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Xu et al. Vessel Plus 2023;7:33 https://dx.doi.org/10.20517/2574-1209.2023.98 Page 5 of 9

Signaling #11948, 1:1,000), transforming growth factor beta (TGFβ) (Cell Signaling #3711, 1:1,000), SMAD
2/3 (Cell Signaling #8685, 1:1,000), mammalian target of rapamycin (mTOR) (Cell Signaling #2983, 1:1,000),
B-cell lymphoma 2 (Bcl-2) (Cell Signaling #3498, 1:1,000), superoxide dismutase 1 (SOD1) (Cell Signaling
#37385, 1:1,000), superoxide dismutase 2 (SOD2) (Cell Signaling #13141, 1:1,000), phospho-endothelial
nitric oxide synthase (p-eNOS, Ser1177) (Cell Signaling #9570, 1:1,000), endothelial nitric oxide synthase
(eNOS) (Cell Signaling #32027, 1:1,000), and beta-actin (Cell Signaling #58169, 1:1,000). The following
secondary antibodies were used: anti-mouse IgG, HRP-linked antibody (Cell Signaling #7076, 1:5,000), and
anti-rabbit IgG, HRP-linked antibody (Cell Signaling #7074, 1:5,000). The band intensities were normalized
by beta-actin and quantified on ImageJ.

Statistical analysis
Continuous data were obtained from echocardiogram, histological, and immunoblotting results. Data
analysis was performed on GraphPad Prism 9.0.2. The Shapiro-Wilk test was done to check for normality.
For pairwise comparisons, either the unpaired t-test (parametric data) or the Mann-Whitney test (non-
parametric data) was used. For comparisons of more than two groups, the one-way ANOVA (parametric)
or Kruskal-Wallis (non-parametric) tests were used, followed by the post-hoc Tukey (parametric) or Dunn
(non-parametric) tests.

RESULTS
No long-term improvements in cardiac recovery were found after HBMSC-EV injection, when
delivered either IM or IV
Preoperatively, all mice had normal cardiac function on echocardiogram. Overall, there were no significant
changes in either LVEF or FS by postoperative day 28 [Figure 2]. The FS of IM-EV was significantly greater
than that of IV-EV on postoperative 7 (P = 0.0038), but these differences disappeared with time.

Quantification of myocardial infarct after Masson-Trichrome staining showed that although there was a
trend in differences in infarct size among the four groups, infarct size differences did not reach significance
by IM or IV administration of HBMSC-EV (P = 0.05620) [Figure 3A]. There were also no significant
changes in border zone interstitial fibrosis (P = 0.6333) [Figure 3B].

Intravenously delivered HBMSC-EV do not affect hepatic inflammation, fibrosis or proliferation in
the absence of liver disease
Hepatic uptake of IV HBMSC-EV after murine MI was demonstrated in previous studies, but this current
study did not demonstrate any long-term effects of the HBMSC-EV in the absence of liver injury
[7]
[Figure 4A] . Hematoxylin and eosin staining showed no leukocyte infiltrates upon review. Masson-
Trichrome staining did not demonstrate differences in fibrosis (P = 0.8167) [Figure 4B]. PCNA
immunofluorescence was negative for both control and HBMSC-EV groups, as expected in the absence of
liver injury or malignancy.

Concurrent immunoblotting of major signaling proteins of inflammatory and fibrosis and proliferative
pathways supported the histological results [Figure 4C]. Expression of tumor necrosis factor-alpha (TNF-α),
a major regulator of the inflammatory process, and some of the key fibrotic pathway markers transforming
growth factor-β (TGF-β) and SMAD 2/3 was not changed after IV HBMSC-EV (P = 0.3944, P = 0.1961,
respectively). The mammalian target of rapamycin (mTOR) is involved with cellular growth and had
increased expression (P = 0.0066) after HBMSC IV injection, but given the complexity of its regulation, this
finding is difficult to interpret. B-cell lymphoma 2 (Bcl-2), an apoptosis regulator, was not significantly
different (P = 0.8055). Proteins involved in oxidative stress were evaluated as well, and although expression
of superoxide dismutase 1 (SOD1) and total endothelial nitric oxide synthase (eNOS) were altered after