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Page 8 of 14 Broadwin et al. Vessel Plus 2023;7:25 https://dx.doi.org/10.20517/2574-1209.2023.103

Figure 3. Network analysis. A “direct interactions” network tool in Metacore was used to plot known connections between the factors
(genes) in a list of DMLs in a comparison of ischemic vs. normal tissue on (A) normal diet, or (B) high-fat diet background.

Our enrichment analysis of methylated loci suggests that in a normal diet, ischemia alone can induce a
change in the methylome. By performing a pairwise comparison, we were further able to investigate the
independent effect of a high-fat diet on epigenetic response to cardiac ischemia. It has been well established
that metabolic syndrome alters the pathologic response to ischemic stress, and our results suggest that
alteration in the cardiac methylome may be a part of this maladaptive response. Treatment with EVs
appeared to have an independent effect on the methylome of normal diet swine and our swine model of
metabolic syndrome.

Previous work using the same experimental animals that were used in this study demonstrated profound
changes in ischemic myocardium in response to EV therapy. Immunofluorescent examination
demonstrated increased expression of arteriolar marker, SMA, and endothelial marker, CD31, in ischemic
myocardium treated with EVs compared to saline controls . This increase in both arteriolar and capillary
[15]
density prompted immunoblotting analysis of angiogenic signaling and revealed increased expression of
VEGR1, MAP kinase, and phosphorylated-MAP kinase, while significant downregulation in VEGFR2 was
[12]
observed . Previously, we also undertook an investigation of transcriptional mRNA expression in
chronically ischemic swine myocardium after EV treatment in HFD and normal diet swine. Interestingly,
EV treatment in ND resulted in gene enrichment in many metabolic pathways, including aerobic
respiration, electron transport chain, and oxidative phosphorylation, while differential expression in HFD
EV-treated swine centered around immune pathways .
[13]
In addition to the aforementioned observations previously published by our group, we have demonstrated
multiple physiologic alterations in response to EV therapy. EV treatment improved cardiac output, stroke
volume, and angiogenesis in our model of ischemic cardiac disease. Despite these profound effects, the full
extent of the mechanism by which they work remains unclear . These results suggest that further
[16]
investigation of epigenetic alterations induced by EV injection is warranted to elucidate the mechanism
behind their effect.

Ischemia effect
When investigating the effect of ischemia, our results demonstrated changes in molecular functions and
pathways involved in various metabolic pathways. Given the known metabolic shifts (i.e., glycolytic shift) in
cardiac ischemic, alteration of DNA methylation may represent one mechanism of metabolic response to