Abstract
Objective-Left ventricular (LV) remodeling after acute myocardial infarction still remains an important issue in cardiovascular 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 the present 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 of vascular endothelial growth factor, endothelial nitric oxide synthase, phosphorylated ERK, and phosphorylated Akt in the infarcted 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, suppressed the 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 mice in vivo, for which mechanotransduction and its downstream pathways may be involved. (Arterioscler Thromb Vasc Biol.

A 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 patient management 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 in cardiovascular 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] [5] [6] [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 treatment of patients with several diseases in orthopedics, dentistry, and brain stimulation. [9] [10] [11] [12] [13] However, it remains to be examined what molecular mechanisms are involved in the LIPUS-induced beneficial effects.
Vascular endothelial cells cover the inner surface of blood vessels 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 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 are organized by caveolins. 15, 16 One of the aspects of caveolae is known to be flow-sensing organelles converting mechanical stimuli into chemical signals transmitted into the cells, socalled mechanotransduction. 15 Caveolins have been shown to bind 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] [18] [19] [20] Caveolar membranes are also enriched in cholesterol, glycosphingolipids, and signaling enzymes, such as Src kinase. 21 In addition, caveolae are reported to respond to 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] [24] [25] Although caveolin-1 has no extracellular component, caveolin-1 may play an important role in sensing mechanical stress or the distortion of the extracellular membranes through interaction with β1-integrin. 19, 20, 26, 27 In the present study, we aimed to demonstrate whether the LIPUS ameliorates post-MI LV remodeling and if so, to elucidate the underlying 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 Post-MI 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 weight or LV wall thickness was noted among the 4 groups during the study period ( Figure IC in the online-only Data Supplement). Infarct size was significantly smaller in the MI-LIPUS group than 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 no-LIPUS group (Figure 2A ). There was no significant difference in 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 was no 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 extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt ( Figure 2C and 2D). In the infarcted area, the LIPUS therapy upregulated the expression of VEGF and eNOS on day 6, but not on days 3 and 28 ( 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 
Effects of the LIPUS Therapy on Signaling Pathways in Human Umbilical Vein Endothelial Cells In Vitro
We then aimed to further elucidate the underlying molecular mechanisms involved in the beneficial effects of the LIPUS therapy in human umbilical vein endothelial cells (HUVECs) in vitro. 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 peak at 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 was correlated 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 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 differentially expressed (upregulation in 381 genes and downregulation in 622 genes). We also analyzed the pooled data of HUVECs and human coronary 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, were significantly 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 showed that 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 the role of focal adhesion pathway in the LIPUS-induced angiogenesis, we preformed studies with small interfering RNA (siRNA) to interfere some molecules that are supposed to be involved in the mechanotransduction 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] [22] [23] [24] suppressed the LIPUSinduced upregulation of VEGF ( Figure 3D ). Knockdown with siRNA of either Fyn or FAK also suppressed the LIPUS-induced upregulation of VEGF ( Figure 3D ). To evaluate the importance of structure of caveolae itself, conformational changes of caveolae were induced with either knockdown of serum deprivation response protein, a caveolar protein required for the formation of characteristic flask-shaped caveolar membrane invaginations, or methyl-β-cyclodextrin, an inhibitor specifically designed to disrupt 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 ). Knockdown with siRNA of each molecule was confirmed with real-time polymerase 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, phosphorylation of 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 the activation state of β1-integrin ( Figure VB in the online-only Data Supplement). 31 To further examine the involvement of each molecule in focal adhesion pathway, siRNA interference studies were performed. Knockdown with siRNA of 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 
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 were treated 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, the beneficial 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 
Expression of Caveolin-1 in MI Mice and in Human Autopsy Samples
Immunohistochemical staining showed that the immunoreactivity of caveolin-1 was enhanced in the infarcted area 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 immunoreactivity of caveolin-1 was significantly enhanced in coronary arterial endothelial cells in the ischemic myocardium (the distal site of the culprit coronary lesion) when compared with the remote area ( Figure 6A-6C ).
Discussion
In the present study, we were able to demonstrate that the LIPUS therapy ameliorates post-MI LV remodeling in mice in 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 effects of 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 the mortality 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 upregulation of 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, and post-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 upregulation of VEGF in response to the LIPUS was prolonged as it was noted 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. 
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 phosphorylation of angiogenesis-related proteins in HUVECs using the Bio-Plex phosphoprotein assay. In this analysis, phosphorylation of ERK1/2 and Akt was enhanced by the LIPUS therapy in vitro. 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 showed that 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 structure of caveolae. 15, 16 The present results with siRNA suggest that caveolin-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 changes 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 that the 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 the endothelial cell-specific caveolin-1 knockout mice, suggesting that endothelial cells play pivotal roles in the angiogenic effects of the LIPUS. Taken together, these results suggest that the acoustic streaming by the LIPUS induces distortion of caveolae on endothelial cells, which then transmits the mechanical stimuli to intracellular signaling pathways with subsequent 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 by mechanical stimuli (eg, shear stress) on the surface of vascular endothelial cells. 15, [19] [20] [21] [22] 24 
Biological Effects of LIPUS Other Than Angiogenesis
The microarray analysis suggested that the LIPUS exerts biological effects on cell cycles, metabolic pathways, RNA transport, DNA replication, mRNA surveillance, mismatch repair, and protein export in addition to its angiogenic effects. In this regard, we have recently reported that the low-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 influx of 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 Ca 2+ levels. This point remains to be examined in 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-mediated activation of ERK1/2 and PI3-Akt pathways is mediated by caveolin-1. 35 In the present study, endothelial expression of caveolin-1 was enhanced in the infarcted area early after AMI 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 those observed 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 (20 minutes×3 levels×3 days). We speculate that the repeated irradiation 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 
Remote Infarcted
LIPUS might be further blunted in Cav-1-KO mice. Third, we did not examine the effects of the LIPUS in genetically modified 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 the present study based on the results of microarray analysis. Although we showed that caveolin-1, β1-integrin, Fyn, and FAK 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 of angiogenic effects of the LIPUS. However, it is possible that 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.
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] [2] [3] Although regenerative therapies, such as gene and cell therapies, have been 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] [42] [43] Because the intensity of ultrasound used in the LIPUS therapy is below the upper 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 to suppress post-MI LV remodeling. It could also be applied to other ischemic disorders, such as ischemic cardiomyopathy, peripheral arterial disease, refractory chronic skin ulcers, and spinal cord injury as in the case with low-energy extracorporeal shock wave therapy. [44] [45] [46] 
Conclusions
In the present study, we were able to demonstrate that the LIPUS therapy ameliorates post-MI LV remodeling in mice in 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 post-MI LV remodeling.

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