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Broadwin et al. Vessel Plus 2023;7:25 https://dx.doi.org/10.20517/2574-1209.2023.103 Page 11 of 14

altered histone methylation patterns in vascular tissue. Specifically, one example of this is alterations in the
methylation of genes coding for eNOS, which have been implicated in atherosclerotic heart disease in
[33]
humans . Interestingly, as with the Dutch Hunger, these epigenetic changes appear to be stable and
[33]
transfer to subsequent generations of mice . While no individual gene reached significance, enrichment
analysis of our nominal data revealed an altered methylation in pathways involved in lipid metabolism, and
angiogenesis. Given that alteration of the methylome of these pathways has been implicated in multiple
tissue types across a variety of animal modules of cardiovascular disease, their epigenetic regulation may
represent a key area of investigation into the physiologic response to cardiovascular insult.

EV effect in ND
Our analysis of the EV effect also demonstrated a significant alteration in methylation of the FN3KRP gene,
which codes for a protein involved in the reversal of glycation, a process known to play a part in arterial
stiffening . It is possible that this housekeeping gene is methylated as a maladaptive response to ischemic
[34]
stress and merits further investigation. Previous studies have investigated alteration in the methylation
[11]
pattern of DNA in EVs of patients with ACS . Furthermore, one group investigated the effect of ACS EVs
on PMBCs and demonstrated that EVs did alter the DNA methylation profile of PMBCs. Enrichment
analysis of pathways with altered methylation patterns included Wnt signaling pathway, cadherin signaling
pathway, angiogenesis, and PI3 Kinase pathways . Much like PMBCs altered by treatment with ACS EVs,
[11]
we observed enrichment analysis of our EV-treated normal diet swine myocytes demonstrated alteration of
the Wnt pathway [Figure 2C]. However, our analysis did not reveal any alteration to the cadherin signaling
pathway, angiogenesis, or the PI3 kinase pathway, suggesting that myocytes and blood may not have the
same epigenetic response to ischemic stress. Given this, we believe methylome alteration may be in part
responsible for tissue-specific response to ischemic insult, though this will require further study.

EV effect in HFD
Previously, our lab demonstrated an improvement in diastolic function and increased expression of eNOS,
VEGFR2, and MAPK ERK1/ERK2 in an EV-treated MetS swine model of chronic ischemia [13,15] . Studies in
mice have also demonstrated EVs can play a critical role in the pathogenesis of insulin resistance via
alteration in AhR signaling . Despite this, the exact mechanisms driving these changes remain unclear. In
[35]
this study, within the EV HFD group, we were able to show alteration in methylation of the PCSK7 gene,
which has been implicated in hyperlipidemia . As hyperlipidemia is a known risk factor of cardiovascular
[36]
disease and this gene was only shown to have a significant difference in methylation with EVs in HFD
swine, epigenetic alteration via methylome manipulation may be a mechanism by which improvement in
cardiac outcomes is seen in EV treated swine. Similarly, we observed significant methylation of the BTC
gene, a member of the EGF family, and our enrichment analysis of genes found to be nominally alerted
prior to Bonferroni correction showed alteration of multiple EGFR-related pathways. Given the known role
of the EGF MAPK pathway in cardiac hypertrophy, this altered methylation of the BTC gene may yet again
represent a mechanism by which the observed benefits of EV treatment may be explained. These alterations
in the methylome invite an intriguing possible mechanism by which the observed effect of EVs is
accomplished via epigenetic regulation; however, this will require further investigation.

EV effect on myocardial perfusion
In the current analysis, which represents a subgroup that underwent methylome analysis of our whole swine
cohort, we did not observe significant differences in myocardial blood flow in the HFD swine treated with
EVs either at rest or during pacing. However, this finding is likely due to the small sample size used in this
study [Figure 4]. Our previous research using an entire cohort that underwent the same protocol revealed a
significant increase in perfusion at the rest of EV-treated swine in both normal diet and HFD groups
compared with saline injection controls .
[13]