Pulsed focused ultrasound (pFUS) technology is being developed for clinical neuro/immune modulation and regenerative medicine. Biological signal transduction of pFUS forces can require mechanosensitive or voltage-gated plasma membrane ion channels. Previous studies suggested pFUS is capable of activating either channel type, but their mechanistic relationship remains ambiguous. We demonstrated pFUS bioeffects increased mesenchymal stem cell tropism (MSC) by altering molecular microenvironments through cyclooxygenase-2 (COX2)-dependent pathways. This study explored specific relationships between mechanosensitive and voltage-gated Ca2+ channels (VGCC) to initiate pFUS bioeffects that increase stem cell tropism.Methods: Murine kidneys and hamstring were given pFUS (1.15 or 1.125 MHz; 4MPa peak rarefactional pressure) under ultrasound or magnetic resonance imaging guidance. Cavitation and tissue displacement were measure by hydrophone and ultrasound radiofrequency data, respectively. Elastic modeling was performed from displacement measurements. COX2 expression and MSC tropism were evaluated in the presence of pharmacological ion channel inhibitors or in transient-receptor-potential-channel-1 (TRPC1)-deficient mice. Immunohistochemistry and co-immunoprecipitation examined physical channel relationships. Fluorescent ionophore imaging of cultured C2C12 muscle cells or TCMK1 kidney cells probed physiological interactions.Results: pFUS induced tissue deformations resulting in kPa-scale forces suggesting mechanical activation of pFUS-induced bioeffects. Inhibiting VGCC or TRPC1 in vivo blocked pFUS-induced COX2 upregulation and MSC tropism to kidneys and muscle. A TRPC1/VGCC complex was observed in plasma membranes. VGCC or TRPC1 suppression blocked pFUS-induced Ca2+ transients in TCMK1 and C2C12 cells. Additionally, Ca2+ transients were blocked by reducing transmembrane Na+ potentials and observed Na+ transients were diminished by genetic TRPC1 suppression.Conclusion: This study suggests that pFUS acoustic radiation forces mechanically activate a Na+-containing TRPC1 current upstream of VGCC rather than directly opening VGCC. The electrogenic function of TRPC1 provides potential mechanistic insight into other pFUS techniques for physiological modulation and optimization strategies for clinical implementation.

BACKGROUND Myocardial contractility, a significant determinant of cardiac function, is valuable for diagnosis and evaluation of treatment in cardiovascular disorders including heart failure. Shear Wave Elasticity Imaging (SWEI) is a newly developed ultrasound-based elastography technique that can directly assess the stiffness of cardiac tissue. The aim of the study was to verify the ability of technique to quantify contractility changes in the myocardium. METHODS In 12 isolated rabbit hearts we made SWEI measurements of systolic stiffness at 5 different coronary perfusion pressures from 0 to 92 mmHg. The changes in coronary perfusion were used to induce acute stepwise reversible changes in cardiac contractility via the Gregg effect. The Gregg effect is the dependency of contractility on coronary perfusion. In 4 of the hearts, we repeated the measurements after delivery of Gadolinium, which is known to block the Gregg effect. RESULTS Systolic stiffness measured by SWEI changed linearly with coronary perfusion pressure with a slope of 0.27 kPa/mmHg (mean of 95% CI, R2=0.73). As expected the change in contractility due to the Gregg effect was blocked by Gadolinium with a significant reduction of the slope to 0.08 kPa/mmHg. CONCLUSION SWEI measurements of systolic stiffness provide an index of contractility in the unloaded isolated rabbit heart. While this study was done under ideal imaging conditions and with non-physiological loading conditions, it reinforces the concept that this ultrasound technique has the potential to provide a direct and noninvasive index of cardiac contractility.

Mesenchymal stromal cells (MSC) are potential renal therapeutics. Clinically, results are mixed partly because MSC tropism to kidneys is minimal following infusion. Ultrasound augmentation of the renal microenvironment is becoming increasingly-important in renal MSC therapies. We demonstrated pulsed-focused-ultrasound (pFUS) increases enhanced homing permeability and retention of MSC in mouse kidneys. Here, we characterized the temporal proteomic response to pFUS in mouse kidneys and its association with MSC tropism. pFUS induced molecular cascades of initial increases in tumor necrosis factor-α (TNFα) and interleukin (IL)-1α, that activated nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and cyclooxygenase-2 (COX2) pathways without cell death. This was followed by a 24–48 hour-long response of increased cell adhesion molecules (CAM), trophic and anti-inflammatory factors. Pretreating animals with anti-inflammatory drugs etanercept (TNFα inhibitor), anakinra (IL-1 receptor antagonist), prednisone (NFκB translocation inhibitor), or ibuprofen (COX inhibitor) suppressed molecular changes and inhibited renal MSC tropism. We further examined the role of COX2 using a COX2-knock-out mouse where pFUS was unable to increase MSC tropism. These results demonstrate that renal micro-environmental changes induce MSC tropism and could influence the therapeutic efficacy of MSC. Optimizing the microenvironment and understanding drug effects will enable improvements in MSC therapies for renal disease.

Mesenchymal stromal cell (MSC) therapies combined with renal pulsed focused ultrasound (pFUS) pretreatment increase MSC homing and improve cisplatin‐induced acute kidney injury (AKI) better than MSC alone. However, mechanisms underlying improved outcomes remain unknown. We hypothesize pFUS up‐regulates renal interferon‐γ (IFNγ) and stimulates MSC to produce interleukin‐10 (IL‐10) after migrating to kidneys. To demonstrate initially, MSC cultured with IFNγ up‐regulated IL‐10. More MSC‐derived IL‐10 was detected in kidneys when IFNγ‐stimulated MSC were infused and they improved AKI better than unstimulated MSC. Next, IFNγ‐knockout mice with AKI received pFUS+MSC, but MSC‐derived IL‐10 expression and AKI were similar to using MSC alone. AKI in wild‐type mice receiving pFUS and IL‐10‐deficient MSC was also unimproved compared to administering IL‐10‐deficient MSC alone. Indoleamine 2,3‐dioxygenase (IDO), an anti‐inflammatory enzyme up‐regulated in MSC by IFNγ, was up‐regulated during AKI, but was not further elevated in MSC from pFUS‐treated kidneys, suggesting that IDO is not involved in improved AKI healing by pFUS+MSC. These data suggest IFNγ is up‐regulated by pFUS and after i.v.‐infused MSC home to pFUS‐treated kidneys, IFNγ stimulates additional IL‐10 production by MSC to improve AKI. Analogous mechanisms of ultrasound‐treated tissue microenvironments stimulating therapeutic MSC may exist in other pathologies where adjuvant ultrasound techniques are successful.

BackgroundImage-guided high intensity focused ultrasound has been used as an extracorporeal cardiac pacing tool and to enhance homing of stem cells to targeted tissues. However, molecular changes in the myocardium after sonication have not been widely investigated. Magnetic-resonance (MR)-guided pulsed focused ultrasound (pFUS) was targeted to the rat myocardium over a range of pressures and the microenvironmental and histological effects were evaluated over time.MethodsEight-to-ten-week-old Sprague–Dawley rats received T2-weighted MR images to target pFUS to the left ventricular and septum without cardiac or respiratory gating. Rats were sonicated through the thoracic wall at peak negative pressures (PNP) from 1 to 8 MPa at a center frequency of 1 MHz, 10 ms pulse duration and 1 Hz pulse repetition frequency for 100 pulses per focal target. Following pFUS, myocardium was harvested over 24 h and subjected to imaging, proteomic, and histological measurements.ResultspFUS to the myocardium increased expression of cytokines, chemokines, and trophic factors characterized by an initial increase in tumor necrosis factor (TNF)-α followed by increases in pro- and anti-inflammatory factors that returned to baseline by 24 h. Immediately after pFUS, there was a transient (< 1 h) increase in N-terminal pro b-type natriuretic peptide (NT-proBNP) without elevation of other cardiac injury markers. A relationship between PNP and expression of TNF-α and NT-proBNP was observed with significant changes (p < 0.05 ANOVA) ≥ 4 MPa compared to untreated controls. Contrast-enhanced ex vivo T1-weighted MRI revealed vascular leakage in sonicated myocardium that was accompanied by the presence of albumin upon immunohistochemistry. Histology revealed infiltration of neutrophils and macrophages without morphological myofibril changes in sonicated tissue accompanied by pulmonary hemorrhage at PNP > 4 MPa.ConclusionsMR-guided pFUS to myocardium induced transient proteomic and histological changes. The temporal proteomic changes in the myocardium indicate a short-lived sterile inflammatory response consistent with ischemia or contusion. Further study of myocardial function and strain is needed to determine if pFUS could be developed as an experimental model of cardiac injury and chest trauma.Electronic supplementary materialThe online version of this article (10.1186/s12967-017-1361-y) contains supplementary material, which is available to authorized users.