{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T19:18:05Z","timestamp":1790104685522,"version":"4.0.1"},"reference-count":49,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2024,11,21]],"date-time":"2024-11-21T00:00:00Z","timestamp":1732147200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100007479","name":"Fondazione Cariparo","doi-asserted-by":"publisher","award":["CALE_ECCE19_01"],"award-info":[{"award-number":["CALE_ECCE19_01"]}],"id":[{"id":"10.13039\/100007479","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007479","name":"Fondazione Cariparo","doi-asserted-by":"publisher","award":["21\/09 IRP"],"award-info":[{"award-number":["21\/09 IRP"]}],"id":[{"id":"10.13039\/100007479","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007363","name":"Fondazione Citt\u00e0 della Speranza","doi-asserted-by":"publisher","award":["CALE_ECCE19_01"],"award-info":[{"award-number":["CALE_ECCE19_01"]}],"id":[{"id":"10.13039\/100007363","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007363","name":"Fondazione Citt\u00e0 della Speranza","doi-asserted-by":"publisher","award":["21\/09 IRP"],"award-info":[{"award-number":["21\/09 IRP"]}],"id":[{"id":"10.13039\/100007363","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Microglia, the brain-resident immune cells, orchestrate neuroinflammatory responses and are crucial in the progression of neurological diseases, including ischemic stroke (IS), which accounts for approximately 85% of all strokes worldwide. Initially deemed detrimental, microglial activation has been shown to perform protective functions in the ischemic brain. Besides their effects on neurons, microglia play a role in promoting post-ischemic angiogenesis, a pivotal step for restoring oxygen and nutrient supply. However, the molecular mechanisms underlying microglia\u2013endothelial cell interactions remain largely unresolved, particularly in humans. Using both in vitro and in vivo models, we investigated the angiogenic signature and properties of extracellular vesicles (EVs) released by human microglia upon hypoxia\u2013reperfusion stimulation. EVs were isolated and characterized in terms of their size, concentration, and protein content. Their angiogenic potential was evaluated using endothelial cell assays and a zebrafish xenograft model. The in vivo effects were further assessed in a mouse model of ischemic stroke. Our findings identified key proteins orchestrating the pro-angiogenic functions of human microglial EVs under hypoxic conditions. In vitro assays demonstrated that hypoxic EVs (hypEVs) promoted endothelial cell migration and tube formation. In vivo, hypEVs induced vessel sprouting in zebrafish and increased microvessel density in the perilesional area of mice following ischemic stroke.<\/jats:p>","DOI":"10.3390\/ijms252312508","type":"journal-article","created":{"date-parts":[[2024,11,21]],"date-time":"2024-11-21T06:11:54Z","timestamp":1732169514000},"page":"12508","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Hypoxic Human Microglia Promote Angiogenesis Through Extracellular Vesicle Release"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-9330-4766","authenticated-orcid":false,"given":"Alessandra Maria","family":"Testa","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy"},{"name":"Laboratory of Immunity, Inflammation and Angiogenesis, Istituto di Ricerca Pediatrica (IRP), Fondazione Citt\u00e0 della Speranza, 35127 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1016-3279","authenticated-orcid":false,"given":"Livia","family":"Vignozzi","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7075-1414","authenticated-orcid":false,"given":"Diana","family":"Corallo","sequence":"additional","affiliation":[{"name":"Laboratory of Target Discovery and Biology of Neuroblastoma, Istituto di Ricerca Pediatrica (IRP), Fondazione Citt\u00e0 della Speranza, 35127 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3886-4360","authenticated-orcid":false,"given":"Sanja","family":"Aveic","sequence":"additional","affiliation":[{"name":"Laboratory of Target Discovery and Biology of Neuroblastoma, Istituto di Ricerca Pediatrica (IRP), Fondazione Citt\u00e0 della Speranza, 35127 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonella","family":"Viola","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5152-8225","authenticated-orcid":false,"given":"Manuela","family":"Allegra","sequence":"additional","affiliation":[{"name":"Laboratory of Neuronal Circuits in Developmental Disorders, Istituto di Ricerca Pediatrica (IRP), Fondazione Citt\u00e0 della Speranza, 35127 Padua, Italy"},{"name":"Neuroscience Institute, National Research Council, 35131 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberta","family":"Angioni","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy"},{"name":"Laboratory of Immunity, Inflammation and Angiogenesis, Istituto di Ricerca Pediatrica (IRP), Fondazione Citt\u00e0 della Speranza, 35127 Padua, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"921","DOI":"10.1007\/s12264-019-00388-3","article-title":"Dual Functions of Microglia in Ischemic Stroke","volume":"35","author":"Qin","year":"2019","journal-title":"Neurosci. Bull."},{"key":"ref_2","unstructured":"GBD 2016 Stroke Collaborators (2019). Global, regional, and national burden of stroke, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol., 18, 439\u2013458."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1007\/s00401-015-1524-y","article-title":"Microglia-blood vessel interactions: A double-edged sword in brain pathologies","volume":"131","author":"Teodorczyk","year":"2016","journal-title":"Acta Neuropathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3574","DOI":"10.1016\/j.celrep.2018.03.011","article-title":"Upregulation of Microglial ZEB1 Ameliorates Brain Damage after Acute Ischemic Stroke","volume":"22","author":"Li","year":"2018","journal-title":"Cell Rep."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Lian, L., Fu, R., Liu, J., Shan, X., Jin, Y., and Xu, S. (2022). Microglia: The Hub of Intercellular Communication in Ischemic Stroke. Front. Cell. Neurosci., 16.","DOI":"10.3389\/fncel.2022.889442"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5965","DOI":"10.1523\/JNEUROSCI.0060-08.2008","article-title":"Microglia cells protect neurons by direct engulfment of invading neutrophil granulocytes: A new mechanism of CNS immune privilege","volume":"28","author":"Neumann","year":"2008","journal-title":"J. Neurosci."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Raffaele, S., and Fumagalli, M. (2022). Dynamics of Microglia Activation in the Ischemic Brain: Implications for Myelin Repair and Functional Recovery. Front. Cell. Neurosci., 16.","DOI":"10.3389\/fncel.2022.950819"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1002\/dneu.22576","article-title":"Microglial interactions with the neurovascular system in physiology and pathology","volume":"78","author":"Zhao","year":"2018","journal-title":"Dev. Neurobiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.pneurobio.2013.11.004","article-title":"Vascular remodeling after ischemic stroke: Mechanisms and therapeutic potentials","volume":"115","author":"Liu","year":"2014","journal-title":"Prog. Neurobiol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Var, S.R., Shetty, A.V., Grande, A.W., Low, W.C., and Cheeran, M.C. (2021). Microglia and Macrophages in Neuroprotection, Neurogenesis, and Emerging Therapies for Stroke. Cells, 10.","DOI":"10.3390\/cells10123555"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.bbi.2014.03.003","article-title":"Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia\/macrophages and increased angiogenesis and neurogenesis following experimental stroke","volume":"40","author":"Jin","year":"2014","journal-title":"Brain Behav. Immun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2224","DOI":"10.1177\/0271678X17694185","article-title":"Depletion of microglia exacerbates postischemic inflammation and brain injury","volume":"37","author":"Jin","year":"2017","journal-title":"J. Cereb. Blood Flow Metab."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"11499","DOI":"10.1038\/ncomms11499","article-title":"Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke","volume":"7","author":"Szalay","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Gabrielli, M., Raffaele, S., Fumagalli, M., and Verderio, C. (2022). The multiple faces of extracellular vesicles released by microglia: Where are we 10 years after?. Front. Cell. Neurosci., 16.","DOI":"10.3389\/fncel.2022.984690"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1038\/nrn.2015.29","article-title":"Extracellular vesicles round off communication in the nervous system","volume":"17","author":"Budnik","year":"2016","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1016\/j.tins.2019.02.007","article-title":"Neuroimmune Crosstalk through Extracellular Vesicles in Health and Disease","volume":"42","author":"Delpech","year":"2019","journal-title":"Trends Neurosci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1186\/s12974-018-1204-7","article-title":"Inflammation leads to distinct populations of extracellular vesicles from microglia","volume":"15","author":"Yang","year":"2018","journal-title":"J. Neuroinflamm."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1186\/s12974-017-0819-4","article-title":"Microglial-derived microparticles mediate neuroinflammation after traumatic brain injury","volume":"14","author":"Kumar","year":"2017","journal-title":"J. Neuroinflamm."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"7268","DOI":"10.4049\/jimmunol.174.11.7268","article-title":"Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia","volume":"174","author":"Bianco","year":"2005","journal-title":"J. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Drago, F., Lombardi, M., Prada, I., Gabrielli, M., Joshi, P., Cojoc, D., Franck, J., Fournier, I., Vizioli, J., and Verderio, C. (2017). ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes. Front. Pharmacol., 8.","DOI":"10.3389\/fphar.2017.00910"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1161\/STROKEAHA.115.011932","article-title":"Modeling Ischemic Stroke In Vitro: Status Quo and Future Perspectives","volume":"47","author":"Holloway","year":"2016","journal-title":"Stroke"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e12404","DOI":"10.1002\/jev2.12404","article-title":"Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches","volume":"13","author":"Welsh","year":"2024","journal-title":"J. Extracell. Vesicles"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/s41590-023-01711-x","article-title":"Analysis of brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke","volume":"25","author":"Shahanoor","year":"2024","journal-title":"Nat. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1286","DOI":"10.1002\/iub.2275","article-title":"How hypoxia regulate exosomes in ischemic diseases and cancer microenvironment?","volume":"72","author":"Yaghoubi","year":"2020","journal-title":"IUBMB Life"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Jiang, H., Zhao, H., Zhang, M., He, Y., Li, X., Xu, Y., and Liu, X. (2022). Hypoxia Induced Changes of Exosome Cargo and Subsequent Biological Effects. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.824188"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e12002","DOI":"10.1002\/jev2.12002","article-title":"Hypoxia and extracellular vesicles: A review on methods, vesicular cargo and functions","volume":"10","author":"Bister","year":"2020","journal-title":"J. Extracell. Vesicles"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1007\/s00018-022-04357-4","article-title":"Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content with potential relevance to inflammation and recovery processes","volume":"79","author":"Bub","year":"2022","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4197","DOI":"10.7150\/thno.84059","article-title":"Preconditioned extracellular vesicles from hypoxic microglia reduce poststroke AQP4 depolarization, disturbed cerebrospinal fluid flow, astrogliosis, and neuroinflammation","volume":"13","author":"Xin","year":"2023","journal-title":"Theranostics"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Lecuyer, M., Pathipati, P., Faustino, J., and Vexler, Z.S. (2021). Neonatal stroke enhances interaction of microglia-derived extracellular vesicles with microglial cells. Neurobiol. Dis., 157.","DOI":"10.1016\/j.nbd.2021.105431"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s10456-008-9101-9","article-title":"Endoglin in angiogenesis and vascular diseases","volume":"11","author":"Goumans","year":"2008","journal-title":"Angiogenesis"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"199","DOI":"10.20517\/2347-8659.2017.18","article-title":"The roles of endoglin gene in cerebrovascular diseases","volume":"4","author":"Zhu","year":"2017","journal-title":"Neuroimmunol. Neuroinflamm."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"43799","DOI":"10.1074\/jbc.M207160200","article-title":"Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth factor-beta pathways","volume":"277","author":"Botella","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"588","DOI":"10.2183\/pjab.86.588","article-title":"The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine","volume":"86","author":"Nakamura","year":"2010","journal-title":"Proc. Jpn. Acad. Ser. B Phys. Biol. Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"S7","DOI":"10.1177\/1758834011422556","article-title":"An overview of the c-MET signaling pathway","volume":"3","author":"Organ","year":"2011","journal-title":"Ther. Adv. Med. Oncol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"283","DOI":"10.3892\/etm.2014.2133","article-title":"Therapeutic potential of hepatocyte growth factor against cerebral ischemia (Review)","volume":"9","author":"Zeng","year":"2015","journal-title":"Exp. Ther. Med."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Gorgun, C., Ceresa, D., Lesage, R., Villa, F., Reverberi, D., Balbi, C., Santamaria, S., Cortese, K., Malatesta, P., and Geris, L. (2021). Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs). Biomaterials, 269.","DOI":"10.1016\/j.biomaterials.2020.120633"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3267","DOI":"10.18632\/oncotarget.6572","article-title":"Artemin is hypoxia responsive and promotes oncogenicity and increased tumor initiating capacity in hepatocellular carcinoma","volume":"7","author":"Zhang","year":"2016","journal-title":"Oncotarget"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1038\/nrn812","article-title":"The GDNF family: Signalling, biological functions and therapeutic value","volume":"3","author":"Airaksinen","year":"2002","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Banerjee, A., Wu, Z.S., Qian, P.X., Kang, J., Liu, D.X., Zhu, T., and Lobie, P.E. (2012). ARTEMIN promotes de novo angiogenesis in ER negative mammary carcinoma through activation of TWIST1-VEGF-A signalling. PLoS ONE., 7.","DOI":"10.1371\/journal.pone.0050098"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1038\/s41419-021-04363-7","article-title":"Extracellular vesicles from hypoxia-preconditioned microglia promote angiogenesis and repress apoptosis in stroke mice via the TGF-\u03b2\/Smad2\/3 pathway","volume":"12","author":"Zhang","year":"2021","journal-title":"Cell Death Dis."},{"key":"ref_41","first-page":"e51729","article-title":"Modeling stroke in mice: Permanent coagulation of the distal middle cerebral artery","volume":"89","author":"Llovera","year":"2014","journal-title":"J. Vis. Exp."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1757900","DOI":"10.1080\/20013078.2020.1757900","article-title":"CD73+ extracellular vesicles inhibit angiogenesis through adenosine A2B receptor signalling","volume":"9","author":"Angioni","year":"2020","journal-title":"J. Extracell. Vesicles"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e12085","DOI":"10.1002\/jev2.12085","article-title":"Biodistribution of extracellular vesicles following administration into animals: A systematic review","volume":"10","author":"Kang","year":"2021","journal-title":"J. Extracell. Vesicles"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"26316","DOI":"10.3402\/jev.v4.26316","article-title":"Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting","volume":"4","author":"Wiklander","year":"2015","journal-title":"J. Extracell. Vesicles"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1186\/s12974-019-1660-8","article-title":"Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke","volume":"16","author":"Song","year":"2019","journal-title":"J. Neuroinflamm."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"421","DOI":"10.17219\/acem\/91826","article-title":"IL-4-polarized BV2 microglia cells promote angiogenesis by secreting exosomes","volume":"28","author":"Tian","year":"2019","journal-title":"Adv. Clin. Exp. Med."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Kanazawa, M., Miura, M., Toriyabe, M., Koyama, M., Hatakeyama, M., Ishikawa, M., Nakajima, T., Onodera, O., Takahashi, T., and Nishizawa, M. (2017). Microglia preconditioned by oxygen-glucose deprivation promote functional recovery in ischemic rats. Sci. Rep., 7.","DOI":"10.1038\/srep42582"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"17406","DOI":"10.1073\/pnas.0703446104","article-title":"High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish","volume":"104","author":"Stoletov","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2918","DOI":"10.1038\/nprot.2007.412","article-title":"The zebrafish\/tumor xenograft angiogenesis assay","volume":"2","author":"Nicoli","year":"2007","journal-title":"Nat. Protoc."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/23\/12508\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:36:43Z","timestamp":1760114203000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/23\/12508"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,21]]},"references-count":49,"journal-issue":{"issue":"23","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["ijms252312508"],"URL":"https:\/\/doi.org\/10.3390\/ijms252312508","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11,21]]}}}