MR‐guided pulsed focused ultrasound improves mesenchymal stromal cell homing to the myocardium

Abstract Image‐guided pulsed focused ultrasound (pFUS) is a non‐invasive technique that can increase tropism of intravenously (IV)‐infused mesenchymal stromal cells (MSC) to sonicated tissues. MSC have shown promise for cardiac regenerative medicine strategies but can be hampered by inefficient homing to the myocardium. This study sonicated the left ventricles (LV) in rats with magnetic resonance imaging (MRI)‐guided pFUS and examined both proteomic responses and subsequent MSC tropism to treated myocardium. T2‐weighted MRI was used for pFUS targeting of the entire LV. pFUS increased numerous pro‐ and anti‐inflammatory cytokines, chemokines, and trophic factors and cell adhesion molecules in the myocardial microenvironment for up to 48 hours post‐sonication. Cardiac troponin I and N‐terminal pro‐B‐type natriuretic peptide were elevated in the serum and myocardium. Immunohistochemistry revealed transient hypoxia and immune cell infiltration in pFUS‐targeted regions. Myocardial tropism of IV‐infused human MSC following pFUS increased twofold‐threefold compared with controls. Proteomic and histological changes in myocardium following pFUS suggested a reversible inflammatory and hypoxic response leading to increased tropism of MSC. MR‐guided pFUS could represent a non‐invasive modality to improve MSC therapies for cardiac regenerative medicine approaches.

inefficient myocardial homing and requires invasive techniques for adequate delivery. pFUS has been applied for various cardiac applications such as treating cardiac arrhythmias, as a cardiac pacing tool, as a contusion model and as a method to mitigate cardiac diseases. [16][17][18][19] The current study investigates whether pFUS could be used to modify the myocardial microenvironment and increase tropism of IV-injected MSC.
Ultrasound exposures have previously demonstrated increased stem cell homing to myocardium, but these indications coupled with intravenous microbubble (MB) infusion can cause ultrasound-targeted MB destruction (UTMB). [17][18][19] UTMB has been shown to cause some molecular changes that are necessary to induce MSC tropism, but UTMB often results in tissue damage making the approaches less attractive as a regenerative medicine technique.
In this study, pFUS without MB was administered to the left ventricle at parameters previously shown not to damage other tissues. We investigated whether pFUS could generate cellular and molecular changes in the heart to ultimately enhance permeability and retention of human mesenchymal stromal cells (MSC) in treated myocardium. The pFUS treatments presented here could provide clear benefit to non-invasively increasing MSC homing to myocardium without causing tissue damage associated with UTMD.

| Animals
All animal experiments were approved by the Animal Care and Use Committee at the NIH Clinical Center and were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory. 20 Eight-to-ten-week-old female Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were provided free access to food and water during the study. The hair on the chest was removed with depilatory cream prior to pFUS treatment, and the average weight of rats was 230.2 ± 9.7 g.

| MR-guided pFUS and MRI
Rats (n = 87) were placed on a pre-clinical MR-compatible imageguided high intensity focused ultrasound (HIFU) system (RK-100, FUS instruments, Ontario, Canada). The left side of the chest submerged in degassed H 2 O maintained at 37°C in order to place the heart perpendicular to the ultrasonic transducer ( Figure 1A). The rats were anesthetized with 1.5% of isoflurane in 100% O 2 during the pFUS treatment ( Figure 1).
MR images performed with multi-slice fast field echo (FFE) sequences with repetition time (TR) = 8.9 ms, echo time (TE) = 4.5 ms, flip angle (FA) = 45° and a slice thickness of 1 mm with an in-plane resolution of 0.14 × 0.14 mm 2 of the heart were acquired using a clinical 3T scanner with a radiofrequency receive coil (Achieva, Philips Healthcare, USA) without cardiac or respiratory gating. A focused ultrasound transducer with a centre frequency = 1.  Figure 1C-J).

| Proteomic analysis of myocardium and serum following pFUS
The pFUS-targeted myocardium and blood were harvested (n = 5-10 rats/time point) at post-0 (SHAM (SH) or Baseline (BL)), 0.25, 1, 3, 6, 12, 18, 24, 36, 48 and 96 hours. The harvested myocardium was frozen immediately in liquid nitrogen. The blood was also harvested to analyse the cardiac injury marker expression following pFUS with centrifugation at speed 500g (3,000 rpm) for 10 minutes to isolate the serum, which was stored in −80°C for further analysis. The SH

| Statistical analyses
All data are presented as mean ± standard deviation, and data analyses were performed with Prism (version 7, GraphPad Software, Inc La Jolla, CA). One-way analysis of variance (ANOVA) with Dunnett post hoc tests were used for multiple comparisons. Means were compared with two-tailed unpaired t test. The P-value less than 0.05 was considered as statistical significance.

| MR-guided pFUS
Rats were placed approximately 90 degree rotated to the left on the MR-compatible pre-clinical FUS system in order to pose the heart F I G U R E 1 MR-guided pFUS. A, Rats placed on a MR-compatible pre-clinical focused ultrasound system. F, Illustration of experimental setup of pFUS treatment to the rat heart. Left side of the rat chest was submerged in degassed water to pose the heart perpendicular to the focused ultrasound transducer. B-E, Sequential T2w coronal MR images were acquired at 1 mm slice thickness for pFUS to target the left ventricle of the rat heart. Blue outline area represents the left ventricle, and red outline area represents the right ventricle. G-J, pFUS targeting spots in red circle based on the MR guidance images perpendicular to the ultrasonic transducer that could minimize the sonication to the lungs ( Figure 1A and F). Under MR guidance, the left ventricle was sonicated with 3 MPa of PNP, which is approximately 1.6 MPa intramyocardially (equivalent to a MI = 1.7) as a result of attenuation at rat chest wall. 22 Following pFUS treatment, no macroscopic and microscopic evidence of pulmonary damage was observed as has been previously reported at sonications at PNP = 6 MPa. 22

| D ISCUSS I ON
MR-guided pFUS is a non-invasive tool for modulating tissue microenvironments and has potential clinical translational in the treatment of cardiac diseases. In this study, pFUS mechanotransductive effects were evaluated based on the molecular changes in the myocardium and the ability to enhance homing permeability and retention (EHPR) of infused MSC to targeted regions. [5][6][7][8][17][18][19]23 MRI guidance for pFUS provided the ability for accurate targeting to the volumes of interest (VOI) in the myocardium that ultimately resulted in the induced molecular responses consistent with increased expression of pro-and anti-inflammatory CCTF between 6 and 36 hours. [4][5][6][7][8]22,24 In comparison with the current study, we previously reported that of hypoxia and molecular signalling cascades. [35][36][37][38] Moreover, the effects of pFUS to induce a sterile inflammatory response in the targeted myocardium does not lead to persistent tissue damage require further examination. 39,40 There are several studies demonstrating the potential use of ultrasound (both focused and unfocused) to the heart coupled with MB infusion that generated molecular changes in the myocardium increased would not be directly compared, we believe that the underlying molecular mechanism that attracts the MSC homing would be similar. In the current study, pFUS induced an EHPR effect on IV-infused human MSC that led the significantly increased numbers of human cells homing to the targeted regions by 24 hours. These results are similar to our previous findings 17,18 in which the increased tropism to murine skeletal muscle and kidney in response to the release of chemoattractants (ie MCP1, GM-CSF) following pFUS at 4 MPa. [4][5][6][7][8][16][17][18][19][20]22,[24][25][26][27][28][30][31][32][33]39,40 These results are consistent with previous observation, which demonstrated the ability of pFUS to induce changes in the tissue microenvironment that would EHPR of infused stem cells into targeted regions of hypoxia. 13 We speculate that MSC homing plays a major role in paracrine signalling rather than in differentiation to myocardium muscle. Moreover, the MSC that were used in this study were expanded from volunteers who had bone marrow biopsies. However, it is possible that more efficient homing and survival capacity may be achieved by using stem cells derived from rats that would obfuscate an immune response and result in prolonged in vivo lifespan of MSC. 41,42 Moreover, we did not examine the possible changes in cellular metabolism such as functional changes by mitochondria induced by pFUS. 18 Future studies will assess cardiac functional changes following pFUS including optimization of sonication parameters to induce necessary MSC tropism without elevating injury biomarkers. The results of this study provide a basis for additional investigation of treatment parameters that could be translatable as part of a regenerative medicine strategy in the treatment of cardiac injury or disease.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

AUTH O R CO NTR I B UTI O N S
KWJ, SRB and JAF: Study design; KWJ, TT, BKL, RBR and SRB: Experiments; KWJ, TT, RR, SRB and JAF: Data analysis and interpretation; KWJ, SRB and JAF: Manuscript drafting. All authors: Manuscript reviewing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author, KWJ The data are not publicly available. F I G U R E 7 fIHC of anti-human mitochondrial staining of pFUS targeted and controls rats (n = 3/group) following 24 h after infusion of human MSC. A and B, the ROI of the SHAM and pFUStargeted myocardium, respectively. C, Quantitative analysis of numbers of human MSC (red) in rat myocardium compared with controls. Asterisks represent the statistical significance set at P < .05 based on ANOVA. Scale bar = 300 µm A B C