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image of Neuroprotective Proteins in Hypoxia-stressed Astrocyte-Derived Extracellular Vesicles

Abstract

Background

Advances in mass spectrometry-based proteomic analysis have generated extensive protein data from cells involved in neurodegenerative diseases. The field of neuroproteomics is expanding to include the study of extracellular vesicles (EVs) to identify potential biomarkers for disease prevention and endogenous factors involved in neuroprotection.

Methods

In this study, rat cortical astrocytes in normoxia were cultured under normoxic conditions and subsequently exposed to hypoxia. Astrocyte-derived EVs released into the supernatant were collected separately from both conditions. Label-free mass spectrometry-based proteomics was then performed to assess the effects of hypoxia on the EV protein cargo. A meta-analysis comparing the results with previously published EV proteomic datasets was also conducted.

Results

This study revealed a differential expression of 83 upregulated proteins under hypoxic conditions and 61 downregulated proteins under normoxic conditions, highlighting the protective protein signatures elicited by astrocytes. The dataset has been deposited in the ProteomeXchange Consortium with the identified PXD050160.

Conclusion

The present study makes a novel contribution by employing proteomic techniques to characterize the protein cargo of EVs isolated from primary rat astrocytes. This approach enables a more refined analysis of astrocyte-specific intercellular signaling under hypoxic conditions and provides valuable insights into the roles of astrocytes in maintaining brain homeostasis and contributing to pathological processes.

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2025-06-19
2025-10-15
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References

  1. Pantazopoulou M. Lamprokostopoulou A. Karampela D.S. Alexaki A. Delis A. Coens A. Samiotaki M. Kriebardis A.G. Melki R. Pagakis S.N. Stefanis L. Vekrellis K. Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes. Cell. Mol. Life Sci. 2023 80 7 193 10.1007/s00018‑023‑04841‑5 37391572
    [Google Scholar]
  2. Khan N.A. Asim M. El-Menyar A. Biswas K.H. Rizoli S. Al-Thani H. The evolving role of extracellular vesicles (exosomes) as biomarkers in traumatic brain injury: Clinical perspectives and therapeutic implications. Front. Aging Neurosci. 2022 14 933434 10.3389/fnagi.2022.933434
    [Google Scholar]
  3. Lizarraga-Valderrama L.R. Sheridan G.K. Extracellular vesicles and intercellular communication in the central nervous system. FEBS Lett. 2021 595 10 1391 1410 10.1002/1873‑3468.14074 33728650
    [Google Scholar]
  4. Vinaiphat A. Sze S.K. Proteomics for comprehensive characterization of extracellular vesicles in neurodegenerative disease. Exp. Neurol. 2022 355 114149 10.1016/j.expneurol.2022.114149 35732219
    [Google Scholar]
  5. Allen S.P. Seehra R.S. Heath P.R. Hall B.P.C. Bates J. Garwood C.J. Matuszyk M.M. Wharton S.B. Simpson J.E. Transcriptomic analysis of human astrocytes in vitro reveals hypoxia-induced mitochondrial dysfunction, modulation of metabolism, and dysregulation of the immune response. Int. J. Mol. Sci. 2020 21 21 8028 10.3390/ijms21218028 33126586
    [Google Scholar]
  6. Vangeison G. Rempe D.A. The Janus-faced effects of hypoxia on astrocyte function. Neuroscientist 2009 15 6 579 588 10.1177/1073858409332405 19359669
    [Google Scholar]
  7. Baumann J. Tsao C.C. Huang S.F. Gassmann M. Ogunshola O.O. Astrocyte-specific hypoxia-inducible factor 1 (HIF-1) does not disrupt the endothelial barrier during hypoxia in vitro. Fluids Barriers CNS 2021 18 1 13 10.1186/s12987‑021‑00247‑2 33736658
    [Google Scholar]
  8. Guo M. Ma X. Feng Y. Han S. Dong Q. Cui M. Zhao Y. In chronic hypoxia, glucose availability and hypoxic severity dictate the balance between HIF-1 and HIF-2 in astrocytes. FASEB J. 2019 33 10 11123 11136 10.1096/fj.201900402RR 31298941
    [Google Scholar]
  9. Schwanhäusser B. Busse D. Li N. Dittmar G. Schuchhardt J. Wolf J. Chen W. Selbach M. Global quantification of mammalian gene expression control. Nature 2011 473 7347 337 342 10.1038/nature10098 21593866
    [Google Scholar]
  10. Kowal J. Arras G. Colombo M. Jouve M. Morath J.P. Primdal-Bengtson B. Dingli F. Loew D. Tkach M. Théry C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl. Acad. Sci. USA 2016 113 8 E968 E977 10.1073/pnas.1521230113 26858453
    [Google Scholar]
  11. Heras-Romero Y. Morales-Guadarrama A. Santana-Martínez R. Ponce I. Rincón-Heredia R. Poot-Hernández A.C. Martínez-Moreno A. Urrieta E. Bernal-Vicente B.N. Campero-Romero A.N. Moreno-Castilla P. Greig N.H. Escobar M.L. Concha L. Tovar-y-Romo L.B. Improved post-stroke spontaneous recovery by astrocytic extracellular vesicles. Mol. Ther. 2022 30 2 798 815 10.1016/j.ymthe.2021.09.023 34563674
    [Google Scholar]
  12. Campero-Romero A.N. Real F.H. Santana-Martínez R.A. Molina-Villa T. Aranda C. Ríos-Castro E. Tovar-y-Romo L.B. Extracellular vesicles from neural progenitor cells promote functional recovery after stroke in mice with pharmacological inhibition of neurogenesis. Cell Death Discov. 2023 9 1 272 10.1038/s41420‑023‑01561‑4 37507361
    [Google Scholar]
  13. Phan N.V. Rathbun E.M. Ouyang Y. Carmichael S.T. Segura T. Biology-driven material design for ischaemic stroke repair. Nat. Rev. Bioeng. 2023 2 1 44 63 10.1038/s44222‑023‑00117‑6
    [Google Scholar]
  14. Percie du Sert N. Hurst V. Ahluwalia A. Alam S. Avey M.T. Baker M. Browne W.J. Clark A. Cuthill I.C. Dirnagl U. Emerson M. Garner P. Holgate S.T. Howells D.W. Karp N.A. Lazic S.E. Lidster K. MacCallum C.J. Macleod M. Pearl E.J. Petersen O.H. Rawle F. Reynolds P. Rooney K. Sena E.S. Silberberg S.D. Steckler T. Würbel H. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 2020 18 7 e3000410 10.1371/journal.pbio.3000410 32663219
    [Google Scholar]
  15. Ríos-Castro E. Souza G.H.M.F. Delgadillo-Álvarez D.M. Ramírez-Reyes L. Torres-Huerta A.L. Velasco-Suárez A. Cruz-Cruz C. Hernández-Hernández J.M. Tapia-Ramírez J. Quantitative proteomic analysis of MARC-145 cells infected with a mexican porcine reproductive and respiratory syndrome virus strain using a label-free based DIA approach. J. Am. Soc. Mass Spectrom. 2020 31 6 1302 1312 10.1021/jasms.0c00134 32379441
    [Google Scholar]
  16. Delgadillo D.M. Céspedes-Cruz A.I. Ríos-Castro E. Maldonado R.M.G. López-Nogueda M. Márquez-Gutiérrez M. Villalobos-Manzo R. Ramírez-Reyes L. Domínguez-Fuentes M. Tapia-Ramírez J. Differential expression of proteins in an atypical presentation of autoimmune lymphoproliferative syndrome. Int. J. Mol. Sci. 2022 23 10 5366 10.3390/ijms23105366 35628184
    [Google Scholar]
  17. Li G.Z. Vissers J.P.C. Silva J.C. Golick D. Gorenstein M.V. Geromanos S.J. Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Proteomics 2009 9 6 1696 1719 10.1002/pmic.200800564 19294629
    [Google Scholar]
  18. Käll L. Storey J.D. MacCoss M.J. Noble W.S. Assigning significance to peptides identified by tandem mass spectrometry using decoy databases. J. Proteome Res. 2008 7 1 29 34 10.1021/pr700600n 18067246
    [Google Scholar]
  19. Perez-Riverol Y. Bai J. Bandla C. García-Seisdedos D. Hewapathirana S. Kamatchinathan S. Kundu D.J. Prakash A. Frericks-Zipper A. Eisenacher M. Walzer M. Wang S. Brazma A. Vizcaíno J.A. The PRIDE database resources in 2022: A hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022 50 D1 D543 D552 10.1093/nar/gkab1038 34723319
    [Google Scholar]
  20. Guo H. Fan Z. Wang S. Ma L. Wang J. Yu D. Zhang Z. Wu L. Peng Z. Liu W. Hou W. Cai Y. Astrocytic A1/A2 paradigm participates in glycogen mobilization mediated neuroprotection on reperfusion injury after ischemic stroke. J. Neuroinflammation 2021 18 1 230 10.1186/s12974‑021‑02284‑y 34645472
    [Google Scholar]
  21. Kyung J.W. Cho I.H. Lee S. Song W.K. Ryan T.A. Hoppa M.B. Kim S.H. Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons. Sci. Rep. 2017 7 1 41620 10.1038/srep41620 28139716
    [Google Scholar]
  22. Jeppesen D.K. Fenix A.M. Franklin J.L. Higginbotham J.N. Zhang Q. Zimmerman L.J. Liebler D.C. Ping J. Liu Q. Evans R. Fissell W.H. Patton J.G. Rome L.H. Burnette D.T. Coffey R.J. Reassessment of exosome composition. Cell 2019 177 2 428 445.e18 10.1016/j.cell.2019.02.029 30951670
    [Google Scholar]
  23. Deng F. Miller J. A review on protein markers of exosome from different bio-resources and the antibodies used for characterization. J. Histotechnol. 2019 42 4 226 239 10.1080/01478885.2019.1646984 31432761
    [Google Scholar]
  24. Morel L. Regan M. Higashimori H. Ng S.K. Esau C. Vidensky S. Rothstein J. Yang Y. Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1. J. Biol. Chem. 2013 288 10 7105 7116 10.1074/jbc.M112.410944 23364798
    [Google Scholar]
  25. Siman R. Roberts V.L. McNeil E. Dang A. Bavaria J.E. Ramchandren S. McGarvey M. Biomarker evidence for mild central nervous system injury after surgically-induced circulation arrest. Brain Res. 2008 1213 1 11 10.1016/j.brainres.2008.03.034 18456245
    [Google Scholar]
  26. Shimada T. Fournier A.E. Yamagata K. Neuroprotective function of 14-3-3 proteins in neurodegeneration. BioMed Res. Int. 2013 2013 1 11 10.1155/2013/564534 24364034
    [Google Scholar]
  27. Semenza G.L. Hypoxia-inducible factors in physiology and medicine. Cell 2012 148 3 399 408 10.1016/j.cell.2012.01.021 22304911
    [Google Scholar]
  28. Hartl F.U. Hayer-Hartl M. Molecular chaperones in the cytosol: From nascent chain to folded protein. Science 2002 295 5561 1852 1858 10.1126/science.1068408 11884745
    [Google Scholar]
  29. Hochrainer K. Yang W. Stroke proteomics: From discovery to diagnostic and therapeutic applications. Circ. Res. 2022 130 8 1145 1166 10.1161/CIRCRESAHA.122.320110 35420912
    [Google Scholar]
  30. Li L. Li R. Zacharek A. Wang F. Landschoot-Ward J. Chopp M. Chen J. Cui X. ABCA1/ApoE/HDL signaling pathway facilitates myelination and oligodendrogenesis after stroke. Int. J. Mol. Sci. 2020 21 12 4369 10.3390/ijms21124369 32575457
    [Google Scholar]
  31. Jong D.O.G. Verhaar M.C. Chen Y. Vader P. Gremmels H. Posthuma G. Schiffelers R.M. Gucek M. Balkom V.B.W.M. Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes. J. Extracell. Vesicles 2012 1 1 9 10.3402/jev.v1i0.18396
    [Google Scholar]
  32. Lee J.Y. Kim E. Choi S.M. Kim D.W. Kim K.P. Lee I. Kim H.S. Microvesicles from brain-extract—treated mesenchymal stem cells improve neurological functions in a rat model of ischemic stroke. Sci. Rep. 2016 6 1 33038 10.1038/srep33038 27609711
    [Google Scholar]
  33. Couch Y. Akbar N. Davis S. Fischer R. Dickens A.M. Neuhaus A.A. Burgess A.I. Rothwell P.M. Buchan A.M. Inflammatory stroke extracellular vesicles induce macrophage activation. Stroke 2017 48 8 2292 2296 10.1161/STROKEAHA.117.017236 28536169
    [Google Scholar]
  34. Dickens A.M. Tovar-y-Romo L.B. Yoo S.W. Trout A.L. Bae M. Kanmogne M. Megra B. Williams D.W. Witwer K.W. Gacias M. Tabatadze N. Cole R.N. Casaccia P. Berman J.W. Anthony D.C. Haughey N.J. Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions. Sci. Signal. 2017 10 473 eaai7696 10.1126/scisignal.aai7696 28377412
    [Google Scholar]
  35. Cosme J. Guo H. Hadipour-Lakmehsari S. Emili A. Gramolini A.O. Hypoxia-induced changes in the fibroblast secretome, exosome, and whole-cell proteome using cultured, cardiac-derived cells isolated from neonatal mice. J. Proteome Res. 2017 16 8 2836 2847 10.1021/acs.jproteome.7b00144 28641008
    [Google Scholar]
  36. Chandran I.V. Welinder C. Gonçalves de Oliveira K. Cerezo-Magaña M. Månsson A.S. Johansson M.C. Marko-Varga G. Belting M. Global extracellular vesicle proteomic signature defines U87-MG glioma cell hypoxic status with potential implications for non-invasive diagnostics. J. Neurooncol. 2019 144 3 477 488 10.1007/s11060‑019‑03262‑4 31414377
    [Google Scholar]
  37. Wang X. Wang J. Shi X. Pan C. Liu H. Dong Y. Dong R. Mang J. Xu Z. Proteomic analyses identify a potential mechanism by which extracellular vesicles aggravate ischemic stroke. Life Sci. 2019 231 116527 10.1016/j.lfs.2019.06.002 31176783
    [Google Scholar]
  38. You Y. Borgmann K. Edara V.V. Stacy S. Ghorpade A. Ikezu T. Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing. J. Extracell. Vesicles 2020 9 1 1706801 10.1080/20013078.2019.1706801 32002171
    [Google Scholar]
  39. Deitmer J.W. Theparambil S.M. Ruminot I. Noor S.I. Becker H.M. Energy dynamics in the brain: Contributions of astrocytes to metabolism and ph homeostasis. Front. Neurosci. 2019 13 1301 10.3389/fnins.2019.01301 31866811
    [Google Scholar]
  40. Li J. Pan L. Pembroke W.G. Rexach J.E. Godoy M.I. Condro M.C. Alvarado A.G. Harteni M. Chen Y.W. Stiles L. Chen A.Y. Wanner I.B. Yang X. Goldman S.A. Geschwind D.H. Kornblum H.I. Zhang Y. Conservation and divergence of vulnerability and responses to stressors between human and mouse astrocytes. Nat. Commun. 2021 12 1 3958 10.1038/s41467‑021‑24232‑3 34172753
    [Google Scholar]
  41. Smolič T. Tavčar P. Horvat A. Černe U. Vasle H.A. Tratnjek L. Kreft M.E. Scholz N. Matis M. Petan T. Zorec R. Vardjan N. Astrocytes in stress accumulate lipid droplets. Glia 2021 69 6 1540 1562 10.1002/glia.23978 33609060
    [Google Scholar]
  42. Yamagata K. Lactate supply from astrocytes to neurons and its role in ischemic stroke-induced neurodegeneration. Neuroscience 2022 481 219 231 10.1016/j.neuroscience.2021.11.035 34843897
    [Google Scholar]
  43. Genc S. Kurnaz I.A. Ozilgen M. Astrocyte - neuron lactate shuttle may boost more ATP supply to the neuron under hypoxic conditions - in silico study supported by in vitro expression data. BMC Syst. Biol. 2011 5 1 162 10.1186/1752‑0509‑5‑162 21995951
    [Google Scholar]
  44. Pantazopoulou V. Jeannot P. Rosberg R. Berg T.J. Pietras A. Hypoxia-induced reactivity of tumor-associated astrocytes affects glioma cell properties. Cells 2021 10 3 613 10.3390/cells10030613 33802060
    [Google Scholar]
  45. Mojsilovic-Petrovic J. Callaghan D. Cui H. Dean C. Stanimirovic D.B. Zhang W. Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes. J. Neuroinflammation 2007 4 1 12 10.1186/1742‑2094‑4‑12 17474992
    [Google Scholar]
  46. Perriot S. Mathias A. Perriard G. Canales M. Jonkmans N. Merienne N. Meunier C. Kassar E.L. Perrier A.L. Laplaud D.A. Schluep M. Déglon N. Pasquier D.R. Human induced pluripotent stem cell-derived astrocytes are differentially activated by multiple sclerosis-associated cytokines. Stem Cell Reports 2018 11 5 1199 1210 10.1016/j.stemcr.2018.09.015 30409508
    [Google Scholar]
  47. Zamanian J.L. Xu L. Foo L.C. Nouri N. Zhou L. Giffard R.G. Barres B.A. Genomic analysis of reactive astrogliosis. J. Neurosci. 2012 32 18 6391 6410 10.1523/JNEUROSCI.6221‑11.2012 22553043
    [Google Scholar]
  48. Khakh B.S. Sofroniew M.V. Diversity of astrocyte functions] and phenotypes in neural circuits. Nat. Neurosci. 2015 18 7 942 952 10.1038/nn.4043 26108722
    [Google Scholar]
  49. Neal M. Richardson J.R. Epigenetic regulation of astrocyte function in neuroinflammation and neurodegeneration. Biochim. Biophys. Acta Mol. Basis Dis. 2018 1864 2 432 443 10.1016/j.bbadis.2017.11.004 29113750
    [Google Scholar]
  50. Thompson J.W. Dave K.R. Young J.I. Perez-Pinzon M.A. Ischemic preconditioning alters the epigenetic profile of the brain from ischemic intolerance to ischemic tolerance. Neurotherapeutics 2013 10 4 789 797 10.1007/s13311‑013‑0202‑9 23868468
    [Google Scholar]
  51. Liddelow S.A. Guttenplan K.A. Clarke L.E. Bennett F.C. Bohlen C.J. Schirmer L. Bennett M.L. Münch A.E. Chung W.S. Peterson T.C. Wilton D.K. Frouin A. Napier B.A. Panicker N. Kumar M. Buckwalter M.S. Rowitch D.H. Dawson V.L. Dawson T.M. Stevens B. Barres B.A. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 2017 541 7638 481 487 10.1038/nature21029 28099414
    [Google Scholar]
  52. Wheeler M.A. Jaronen M. Covacu R. Zandee S.E.J. Scalisi G. Rothhammer V. Tjon E.C. Chao C.C. Kenison J.E. Blain M. Rao V.T.S. Hewson P. Barroso A. Gutiérrez-Vázquez C. Prat A. Antel J.P. Hauser R. Quintana F.J. Environmental control of astrocyte pathogenic activities in cns inflammation. Cell 2019 176 3 581 596.e18 10.1016/j.cell.2018.12.012 30661753
    [Google Scholar]
  53. Kaur C. Sivakumar V. Zhang Y. Ling E.A. Hypoxia-induced astrocytic reaction and increased vascular permeability in the rat cerebellum. Glia 2006 54 8 826 839 10.1002/glia.20420 16977604
    [Google Scholar]
  54. Wang Y. Fu A.K.Y. Ip N.Y. Instructive roles of astrocytes in hippocampal synaptic plasticity: Neuronal activity-dependent regulatory mechanisms. FEBS J. 2022 289 8 2202 2218 10.1111/febs.15878 33864430
    [Google Scholar]
  55. Wang Y. Fu W.Y. Cheung K. Hung K.W. Chen C. Geng H. Yung W.H. Qu J.Y. Fu A.K.Y. Ip N.Y. Astrocyte-secreted IL-33 mediates homeostatic synaptic plasticity in the adult hippocampus. Proc. Natl. Acad. Sci. USA 2021 118 1 e2020810118 10.1073/pnas.2020810118 33443211
    [Google Scholar]
  56. Ito M. Aswendt M. Lee A.G. Ishizaka S. Cao Z. Wang E.H. Levy S.L. Smerin D.L. McNab J.A. Zeineh M. Leuze C. Goubran M. Cheng M.Y. Steinberg G.K. RNA-sequencing analysis revealed a distinct motor cortex transcriptome in spontaneously recovered mice after stroke. Stroke 2018 49 9 2191 2199 10.1161/STROKEAHA.118.021508 30354987
    [Google Scholar]
  57. Quiroz-Baez R. Hernández-Ortega K. Martínez-Martínez E. Insights into the proteomic profiling of extracellular vesicles for the identification of early biomarkers of neurodegeneration. Front. Neurol. 2020 11 580030 10.3389/fneur.2020.580030 33362690
    [Google Scholar]
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  • Article Type:
    Research Article
Keywords: neuroprotection ; EV cargo ; Astrocyte ; hypoxia ; stroke ; proteomics ; extracellular vesicles
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