Extracellular vesicular microRNAs as potential biomarker for early detection of doxorubicin-induced cardiotoxicity

Figures

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  Figure 1. Linear mixed model showed significant change in cTnI values over time for the 9 dogs enrolled in the study (P = .04). However, pairwise comparison between time points showed only statistically significant difference (P = .01) between pre‐DOX and post‐DOX. One month after treatment completion (post‐DOX), 1 patient had the highest cTnI level at 6.86 ng/mL. The dash line indicates the upper limit of normal for canine cTnI level. Large asterisk denotes significant difference. Open circles and small asterisk denote outliers

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  Figure 2. A, Western blot results showed that these EVs were positive for CD9. Canine MDCK cell‐derived EVs were used as positive control. B, Extracellular vesicle isolation was confirmed by transmission electron microscopy and by the presence of the typical “cup shaped” morphology. A higher resolution image is shown in the upper right hand corner

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  Figure 3. Venn diagram showing the miRNAs that were significantly up‐ or down‐regulated when comparing between pre‐DOX and during DOX (blue circle), and between pre‐DOX and post‐DOX (yellow circle). Down‐ and up‐regulating are represented by the up or down arrows. Overlapping region lists the shared miRNAs that were up‐ or down‐regulated for both time point comparisons. miRNAs that also showed significant change using linear mixed model analysis over all 3 time points are highlighted in red (n = 8). The table ranks (from lowest to highest) the 10 miRNAs with the lowest P‐values for each of the pairwise comparison

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  Figure 4. Box plot for the 3 miRNAs (miR‐107: top, P = .03; miR‐146a: middle, P = .02; miR‐502: bottom, P = .02) that showed statistically significant changes over time using both linear mixed model analysis and DESeq2 analysis. Pairwise comparison showed that the post‐DOX level of miR‐107 is significantly decreased from pre‐DOX level (P = 0.04), and similar changes were seen for miR‐146a (P = .01). On the other hand, miR‐502 levels increased significantly during DOX (P = .01) and post‐DOX (P = .003). Large asterisks denote statistical significance. Open circle and small asterisks denote outliers (n = 8)

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  Figure 5. Box plot representing miR‐181d level for all dogs with decreased LVEF (left, n = 5) and those with greater than 10% decrease in LVEF (right, n = 3) secondary to DOX treatment. Linear mixed model analysis showed significant upregulation over time (P = .01) for all dogs with decreased LVEF and also for dogs with greater than 10% decrease in LVEF (P = .02). Pairwise comparison also showed significant increase in miR‐181d when comparing pre‐DOX to post‐DOX dogs (P = .32). Large asterisk denotes statistical significance. Open circles denote outliers

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  Figure 6. Hierarchical cluster analysis (A) and principal component analysis (B) of miRNA levels as a function of treatment time points. Hierarchical cluster analysis showed greater separation of miRNA profiles between post‐DOX (time 3) and pre‐DOX (time 1) than between during DOX (time 2) and pre‐DOX (time 1). Principal component analysis showed that DOX treatment led to clustering of the miRNA profiles at both subsequent time points (n = 8)

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  Figure 7. Cardiac histopathology from DOX‐treated dogs shows lesions with characteristics of doxorubicin toxicity. A, Ventricular cardiomyocyte vacuolation (closed arrowheads) (hematoxylin and eosin), B, atrophic cardiomyocytes [hematoxylin and eosin], C, (phosphotungtic acid‐hematoxylin), D, cardiomyocyte loss with fatty change (between closed arrows) (hematoxylin and eosin), E, atrial interstitial fibrosis (open arrow) and cardiomyocyte vacuolation (closed arrowheads) (Trichrome), F, cellular disorganization, hypertrophic and hyperplastic arteriolar wall (between open arrowheads) (hematoxylin and eosin), G, H, normal cardiomyocytes shown for comparison (hematoxylin and eosin)