Endothelial extracellular vesicles contain protective proteins and rescue ischemia-reperfusion injury in a human heart-on-chip
Extracellular vesicles (EVs) derived from various stem cell sources induce cardioprotective effects during ischemia-reperfusion injury (IRI). These have been attributed mainly to the antiapoptotic, proangiogenic, microRNA (miRNA) cargo within the stem cell–derived EVs. However, the mechanisms of EV-mediated endothelial signaling to cardiomyocytes, as well as their therapeutic potential toward ischemic myocardial injury, are not clear. EV content beyond miRNA that may contribute to cardioprotection has not been fully illuminated. This study characterized the protein cargo of human vascular endothelial EVs (EEVs) to identify lead cardioactive proteins and assessed the effect of EEVs on human laminar cardiac tissues (hlCTs) exposed to IRI. They mapped the protein content of human vascular EEVs and identified proteins that were previously associated with cellular metabolism, redox state, and calcium handling, among other processes. Analysis of the protein landscape of human cardiomyocytes revealed corresponding modifications induced by EEV treatment. To assess their human-specific cardioprotection in vitro, they developed a human heart-on-a-chip IRI assay using human stem cell–derived, engineered cardiac tissues. They found that EEVs alleviated cardiac cell death as well as the loss in contractile capacity during and after simulated IRI in an uptake- and dose-dependent manner. Moreover, they found that EEVs increased the respiratory capacity of normoxic cardiomyocytes. These results suggest that vascular EEVs rescue hlCTs exposed to IRI possibly by supplementing injured myocytes with cargo that supports multiple metabolic and salvage pathways and therefore may serve as a multitargeted therapy for IRI.
(A) Proteomaps illustrating the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology terms and relative differences in mass abundance for the 1820 proteins identified in normoxic (left, N = 4) and hypoxic (right, N = 2) EVs. (B) Samplewise Pearson’s correlation analysis revealed a high degree of similarity between the global expression profiles for the normoxic (Norm) and hypoxic (Hypo) EV samples. Correlation coefficients between the four normoxic samples were greater than 0.9 and 0.87 for the two hypoxic samples. GO enrichment analysis identified statistically overrepresented (C) cellular localization and (D) biological function in the protein expression dataset. Black vertical lines indicate P value of 0.05.
(A) Proteomap illustrating the KEGG orthology terms and relative differences in mass abundance for the significantly (P < 0.05) differentially expressed proteins (DEPs) in EEV-treated (N = 3 samples), compared with untreated (N = 2 samples), hCMs. (B) Volcano plot presenting the fold change and P value of the DEPs. The red dots represent proteins that were significantly overexpressed in the EEV-treated myocytes and also enriched in the EEVs. (C) Venn diagram demonstrating the amount of the DEPs in the EEV-treated myocytes, relative to the normoxic EEVs’ proteome, and the overlap. (D) Cellular localization in the protein expression dataset. The numbers next to the bars represent the number of significantly (P ≤ 0.05) enriched proteins related to each of the depicted cellular compartments. (E) Dominant biological processes in the DEPs. Each dot represents a single protein, whereas the clusters represent biological processes. The red color scale represents the individual P value for the enrichment of each protein in the dataset. The dashed horizontal line represents 0-fold change, whereas all the dots above the line represent overexpressed proteins. The numbers above each cluster represent the false discovery rate, namely, the confidence level in the representation of the specific biological process. (F) Oxygen consumption rate (OCR; mean ± SEM) of hCMs treated with vehicle (N = 6), normoxic (Norm) EEVs (N = 6), and hypoxic (Hyp) EEVs (N = 6) was measured using Seahorse extracellular flux analyzer while altering mitochondrial function to delineate the different components of respiration. (G) Spare respiratory capacity (mean ± SEM) was calculated as the difference between the maximum and baseline OCR. Significance relative to untreated control was tested with one-way analysis of variance (ANOVA). Statistical significance was presented by *P ≤ 0.05, **P ≤ 0.01.
BY MORAN YADID, JOHAN U. LIND, HERDELINE ANN M. ARDOÑA, SEAN P. SHEEHY, LAUREN E. DICKINSON, FEYISAYO EWEJE, MAARTJE M.C. BASTINGS, BENJAMIN POPE, BLAKELY B. O’CONNOR, JUERG R. STRAUBHAAR, BOGDAN BUDNIK, ANDRE G. KLEBER, KEVIN KIT PARKER
SCIENCE TRANSLATIONAL MEDICINE 14 OCT 2020
DOI: 10.1126/scitranslmed.aax8005
Endothelial extracellular vesicles containing cardioprotective proteins rescue engineered human cardiac tissue in an ischemia-reperfusion injury model.