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 2

ECFCs with high CD44 expression and their shed EVs rescue OIR mice.

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ECFCs with high CD44 expression and their shed EVs rescue OIR mice. (A a...
(A and B) CD44hi ECFCs rescued OIR mice. (A) Representative images and (B) quantification of NV (red) and VO (yellow) of retinal flat mounts from OIR mice. One-way ANOVA with Tukey’s; n = 7 retinas for CD44hi ECFCs, n = 7 retinas for CD44lo ECFCs, n = 5 retinas for HUVECs. (C and D) EVshi rescued OIR mice. (C) Representative images and (D) quantification of retinal flat mounts. Inserts in A and C depict the original unquantified images; scale bars: 1 mm. Additional controls included EVslo, HUVEC EVs, nonconditioned ECFC and HUVEC media subjected to UF-SEC-UF (XFM UF-SEC-UF and M200 UF-SEC-UF, respectively), and EVshi sample depleted of vesicles via overnight UC (EVshi depleted). Data in D are represented as a box-and-whisker plot where the top and bottom of the box represent mean of the upper and lower quartiles, horizontal line within the box represents the mean, and bars outside the box represent the min and max data points. One-way ANOVA with Tukey’s; n = 100 retinas for EVshi, n = 102 retinas for EVslo, n = 10 retinas for HUVEC EVs, n = 11 retinas for XFM UF-SEC-UF, n = 11 retinas for M200 UF-SEC-UF, n = 12 retinas for EVshi depleted. (E) Pharmacologic exosome inhibition of CD44hi ECFCs via GW4869 (20 μM) in DMSO (+GW4869, n = 12 retinas) attenuated the effects of CD44hi ECFCs compared with cells incubated with DMSO alone (-GW4869, n = 12 retinas). Two-tailed Student’s t test. (F and G) ECFCs-shCD44 and their EVs failed to rescue OIR mice. Quantification of NV and VO in mice injected with (F) ECFCs-scrRNA (n = 10 retinas) versus ECFCs-shCD44 (n = 11 retinas) and (G) EVs from ECFCs-scrRNA (n = 10 retinas) versus EVs from ECFCs-shCD44 (n = 11 retinas). Two-tailed Student’s t test. (H) Dose-response curve of OIR mice injected with EVshi. Mice were treated with a starting dose of 1.25 × 106 particles/0.5 μL/eye and serial 10-fold dilutions. Kruskal-Wallis test with Dunn’s multiple-comparison test; n = 6–9 retinas per group. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.