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Broadwin et al. Vessel Plus 2023;7:25 https://dx.doi.org/10.20517/2574-1209.2023.103 Page 3 of 14

upregulated expression of angiogenic markers [12,13] . Furthermore, EVs have been shown to alter the
epigenetic landscape in several ways, from altering mRNA expression to altering histone methylation [11,14] .
However, there remains a paucity of information regarding the effect of EV treatment on the methylome of
ischemic myocardium.

These studies highlight the importance of investigating the molecular and genetic mechanisms underlying
the physiologic and pathophysiologic response to cardiac ischemia. Here, we present a subgroup analysis
from our prior swine EV studies on the epigenome-wide DNA methylation profiles of porcine myocardium
after ischemic insult in the setting of treatment with EV therapy and in normal vs. high-fat diet pigs.

METHODS
Isolation and characterization of extracellular vesicles
In a technique previously reported by our lab, EVs were derived from human bone marrow-derived
mesenchymal stem cells (Lonza, Allendale, NJ). Cells were cultured in MSCGM BulletKit media (Lonza)
and incubated until reaching 80% confluence per manufacturers’ recommendations. After reaching the
desired confluence, culture media was replaced with serum-free RPMI media for 24 h. Mesenchymal stem
cell cultures then underwent ultracentrifugation (ThermoScientific Sorvall WX Ultra Series Centrifuge with
a Sorvall Surespin rotor, ThermoScientific, Waltham, MA) at 100,000 g for 75 min before washing with
sterile PBS and repeating ultracentrifugation at 100,000 g for 30 min in order to isolate EVs which were
resuspended in 10% DMSO. For the administration of EVs to porcine myocardium, a solution of 50 ug of
EVs suspended in 2mL of sterile 0.9% saline was created on the day of surgery [12,13] .

Confirmation studies of EV characteristics were undertaken and previously reported. In brief, qualitative
proteomic analysis was performed and confirmed the presence of EV biomarker proteins “(CD44, CD81,
CDC42, EGFR, CTNNB1, CAMK2D, CAMK2G, COL1A2 and FLNA)” . Furthermore, morphological
[15]
conformation via electron microscopy was undertaken, the results of which were previously published by
our lab and can be viewed in citation 12, Scrimgeour et al. PLoS One. 2020 Sep 11 .
[12]

Swine model of chronic cardiac ischemia
All animal procedures were conducted in accordance with protocols approved by the Institutional Animal
Care and Use Committee of Rhode Island Hospital (#505821). We selected a well-characterized, high-
fidelity, large animal model of chronic myocardial ischemia [12,13,15-19] . In a technique previously described by
our lab, twelve 11-week-old Yorkshire swine were divided into two groups, either normal diet control
(NDC) (n = 6) or high-fat diet (HFD) (n = 6). At 11 weeks of age, pigs were anesthetized with intramuscular
telazol to facilitate endotracheal intubation by veterinary staff, at which point anesthesia was maintained
with inhaled isoflurane. While maintaining sterile technique, a left thoracotomy was performed in order to
expose the heart, then opened the pericardium and dissected out and isolated the left circumflex artery at
the point of its takeoff from the left coronary artery. After isolation of the circumflex artery, an ameroid
constrictor (Research Instruments SW, Escondido, CA) was placed around the left circumflex artery in
order to induce chronic ischemia. The pericardium and thoracotomy were then closed in layers .
[13]
EV treatment
Two weeks after the placement of the ameroid constrictor, the swine underwent redo thoracotomy and
injection of EVs (3 NDC and 3 HFD) or vehicle control (3 NDC and 3 HFD). For this operation, the same
anesthesia protocols listed above were used. After anesthesia, a left thoracotomy was performed via an
incision 2-3 cm below the site of the prior thoracotomy to avoid any interference with tissues that had
adhered to the thoracic wall at the site of the prior incision. Once in the thoracic cavity, the pericardium was
opened and the area of the left ventricle supplied by the left circumflex artery was visualized. Ten injections