
positive regulation of cell migration | 0.012958161 |
response to hypoxia | 0.015327033 |
extracellular matrix organization | 0.015939502 |
cell migration | 0.015939502 |
cell motility | 0.023452526 |
negative regulation of apoptotic process | 0.034158772 |
cell adhesion | 0.045940676 |
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Chinnici, C.M.; Iannolo, G.; Cittadini, E.; Carreca, A.P.; Nascari, D.; Timoneri, F.; Bella, M.D.; Cuscino, N.; Amico, G.; Carcione, C.; et al. Extracellular Vesicle-Derived microRNAs of Human Wharton’s Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis. Int. J. Mol. Sci. 2021, 22, 2045. https://doi.org/10.3390/ijms22042045
Chinnici CM, Iannolo G, Cittadini E, Carreca AP, Nascari D, Timoneri F, Bella MD, Cuscino N, Amico G, Carcione C, et al. Extracellular Vesicle-Derived microRNAs of Human Wharton’s Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis. International Journal of Molecular Sciences. 2021; 22(4):2045. https://doi.org/10.3390/ijms22042045
Chicago/Turabian StyleChinnici, Cinzia Maria, Gioacchin Iannolo, Ettore Cittadini, Anna Paola Carreca, David Nascari, Francesca Timoneri, Mariangela Di Bella, Nicola Cuscino, Giandomenico Amico, Claudia Carcione, and et al. 2021. "Extracellular Vesicle-Derived microRNAs of Human Wharton’s Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis" International Journal of Molecular Sciences 22, no. 4: 2045. https://doi.org/10.3390/ijms22042045