Low-Intensity Pulsed Ultrasound Induces Angiogenesis
and Ameliorates Left Ventricular Dysfunction in a
Porcine Model of Chronic Myocardial Ischemia
Kenichiro Hanawa1, Kenta Ito1, Kentaro Aizawa1, Tomohiko Shindo1, Kensuke Nishimiya1, Yuhi Hasebe1,
Ryuji Tuburaya1, Hideyuki Hasegawa2,3, Satoshi Yasuda4, Hiroshi Kanai2,3, Hiroaki Shimokawa1*
1 Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan, 2 Division of Biomedical Measurements and Diagnostics,
Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan, 3 Department of Electronic Engineering, Graduate School of Engineering, Tohoku
University, Sendai, Japan, 4 National Cerebral and Cardiovascular Center, Osaka, Japan

Abstract
Background: Although a significant progress has been made in the management of ischemic heart disease (IHD), thenumber of severe IHD patients is increasing. Thus, it is crucial to develop new, non-invasive therapeutic strategies. In thepresent study, we aimed to develop low-intensity pulsed ultrasound (LIPUS) therapy for the treatment of IHD.
Methods and Results: We first confirmed that in cultured human endothelial cells, LIPUS significantly up-regulated mRNA
expression of vascular endothelial growth factor (VEGF) with a peak at 32-cycle (P,0.05). Then, we examined the in vivo
effects of LIPUS in a porcine model of chronic myocardial ischemia with reduced left ventricular ejection fraction (LVEF)(n = 28). The heart was treated with either sham (n = 14) or LIPUS (32-cycle with 193 mW/cm2 for 20 min, n = 14) at 3
different short axis levels. Four weeks after the treatment, LVEF was significantly improved in the LIPUS group (4664 to
5765%, P,0.05) without any adverse effects, whereas it remained unchanged in the sham group (4665 to 4766%,
P = 0.33). Capillary density in the ischemic region was significantly increased in the LIPUS group compared with the control
group (10846175 vs. 8586151/mm2, P,0.05). Regional myocardial blood flow was also significantly improved in the LIPUS
group (0.7860.2 to 1.3960.4 ml/min/g, P,0.05), but not in the control group (0.8460.3 to 0.9760.4 ml/min/g). Western
blot analysis showed that VEGF, eNOS and bFGF were all significantly up-regulated only in the LIPUS group.
Conclusions: These results suggest that the LIPUS therapy is promising as a new, non-invasive therapy for IHD.
Citation: Hanawa K, Ito K, Aizawa K, Shindo T, Nishimiya K, et al. (2014) Low-Intensity Pulsed Ultrasound Induces Angiogenesis and Ameliorates Left Ventricular
Dysfunction in a Porcine Model of Chronic Myocardial Ischemia. PLoS ONE 9(8): e104863. doi:10.1371/journal.pone.0104863
Editor: Costanza Emanueli, University of Bristol, United Kingdom
Received March 12, 2014; Accepted July 11, 2014; Published August 11, 2014
Copyright: ß 2014 Hanawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the manuscriptand supporting information files.
Funding: This study was supported in part by the Grant-in-Aid (Regional Innovation Strategy Support Program) and the Special Funds for Education andResearch from the Japanese Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: shimo@cardio.med.tohoku.ac.jp

used for therapeutic applications, including tumor ablation,
thrombolysis, bone regeneration, and facilitated drug delivery[9]. Recently, therapeutic angiogenic effects of low-intensity
ultrasound have been reported in endothelial cells, chickchorioallantoic membrane, and a rat model of hind limb ischemia[10]–[12]. In the present study, we thus examined whether lowintensity pulsed ultrasound (LIPUS) enhances endothelial regeneration in vitro and whether it ameliorates ischemia-induced
myocardial dysfunction in a porcine model of chronic myocardialischemia in vivo, and if so, what molecular mechanisms are
involved. In addition, we also examined whether we can modify
the current diagnostic ultrasound devices into the LIPUS therapy
apparatus, in order to facilitate this LIPUS-mediated angiogenic
therapy in the real world practice of IHD.

Introduction
Ischemic heart disease (IHD) is one of the major causes of deathin developed countries, and its morbidity is also increasing indeveloping countries [1]–[3]. Although recent advances intherapeutic strategies have reduced the mortality of patients with
IHD [1], the number of severe IHD patients is increasing as the
population is rapidly aging. Thus, non-invasive therapeuticstrategies for severe IHD remain to be developed. We havepreviously demonstrated that low-energy extracorporeal cardiac
shock wave (SW) therapy improves myocardial ischemia in a
porcine model of chronic myocardial ischemia and patients withsevere angina pectoris [4]–[8].
Ultrasound is a form of sound whose frequency is higher than
the natural audible range for humans (.20 kHz) and ultrasonography has been widely used as diagnostic devices for several
decades. In addition to diagnostic purposes, ultrasound is clinically
PLOS ONE | www.plosone.org

1

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundEffects of LIPUS on Human Umbilical Vein Endothelial
Cells in Vitro

Materials and Methods
All procedures were performed according to the protocols
approved by the Institutional Committee for Use and Care ofLaboratory Animals of Tohoku University (25MdA-36).

We purchased single-donor human umbilical vein endothelial
cells (HUVEC) (Lonza, Basel, Switzerland) and cultured them in acomplete endothelial medium (EGM-2 BulletKit, Lonza). HUVEC were subcultured and used at passages 3 to 5 and weremaintained in EGM-2. For the LIPUS treatment, we used adiagnostic ultrasound device (Prosound a10; HITACHI Aloka
Medical, Ltd., Mitaka, Japan) whose irradiation conditions could
be modified by software modification. Twenty-four hours before
the LIPUS treatment, HUVEC (16105) were re-suspended in a 2ml tube with EGM (Lonza). The cells were exposed to LIPUS ofvarious conditions [frequency, 1.875 MHz; pulse repetition
frequency, 7.10 kHz; number of cycles, 1, 16, 32, 48 and 64
cycles; voltage applied to each transducer element, 11.30,88.18
volts (V); Ispta, 151,193 mW/cm2] for 20 minutes (n = 9–11each). After irradiation, the cells were stored for 3 hours in thesame medium before RNA extraction and for 20 minutes before
protein extraction.

Measurement of Acoustic Pressure of LIPUS
We measured acoustic pressure of LIPUS under various
conditions [frequency, 1.875 MHz; pulse repetition frequency,
7.10 kHz; number of cycles, 1, 16, 32, 48 and 64; voltage applied
to each transducer element, 11.30,88.18 volts (V); Ispta,
151,193 mW/cm2] by using a needle hydrophone. The numberof cycles of pulsed ultrasound means the number of acoustic waves
per 1 pulse, while 1 cycle is used for diagnostic ultrasound devices.
In the present study, however, we tested larger numbers of cycles
from 16 to 64 (Figure 1). The voltage applied to each transducer
element was controlled to keep the estimated spatial peak temporal
average intensity (Ispta) of LIPUS below the upper limit of
acoustic output standards (,720 mW/cm2) for diagnostic ultrasound devices (U.S. Food and Drug Administration’s Track 3
Limits) and to prevent the ultrasound probe from temperature rise.

Real-Time Polymerase Chain Reaction and Western Blot
Analysis in vitro
Total RNA was extracted using the RNeasy Micro Kit (Qiagen,Hilden, Germany). Total RNA (600 ng) was reverse-transcribed

Figure 1. In vitro study. (A) Acoustic pressure at various cycle numbers. (B) LIPUS treatment up-regulated mRNA expression of VEGF in HUVECs
in vitro with a maximum effect noted at 32-cycle. Results are expressed as mean 6 SEM (n = 9–11 each).
doi:10.1371/journal.pone.0104863.g001

PLOS ONE | www.plosone.org

2

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with Ultrasoundusing a QuantiTect Reverse Transcription Kit (Qiagen). Realtime polymerase chain reaction (RT-PCR) was performed using
the Real-Time Detection System (Bio-Rad Laboratories, Hercules,
CA). Sequences of the primers were (forward, reverse) 59GAGCCTTGCCTTGCTGCTCTA-39,
59-CACCAGGGTCTCGATTGGATG-39 for vascular endothelial
growth factor (VEGF), 59- GTGTGCTAACCGTTACCTGGCTATG-39, 59- CCAGTTCGTTTCAGTGCCACA-39 for basic
fibroblast growth factor (bFGF), 59- TTCTGACTGCACAAACCAGCTTC-39, 59-TTTGACACCACACACAGCTTCAC-39 for
Flk-1,
and
59-GCACCGTCAAGGCTGAGAAC-39,
59TGGTGAAGACGCCAGTGGA-39 for b-actin, all of whichwere designed by the Perfect Real Time Support System (Takara
Bio Inc., Otsu, Japan). The b-actin was used as an internal control
and SYBR Premix Ex Taq I and II (Takara Bio Inc.) were used forthe detection of VEGF cDNA.
Western blot analysis was performed using antibodies thatspecifically recognize proteins, including eNOS (total-eNOS; Cell
Signaling Technology, MA, USA) and phospho-eNOS at Ser1177(p-eNOS; Cell Signaling Technology). The regions containing peNOS and eNOS proteins were visualized by electrochemiluminescence Western blotting luminal reagent (Santa Cruz Biotechnology, Dallas, TX). The extent of p-eNOS was normalized bythat of eNOS.

an ameroid constrictor (LIPUS group, 3 times a week), while wetreated the remaining 14 pigs with the same anesthesia proceduresfor 70 min but without the LIPUS treatment (control group, 3
times a week).

Coronary Angiography and Left Ventriculography
After systemic heparinization (30,000 U/body IV), we performed coronary angiography and left ventriculography in a left
oblique view with the use of a cineangiography system (Toshiba
Medical Systems, Ohtawara, Japan) (n = 9 each) [4]. We semiquantitatively evaluated the extent of collateral flow to the LCx
region by the Rentrop score (0, no visible collateral vessels; 1, faintfilling of side branches of the main epicardial vessel without fillingthe main vessel; 2, partial filling of the main epicardial vessel; 3,complete filling of the main vessel) [15]. We also counted thenumber of visible coronary arteries in the LCx region. To
compare the extent of collateral development at a given time, we
selected the frame in which the whole left anterior descending
coronary artery (LAD) was first visualized [4].

Echocardiographic Evaluation
We performed transthoracic echocardiographic studies before
ameroid implantation (baseline) and at 4 and 8 weeks after theprocedure (n = 9 each). To evaluate regional myocardial function,
we calculated wall thickening fraction (WTF) by using thefollowing formula; WTF = 1006(end-systolic wall thickness 2
end-diastolic wall thickness)/end-diastolic wall thickness [4].

Porcine Model of Chronic Myocardial Ischemia
We used a total of 60 domestic pigs (25 to 30 kg in body weight)
in the present study. After sedation with medetomidine (0.05 mg/
kg, IM) and midazolam (0.3 mg/kg, IM), we maintained theanesthesia with an inhalation of 2–5% sevoflurane for implantation of an ameroid constrictor, LIPUS treatment, and euthanization by intravenous injection of 5% potassium chloride at 8 weeks
after implantation of an ameroid constrictor (Figure 2). We
opened the chest, suspended the pericardium and left atrial
appendage, revealed the left circumflex coronary artery (LCx), andput an ameroid constrictor (Research Instruments NW, Lebanon,
OR) around the proximal LCx to gradually induce a total
occlusion of the artery in 4 weeks without causing myocardial
infarction [4]. This model is widely used to examine the effect ofan angiogenic therapy on the ischemic hibernating myocardium[4], [13], [14]. In the present study, animals that were suspected ofdeveloping myocardial infarction (e.g. severe segmental LV wall
thinning) or showed left ventricular ejection fraction (LVEF) of
more than 55% based on echocardiographic studies at 4 weeks
were excluded before randomization. Finally, 28 pigs wereassigned to the 2 groups with (LIPUS group) or without (control
group) the LIPUS therapy using block randomization with a block
size of 6. We defined absolute change in LVEF at 8 weeks between
the control and the LIPUS group as the primary end-point, withan unpaired t test and a = 5%. Assuming that the LIPUS therapy
exerts similar effects as in our prior SW therapy, the absolute
improvement of 10% in LVEF at 8 weeks was the assumed effectsize for our sample size calculation. Power analysis was conductedin R version 3.0.3. A total of 8 pigs ensured a statistical power of
90% based on our assumption. Thus, the present study (n = 9each) has a sufficient statistical power.

Measurement of Regional Myocardial Blood Flow
We evaluated regional myocardial blood flow (RMBF) withcolored microspheres (Dye-Trak; Triton Technology, San Diego,CA) at 4 and 8 weeks after implantation (n = 7 each) [4]. Due totechnical problems, 2 samples in each group were excluded. We
injected microspheres through the left atrium and aspirated areference arterial blood sample from the descending aorta at aconstant rate of 20 ml/min for 60 seconds using a withdrawal
pump. We extracted microspheres from the left ventricular (LV)wall and blood samples by potassium hydroxide digestion,
extracted the dyes from the spheres with dimethylformamide(200 ml), and determined their concentrations by spectrophotometry [16]. We calculated myocardial blood flow (ml ? min21 ? g21)of the LCx region and the IVS region [4].

Factor VIII Staining
We treated paraffin-embedded sections with a rabbit anti-factor
VIII antibody (N1505; Dako, Copenhagen, Denmark). We
counted the number of factor VIII-positive cells in 10 fields ofthe ischemic LV lateral wall (LCx region) and the non-ischemic
interventricular septum (IVS) (LAD region) in each heart at x400
magnification (n = 7 each) [4].

Western Blot Analysis in Vivo
The sections from the ischemic LCx region and the nonischemic IVS region were used for Western blot analysis (n = 5each). The regions containing VEGF, eNOS, and bFGF proteins
were visualized by electrochemiluminescence Western blotting
luminal reagent (Santa Cruz Biotechnology). The extent of the
VEGF, eNOS, and bFGF was normalized by that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [4].

LIPUS Therapy to Chronic Ischemic Myocardium
On the basis of the in vitro experiment, LIPUS (20-min
irradiation at 32-cycle) was applied to the lateral wall (ischemic
region) by 2-dimensional scan at 3 different short-axis levels,
including (1) basal LV, (2) papillary muscle level, and (3) near theapex of a heart (Figure 2). Among the 28 pigs used, we treated 14
pigs with the LIPUS treatment at 4 weeks after the implantation of
PLOS ONE | www.plosone.org

Statistical Analysis
Results are expressed as mean 6 SEM. Comparisons ofparameters between 2 groups were performed with unpaired
3

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundFigure 2. Experimental protocol. (A) Protocol, (B) LIPUS therapy in action, and (C) schematic images of LIPUS application. UCG, ultrasoundcardiography; CAG, coronary angiography; LVG, left ventriculography.
doi:10.1371/journal.pone.0104863.g002

LIPUS therapy at 32 cycles did not up-regulated mRNA
expression of bFGF, the ratio of phospho-eNOS to total-eNOS(p-eNOS/total-eNOS), a marker of eNOS activation, wassignificantly increased in HUVECs (1.43-fold increase, P,0.05)(Figure S2).

Student’s t-test. Data were analyzed by one-way ANOVA (in vitro
study) or two-way ANOVA (in vitvo study) followed by Tukey’s
HSD test for multiple comparisons. A value of P,0.05 wasconsidered to be statistically significant.

Results
Effects of the LIPUS Therapy on Ischemia-Induced
Myocardial Dysfunction

Acoustic Pressure of LIPUS
When the number of cycles was increased from 1 to 16, 32, 48
and 64, peak acoustic pressure generated by a 1.875 MHz
transducer was decreased from 1.59 to 0.77, 0.56, 0.43 and0.36 MPa, respectively (Table S1). The relationships between
voltage applied to each transducer element and acoustic pressure
are shown in Figure S1. Among the conditions tested, estimatedIspta was highest when the cycle number was 32 (Table S1).

At 4 weeks after ameroid implantation (Pre-Tx), left ventriculography demonstrated reduced LVEF in both groups (Figure 3A
and 3B). At 8 weeks (Post-Tx), while LVEF remained unchangedin the control group (45.664.6 to 46.965.6%, P = 0.33)(Figure 3C), it was normalized in the LIPUS group (46.064.1
to 56.665.2%, P,0.05) (Figure 3D). Absolute improvement of
LVEF 4 weeks after the therapy, the primary end-point of thepresent study, was significantly greater in the LIPUS than in thecontrol group (46.965.6 to 56.665.2%, P,0.05) (Figure 3E).
We also serially measured WTF of the LCx region (lateral wall of
the LV) by transthoracic echocardiography. While WTF was
equally reduced at 4 weeks in both groups (Control, 21.662.7 vs.
LIPUS, 20.669.4%), WTF was markedly improved in the LIPUS
group, but remained unchanged in the control group at 8 weeks(Control, 18.268.0 vs. LIPUS, 34.869.8%, P,0.05) (Figure 4).

Effects of LIPUS on mRNA Expression of VEGF, Flk-1 andbFGF, and eNOS Activity in HUVECs
In accordance with the preliminary data mentioned above(Table S1), the LIPUS treatment significantly up-regulatedmRNA expression of VEGF in HUVECs, with a maximum effectnoted at 32 cycles (1.33-fold increase, P,0.05) (Figure 1) while
the expression of its receptor Flk-1 was not enhanced (Control,
1.0060.06 vs. LIPUS, 1.0160.10; at 32 cycles). Although the
PLOS ONE | www.plosone.org

4August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundPLOS ONE | www.plosone.org

5

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundFigure 3. LIPUS therapy improved ischemia-induced myocardial dysfunction in vivo. (A, B) Four weeks after the implantation of anameroid constrictor (pre-Tx), LV wall motion of the LCx region was reduced in both the control (A) and the LIPUS group (B). (C, D) Four weeks after
the first left ventriculography (post-Tx), no significant change in LV wall motion was noted in the control group (C), whereas marked recovery was
noted in the LIPUS group (D). E, The LIPUS therapy normalized left ventricular ejection fraction in the LIPUS group but not in the control group.
Results are expressed as mean 6 SEM (n = 9 each).
doi:10.1371/journal.pone.0104863.g003

Detailed data of left ventriculography and echocardiography are
shown in Table S2.

(Control, 751620 vs. LIPUS, 785645/mm2, P = 0.27) (Figure 5C).

Angiogenic Effects of the LIPUS Therapy

Effects of the LIPUS Therapy on Regional Myocardial
Blood Flow

There was no difference in the coronary artery dominancebetween the 2 groups; in the control group, right dominant in 0,balanced type in 7, left dominant in 2 and in the LIPUS group,
right dominant in 0, balanced type in 7, and left dominant in 2[17]. At 4 weeks after ameroid implantation, coronary angiography demonstrated a total occlusion of the LCx, which was
perfused via collateral vessels with severe delay in both the control(Figure S3A) and the LIPUS groups (Figure S3B). However, at
8 weeks, the LIPUS group (Figure S3D), but not the control
group (Figure S3C), showed a marked development of coronary
collateral vessels in the ischemic LCx region, with an increased
Rentrop score (Figure S3E) and an increased number of visible
coronary arteries in the region (Figure S3F). Factor VIII staining
showed that the number of factor VIII-positive capillaries wasincreased only in the LIPUS group (Figure 5A and 5B).
Quantitative analysis demonstrated that the number of capillaries
in the ischemic lateral wall region at 8 weeks was significantly
higher in the LIPUS group than in the control group (Control,
8586151 vs. LIPUS, 10846175/mm2, P,0.05), whereas it was
unchanged in the non-ischemic IVS region in both groups

At 4 weeks, RMBF was equally decreased in the lateral wall inboth groups (Control, 0.8460.29 vs. LIPUS, 0.7860.22 ml/min/
g, P = 0.35), but remained normal in the IVS (Control, 1.9160.59
vs. LIPUS, 1.8960.53 ml/min/g, P = 0.47). At 8 weeks, the
LIPUS treatment significantly improved RMBF in the lateral wall(Control, 0.9760.36 vs. LIPUS, 1.3960.35 ml/min/g, P,0.05)(Figure 6A), whereas it remained unchanged in the non-ischemic
IVS in both groups despite the LIPUS treatment (Control,
2.0460.69 vs. LIPUS, 2.1960.77 ml/min/g, P = 0.36) (Figure 6B).

Effects of the LIPUS Therapy on Expression of VEGF,eNOS and bFGF
Western blot analysis at 5 weeks demonstrated that protein
expressions of VEGF, eNOS and bFGF were significantly upregulated in the lateral wall in the LIPUS group, whereas they
were unchanged in the non-ischemic IVS region in both groups
despite the LIPUS treatment (Figure 7).

Figure 4. LIPUS therapy improved regional myocardial function in vivo. LIPUS therapy induced a complete recovery of WTF of the ischemic
lateral wall. Results are expressed as mean 6 SEM (n = 9 each).
doi:10.1371/journal.pone.0104863.g004

PLOS ONE | www.plosone.org

6

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundFigure 5. LIPUS therapy increased the density of factor VIII-positive capillaries in the ischemic myocardium. (A, B) Factor VIII stainingof the LCx region from the control (A) and the LIPUS group (B). Scale bar represents 20 mm. C. Capillary density was significantly higher in the LIPUS
group than in the control group in the ischemic lateral wall. Results are expressed as mean 6 SEM (n = 7 each).
doi:10.1371/journal.pone.0104863.g005

pressure. As a result, the highest estimated Ispta was obtained
when the LIPUS was applied at 32 cycles in the present study. The
intensity of the ultrasound used in the present study is equivalent
to that of ultrasound diagnostic devices while higher intensity
ultrasound is used for thrombolysis or tumor ablation (highintensity focused ultrasound, HIFU) [9]. Although we confirmed
the beneficial effect of the LIPUS at 32 cycles in vivo withenhanced angiogenesis and improved LV myocardial dysfunction,
it remains to be further examined which was more important,higher Ispta or the cycle number of 32 itself. This point needs to be
examined in future studies.

Safety of the LIPUS Therapy
No adverse hemodynamic changes or no histological damage inthe skin or lung were noted by the LIPUS.

Discussion
The novel finding of this study is that the LIPUS therapy
induces angiogenesis in the ischemic myocardium and normalizes
myocardial function in a porcine model of chronic myocardialischemia in vivo. To the best of our knowledge, this is the firstreport that demonstrates the potential usefulness of the LIPUS
therapy as a non-invasive treatment of IHD.

Beneficial Effects of the LIPUS Therapy on IHD
Enhancing Effect of the LIPUS on Endothelial
Regeneration in Vitro

In the present study, we demonstrated that the LIPUS therapy(1) normalized global and regional myocardial function in vivo, (2)increased capillary density and RMBF of the chronically ischemic
region without any adverse effects, and (3) enhanced protein levelsof VEGF, eNOS, and bFGF in the ischemic myocardium without
affecting those in the non-ischemic myocardium. These results
indicate that the LIPUS therapy is an effective and safe
therapeutic strategy for ischemia-induced myocardial dysfunction.

The number of cycles of pulsed ultrasound of our equipment indiagnostic use is 1 cycle while we tested larger numbers of cycles(16, 32, 48 and 64) in the present study for therapeutic purpose.
When the LIPUS was applied to HUVECs, the maximal effect to
up-regulate mRNA expression of VEGF was achieved at 32 cycles.
The peak acoustic pressure was reduced with increasing number ofcycles while elevation of voltage applied to each transducer
element was limited by temperature rise of the ultrasound probe
although higher the voltage could induce higher peak acoustic

PLOS ONE | www.plosone.org

7

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundFigure 6. LIPUS therapy improved RMBF in the ischemic region in vivo. LIPUS therapy significantly increased RMBF, assessed by coloredmicrospheres in the lateral wall (A), while it was not changed in the both groups in the non-ischemic IVS region (B). Results are expressed as mean 6
SEM (n = 7 each).
doi:10.1371/journal.pone.0104863.g006

is possible that the LIPUS directly and/or indirectly affects thefunction and dynamics of immature cells, such as bone marrowderived mononuclear cells and residential cardiac stem cells [24].

Potential Mechanisms for LIPUS-induced Angiogenesis
We demonstrated that the LIPUS up-regulated VEGF expression and eNOS activity in vitro and enhanced angiogenesis in
association with an enhanced protein expression of VEGF, eNOS,
and bFGF in pigs in vivo. Barzelai et al. also reported that LIPUS
induces angiogenesis and up-regulation of VEGF in hind-limb
ischemia in rats [10]. eNOS has vasodilating and angiogenic
effects [18], while bFGF is known to mediate new blood vessel
formation in wound healing [19]. Thus, it is suggested that the
LIPUS therapy improved myocardial function by enhancing
multiple angiogenic pathways. However, it is unclear how LIPUS
regulates the expression of angiogenic factors in the ischemic
myocardium alone.
In the present study, the extent of an increase in VEGF mRNA
expression by a single LIPUS therapy for 20 min was rather smallin HUVECs in vitro, whereas the protein expression of VEGF
increased by more than twice after the LIPUS therapy (3 times aweek) in vivo (2.19-fold increase, P,0.05; Figure 7B). Also, the
LIPUS therapy did not up-regulate the mRNA expression ofbFGF in vitro, whereas the LIPUS therapy significantly enhanced
the protein expression of bFGF in vivo. These results suggest that
repeated therapies up-regulate angiogenic factors more than single
therapy. Another possibility is that cell-to-cell interaction between
endothelial cells and other types of cells is important in enhancing
multiple angiogenic pathways [20]. Bone marrow-derived progenitor cells and residential stem cells have been shown tocontribute to angiogenesis process [21]. Xu et al. reported that
LIPUS stimulated hematopoietic stem/progenitor cell viability,
proliferation and differentiation in vitro [22]. Toyama et al. also
reported that application of LIPUS to circulating angiogenic cells
augmented their generation and migration capacities [23]. Thus, it
PLOS ONE | www.plosone.org

Other Potential Mechanisms of LIPUS-induced Beneficial
Effects on LV dysfunction
Previous studies have shown that LIPUS stimulates the
production of angiogenic cytokines (e.g. IL-8, bFGF, and VEGF)[25] and reduces COX-2 and PGI2 production [26], and that thelow-energy SW therapy exerts multiple effects, including angiogenic, anti-inflammatory, anti-oxidant and anti-apoptotic effects[4]–[8], [27]. Thus, as in the case with the low-energy SW
therapy, the mechanisms other than angiogenesis may also
contribute to the beneficial effects of LIPUS in the present study.
This point remains to be examined in future studies.

The Effects of LIPUS on the Single-cell Level
Mechanical stimuli are known to affect endothelial cell function
and mechanosensors on endothelial cell membranes, such as
integrins and caveolins [28], [29]. Ultrasound irradiation has beenreported to produce shear stress on endothelial cells [11]. Also,mechanical stimulation on cultured endothelial cells induced byshear stress has been reported to enhance endothelial cellproliferation, migration and sprouting [30]. In addition, ultrasound has been shown to induce sonoporation on endothelial cell
membrane leading to influx of calcium ion [31]. Thus, it isconceivable that several mechanisms, such as triggering of
mechanosensors, activation of endothelial cell growth andsonoporation-activated alterations, contribute to the LIPUSinduced angiogenesis. Further studies are needed.

8

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundFigure 7. LIPUS treatment up-regulated protein expression of VEGF, eNOS, and bFGF in the ischemic myocardium (n = 5 each). (A)Western blots of VEGF, eNOS, and bFGF. (B,D) Quantification of VEGF, eNOS, and bFGF expression.
doi:10.1371/journal.pone.0104863.g007

The intensity of the ultrasound used in the present study is
within the limits of acoustic output standards for diagnostic
ultrasound devices defined by U.S. Food and Drug Administration. Since the present LIPUS system is characterized by the use of
the same probe for both diagnostic purpose and for LIPUS
therapy, which makes the LIPUS therapy device much easier tohandle than the SW therapy device, and thus, the LIPUS therapy
is expected as a new, non-invasive angiogenic therapy because
ultrasound diagnostic devices have already been widely used in theworld.

Comparison of SW Therapy and LIPUS Therapy
We have previously demonstrated that low-energy cardiac SW
therapy effectively induces neovascularization and improves
myocardial function in a porcine model of chronic myocardialischemia [4]. Based on the promising results in animal studies, wehave developed the low-energy SW therapy, where the low-energy
SW therapy improves symptoms and myocardial perfusion inpatients with severe angina pectoris [5]–[8]. We also havedemonstrated that the low-energy SW therapy improves thewalking ability of patients with peripheral artery disease (PAD) andintermittent claudication [32]. Furthermore, we have recently
demonstrated that the low-energy SW therapy induces therapeutic
lymphangiogenesis, enhances skin wound healing and improves
locomotor recovery after spinal cord injury [33]–[35]. Since SW
and ultrasound share similarities as they similarly travel straight
through body tissue (fat, muscles, body fluid etc.), we tested
potential usefulness of LIPUS for the treatment of ischemic heartdisease as a non-invasive angiogenic therapy. In the present study,
we were able to demonstrate that the LIPUS at a certain
irradiation condition effectively enhances the expression ofangiogenic cytokines and significantly improves ischemia-induced
myocardial dysfunction in vivo. Since the LIPUS therapy on
ischemic myocardium was equally effective to the low-energy SW
therapy [4], the LIPUS therapy and the low-energy SW therapy
may activate common signaling pathways.

PLOS ONE | www.plosone.org

Clinical Implications
As mentioned above, the LIPUS therapy is expected to beeffective in patients with IHD. Unlike SW that is applied tofocused region, LIPUS is irradiated not only to ischemic region
but also to non-ischemic region. However, the LIPUS therapy
enhanced angiogenesis only in the ischemic region without adverse
effects. We consider that this new LIPUS therapy should be first
applied for patients with symptomatic angina pectoris without
indication for PCI or CABG. The LIPUS therapy is also expected
to be effective in PAD patients with intermittent claudication or
refractory skin ulcer like the low-energy SW therapy [32]–[34].

9August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with UltrasoundeNOS (p-eNOS/total-eNOS), a marker of eNOS activation at 32cycle in vitro. Results are expressed as mean 6 SEM (n = 6 each).
(TIF)

Study Limitations
Several limitations should be mentioned for the present study.
First, we optimized the setting of ultrasound within the intensity of
acoustic output standards for diagnostic devices because higher
intensity of ultrasound involves temperature rise in the probe
which may cause burn injury. To give priority to safety, we did not
examine higher intensity of ultrasound to prevent skin burn due togeneration of heat. Second, in the present study, we performed the
LIPUS therapy 3 times a week based on our previous studies withcardiac SW therapy [4], [5], [7], [36], [37] and observed
beneficial effects of the LIPUS therapy on ischemia-induced LV
dysfunction. However, it is still not clear whether the 3 times is the
optimal number of therapies or not. This point remains to beaddressed in future studies.

Figure S3 LIPUS therapy enhanced coronary angiogenesis
in vivo. (A, B) Four weeks after the implantation of an ameroid
constrictor (pre-Tx) LCx was totally occluded and was perfused via
collateral vessels with severe delay in both the control group (A)and the LIPUS group (B). (C, D) Four weeks after the firstcoronary angiography (post-Tx), no significant change in coronary
vessels was noted in the control group (C), whereas a marked
development of visible coronary vessels was noted in the LIPUS
group (D). (E, F) Four weeks after the first coronary angiography,
no significant increase in the Rentrop score (E) or visible coronary
arteries from LCx (F) was noted in the control group, whereas
increased Rentrop score and a marked development of visible
coronary vessels were noted in the LIPUS group. Results are
expressed as mean 6 SEM (n = 9 each).
(TIF)

Conclusions
In the present study, we were able to demonstrate that the noninvasive LIPUS therapy enhances multiple angiogenic pathways,
effectively increases capillary density and regional myocardial
blood flow (RMBF), and normalizes ischemia-induced myocardial
dysfunction without any adverse effects. Thus, these results
indicate that the LIPUS therapy may be an effective, safe, andnon-invasive therapy for IHD.

The relationships between voltage and acoustic pressure.
(DOC)

Table S1

Table S2 Findings of left ventriculography and echocardiography.
(DOC)

Supporting Information
Figure S1 The relationship between voltage applied to eachtransducer element and acoustic pressure at various cycle
numbers. The each circle represents the highest acoustic pressure
achieved at each cycle number. Elevation of voltage applied toeach transducer element was limited by temperature rise of theultrasound probe.
(TIF)

Acknowledgments
We thank HITACHI Aloka Medical, Ltd. for their technical assistance andvaluable comments on our study. The authors also thank Akemi Saito,
Teru Hiroi, Yumi Watanabe, Ai Nishihara and Hiromi Yamashita for their
excellent technical assistance.

Author Contributions

Figure S2 In vitro study. (A) LIPUS therapy did not upregulated mRNA expression of eNOS or bFGF at 32-cycle.
Results are expressed as mean 6 SEM (n = 8 each). (B) LIPUS
therapy significantly increased the ratio of phospho-eNOS to total-

Conceived and designed the experiments: KI HH SY HK HS. Performed
the experiments: KH KA TS KN YH RT. Analyzed the data: KH KI KA
TS. Wrote the paper: KH KI HS.

References
13. O’Konski MS, White FC, Longhurst J, Roth D, Bloor CM. (1987) Ameroid
constriction of the proximal left circumflex coronary artery in swine: a model oflimited coronary collateral circulation. Am J Cardiovasc Pathol 1: 69–77.
14. Roth DM, Maruoka Y, White FC, Longhurst JC, Bloor CM, et al. (1987)
Development of coronary collateral circulation in left circumflex ameroidoccluded swine myocardium. Am J Physiol 253: H1279–1288.
15. Rentrop KP, Cohen M, Blanke H, Phillips RA. (1985) Changes in collateralchannel filling immediately after controlled coronary artery occlusion by anangioplasty balloon in human subjects. J Am Coll Cardiol 5: 587–592.
16. Prinzen FW, Bassingthwaighte JB. (2000) Blood flow distributions by
microsphere deposition methods. Cardiovasc Res 45: 13–21.
17. Weaver ME, Pantely GA, Bristow JD, Ladley HD. (1986) A quantitative study of
the anatomy and distribution of coronary arteries in swine in comparison withother animals and man. Cardiovasc Res 20: 907–17.
18. Bir SC, Xiong Y, Kevil CG, Luo J. (2012) Emerging role of PKA/eNOS
pathway in therapeutic angiogenesis for ischaemic tissue diseases. Cardiovasc
Res 95: 7–18.
19. Przybylski M. (2009) A review of the current research on the role of bFGF and
VEGF in angiogenesis. J Wound Care 18: 516–519.
20. Zhang M, Shah AM. (2014) ROS signalling between endothelial cells andcardiac cells. Cardiovasc Res 102: 249–57.
21. Garin G, Mathews M, Berk BC. (2005) Tissue-resident bone marrow-derived
progenitor cells: key players in hypoxia-induced angiogenesis. Circ Res 97: 955–
957.
22. Xu P, Gul-Uludag H, Xing J, Swanson E, Chen J, et al. (2012) Low-intensity
pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell
viability, proliferation and differentiation in vitro. Biotechnol Lett 34: 1965–
1973.
23. Toyama Y, Sasaki K, Chibana H, Itaya N, Imaizumi T, et al. (2012) Ultrasoundstimulation restores impaired neovascularization-related capacities of human
circulating angiogenic cells. Cardiovasc Res 95: 448–459.

1. Ruff CT, Braunwald E. (2011) The evolving epidemiology of acute coronary
syndromes. Nat Rev Cardiol 8: 140–147.
2. Japanese Coronary Artery Disease (JCAD) Study Investigators. (2006) Current
status of the background of patients with coronary artery disease in Japan. Circ J
70: 1256–1262.
3. Hata J, Kiyohara Y. (2013) Epidemiology of stroke and coronary artery disease
in Asia. Circ J 77: 1923–1932.
4. Nishida T, Shimokawa H, Matsumoto Y, Takeshita A, Sunagawa K, et al.
(2004) Extracorporeal cardiac shock wave therapy markedly ameliorates
ischemia-induced myocardial dysfunction in pigs in vivo. Circulation 110:3055–3061.
5. Fukumoto Y, Ito A, Takeshita A, Sunagawa K, Shimokawa H, et al. (2006)
Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia inpatients with severe coronary artery disease. Coron Artery Dis 17: 63–70.
6. Ito K, Fukumoto Y, Shimokawa H. (2009) Extracorporeal shock wave therapy as
a new and non-invasive angiogenic therapy. Tohoku J Exp Med 219: 1–9.
7. Kikuchi Y, Ito K, Kuriyama S, Tsuji I, Shimokawa H, et al. (2010) Double-blindand placebo-controlled study of the effectiveness and safety of extracorporeal
cardiac shock wave therapy for severe angina pectoris. Circ J 74: 589–591.
8. Ito K, Fukumoto Y, Shimokawa H. (2011) Extracorporeal shock wave therapy
for ischemic cardiovascular disorders. Am J Cardiovasc Drugs 11: 295–302.
9. ter Haar G. (2007) Therapeutic applications of ultrasound. Prog Biophys Mol
Biol 93: 111–129.
10. Barzelai S, Sharabani-Yosef O, Walden R, Engelberg S, Scheinowitz M, et al.
(2006) Low-intensity ultrasound induces angiogenesis in rat hind-limb ischemia.
Ultrasound Med Biol 32: 139–145.
11. Mizrahi N, Seliktar D, Kimmel E. (2007) Ultrasound-induced angiogenic
response in endothelial cells. Ultrasound Med Biol 33: 1818–1829.
12. Ramli R, Reher P, Harris M, Meghji S. (2009) The effect of ultrasound on
angiogenesis: an in vivo study using the chick chorioallantoic membrane. Int J
Oral Maxillofac Implants. 24: 591–596.

PLOS ONE | www.plosone.org

10

August 2014 | Volume 9 | Issue 8 | e104863

Cardiac Angiogenic Therapy with Ultrasound

24. Leri A, Kajstura J, Anversa P. (2011) Role of cardiac stem cells in cardiac
pathophysiology: a paradigm shift in human myocardial biology. Circ Res 109:
941–961.
25. Reher P, Doan N, Bradnock B, Meghji S, Harris M. (1999) Effect of ultrasoundon the production of IL-8, basic FGF and VEGF. Cytokine 11: 416–423.
26. Nakamura T, Fujihara S, Katsura T, Inubushi T, Tanaka E, et al. (2011) Lowintensity pulsed ultrasound reduces the inflammatory activity of synovitis. Ann
Biomed Eng 39: 2964–2971.
27. Fu M, Sun CK, Shao PL, Leu S, Yip HK, et al. (2011) Extracorporeal shock
wave therapy reverses ischemia-related left ventricular dysfunction andremodeling: molecular-cellular and functional assessment. PLoS One 6: e24342.
28. Shyy JY, Chien S. (2002) Role of integrins in endothelial mechanosensing ofshear stress. Circ Res 91: 769–775.
29. Yu J, Bergaya S, Kurzchalia TV, Stan RV, Sessa WC, et al. (2006) Direct
evidence for the role of caveolin-1 and caveolae in mechanotransduction andremodeling of blood vessels. J Clin Invest 116: 1284–1291.
30. Zaragoza C, Márquez S, Saura M. (2012) Endothelial mechanosensors of shear
stress as regulators of atherogenesis. Curr Opin Lipidol 23: 446–452.
31. Hassan MA, Campbell P, Kondo T. (2010) The role of Ca2+ in ultrasoundelicited bioeffects: progress, perspectives and prospects. Drug Discov Today 15:
892–906.

PLOS ONE | www.plosone.org

32. Serizawa F, Ito K, Sato A, Shimokawa H, Satomi S, et al. (2012) Extracorporeal
shock wave therapy improves the walking ability of patients with peripheralartery disease and intermittent claudication. Circ J 76: 1486–1493.
33. Serizawa F, Ito K, Sato A, Shimokawa H, Satomi S, et al. (2011) Extracorporeal
shock wave therapy induces therapeutic lymphangiogenesis in a rat model ofsecondary lymphoedema. Eur J Vasc Endovasc Surg 42: 254–260.
34. Hayashi D, Kawakami K, Ito K, Shimokawa H, Tachi M, et al. (2012) Lowenergy extracorporeal shock wave therapy enhances skin wound healing indiabetic mice: a critical role of endothelial nitric oxide synthase. Wound Repair
Regen 20: 887–895.
35. Yamaya S, Ozawa H, Ito K, Shimokawa H, Itoi E, et al. (2014) Low-energy
extracorporeal shock wave therapy promotes VEGF expression and neuroprotection and improves locomotor recovery after spinal cord injury. J Neurosurg(in press).
36. Uwatoku T, Ito K, Hizume T, Sunagawa K, Shimokawa H, et al. (2007)
Extracorporeal cardiac shock wave therapy improves left ventricular remodelingafter acute myocardial infarction in pigs. Coron Artery Dis 18: 397–404.
37. Ito Y, Ito K, Fukumoto Y, Yasuda S, Shimokawa H, et al. (2010) Cardiac shock
wave therapy ameliorates left ventricular remodeling after myocardial ischemiareperfusion injury in pigs in vivo. Coron Artery Dis 21: 304–11.

11

August 2014 | Volume 9 | Issue 8 | e104863