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. 2023 Mar 21:32:247-262.
doi: 10.1016/j.omtn.2023.03.006. eCollection 2023 Jun 13.

Circulating extracellular vesicles promote recovery in a preclinical model of intracerebral hemorrhage

Fernando Laso-García  1   2 , Laura Casado-Fernández  1 , Dolores Piniella  1   3 , Mari Carmen Gómez-de Frutos  1 , Jone Karmele Arizaga-Echebarria  1 , María Pérez-Mato  1 , Elisa Alonso-López  1 , Laura Otero-Ortega  1 , Susana Belén Bravo  4 , María Del Pilar Chantada-Vázquez  4 , José Avendaño-Ortiz  5 , Eduardo López-Collazo  5 , María Isabel Lumbreras-Herrera  6 , Angelo Gámez-Pozo  6 , Blanca Fuentes  1 , Exuperio Díez-Tejedor  1 , María Gutiérrez-Fernández  1 , María Alonso de Leciñana  1
Affiliations

Circulating extracellular vesicles promote recovery in a preclinical model of intracerebral hemorrhage

Fernando Laso-García et al. Mol Ther Nucleic Acids. .

Abstract

Circulating extracellular vesicles (EVs) are proposed to participate in enhancing pathways of recovery after stroke through paracrine signaling. To verify this hypothesis in a proof-of-concept study, blood-derived allogenic EVs from rats and xenogenic EVs from humans who experienced spontaneous good recovery after an intracerebral hemorrhage (ICH) were administered intravenously to rats at 24 h after a subcortical ICH. At 28 days, both treatments improved the motor function assessment scales score, showed greater fiber preservation in the perilesional zone (diffusion tensor-fractional anisotropy MRI), increased immunofluorescence markers of myelin (MOG), and decreased astrocyte markers (GFAP) compared with controls. Comparison of the protein cargo of circulating EVs at 28 days from animals with good vs. poor recovery showed down-expression of immune system activation pathways (CO4, KLKB1, PROC, FA9, and C1QA) and of restorative processes such as axon guidance (RAC1), myelination (MBP), and synaptic vesicle trafficking (SYN1), which is in line with better tissue preservation. Up-expression of PCSK9 (neuron differentiation) in xenogenic EVs-treated animals suggests enhancement of repair pathways. In conclusion, the administration of blood-derived EVs improved recovery after ICH. These findings open a new and promising opportunity for further development of restorative therapies to improve the outcomes after an ICH.

Keywords: MT: Special Issue - Exploiting Extracellular Vesicles as Therapeutic Agents; brain repair; extracellular vesicles; intracerebral hemorrhage; preclinical studies; proteomics; safety; treatment.

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Conflict of interest statement

A.G.-P. is a shareholder of Biomedica Molecular Medicine SL.

Figures

None
Graphical abstract
Figure 1
Figure 1
Extracellular vesicles (EVs) characterization (A) Presence of specific EVs markers (Alix, CD63, and CD81) and albumin as a purity control by western blot analysis after isolation. (B) Transmission electron microscopy (TEM) images showing the characteristic size and form of the EVs. (C) NTA showing the size and concentration of the EVs. AEVs, allogeneic extracellular vesicles; NTA, nanoparticle tracking analysis; XEVs, xenogenic extracellular vesicles.
Figure 2
Figure 2
Functional evaluation and brain imaging (A) Functional evaluation tests along the study time points. “Δ” indicates p < 0.05 in AEV-treated animals compared with the control group. “O” indicates p < 0.05 in the XEVs-treated animals compared with the control group. (B) T2-MRI images. Colored lines mark the total volume of lesion at 48 h and the residual lesion at 28 days surrounded by dilated cisterns as a result of tissue retraction. ∗ indicates p < 0.05. Data are shown as mean ± SD. AEVs, allogeneic extracellular vesicles; FA, fractional anisotropy; ICH, intracerebral hemorrhage; XEVs, xenogenic extracellular vesicles.
Figure 3
Figure 3
Signal detection of brain markers by immunofluorescence (A) Myelin (MOG), oligodendrocytes (OLIG-2), astrocytes (GFAP), and vascular (VEGF) antibody expression. (B) Quantification of brain marker expression. ∗ indicates p < 0.05. Data are shown as mean ± SD. AEVs, allogeneic extracellular vesicles; XEVs, xenogenic extracellular vesicles.
Figure 4
Figure 4
Immune response evaluation (A) Cytokine levels in rat plasma at baseline and at 3 h, 6 h, 24 h, and 48 h after treatment. (B) Macroscopic and microscopic examination of organs showed no signs of malignancy or neoformations. ∗ indicates p < 0.05. Data are shown as mean ± SD. AEVs, allogeneic extracellular vesicles; XEVs, xenogenic extracellular vesicles.
Figure 5
Figure 5
Protein content in EVs and outlining of their metabolic pathways Proteins and pathways up-expressed are marked in green, and those down-expressed in red. The volcano plots represent proteins present in EVs by study group comparisons. Biological pathways in which the differential proteins appear to be involved are shown in the pie charts on the right. Some biological pathways considered to be theoretically less involved in damage or repair mechanisms were grouped as “others” to ease the graphic representation of data (e.g., erythrocyte development, integrin signaling). (A) Control animals with good vs. poor spontaneous recovery. (B) Treated animals with AEVs vs. controls. (C) XEVs-treated vs. control animals. (D) AEVs-treated vs. XEVs-treated animals. AEVs, allogeneic extracellular vesicles; FC, fold change; XEVs, xenogenic extracellular vesicles.
Figure 6
Figure 6
Experimental procedure schemes (A) Design of the efficacy study. (B) Design of the safety study. AEVs, allogeneic EVs; EVs, extracellular vesicles; FE, functional evaluation; IF, immunofluorescence; MRI, magnetic resonance imaging; XEVs, xenogenic EVs.
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