Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration
Kyle V. Marra, … , Susumu Sakimoto, Martin Friedlander
Kyle V. Marra, … , Susumu Sakimoto, Martin Friedlander
Published May 31, 2022
Citation Information: JCI Insight. 2022;7(12):e155928. https://doi.org/10.1172/jci.insight.155928.
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Research Article Ophthalmology Vascular biology

Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

Abstract

Disruption of the neurovascular unit (NVU) underlies the pathophysiology of various CNS diseases. One strategy to repair NVU dysfunction uses stem/progenitor cells to provide trophic support to the NVU’s functionally coupled and interdependent vasculature and surrounding CNS parenchyma. A subset of endothelial progenitor cells, endothelial colony-forming cells (ECFCs) with high expression of the CD44 hyaluronan receptor (CD44hi), provides such neurovasculotrophic support via a paracrine mechanism. Here, we report that bioactive extracellular vesicles from CD44hi ECFCs (EVshi) are paracrine mediators, recapitulating the effects of intact cell therapy in murine models of ischemic/neurodegenerative retinopathy; vesicles from ECFCs with low expression levels of CD44 (EVslo) were ineffective. Small RNA sequencing comparing the microRNA cargo from EVshi and EVslo identified candidate microRNAs that contribute to these effects. EVshi may be used to repair NVU dysfunction through multiple mechanisms to stabilize hypoxic vasculature, promote vascular growth, and support neural cells.

Authors

Kyle V. Marra, Edith Aguilar, Guoqin Wei, Ayumi Usui-Ouchi, Yoichiro Ideguchi, Susumu Sakimoto, Martin Friedlander

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Figure 1

ECFC culture and EV isolation.

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ECFC culture and EV isolation. (A–D) ECFC characterization and CD44 sort...
(A–D) ECFC characterization and CD44 sorting and KD. (A) Representative images of confluent ECFC colonies taken at 5× (top) and 10× (bottom) original magnification. Scale bar: 500 μm (top), 200 μm (bottom). (B) Immunophenotypic characterization of ECFCs. Representative flow cytometry histograms of ECFCs demonstrated positive expression of CD13, CD31, CD105, and HLA-ABC and negative expression of hematopoietic markers CD14 and CD45, mesenchymal stem cell marker CD90, as well as HLA-DR (right-shifted, black-filled curves in comparison with gray-filled curves representing the appropriate isotype controls; n = 3 replicates). (C) Representative gating strategy to sort CD44hi and CD44lo ECFCs using FACS. (D) Flow cytometric analysis of CD44 in ECFCs-shCD44 (black-filled curves) and ECFCs-scrRNA (gray-filled curves) following lentivirus-mediated transduction of ECFCs with shCD44. (E–H) EV isolation protocol, yield, morphology, and immunophenotype. (E) Schematic of UF-SEC-UF protocol. (F) UF-SEC-UF obtained a significantly higher EV yield than differential UC. Two-tailed Student’s t test; n = 4 EV isolations. Error bars represent SEM. (G) TEM of EV samples isolated via differential UC (left) or UF-SEC-UF (right). Differential UC samples demonstrated aggregation of macromolecules (red arrows) and EVs (yellow arrow). UF-SEC-UF produced EV samples devoid of contaminating aggregates. Scale bars: 0.2 μm. (H) Representative magnetic bead–assisted flow cytometry histograms of EVshi and EVslo. Both populations positively expressed tetraspanins CD9, CD63, and CD81, as well as endothelial marker CD31 (right-shifted, black filled curves compared with gray-filled curves of negative control samples; n = 3 replicates). *P < 0.05. MWCO, molecular weight cutoff.