Low-Intensity Pulsed Ultrasound Enhances Angiogenesis
and Ameliorates Left Ventricular Dysfunction in a Mouse
Model of Acute Myocardial Infarction
Tomohiko Shindo, Kenta Ito, Tsuyoshi Ogata, Kazuaki Hatanaka, Ryo Kurosawa,
Kumiko Eguchi, Yuta Kagaya, Kenichiro Hanawa, Kentaro Aizawa, Takashi Shiroto,
Sachie Kasukabe, Satoshi Miyata, Hirofumi Taki, Hideyuki Hasegawa,
Hiroshi Kanai, Hiroaki Shimokawa

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Objective—Left ventricular (LV) remodeling after acute myocardial infarction still remains an important issue incardiovascular medicine. We have recently demonstrated that low-intensity pulsed ultrasound (LIPUS) therapy improves
myocardial ischemia in a pig model of chronic myocardial ischemia through enhanced myocardial angiogenesis. In thepresent study, we aimed to demonstrate whether LIPUS also ameliorates LV remodeling after acute myocardial infarction
and if so, to elucidate the underlying molecular mechanisms involved in the beneficial effects of LIPUS.
Approach and Results—We examined the effects of LIPUS on LV remodeling in a mouse model of acute myocardial infarction,
where the heart was treated with either LIPUS or no-LIPUS 3 times in the first week (days 1, 3, and 5). The LIPUS improved
mortality and ameliorated post–myocardial infarction LV remodeling in mice. The LIPUS upregulated the expression ofvascular endothelial growth factor, endothelial nitric oxide synthase, phosphorylated ERK, and phosphorylated Akt in theinfarcted area early after acute myocardial infarction, leading to enhanced angiogenesis. Microarray analysis in cultured
human endothelial cells showed that a total of 1050 genes, including those of the vascular endothelial growth factor
signaling and focal adhesion pathways, were significantly altered by the LIPUS. Knockdown with small interfering
RNA of either β1-integrin or caveolin-1, both of which are known to play key roles in mechanotransduction, suppressedthe LIPUS-induced upregulation of vascular endothelial growth factor. Finally, in caveolin-1–deficient mice, the beneficial
effects of LIPUS on mortality and post–myocardial infarction LV remodeling were absent.
Conclusions—These results indicate that the LIPUS therapy ameliorates post–myocardial infarction LV remodeling in micein vivo, for which mechanotransduction and its downstream pathways may be involved.   (Arterioscler Thromb Vasc
Biol. 2016;36:1220-1229. DOI: 10.1161/ATVBAHA.115.306477.)
Key Words: caveolae ◼ caveolin-1 ◼ integrins ◼ myocardial infarction ◼ myocardial ischemiaA

mong the public health issues during the past 50 years,
ischemic heart disease has been one of the world’s top
killer diseases.1 Recent progress in emergency care and patientmanagement has improved the prognosis of patients with acute
myocardial infarction (AMI).2,3 However, left ventricular (LV)
remodeling after AMI still remains an unsolved problem incardiovascular medicine. Thus, it is crucial to develop new
therapeutic strategies to ameliorate post–myocardial infarction (MI) LV remodeling. We have previously demonstrated
that low-energy extracorporeal cardiac shock wave therapy
improves myocardial ischemia in a porcine model of chronic
myocardial ischemia and patients with severe angina pectoris through enhanced myocardial angiogenesis.4–7 Recently,

we have further demonstrated that low-intensity pulsed ultrasound (LIPUS) therapy also induces angiogenesis and ameliorates myocardial ischemia in a porcine model of chronic
myocardial ischemia.8 LIPUS is also used for the treatmentof patients with several diseases in orthopedics, dentistry, andbrain stimulation.9–13 However, it remains to be examined whatmolecular mechanisms are involved in the LIPUS-induced
beneficial effects.
Vascular endothelial cells cover the inner surface of bloodvessels and are directly subjected to blood flow–induced
mechanical stimuli including shear stress. These stimuli
invoke specific responses within the cells, leading to changes
in their intrinsic structure and function.14 Endothelial cells

Received on: August 26, 2015; final version accepted on: April 3, 2016.
From the Department of Cardiovascular Medicine, Graduate School of Medicine (T. Shindo, K.T., T.O., K. Hatanaka, R.K., K.E., Y.K., K. Hanawa, K.A.,
T. Shiroto, S.K., S.M., H.S.) and Department of Electronic Engineering, Graduate School of Engineering and Division of Biomedical Measurements andDiagnostics, Graduate School of Biomedical Engineering (H.T., H.H., H.K.), Tohoku University, Sendai, Japan.
This manuscript was sent to Qingbo Xu, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.306477/-/DC1.
Correspondence to Kenta Ito, MD, PhD, Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi,
Aoba-ku, Sendai 980-8574, Japan. E-mail ito-kenta@cardio.med.tohoku.ac.jp© 2016 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org

1220

DOI: 10.1161/ATVBAHA.115.306477

Shindo et al   LIPUS Ameliorates LV Dysfunction After AMI   1221

Nonstandard Abbreviations and Acronyms
AMI
eNOS
ERK1/2
FAK
HUVEC
LIPUS
MI
siRNA
VEGF

acute myocardial infarction
endothelial nitric oxide synthase
extracellular signal-regulated kinase 1/2
focal adhesion kinase
human umbilical vein endothelial cells
low-intensity pulsed ultrasoundmyocardial infarction
small interfering RNA
vascular endothelial growth factor

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may sense these stimuli and convert them into a sequence of
biological responses. Caveolae are flask-like invaginations of
the plasma membrane with 40 to 80 nm in diameter and areorganized by caveolins.15,16 One of the aspects of caveolae isknown to be flow-sensing organelles converting mechanicalstimuli into chemical signals transmitted into the cells, socalled mechanotransduction.15 Caveolins have been shown tobind to a variety of proteins involved in signaling pathways,
such as G-protein subunits, tyrosine kinases, nitric oxide
synthase, small guanosine triphosphatases, and growth factor receptors.17–20 Caveolar membranes are also enriched incholesterol, glycosphingolipids, and signaling enzymes, suchas Src kinase.21 In addition, caveolae are reported to respondto cell stretch and to contribute to stretch-induced signaling.22
Integrins are reported to regulate multiple pathways, including
ERK, PI3K, focal adhesion kinase (FAK), Src, and Rho guanosine triphosphatases.23–25 Although caveolin-1 has no extracellular component, caveolin-1 may play an important role insensing mechanical stress or the distortion of the extracellular
membranes through interaction with β1-integrin.19,20,26,27 In thepresent study, we aimed to demonstrate whether the LIPUS
ameliorates post-MI LV remodeling and if so, to elucidate theunderlying molecular mechanisms involved in the beneficial
effects of the LIPUS.

Materials and Methods
Materials and Methods are available in the online-only Data
Supplement.

Results
Effects of the LIPUS Therapy on PostMI LV Remodeling In Vivo
To test whether the LIPUS therapy ameliorates LV dysfunction after AMI, wild-type mice were subjected to left anterior
descending coronary artery ligation, and cardiac function was
followed up for 2 months (Figure 1A). Mortality was significantly lower in the LIPUS group than in the no-LIPUS group(Figure 1B). In the no-LIPUS group, LV contractile function was progressively depressed 8 weeks after AMI, which
was significantly ameliorated in the LIPUS group (fractional
shortening at 8 weeks, 4.8±2.1% versus 12.7±1.6%; P<0.01;
P<0.0001 by 2-way ANOVA; Figure 1C). In contrast, LIPUS
therapy had no effects on cardiac function in the sham-operated
animals (Figure 1C). Similarly, no difference in body weightor LV wall thickness was noted among the 4 groups during thestudy period (Figure IC in the online-only Data Supplement).
Infarct size was significantly smaller in the MI-LIPUS groupthan in the MI-no-LIPUS group (Figure 1D). No complications, such as arrhythmias or skin burn, were noted during or
after the treatment.

Effects of the LIPUS Therapy on
Capillary Density and Signaling Pathways
in the Infarcted Hearts In Vivo
Heart weight/tibia length ratio was significantly lower in the
MI-LIPUS group than in the MI-no-LIPUS group on day 28(Figure IIA in the online-only Data Supplement). Although
there was no difference in capillary density on day 3 or day
6 between the 2 groups (Figure IIB in the online-only Data
Supplement), capillary density in the border area on day 28
was significantly higher in the LIPUS group than in the noLIPUS group (Figure 2A). There was no significant differencein heart weight/tibia length ratio or capillary density in the LV
between the 2 groups on day 56. We then aimed to elucidate
the molecular mechanisms involved in the beneficial effects of
the LIPUS therapy. First, we examined the expression of angiogenic molecules in the infarcted, border, and remote areas of the
LV in mice in vivo. mRNA expression of vascular endothelial
growth factor (VEGF) and endothelial nitric oxide synthase(eNOS) in the infarcted area was upregulated in the LIPUS
group compared with the no-LIPUS group on day 6, but not on
day 3 (Figure 2B). Although mRNA expression of transforming
growth factor-β1 was enhanced in the infarcted area, there wasno difference in the expression levels between the MI-no-LIPUS
and the MI-LIPUS groups (Figure IIC in the online-only Data
Supplement). We also examined protein levels of VEGF, eNOS,
and caveolin-1 and the extent of phosphorylation of extracellularsignal-regulated kinase 1/2 (ERK1/2) and Akt (Figure 2C and
2D). In the infarcted area, the LIPUS therapy upregulated theexpression of VEGF and eNOS on day 6, but not on days 3 and28 (Figure 2C). The LIPUS therapy also enhanced phosphorylation of ERK1/2 (Thr202-Tyr204) and Akt (Ser473; Figure 2D).
We also found that expression of caveolin-1 was significantly
upregulated in the remote area on day 3, but not on day 6 or 28(Figure IID in the online-only Data Supplement).

Effects of the LIPUS Therapy on
Signaling Pathways in Human Umbilical
Vein Endothelial Cells In Vitro
We then aimed to further elucidate the underlying molecularmechanisms involved in the beneficial effects of the LIPUS
therapy in human umbilical vein endothelial cells (HUVECs) invitro. First, we performed scratch assay to study the effects of
the LIPUS on proliferation of HUVECs. The LIPUS significantly
enhanced endothelial proliferation (Figure IIIB in the online-only
Data Supplement). We confirmed that the LIPUS (for 20 minutes) upregulated the mRNA expression of VEGF with a peakat 6 hours after irradiation (6 hours, 1.42±0.28-fold versus controls; P<0.05; Figure 3A; Figure IIIC in the online-only Data
Supplement). And, the mRNA expression level of caveolin-1 wascorrelated to that of VEGF in HUVECs 6 hours after LIPUS irradiation (Figure 3A). The LIPUS did not affect the expression of
VEGF in human cardiac fibroblasts, whereas it slightly enhanced

1222   Arterioscler Thromb Vasc Biol   June 2016

C

Sham

A
(mm)

B

(mm)
8

LVIDd

8
6

6

4

4

2

2

0

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1D

(%)
40

2W

4W

6W

8W

LVFS

1D

*

*

*

*

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LVEF

60

20

*

*

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10

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D

Pre

80

30

0

0
(%)

LVIDs

Pre

1D

2W

4W

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**

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(n=12)

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*
**

20
0

(n=10)(n=10)

Pre

1D

2W

4W

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8W

Sham

Infarct size(%)
40

P<0.05
P<0.05(n=10)(n=10)

30
20
10
0

4W

8W

Figure 1. Low-intensity pulsed ultrasound (LIPUS) therapy ameliorates post–myocardial infarction (MI) left ventricular (LV) remodeling in mice invivo. A, Study protocol. B, Survival rate. C, Representative M-mode echocardiographic images 8 wk after acute myocardial infarction. Graphs
showing the time course of LV internal dimension at end diastole (LVIDd), LVID at end systole (LVIDs), LV fractional shortening (LVFS), and LV
ejection fraction (LVEF). D, Representative photographs of heart sections stained with Masson trichrome staining on day 28. A graph shows theinfarct size. Results are expressed as mean±SD. LAD indicates left anterior descending coronary artery; and UCG, ultrasoundcardiography.

the expression of VEGF in human cardiac myocytes (24 hours,
1.16±0.13-fold versus controls; P<0.05; Figure 3A). The protein expression of VEGF was enhanced at 24 hours after irradiation in the LIPUS group when compared with the control group(Figure 3B). Microarray analysis was performed in the harvested
HUVECs and human coronary artery endothelial cells 6 hours
after irradiation. In HUVECs, 31 385 probe sets were analyzed,
where 1260 genes were found to be differentially expressed(upregulation in 224 gens and downregulation in 1036 genes).
In human coronary artery endothelial cells, 31 710 probe sets
were analyzed, where 1003 genes were found to be differentiallyexpressed (upregulation in 381 genes and downregulation in 622
genes). We also analyzed the pooled data of HUVECs and humancoronary artery endothelial cells after revising with ComBat analysis to eliminate the batch effects between the 2 cell types.28 The
ComBat analysis showed that a total of 1050 genes, including
those of the VEGF signaling and focal adhesion pathways, weresignificantly altered by the LIPUS treatment (Figure 3C; Table
II in the online-only Data Supplement). The data analyzed by

Ingenuity Pathway Analysis tools (Ingenuity Systems, Mountain
View, CA) represent the possible involvements of focal adhesion
pathway and its downstream signaling pathways (Figure IIID in
the online-only Data Supplement). Microarray analysis showedthat the expression level of VEGF, eNOS, β1-integrin–like protein, and SIRT1 significantly upregulated by the LIPUS treatment, whereas that of caveolin-1 and β1-integrin unchanged(Figure IIIE in the online-only Data Supplement). To examine therole of focal adhesion pathway in the LIPUS-induced angiogenesis, we preformed studies with small interfering RNA (siRNA) tointerfere some molecules that are supposed to be involved in themechanotransduction cascade. Knockdown with siRNA of either
β1-integrin or caveolin-1, both of which are known to play key
roles in the mechanotransduction,16,21–24 suppressed the LIPUSinduced upregulation of VEGF (Figure 3D). Knockdown withsiRNA of either Fyn or FAK also suppressed the LIPUS-induced
upregulation of VEGF (Figure 3D). To evaluate the importanceof structure of caveolae itself, conformational changes of caveolae were induced with either knockdown of serum deprivation

Shindo et al   LIPUS Ameliorates LV Dysfunction After AMI   1223

BorderInfarcted

A

B

RemoteVEGF mRNA
P<0.05

1.5

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Sham

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LIPUS

Infarcted Remote

Infarcted Remote
Day 6

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100µm

100µm

eNOS mRNA

100µm

1.2

Capillary density(/mm2)
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P<0.05

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Day 3

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Day 6

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Day 3

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-tubulin

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eNOS / -tubulin

Infarcted Remote
Day 6

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6
Infarcted Remote
Day 3

Infarcted Remote
Day 6

Infarcted Remote
Day 284
2
0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

Figure 2. Low-intensity pulsed ultrasound (LIPUS) therapy enhances angiogenesis in the border area and upregulates vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) signaling pathways via phosphorylation of extracellular signalregulated kinase 1/2 (ERK1/2) and Akt in the left ventricle in mice in vivo. A, Representative images of CD31 staining at day 28. Capillarydensity evaluated by CD31 staining. B, mRNA expression of VEGF and eNOS. C, Protein levels of VEGF and eNOS. D, Phosphorylationof ERK1/2 at Thr202-Tyr204 and Akt at Ser473. Results are expressed as mean±SD. MI indicates myocardial infarction; pAkt, phosphorylated Akt; and pERK, phosphorylated ERK.

response protein, a caveolar protein required for the formationof characteristic flask-shaped caveolar membrane invaginations,or methyl-β-cyclodextrin, an inhibitor specifically designed todisrupt lipid rafts in cells by depleting the cholesterol component.29,30 Knockdown with siRNA of serum deprivation response
protein suppressed the LIPUS-induced upregulation of VEGF
(Figure 3D). In the cell lysate treated with methyl-β-cyclodextrin
30 minutes before irradiation, the LIPUS-induced upregulation of VEGF was also suppressed (Figure 3D). Knockdownwith siRNA of each molecule was confirmed with real-timepolymerase chain reaction (Figure IV in the online-only Data
Supplement).

Effects of the LIPUS Therapy on Protein
Phosphorylation in HUVEC In Vitro
To further examine the intracellular responses induced by the
LIPUS, a comprehensive screening of protein phosphorylation

was performed using the Bio-Plex system in HUVECs(Figure 4A; Figure VA in the online-only Data Supplement).
Immediately after the LIPUS irradiation, phosphorylationof ERK1/2 (Thr202-Tyr204) and Akt (Ser473) was significantly enhanced in the LIPUS group compared with the control (no-LIPUS) group (Figure 4B). Phosphorylation of FAK(Tyr397), but not that of Fyn, was also significantly higher
in the LIPUS group than in the control group (Figure 4B).
The LIPUS did not enhance HUTS-4 expression in HUVECs,
suggesting that the LIPUS has no significant effects on theactivation state of β1-integrin (Figure VB in the online-only
Data Supplement).31 To further examine the involvementof each molecule in focal adhesion pathway, siRNA interference studies were performed. Knockdown with siRNAof either β1-integrin or caveolin-1 suppressed the LIPUSinduced phosphorylation of ERK1/2 and Akt (Figure 4C
and 4D). Knockdown with siRNA of either Fyn or FAK also

1224   Arterioscler Thromb Vasc Biol   June 2016

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B

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Figure 3. Focal adhesion pathway is crucial for the low-intensity pulsed ultrasound (LIPUS)–induced upregulation of vascular endothelial
growth factor (VEGF) in vitro. A, mRNA expression of VEGF in human umbilical vein endothelial cells (HUVECs), human cardiac fibroblasts (HCFs), and human cardiac myocytes (HCMs) in response to LIPUS. And, a positive correlation between caveolin-1 (Cav-1) mRNA
expression and VEGF mRNA expression in vitro. B, Protein levels of VEGF at 12 and 24 h after LIPUS irradiation. C, Heat map of the
microarray clustering analysis of LIPUS-irradiated HUVECs and human coronary artery endothelial cells. D, Effects of knockdown of
Cav-1, β1-integrin (β1-Itg), Fyn, and FAK with small interfering RNA (siRNA) on VEGF expression. Effects of knockdown of serum deprivation response protein (SDPR) with siRNA on VEGF expression. Effects of methyl-β-cyclodextrin (MβCD) on VEGF expression. The samples used for the analysis were HUVECs harvested 6 h after the LIPUS irradiation. Results are expressed as mean±SD.

suppressed the LIPUS-induced phosphorylation of ERK1/2
and Akt (Figure 4C and 4D). Knockdown of each moleculewith siRNA was confirmed with Western blotting (Figure VC
in the online-only Data Supplement).

Effects of the LIPUS Therapy on Post-MI LV
Remodeling in Caveolin-1 Knockout Mice In Vivo
To confirm the contribution of caveolin-1 to the LIPUSinduced beneficial effects on post-MI LV remodeling in vivo,
we examined the effects of the LIPUS therapy in caveolin-1
knockout (Cav-1-KO) mice. Cav-1-KO mice were subjected
to left anterior descending coronary artery ligation and weretreated with the LIPUS therapy. Unlike in wild-type mice
(Figure 1B), the LIPUS therapy had no beneficial effects on
the mortality in Cav-1-KO mice (Figure 5A). Similarly, thebeneficial effects of the LIPUS therapy on contractile function, infarct size, and capillary density noted in wild-type mice

were all absent in Cav-1-KO mice (Figure 5B–5D; Figure VI inthe online-only Data Supplement). Furthermore, the LIPUSinduced upregulation of VEGF and eNOS mRNA expression
and phosphorylation of ERK1/2 and Akt noted in wild-typemice were absent in Cav-1-KO mice (Figure VII in the onlineonly Data Supplement). The LIPUS-induced upregulation of
VEGF and eNOS and LIPUS-enhanced phosphorylation of
ERK1/2 and Akt were all blunted in endothelial cell–specificCav-1-KO mice as in systemic Cav-1-KO mice (Figure VIIIin the online-only Data Supplement). These results suggestthat caveolin-1 plays a pivotal role in the beneficial effects of
LIPUS on post-MI LV remodeling in mice in vivo.

Expression of Caveolin-1 in MI Miceand in Human Autopsy Samples
Immunohistochemical staining showed that the immunoreactivity of caveolin-1 was enhanced in the infarcted area

Shindo et al   LIPUS Ameliorates LV Dysfunction After AMI   1225

A

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Control (n=6)0(-)siRNA Cav-1
siRNA

1-Itg
siRNA

Fyn
siRNA

FAK Scrambled
siRNA siRNA

LIPUS (n=6)

Figure 4. Low-intensity pulsed ultrasound (LIPUS) enhances extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation
in a focal adhesion pathway–dependent manner in vitro. A, The phosphoprotein assay with Bio-Plex in LIPUS-irradiated human umbilicalvein endothelial cells. B, Phosphorylation of ERK1/2 at Thr202-Tyr204, Akt at Ser473, focal adhesion kinase (FAK) at Tyr397, and Fyn (Src)
at Tyr416. Knockdown with small interfering RNA (siRNA) of caveolin-1 (Cav-1), β1-integrin (β1-Itg), Fyn, and FAK suppressed the LIPUSinduced ERK1/2 and Akt phosphorylation. C, Effects of knockdown of Cav-1, β1-Itg, Fyn, and FAK with siRNA on ERK1/2 at Thr202Tyr204. D, Effects of knockdown with siRNA on Akt at Ser473. Results are expressed as mean±SD. MEK1 indicates MAPK kinase; andpAkt, phosphorylated Akt.

when compared with the remote area on day 3 in mice invivo (Figure IX in the online-only Data Supplement). We
also examined the expression of caveolin-1 in autopsy samples from patients who died of AMI. The immunoreactivityof caveolin-1 was significantly enhanced in coronary arterial
endothelial cells in the ischemic myocardium (the distal siteof the culprit coronary lesion) when compared with the remotearea (Figure 6A–6C).

Discussion
In the present study, we were able to demonstrate that the
LIPUS therapy ameliorates post-MI LV remodeling in micein vivo, where the mechanotransduction system, including
β1-integrin and caveolin-1 and its downstream pathways,
plays pivotal roles in the beneficial effects of the LIPUS(Figure 6D). This study demonstrates the beneficial effectsof the LIPUS therapy on post-MI LV remodeling and its
intracellular signaling pathways.

Beneficial Effects of the LIPUS Therapy on
Post-MI LV Remodeling in Mice In Vivo
In the present study, the LIPUS therapy enhanced angiogenesis, ameliorated post-MI LV remodeling, and improved themortality in mice in vivo. We treated the animals with the
LIPUS on days 1, 3, and 5, resulting in the enhanced phosphorylation of ERK1/2 and Akt on day 3 and the upregulationof VEGF and eNOS on day 6. Although the LIPUS therapy
was performed only in the acute phase with resultant upregulation of VEGF and eNOS, capillary density was enhanced, andpost-MI LV remodeling was ameliorated in the chronic phase.
These results suggest that it is important to turn on the angiogenic pathways immediately after the onset of AMI to suppress
post-MI LV remodeling. In the present study, the upregulationof VEGF in response to the LIPUS was prolonged as it wasnoted at day 6. We consider that the prolonged upregulation of
VEGF was caused by the repetitive LIPUS treatment because
we applied the LIPUS to the heart at 1, 3, and 5 days after MI.

1226   Arterioscler Thromb Vasc Biol   June 2016

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Figure 5. Absence of the beneficial effects of
low-intensity pulsed ultrasound (LIPUS) on
post–myocardial infarction (MI) left ventricular (LV) remodeling in caveolin-1 knockout
mice. A, Survival rate. B, Echocardiographic
evaluation. Graph showing the time course of
LV internal dimension at end diastole (LVIDd),
LVID at end systole (LVIDs), LV fractional
shortening (LVFS), and LV ejection fraction(LVEF). C, Infarct size. D, Capillary density.
Results are expressed as mean±SD.

8W

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Potential Mechanisms for Anginogenic
Effects of the LIPUS Therapy After AMI
The degree of the LIPUS-induced upregulation of mRNA
was higher in HUVECs than in human cardiac myocytes,
suggesting that vascular endothelial cells may be the main
player for the LIPUS-induced angiogenesis (Figure 3A).
We performed comprehensive analysis of phosphorylationof angiogenesis-related proteins in HUVECs using the BioPlex phosphoprotein assay. In this analysis, phosphorylationof ERK1/2 and Akt was enhanced by the LIPUS therapy invitro. The LIPUS therapy also enhanced the expression of
VEGF and eNOS, followed by enhanced phosphorylation of
ERK1/2 and Akt in the acute phase of AMI. Capillary density was higher in the LIPUS group than in the no-LIPUS
group in the chronic phase. Microarray analysis showedthat not only VEGF signaling pathway but also focal adhesion pathway was significantly affected by the LIPUS. Focal
adhesion pathway contains several proteins on the cell membrane, including caveolin-1 and β1-integrin, both of which
are important components of the caveolae and are known
to play key roles in the mechanotransduction process.15,16
Especially, caveolin-1 is required to maintain the structureof caveolae.15,16 The present results with siRNA suggest thatcaveolin-1, β1-integrin, Fyn, FAK, ERK1/2, and Akt are
all involved in the LIPUS-induced upregulation of VEGF
(Figure 6D). In addition, we found that the conformational

8Wchanges of caveolae by either knockdown of serum deprivation protein response with siRNA or administration of
methyl-β-cyclodextrin suppressed the LIPUS-induced
upregulation of VEGF. Furthermore, we demonstrated thatthe beneficial effects of the LIPUS therapy on post-MI LV
remodeling were blunted in Cav-1-KO mice. LIPUS-induced
upregulation of angiogenic molecules was also blunted in theendothelial cell–specific caveolin-1 knockout mice, suggesting that endothelial cells play pivotal roles in the angiogenic
effects of the LIPUS. Taken together, these results suggestthat the acoustic streaming by the LIPUS induces distortionof caveolae on endothelial cells, which then transmits themechanical stimuli to intracellular signaling pathways withsubsequent phosphorylation of Fyn, FAK, ERK1/2, and Akt
and resultant enhanced the expression of VEGF and angiogenesis (Figure 6D). These molecules are also known to play
key roles in cell proliferation and angiogenesis induced bymechanical stimuli (eg, shear stress) on the surface of vascular endothelial cells.15,19–22,24

Biological Effects of LIPUS Other
Than Angiogenesis
The microarray analysis suggested that the LIPUS exerts
biological effects on cell cycles, metabolic pathways, RNAtransport, DNA replication, mRNA surveillance, mismatch
repair, and protein export in addition to its angiogenic

Shindo et al   LIPUS Ameliorates LV Dysfunction After AMI   1227

CD31

A

1-integrin

Caveolin-1aRemote
1.0mm

1.0mm

1.0mm

b

Infarcted
1.0mm

B

1.0mm

Infarcted

Remote
a

b

1.0mm

C

4

Caveolin-1
P<0.05

3
2

Caveolin-1

1

Downloaded from http://atvb.ahajournals.org/ by guest on April 15, 2017

100µm

100µm

0

D

effects. In this regard, we have recently reported that thelow-energy shock wave therapy suppresses post-MI LV
remodeling in rats through anti-inflammatory effects in addition to its angiogenic effects,32 suggesting that the LIPUS
may also exert anti-inflammatory effects. Ultrasound has
been reported to induce sonoporation and subsequent influxof calcium ion, which was correlated to the LIPUS-induced
bioeffects in cultured cells.33 Thus, it is possible that the
LIPUS exerts several biological effects through alteration of
intracellular Ca2+ levels. This point remains to be examinedin future studies.

Role of Caveolin-1 in the Ischemic Heart
It is reported that the shear stress–induced intracellular signaling is mediated by the activation of β1-integrin and concurrent caveolin-1 phosphorylation.34 β1-integrin–mediatedactivation of ERK1/2 and PI3-Akt pathways is mediated bycaveolin-1.35 In the present study, endothelial expression of
caveolin-1 was enhanced in the infarcted area early after AMI

Remote Infarcted

Figure 6. Expression of caveolin-1 in
autopsy samples from patients with AMI
and molecular mechanisms for the mechanotransduction in the low-intensity pulsed
ultrasound (LIPUS)–induced angiogenesis.
A, Representative images of immunostaining of CD31, caveolin-1, and β1-integrin in
the infarcted and the remote areas. B, Highpower fields of immunostaining of caveolin-1
from A. C, Expression of caveolin-1 by asemi-quantitative scoring system. D, Possible molecular mechanisms for the LIPUSinduced angiogenesis are shown. ERK1/2
indicates extracellular signal-regulated
kinase 1/2; eNOS, endothelial nitric oxide
synthase; and VEGF, vascular endothelial
growth factor.

in mice and in autopsy samples of patients with AMI, suggesting that the abrupt reduction of coronary flow and shear
stress affects endothelial cells in the ischemic myocardium
to upregulate caveolin-1, leading to an increased sensitivity
to LIPUS.

Study Limitations
Several limitations should be mentioned for the present study. First, the effects of LIPUS on VEGF expression
were rather small in in vitro studies compared with thoseobserved in in vivo studies. In the present study, the LIPUS
was applied to the cells for 20 minutes in in vitro studies,
whereas it was applied to the heart at 3 different short-axis
levels on 1, 3, and 5 days after AMI in in vivo studies (20minutes×3 levels×3 days). We speculate that the repeatedirradiation enhanced the effects of LIPUS on angiogenesis.
Second, it has been reported that Cav-1-KO mice develop
cardiac hypertrophy and impaired coronary collateral growth
in response to ischemia.36 Thus, the beneficial effects of1228   Arterioscler Thromb Vasc Biol   June 2016
LIPUS might be further blunted in Cav-1-KO mice. Third,
we did not examine the effects of the LIPUS in geneticallymodified animals other than Cav-1-KO mice because β1integrin knockout mice are embryonic lethal.37 Fourth, about
the molecular mechanisms for the LIPUS-induced angiogenic effects, we focused on focal adhesion pathway in thepresent study based on the results of microarray analysis.
Although we showed that caveolin-1, β1-integrin, Fyn, andFAK play pivotal roles in the LIPUS-induced signaling pathway, other subcellular structures related to mechanotransduction, such as cytoskeleton and cell adhesion complexes,
might also be involved.38 Further studies are needed. Fifth,in the siRNA experiments, we used VEGF as an index ofangiogenic effects of the LIPUS. However, it is possiblethat other mechanisms related to angiogenesis, such as antiinflammatory and antifibrotic effects, may also contribute to
the beneficial effects of the LIPUS. This point also remains
to be examined in future studies.
Downloaded from http://atvb.ahajournals.org/ by guest on April 15, 2017

Clinical Implications
Despite recent progress in emergency care and management of AMI, post-MI remodeling and heart failure remain
an important issue in cardiovascular medicine.1–3 Althoughregenerative therapies, such as gene and cell therapies, havebeen under development to suppress worsening heart failure,39,40 most of them are invasive in nature, and their efficacy
and safety have not been established yet.41–43 Because theintensity of ultrasound used in the LIPUS therapy is below theupper limit of acoustic output standards for diagnostic ultrasound devices, the therapy does not cause compression, heat,or discomfort. For this noninvasive nature, our LIPUS therapy
can be used as an adjunctive therapy in patients with AMI tosuppress post-MI LV remodeling. It could also be applied toother ischemic disorders, such as ischemic cardiomyopathy,
peripheral arterial disease, refractory chronic skin ulcers, andspinal cord injury as in the case with low-energy extracorporeal shock wave therapy.44–46

Conclusions
In the present study, we were able to demonstrate that the
LIPUS therapy ameliorates post-MI LV remodeling in micein vivo, where mechanotransduction and its downstream pathways may be involved. Thus, the LIPUS therapy may be a
promising, new, noninvasive strategy for the treatment of postMI LV remodeling.

Acknowledgments
We thank Yumi Watanabe, Ai Nishihara, and Hiromi Yamashita for
their excellent technical assistance.

Sources of Funding
This study was supported in part by Grants-in-Aid for Scientific
Research from the Japanese Ministry of Education, Culture, Sports,
Science, and Technology, Tokyo, Japan, and the Japanese Ministry of
Health, Labor, and Welfare, Tokyo, Japan.

Disclosures
None.

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Highlights• The low-intensity pulsed ultrasound (LIPUS) therapy ameliorates post–myocardial infarction left ventricular remodeling in mice in vivo.
• The mechanotransduction system, including β1-integrin and caveolin-1 and its downstream pathways, plays pivotal roles in the beneficial
effects of the LIPUS therapy.
• Our current study provides a promising, new, noninvasive strategy for the patients with acute myocardial infarction and also clarifies theimportance of mechanotransduction.

Downloaded from http://atvb.ahajournals.org/ by guest on April 15, 2017

Low-Intensity Pulsed Ultrasound Enhances Angiogenesis and Ameliorates Left
Ventricular Dysfunction in a Mouse Model of Acute Myocardial Infarction
Tomohiko Shindo, Kenta Ito, Tsuyoshi Ogata, Kazuaki Hatanaka, Ryo Kurosawa, Kumiko
Eguchi, Yuta Kagaya, Kenichiro Hanawa, Kentaro Aizawa, Takashi Shiroto, Sachie Kasukabe,
Satoshi Miyata, Hirofumi Taki, Hideyuki Hasegawa, Hiroshi Kanai and Hiroaki Shimokawa
Arterioscler Thromb Vasc Biol. 2016;36:1220-1229; originally published online April 14, 2016;
doi: 10.1161/ATVBAHA.115.306477
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2016 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636

The online version of this article, along with updated information and services, is located on theWorld Wide Web at:
http://atvb.ahajournals.org/content/36/6/1220

Data Supplement (unedited) at:
http://atvb.ahajournals.org/content/suppl/2016/04/13/ATVBAHA.115.306477.DC1
http://atvb.ahajournals.org/content/suppl/2016/04/13/ATVBAHA.115.306477.DC2

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SUPPLEMENTAL MATERIAL

Low-intensity Pulsed Ultrasound Enhances Angiogenesis and Ameliorates
Left Ventricular Dysfunction in a Mouse Model of
Acute Myocardial Infarction
Tomohiko Shindo,1 Kenta Ito,1 Tsuyoshi Ogata, 1 Kazuaki Hatanaka,1 Ryo Kurosawa,1Kumiko Eguchi,1 Yuta Kagaya,1 Kenichiro Hanawa,1 Kentaro Aizawa,1 Takashi Shiroto,1Sachie Kasukabe,1 Satoshi Miyata,1 Hirofumi Taki,2,3 Hideyuki Hasegawa,2,3
Hiroshi Kanai,2,3 Hiroaki Shimokawa.1
1

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine,
Sendai, Japan, 2Department of Electronic Engineering, Graduate School of Engineering,Tohoku University, Sendai, Japan, and 3Division of Biomedical Measurements andDiagnostics, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.
Running title: LIPUS Ameliorates LV Dysfunction after AMI

Key words: angiogenesis, mechanotransduction, cell signaling, contractile reserve,
infarct remodeling
Subject Codes: [27] Other Treatment, [105] Contractile function, [129] Angiogenesis,[138] Cell signaling/signal transduction, [151] Ischemic biology - basic studies.
TOC category: Basic study
TOC subcategory: Vascular Biology
Address for Correspondence:
Kenta Ito, MD, PhD.
Associate Professor
Department of Cardiovascular Medicine
Tohoku University Graduate School of Medicine
1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
TEL: +81-22-717-7153, FAX: +81-22-717-7156
E-mail: ito-kenta@cardio.med.tohoku.ac.jp

SUPPLEMENTAL MATERIAL

Shindo et al., ATVB/2015/306477/R2, Page 2

Supplemental Tables

Table I.

List of Antibodies Used in the Present Study

Antibody

Application

Dilution

Company

VEGF

Immunoblot

1:1000Santa Cruz#sc-152

eNOS

Immunoblot

1:1000

Enzo#ADI-905-386

HUTS-4

Immunoblot

1:1000

LSP#LS-B2861-50

α-tubulin

Immunoblot

1:1000Sigma#T5168

Phospho-ERK1/2

Immunoblot

1:1000Cell Signaling#9106

Total- ERK1/2

Immunoblot

1:1000Cell Signaling#9102

Phospho-Akt

Immunoblot

1:1000Cell Signaling#9271

Total-Akt

Immunoblot

1:1000Cell Signaling#9272

Phospho-Src

Immunoblot

1:1000Cell Signaling#6943

Total-Fyn

Immunoblot

1:1000Cell Signaling#4023

Phospho-FAK

Immunoblot

1:1000Cell Signaling#8556

Total-FAK

Immunoblot

1:1000Cell Signaling#3285

Caveolin-1

Immunoblot

1:1000Cell Signaling#3238

β1-integrin

Immunoblot

1:1000Cell Signaling#4706

CD31

IP

1:1000

Dako#M0823

Caveolin-1

IP

1:400

Cell Signaling#3238

β1-integrin

IP

1:100

Cell Signaling#4706

SUPPLEMENTAL MATERIAL
Table II.

Shindo et al., ATVB/2015/306477/R2, Page 3

Results of the Microarray Clustering Analysis of
LIPUS-irradiated HUVECs and HCAECs

Signaling pathway

P value

Cell cycle

0.00001

Metabolic pathway

0.00001

RNA transport

0.00001

DNA replication

0.00001

mRNA surveillance pathway

0.00001

Mismatch repair

0.00001

VEGF signaling pathway

0.00001

Protein export

0.0005Glutathione metabolism

0.0017

Notch signaling pathway

0.003

Focal adhesion

0.0049Phosphatidylinositol signaling system 0.0117
GnRH signaling pathway

0.0177

ErbB signaling pathway

0.0201

Supplemental Figure I

A

(kPa)
3000

Pressure(kPa)

1 cycle

3000

2000

2000

1000

1000

Pressure

0

0

-1000

-1000

-2000

-2000

-3000

-3000
0

1

2

3

Time

32 cycle

45(µsec)

0

5

10

15

20

25 (µsec)

Time

Supplemental Figure I. Effects of the LIPUS Therapy on BW and LV Wall Thickness after AMI in Mice in Vivo
A. Acoustic pressure at 1 and 32 cycles. B. Schematic illustration of in vivo study.
C. Body weight (BW) was comparable among the 4 groups. Graph showing the time course of left ventricular anteriorwall dimension at end-diastole (LVAWDd), and left ventricular posterior wall dimension at end-diastole (LVPWDd).
Results are expressed as mean ± SD.

BSupplemental Figure I

Transducer

Ultrasound

Mouse

Agar Phantom

Supplemental Figure I

C (g)

BW

40

Sham – no-LIPUS (n=10)(n=10)
Sham – LIPUS(n=12)
MI – no-LIPUS

30
20(n=12)

MI – LIPUS
10
0
Pre

2W

(mm)
1.5

4W

6W

8W

LVAWDd

(mm)
1.5

1.0

1.0

0.5

0.5

0.0

0.0
Pre

1D

2W

4W

6W

8W

LVPWDd

Pre

1D

2W

4W

6W

8W

Supplemental Figure IIA(mg/mm)Heart weight / Tibia length

16
P<0.0512■ Sham(n=6)■ AMI – no-LIPUS (n=6)

8■ AMI – LIPUS

4
0

Day 3

Day6

Day 28

Day 56

Lung weight / Tibia length(mg/mm)
20
15
10
5
0

Day 3

Day6

Day 28

Day 56(n=6)

Supplemental Figure II

B
Capillary density(/mm2)
4000
3000■ Sham(n=6)■ AMI – no-LIPUS (n=6)

2000■ AMI – LIPUS

1000
0Infarcted Border Remote

Infarcted Border Remote

Day 3

C

P<0.052.0

Day 6

TGFβ1 mRNA

P<0.05■ Sham(n=6)■ AMI – no-LIPUS (n=6)

1.5■ AMI – LIPUS

1.0
0.5
0.0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

(n=6)

(n=6)

Supplemental Figure II

D

Caveolin-1 / α-tubulin
Caveolin-1α-tubulin
P<0.05

1.5

P<0.05

1.0

0.5

0.0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

Infarcted Remote
Day 28■ Sham■ AMI – no-LIPUS

(n=6)
(n=6)

■ AMI – LIPUS(n=6)

Supplemental Figure II. Effects of the LIPUS Therapy on Histopathological Findings and Molecular
Expressions after AMI in Mice
A. The time course of heart weight/tibia length (HW/TL) ratio and lung weight/tibia length (LW/TL) ratio.
B. Capillary density at 3 and 6 days after AMI. C. TGF-β1 mRNA expression at 3 and 6 days after AMI.
D. Protein levels of caveolin-1. Results are expressed as mean ± SD.

ASupplemental Figure III

Transducer

UltrasoundAgar Phantom

Cultured Cell
Teflon Plate

Pre

B

Day 2

Supplemental Figure III

Control

LIPUS

Scratch assay
250
200

Pixel

150

P<0.05■ LIPUS100
50
0■ Control

Pre
0

Day
1 1

Day
2 2

3

(n=6)
(n=6)

Supplemental Figure III

C(fold)
2.5

HUVECseNOS mRNA
P<0.05

2.0
1.5
1.0
0.5
0.0

3Hr 6Hr 12Hr 24Hr
LIPUS

D

Supplemental Figure III

Supplemental Figure III

E

Gene expression
2.0

P<0.05

P<0.05
P<0.05

P<0.05

1.5

P<0.05■ Control (n=3)■ LIPUS (n=3)

1.0
0.5
0.0

VEGF-AeNOS

TGF-β1 Caveolin-1 β1-integrin

ItgβL1SIRT1Supplemental Figure III. Effects of the LIPUS on MAPK and PI3K-Akt Signaling
A. Schematic illustration of in vivo study. B. The results of scratch assay that shows the effect of LIPUS on
proliferation in HUVECs. C. mRNA expression of eNOS in HUVECs in response to LIPUS. D. To examine
gene ontology, including biological processes, cellular components, molecular functions and genetic
networks, the obtained microarray data were analyzed using Ingenuity Pathway Analysis tools (Ingenuity
Systems, Mountain View, CA, USA), a web-delivered application that enables the identification,
visualization and exploration of molecular interaction networks from gene expression data. The schematic
illustration represents LIPUS-induced molecular interactions including focal adhesion pathway, MAPK
signaling pathway and PI3K-Akt signaling pathway. E. The part of expression data of microarray, showingthat VEGF-A, eNOS, Integrin β1-like protein, SIRT1 were significantly up-regulated in response to the
LIPUS. Results are expressed as mean ± SD.

Supplemental Figure IV

mRNA expression
1.5

P<0.05

P<0.05

P<0.05

P<0.05

P<0.051.0■ (-) siRNA (n=12)■ (+) siRNA (n=12)

0.5
0.0

Cav1

Cav1

β1Itg

β1Itg

KD(-) KD(+)
KD(-) KD(+)
Caveolin-1
β1-integrin
siRNA
siRNA

Fyn
KD(-)

Fyn

KD(+)
Fyn
siRNA

FAK
KD(-)

FAK

KD(+)
FAK
siRNA

SDPR
KD(-)

SDPR

KD(+)
SDPR
siRNA

Supplemental Figure IV. Confirmation of Knockdown with siRNA of Each Molecule by Real-time PCR
Knockdown of caveolin-1, β1-integrin, Fyn, FAK and SDPR with siRNA was confirmed. Results are expressed
as mean ± SD.

Supplemental Figure VA

Relative phosphorylation

2.0

(n=6)
■ LIPUS (20min) (n=6)■ Control■ LIPUS (40min) (n=6)

1.5
1.0
0.5
0.0

EGFR VEGFR-2 PDGFR-a PDGFR-b IGF-1R
EGFR(T1173) (T1068)

HER-2

Smad2 GSK-3a/b

Stat1

Stat3
(T705)

Stat3(S727)

Supplemental Figure V

B

HUTS-4
HUTS-4β1-integrin
0.8
0.6
0.4
0.2
0.0

■ LIPUS (20min)

(n=6)
(n=6)

■ LIPUS (40min)(n=6)■ ControlSupplemental Figure V

C

Protein expression

1.5
P<0.05

P<0.05

P<0.05

P<0.051.0■ (-) siRNA (n=12)■ (+) siRNA (n=12)

0.5

0.0

Cav1

Cav1

KD(-)
KD(+)
Caveolin-1
siRNA

β1Itg

β1Itg

KD(-)
KD(+)
β1-integrin
siRNA

Fyn KD(-) Fyn KD(+)

Fyn
siRNA

FAK KD(-)

FAKFAKKD(+)
siRNA

Supplemental Figure V. Bio-Plex Analysis of Phosphorylation of Growth Factors and Second Messengers
A. The results of the phosphoprotein assay with Bio-Plex of LIPUS-irradiated HUVECs at 20 or 40 min are shown.
B. The results of Western blot analysis of HUTS-4 which related to activation of β1-integrin. C. Confirmation ofknockdown with siRNA of each molecule by Western blotting. Knockdown with siRNA of caveolin-1, β1-integrin,
Fyn, FAK with siRNA was performed. Results are expressed as mean ± SD.

Supplemental Figure VI

A (g)

BW

40
30

Sham – no-LIPUS (n=8)(n=8)
Sham – LIPUS

20

MI – no-LIPUS(n=10)

MI – LIPUS(n=10)

10
0
Pre

2W

(mm)

4W

6W

8W

1.6

(mm)
1.6

1.2

1.2

0.8

0.8

0.4

0.4

0.0

0.0

LVAWDd

Pre

1D

2W

4W

6W

8W

LVPWDd

Pre

1D

2W

4W

6W

8W

Supplemental Figure VI

B(mg/mm)Heart weight / Tibia length20
15■ Sham(n=6)■ AMI – no-LIPUS (n=6)

10■ AMI – LIPUS

50(mg/mm)

Day 3

Day6

Day 28

Day 56

Lung weight / Tibia length

30
20
10
0

Day 3

Day6

Day 28

Day 56(n=6)

Supplemental Figure VI

C
Capillary density(/mm2)
3000
2000
1000
0

Infarcted Border Remote
Day 3

Infarcted Border Remote
Day 6

Supplemental Figure VI. Effects of the LIPUS Therapy on BW, LV Wall Thickness, and Histopathological
Findings after AMI in Cav-1-KO Mice in Vivo
A. Graphs showing the time course of the BW, left ventricular anterior wall dimension at end-diastole (LVAWDd), left
ventricular posterior wall dimension at end-diastole (LVPWDd). B. The time course of heart weight/tibia length(HW/TL) ratio and lung weight/tibia length (LW/TL) ratio. C. Capillary density at 3 and 6 days after MI in each zone.
Results are expressed as mean ± SD.

Supplemental Figure VII

A

VEGF mRNA

3
2■ Sham(n=6)■ AMI – no-LIPUS (n=6)

1■ AMI – LIPUS

0

Infarcted Remote

Infarcted Remote

Day 3

Day 6

eNOS mRNA

P<0.05

3

1.5

2

1.0

1

0.5

0

Infarcted Remote
Day 3

(n=6)

Infarcted Remote
Day 6

0.0

TGFβ1 mRNA

P<0.05

Infarcted Remote
Day 3

Infarcted Remote
Day 6

VEGF / α-tubulin

B

Supplemental Figure VII
VEGF
α-tubulin

0.8

(n=6)
■ Sham■ AMI – no-LIPUS (n=6)(n=6)■ AMI – LIPUS

0.6
0.4
0.2
0.0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

Infarcted Remote
Day 28eNOS / α-tubulin
eNOS
α-tubulin

0.5
0.4
0.3
0.2
0.1
0.0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

Infarcted Remote
Day 28

C

Supplemental Figure VII

p/t ERK1/2
pERK1/2
tERK1/2

2.0
1.5■ Sham(n=6)■ AMI – no-LIPUS (n=6)

1.0■ AMI – LIPUS

0.5
0.0

Infarcted Remote
Day 3

(n=6)

Infarcted Remote
Day 6

p/t Akt
pAkt
tAkt

1.2
0.8
0.4
0.0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

Supplemental Figure VII. No Effects of the
LIPUS Therapy on Expression of VEGF andeNOS and Phosphorylation of ERK1/2 and
Akt in Cav-1-KO Mice in Vivo
A. Left ventricular lysates from infarcted(includes border) and remote regions wereanalyzed for mRNA expression of VEGF,eNOS and TGFβ1 at 3 and 6 days after AMI.
B-C. Western blot analysis was performed at
3, 6 and 28 days after AMI to determine theprotein expression of VEGF, eNOS, andphosphorylation of ERK1/2 (C) and Akt (C)after MI. Results are expressed as mean ± SD.

ASupplemental Figure VIII

VEGF

VEGF
α-tubulin
1.2

P<0.05

0.8
0.4
0.0

Infarcted Remote

Infarcted Remote
Day 6

Day 3

Infarcted Remote

Infarcted Remote

Day 3

Day 6

WT

eCav-1 KO

eNOS
eNOS
α-tubulin

1.5
P<0.051.0
0.5
0.0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

WT

Infarcted Remote
Day 3

Infarcted Remote
Day 6

eCav-1 KO

B

Supplemental Figure VIII

p/t ERK1/2

1.2

pERK1/2
tERK1/2
P<0.05

0.8
0.4
0.0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

WT

Infarcted Remote

Infarcted Remote

Day 3

Day 6

p/t Akt

eCav-1 KO
pAkt
tAkt

8

P<0.05

6
4
2
0

Infarcted Remote
Day 3

Infarcted Remote
Day 6

Infarcted Remote
Day 3

Infarcted Remote
Day 6

WT
eCav-1 KO
Supplemental Figure VIII. LIPUS-induced up-regulation of angiogenic molecules were also blunted in eCav-1-KO mice
A. Protein levels of VEGF and eNOS. B. Phosphorylation of ERK1/2 and Akt. Results are expressed as mean ± SD.

Supplemental Figure IX

ASham

AMI-Infarcted

400µm

B

400µm

AMI-Infarcted

Sham

100µm

100µm

AMI-Border

400µm

AMI-Border

100µm

AMI-Remote

400µm

AMI-Remote

100µmSupplemental Figure IX

C

Caveolin-1-positive area
P<0.05(%)
15

P<0.05
P<0.05■ Sham (n=6)
(n=6)
■ AMI

10

5

0

D3-Sham

D3-Infarcted Border
D3-Border
Infarcted

D3-Remote
RemoteSupplemental Figure IX. Rapid response of caveolin-1 expression was found in histologicalexamination after MI in vivo.
A. Representative photomicrographs of the immunostaining for caveolin-1 in the site of infarcted,
border and remote area of WT mice 3 days after AMI. B. High-power field of the images shown in A.
C. %area of the caveolin-1-positive cells analyzed by ImageJ Software (NIH).
Results are expressed as mean ± SD.

Materials and Methods

Low-intensity Pulsed Ultrasound Enhances Angiogenesis and Ameliorates
Left Ventricular Dysfunction in a Mouse Model of
Acute Myocardial Infarction
Tomohiko Shindo,1 Kenta Ito,1 Tsuyoshi Ogata, 1 Kazuaki Hatanaka,1 Ryo Kurosawa,1Kumiko Eguchi,1 Yuta Kagaya,1 Kenichiro Hanawa,1 Kentaro Aizawa,1 Takashi Shiroto,1Sachie Kasukabe,1 Satoshi Miyata,1 Hirofumi Taki,2,3 Hideyuki Hasegawa,2,3
Hiroshi Kanai,2,3 Hiroaki Shimokawa.1
1

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine,
Sendai, Japan, 2Department of Electronic Engineering, Graduate School of Engineering,Tohoku University, Sendai, Japan, and 3Division of Biomedical Measurements andDiagnostics, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.
Running title: LIPUS Ameliorates LV Dysfunction after AMI

Key words: angiogenesis, mechanotransduction, cell signaling, contractile reserve,
infarct remodeling
Subject Codes: [27] Other Treatment, [105] Contractile function, [129] Angiogenesis,[138] Cell signaling/signal transduction, [151] Ischemic biology - basic studies.
TOC category: Basic study
TOC subcategory: Vascular Biology
Address for Correspondence:
Kenta Ito, MD, PhD.
Associate Professor
Department of Cardiovascular Medicine
Tohoku University Graduate School of Medicine
1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
TEL: +81-22-717-7153, FAX: +81-22-717-7156
E-mail: ito-kenta@cardio.med.tohoku.ac.jp

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 2

Materials and Methods

The present study confirms to the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health and was performed according to theprotocols approved by the Institutional Committee for Use and Care of Laboratory Animals of
Tohoku University (2013Ikumikae-135, 2013Idou-516) and the Ethics Committee of Tohoku
University Graduate School of Medicine (2014-1-048).

Detailed methods are described in

the supplemental data.

Animal Models
Male C57BL/6 wild-type (WT) mouse (8-week-old, 20-30g in body weight),
caveolin-1-deficient (Cav-1-KO) mice (8-week-old, 22-30g in body weight) and endothelial
cell specific caveolin-1-deficient (eCav-1-KO) mice were used in the present study.
Cav-1-KO mice were developed by breeding pairs of heterozygous mice (Jackson Laboratory,
Barharbor, ME, USA) and were maintained in our institute.1

The Cav-1-KO mice were

backcrossed to C57BL/6 mice over 10 generations and thus C57BL/6 mice were used as a
WT control.

We newly generated Cav-1-floxed (Cav-1flox/flox) mice, in which Cav-1 exon3

is flanked by two loxP sites on the C57BL6 background (UNITECH CO, Kashiwa, Japan).
To generate eCav-1-KO mice, Cav-1flox/flox mice were cross-bred with Tie2-Cre mice (JacksonLaboratory, Bar Harbor, ME, USA).2

The mice were subjected to ligation of the proximal

left anterior descending coronary artery (LAD) to induce AMI.3

They were randomly

assigned to the 2 groups with (LIPUS group) or without (no-LIPUS group) the LIPUS therapy.
In addition to the AMI groups, the sham-operated groups (with or without LIPUS therapy)
were also made with the same procedure but without the LAD ligation.

Animals were

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 3

excluded from the present study when LV fractional shortening (FS) exceeded 30 % at day 1.
We stored the heart samples at 3, 6, 28, and 56 days after AMI.

Animals were euthanized bycervical dislocation under anesthetic inhalation overdose with isoflurane.

For mRNA andprotein analysis, the heart was divided into infarcted (including adjacent border area) andremote area.

For histological analysis, the heart was divided into infarcted, border andremote areas.

LIPUS Therapy
For the LIPUS therapy, we used a diagnostic ultrasound device (Prosound α10; HITACHI
Aloka Medical, Ltd., Mitaka, Japan) whose irradiation conditions could be modified bysoftware modification.

Based on our previous studies,4 we performed LIPUS therapy under

the following conditions; frequency 1.875 MHz, pulse repetition frequency 4.90 kHz, numberof cycles 32, voltage applied to each transducer element, 17.67-22.38 volts (V), and Ispta(spatial peak temporal average intensity) 117-174 mW/cm2.

The power of LIPUS was 0.25

W/cm2, the beams were irradiated from the sector-shaped probe and were focused at 6 cm
depth.4

The number of cycles of pulsed ultrasound represents that of acoustic waves per 1

pulse, while 1 cycle is used for diagnostic ultrasound devices (Supplemental Figure IA).
The voltage applied to each transducer element was controlled to keep estimated Ispta of
LIPUS below the upper limit of acoustic output standards (<720 mW/cm2) for diagnostic
ultrasound devices (US Food and Drug Administration’s Track 3 Limits) and to prevent theultrasound probe from temperature rise.4

LIPUS was applied to mice or cultured cells

through an agar phantom gel (Supplemental Figure IB, IIIA).

The attenuation coefficientof the phantom gel was almost comparable to that of living cells (muscles, fat, blood etc.).
In in vivo studies, LIPUS was applied to the heart by 2-dimensional scan at 3 different shortaxis levels (aortic valve, mitral valve and papillary muscle levels for 20 min each) in a day

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 4

under inhalation anesthesia with 1.2% isoflurane.4

The LIPUS group was subjected to the

LIPUS therapy 3 times in the first week (1, 3 and 5 days after AMI), whereas the no-LIPUS
group underwent the same procedures 3 times including anesthesia but without the LIPUS
therapy (Figure 1A).

Echocardiographic Evaluation
In order to follow up the time course of LV function after AMI, we performed transthoracic
two-dimensional echocardiography (Vevo2100, Visual Sonics, Ontario, Canada) on miceanesthetized by inhalation of isoflurane (1-1.5%) before and at 1, 14, 28, 42 and 56 days afterAMI (Figure 1A).

Histopathological Analysis
Excised hearts were fixed with 4% paraformaldehyde for histological andimmunohistochemical examination.

After 24-48 hours of fixation and dehydration throughincreasing concentrations of ethanol, the tissue specimens were embedded in paraffin andsliced at 3µm in thickness. The sections were used for hematoxylin-eosin (HE,
Sigma-Aldrich, Shinagawa, Japan), Masson-trichrome (MT, Sigma-Aldrich),
immunohistochemical stainings for CD31 (anti-CD31, 1:400, Abcam, Cambridge, UK),immunodetection was accomplished using a Histofine kit (Nichirei, Tsukiji, Japan).

Theextent of infarct area was calculated as a rate of fibrotic area using the following formula:fibrotic area/(LV free wall + interventricular septum)×100 (%).

Cross-sectional area of MT

stained image was used to analyze the infarct area with the use of Image J Software (NIH).
The number of immunopositive cells was counted in the infarcted, border and remote areas,
where 6 random fields were examined in each sample at × 400 magnification in a blindedmanner.

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 5

Real-time PCR in Vivo
We measured mRNA expression of vascular endothelial growth factor (VEGF), endothelial
nitric oxide synthase (eNOS), and caveolin-1 in the LV.

The heart samples were

homogenized and used for mRNA extraction with an RNeasy Plus Universal kit (QIAGEN,
Hilden, Germany).

cDNA was synthesized by using PrimeScript RT Master Mix (Takara

Bio Inc., Otsu, Japan).

The primer sequences were as follows; VEGFA, (Forward)

5’-ACATTGGCTCACTTCCAGAAACAC-3’ and (Reverse)
5’-TGGTTGGAACCGGCATCTTTA-3’; eNOS, (Forward)
5’-ATTCTGGCAAGACAGACTACACGA-3’ and (Reverse)
5’-TCCCGGTAGAGATGGTCCAG-3’; caveolin-1, (Forward)
5’-TTTCACCTGTGTACCTGAGTCTCCA-3’ and (Reverse)
5’-AAACAAGTCAAGCAGGGTTCCAA-3’; Klf2, (Forward)
5’-AGCTACACCAACTGCGGCAAG-3’ and (Reverse)
5’-TCGCACAAGTGGCACTGAAAG-3’; GAPDH, (Forward)
5’-TGTGTCCGTCGTGGATCTGA-3’ and (Reverse)5’-TTGCTGTTGAAGTCGCAGGAG-3’.
After reverse transcription, real-time PCR was performed with SYBR Premix Ex Taq II(Takara Bio Inc) and CFX96TM Real-Time system C1000TM Thermal Cycler (Bio-Rad,
Hercules CA, USA).

The PCR conditions were 40 cycles of 2 sec at 98℃ and 5 sec at 55℃.

mRNA expression levels were compared between the control and the LIPUS groups.
Results are reported as the quotients of the copy number of the gene of interest relative to thatof glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a housekeeping gene.

Western Blot Analysis in Vivo
A detailed list of antibodies used in the current study is supplied as supplemental material(Supplemental Table I).

Protease and phosphatase inhibitor cocktails were from Sigma, St.

Materials and Methods
Louis, MO.

Shindo et al., ATVB/2015/306477/R2, Page 6

Homogenized heart with the protease inhibitor cocktail and phosphataseinhibitor cocktail were centrifuged at 15,000 g for 15 min and protein concentration in the
supernatant was determined with the bicinchoninic acid protein assay (#23225, Pierce,
Rockford, IL).

Equal amounts of proteins were subjected to SDS-PAGE and subsequently

were electrotransferred to nitrocellulose membranes.

The membranes were blocked for 1

hour at room temperature in 5% BSA in Tris-buffered saline with Tween 20 (TBS-T).
Primary antibody incubations were performed at different dilutions as described in antibody
list.

All incubations for primary antibodies were done for overnight at 4℃.

antibody was used at 1:4,000 dilutions for 1 hour at room temperature.

The secondary

Proteins werevisualized by the enhanced chemiluminescence system (ECL Western Blotting Detection Kit,
GE Healthcare) and quantified with the Image J Software (NIH).

We measured protein

levels of VEGF, eNOS, and phosphorylated ERK1/2 (pERK1/2) at Thr202 and Tyr204,
total-ERK1/2 (tERK1/2), phosphorylated Akt (pAkt) at Ser473, total-Akt (tAkt), α-tubulin in
the LV.

The extents of expression of VEGF, eNOS, pERK1/2, pAkt were normalized bythat of α-tubulin, tERK1/2, tAkt, respectively.

Effects of LIPUS on Human Endothelial Cells in Vitro
Human umbilical vein endothelial cells (HUVEC) from a single donor and human coronary
artery endothelial cells (HCAEC) (Lonza, Basel, Switzerland) were cultured in a complete
endothelial medium (EGM-2 BulletKit; Lonza).
were maintained in EGM-2.

The cells were used at passages 3 to 5 andTwenty-four hours before the LIPUS treatment, the cells(1×105) were re-suspended in a 2-ml tube with EGM (Lonza).

They were exposed to LIPUS

of 32 cycles (frequency, 1.875 MHz; pulse repetition frequency, 4.90 kHz; voltage, 22.38
volts (V); Ispta, 238 mW/cm2) for 20 min (n=12 each).

After irradiation, the cells werestored for 3, 6, 12 and 24 hours in the same medium before RNA extraction and for 0, 20 min,and 6, 12, 24, 36, 48 and 72 hours before protein extraction.

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 7

Scratch Assay
HUVECs were scratched with a pipette tip 24 hours after dissemination in the culture medium.
After scratching HUVECs, the LIPUS was irradiated to the cells in the same manner as
previously described.

HUVECs were followed up for 48 hours after the irradiation.

The

length (pixel) of proliferation cells was counted.5, 6

Real-time PCR in Vitro
mRNA was extracted using the RNeasy Plus Mini kit (QIAGEN).

mRNA (600ng) was

reverse-transcribed using a QuantiTect Reverse Transcription kit (QIAGEN).

Real-time

PCR was performed using the Real-Time Detection System (Bio-Rad Laboratories).
was synthesized by using PrimeScript RT Master Mix (Takara Bio Inc).

cDNAThe primer

sequences were as follows;
VEGFA, (Forward) 5’-GAGCCTTGCCTTGCTGCTCTA-3’ and (Reverse)
5’-CACCAGGGTCTCGATTGGATG-3’; eNOS, (Forward)
5’-AAAGACAAGGCAGCAGTGGAAAT-3’ and (Reverse)
5’-TCCACGATGGTGACTTTGGCTA-3’; caveolin-1, (Forward)
5’-TTCTGGGCTTCATCTGGCAAC-3’ and (Reverse)
5’-GCTCAGCCCTATTGGTCCACTTTA-3’; β1-integrin, (Forward)
5’-GGTTTCACTTTGCTGGAGATGG-3’ and (Reverse)
5’-CAGTTTCTGGACAAGGTGAGCAATA-3’; Fyn, (Forward)
5’-CGGATTGGCCCGATTGATAG-3’ and (Reverse)
5’-CACGTCAGACTTGATTGTGAACCTC-3’; FAK, (Forward)
5’-ACATTATTGGCCACTGTGGATGAG-3’ and (Reverse)
5’-GGCCAGTTTCATCTTGTTGATGAG-3’; SDPR, (Forward)
5’-CTCCGACGCAACCATTTCA-3’ and (Reverse) 5’-AAACACGCTGGCAGGGATCT-3’;

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 8

GAPDH, (Forward) 5’-GCACCGTCAAGGCTGAGAAC-3’ and (Reverse)
5’-TGGTGAAGACGCCAGTGGA-3’, all of which were designed by the Perfect Real Time
Support System (Takara Bio Inc).

Microarray Analysis in Vitro
To identify genes that are affected by LIPUS, we carried out Agilent SurePrint G3 Human GE
8×60K v2 Microarray analysis (Takara Bio Inc) of cells cultured 6 hours after LIPUS at 0.25
W/cm2 for 20 min.

Genes that were up- or down-regulated by >1.5-fold were examinedusing Agilent Feature Extraction software (Takara Bio Inc.).

After the analysis of each

HUVECs and HCAECs, we analyzed the pooled data of both cell types after revising with
combat analysis to eliminate the batch effects lay between the two cell types.

After thecombat analysis, we performed the functional and pathway analysis of differentiallyexpressed genes in the endothelial cells exposed to LIPUS using the Gene Ontology and
KEGG Pathway database.

Significant genetic networks were identified in the differentiallyexpressed genes affected by LIPUS.

The data are summarized in Supplemental Table II.

Results were also analyzed through the use of QIAGEN’s Ingenuity® Pathway Analysis(IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity).

Studies with siRNA
Pre-designed siRNA targeting human caveolin-1, β1-integrin, Fyn, focal adhesion kinase(FAK) and serum deprivation protein response (SDPR), and negative control siRNA wereobtained from QIAGEN (caveolin-1 siRNA #S100299614, β1-integrin siRNA #S100300573,
Fyn siRNA #S100605451, FAK siRNA #S100301532, SDPR siRNA #S103246789;
QIAGEN, Tokyo, Japan).

siRNA transfection was performed using HiPerFect Transfection

Reagent (#301702; QIAGEN).

Briefly, 0.5×106 cells were transfected in 2 ml of EGM-2

containing 500µl of HiPerFect Transfection Reagent and 100nM of siRNA.

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Shindo et al., ATVB/2015/306477/R2, Page 9

Measurement of Phospho-protein by Bio-Plex System in Vitro
We also measured phosphorylated proteins using Bio-Plex Pro Human Phospho-protein
custom plate and Bio-Plex 200 system according to the manufacturer’s instructions (Bio-Rad
Laboratories).

We measured the phosphorylated proteins of the cultured cells harvested with

Bio-Plex Pro Signaling Reagent kit (Bio-Rad Laboratories, #171-304006M) andphosphatase-treated HeLa (Bio-Rad Laboratories, #171-YZB001).

We evaluatedphosphorylated MEK1 at Ser217/Ser221, phosphorylated Erk1/2 at Thr202/Tyr204 andThr185/Tyr187, phosphorylated p38 MAPK at Thr180/Tyr182, phosphorylated JNK at
Thr183/Tyr185, phosphorylated c-Jun at Ser63, phosphorylated Akt at Ser473 and Thr308,
phosphorylated p70 S6 kinase at Thr421/Ser424 and Thr389, phosphorylated NF-kB p65 at
Ser536, phosphorylated IkB-a at Ser32/Ser36, phosphorylated EGFR at Tyr1173 and Tyr1068,
phosphorylated VEGFR-2 at Tyr1175, phosphorylated PDGFR-a at Tyr754, phosphorylated
PDGFR-b at Tyr751, phosphorylated IGF-1R at Tyr1131, phosphorylated HER-2 at Tyr1248,
phosphorylated Smad2 at Ser465/Ser467, phosphorylated GSK-3a/b at Ser21/Ser9,phosphorylated Stat1 at Tyr701, phosphorylated Stat3 at Tyr705 and Ser727.
analyzed using the Bio-Plex Manager 4.1.1 software.

Results were

All measurements were performed by

Bio-Rad Research Solution Center with blinded manner.

Western Blot Analysis in Vitro
Cultured cells were harvested with cell lysis buffer (50mM Tris-HCl (pH 7.4), 150mM NaCl,
1mM EDTA, 0.25% sodium deoxycholate, 1% NP-40, with the protease inhibitor cocktail
and phosphatase inhibitor cocktail.

Cell lysates were centrifuged at 15,000 g for 15 min andprotein concentration in the supernatant was determined with the bicinchoninic acid protein
assay (#23225, Pierce).

Equal amounts of proteins were subjected to SDS-PAGE andsubsequently were electrotransferred to nitrocellulose membranes.

The membranes were

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 10

blocked for 1 hour at room temperature in 5% BSA in Tris-buffered saline with Tween 20(TBS-T).

Primary antibody incubations were performed at different dilutions as described in

antibody list.

All incubations for primary antibodies were done for overnight at 4℃.

secondary antibody was used at 1:4,000 dilutions for 1 hour at room temperature.

The

Proteins

were visualized by the enhanced chemiluminescence system (ECL Western Blotting
Detection Kit, GE Healthcare) and quantified with the Image J Software (NIH).

We

measured protein levels of VEGF, eNOS, and phosphorylated ERK1/2 (pERK1/2) at Thr202
and Tyr204, total-ERK1/2 (tERK1/2), phosphorylated Akt (pAkt) at Ser473, total-Akt (tAkt),phosphorylated Src (pSrc) at Tyr416, total-Fyn (tFyn), phosphorylated FAK (pFAK) at
Tyr397, total-FAK (tFAK), α-tubulin.

The protein levels of VEGF, eNOS and HUTS-4

were normalized to the levels of α-tubulin.

The levels of pERK1/2, pAkt, pSrc and pFAK

were normalized to those of tERK1/2, tAkt, tFyn, and tFAK.

Human Autopsy Samples
Human autopsy samples of coronary arteries were obtained from patients who died of AMI(N=15, 66±12 year-old, 11 males/4 females).

Experimental samples were taken from the

distal site of the culprit coronary artery, while samples from non-culprit coronary artery werealso used as control samples.

Immunohistochemical stainings for CD31 (#M0823; Dako,

Tokyo, Japan), caveolin-1 (#3238; Cell Signaling, Danvers, USA) and β1-integrin (#4706;
Cell Signaling) were performed.

The expression of caveolin-1 was evaluated in a blindedmanner by a semi-quantitative scoring system; no staining 0, weak staining 1, moderate
staining 2, and strong staining 3.

Statistical Analysis

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Shindo et al., ATVB/2015/306477/R2, Page 11

Continuous results are expressed as mean ± SD. We utilized Student’s t-test followed by
Bonferroni type multiple comparisons and 2-way ANOVA with Turkey’s HSD multiple
comparison test to compare mean values (GraphPad Prism Software Inc, San Diego, CA).
Survival analysis was performed by the Kaplan-Meier method, and between-groupdifferences in survival were tested by the log-rank (Mantel-Cox) test.
< 0.05 were considered to be statistically significant.

For all tests, P-values

Materials and Methods

Shindo et al., ATVB/2015/306477/R2, Page 12

Supplemental References

1.

Ohashi J, Sawada A, Nakajima S, Noda K, Takaki A, Shimokawa H.

Mechanisms

for enhanced endothelium-derived hyperpolarizing factor-mediated responses in
microvessels in mice.
2.

Circ J. 2012;76:1768-1779.

Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M.
Tie2-cre transgenic mice: A new model for endothelial cell-lineage analysis in vivo.
Dev Biol. 2001;230:230-242.

3.

Aaron AM, Hermann JL, Weil BR, Wang Y, Tan J, Moberly SP, Fiege JW, Meldrum
DR. Animal models of myocardial and vascular injury.

J Surg Res.

2010;162:239-49.
4.

Hanawa K, Ito K, Aizawa K, Shindo T, Nishimiya K, Hasebe Y, Tuburaya R,
Hasegawa H, Yasuda S, Kanai H, Shimokawa H.

Low-intensity pulsed ultrasoundinduces angiogenesis and ameliorates left ventricular dysfunction in a porcine modelof chronic myocardial ischemia.
5.

PLoS One. 2014;9:e104863.

Shimizu T, Fukumoto Y, Tanaka S, Satoh K, Ikeda S, Shimokawa H.

Crucial role ofROCK2 in vascular smooth muscle cells for hypoxia-induced pulmonary hypertension
in mice.
6.

Arterioscler Thromb Vasc Biol. 2013;33:2780-91.

Liang CC, Park AY, Guan JL.

In vitro scratch assay: a convenient and inexpensive

method for analysis of cell migration in vitro.

Nat Protoc. 2007;2:329-33.

Acoustic streaming
Extracellular
Intracellular
Caveolae
β1-integrin 

α-integrin
Caveolin-1
Fyn

SH3
and

P
P

Erk 1/2

Focal adhesion kinaseP

Caveolin-1
eNOS

Akt
Nucleus

VEGF