Stimulation of adult resident cardiac progenitor cells by durable myocardial expression of thymosin beta 4 with ultrasound-targeted microbubble delivery.

It has been proposed that thymosin beta 4 (TB4)-protein delivery stimulates differentiation of resident adult WT1-positive cardiac progenitor cells, but with very low efficiency. We determined whether gene therapy with human TB4 stimulates proliferation of resident adult cardiac progenitor cells in normal rat heart. Ultrasound-targeted microbubble destruction (UTMD) was used to deliver the human TB4 gene under a piggybac transposon plasmid to normal rat heart. The rat hearts were assayed by quantitative reverse transcription-PCR and immunohistology with a confocal microscope at 1, 2, 3, 4 and 12 weeks after UTMD. Exogenous TB4 stimulation resulted in the presence of WT1-positive cardiac progenitor cells from epicardium to endocardium. TB4 stimulated angiogenesis and arteriogenesis. One month after TB4 gene therapy by UTMD, the percentage of NKX2.5-positive cardiomyocytes was 5.5±1.0% and NKX2.5 mRNA was 24-fold higher than in the control groups (P<0.001). Similar results were found for ISL-1, BrDu, Ki-67, PHH3 and aurora B (P<0.001). Cardiac-specific delivery of exogenous human TB4 gene efficiently stimulates proliferation and differentiation of resident WT1-positive adult cardiac progenitor cells into three intact cardiac cell lineages-vascular endothelial cells, coronary artery smooth muscle cells and cardiac muscle cells in normal adult rat heart.

An effective method to release human islets from surrounding acinar cells with agitation in high osmolality solution.

INTRODUCTION: Islet purification is mainly performed by the density gradient method. However, purification of the embedded islets that are surrounded by exocrine tissue should be difficult, because their density is similar to exocrine tissue. In this study, we performed chart review to assess the relationship between the ratio of embedded islets and efficacy of purification. Then, we tested several conditions of a new method to free the islets from surrounded exocrine tissues using high osmolality solution with gentle agitation. MATERIALS AND METHODS: First, we performed chart review of our human islet isolation. Second, embedded islet-enriched human islet fractions (embedded islets >50%) were suspended in University of Wisconsin (UW) solution (UW group, 320 mOsm/kg/H(2)0) or osmolality-adjusted UW solution (400, 500, and 600 mOsm/kg/H(2)0; 400 group, 500 group, and 600 group, respectively). Each tube was gently shaken at 4°C. The tissue samples were taken before shaking and after 15, 30, and 60 minutes. Islet yield, percentage of embedded islets, and viabilities were assessed. RESULTS: The chart review revealed that high ratio of embedded islets deteriorated the efficacy of islet purification. The islet yield in all groups except for the 600 group did not change at 15 minutes, but it decreased in all groups at 60 minutes. The average percentage of embedded islets before shaking was 62.6%. Although percentage of embedded islets were decreasing in all groups, it was < 20% at 15 minutes in the 500 and 600 groups whereas it was >44% in the UW group, which indicated that higher osmolality would have a greater effect. Viability was >95% in all groups at 30 minutes. CONCLUSIONS: The embedded islets deteriorated the efficacy of islet purification. Gentle agitation of embedded islets in high osmolality (500 mOsm/kg/H(2)O, 15 minutes) could release islets from surrounded exocrine tissue.

Assessment of human islet isolation with four different collagenases.

BACKGROUND: The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein. METHODS: The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI). RESULTS: There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group (P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others (P = .0003 vs MTF, .0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group (P = .006). The viability in the NB1 group was significantly greater than the HI group (P = .003). CONCLUSION: Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.

Improved method of human islet isolation for young donors.

BACKGROUND: Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield. METHODS: We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n = 3) or 100 mL of solution (5 mg/mL, new group, n = 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol. RESULTS: Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume. CONCLUSION: Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors.

Neurogenic differentiation 1 directs differentiation of cytokeratin 19-positive human pancreatic nonendocrine cells into insulin-producing cells.

BACKGROUND: It has been reported that the human pancreatic nonendocrine fraction, which remains after islet isolation, can be differentiated toward beta cells. However, the optimal method to accomplish this goal has not been established. In this study, we introduced the human neurogenic differentiation 1 (NeuroD1) gene into human nonendocrine pancreatic epithelial cells (NEPECs) and promoted insulin-producing cells in vitro. METHODS: The human pancreatic nonislet fractions were obtained from brain-dead donors and cultured in suspension for 2-3 days followed by culture with G418 for 4 days. These cells (NEPECs) were then plated on dishes. The NEPECs spread into a cell monolayer within 7 days and all of the cells were cytokeratin-19 (CK19) positive. Seven days after plating, plasmids encoding human NeuroD1 gene under human CK19 promoter were transfected 3 times every other day (termed NEPEC+ND). Seven days after starting induction, these cells were characterized. RESULTS: Seven days after starting the induction of human NeuroD1, NEPEC+ND strongly expressed NeuroD1 and insulin mRNA. The ratio of NeuroD1-positive cells in NEPEC+ND was significantly higher than in NEPEC. Human insulin-positive cells in NEPEC+ND were also significantly greater than in NEPEC. Human insulin and C-peptide levels in culture medium in NEPEC+ND were significantly higher than in NEPEC. CONCLUSIONS: These findings demonstrated that human NeuroD1 under control of the CK19 promoter can induce the differentiation of CK19-positive NEPECs into insulin-producing cells.

Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy.

This study uses a novel approach to gene therapy in which plasmid DNA is targeted to the pancreas in vivo using ultrasound-targeted microbubble destruction (UTMD) to achieve islet regeneration. Intravenous microbubbles carrying plasmids are destroyed within the pancreatic microcirculation by ultrasound, achieving local gene expression that is further targeted to ß-cells by a modified rat insulin promoter (RIP3.1). A series of genes implicated in endocrine development were delivered to rats 2 days after streptozotocin-induced diabetes. The genes, PAX4, Nkx2.2, Nkx6.1, Ngn3 and Mafa, produced α-cell hyperplasia, but no significant improvement in ß-cell mass or blood glucose level 30 days after UTMD. In contrast, RIP3.1-NeuroD1 promoted islet regeneration from surviving ß-cells, with normalization of glucose, insulin and C-peptide levels at 30 days. In a longer-term experiment, four of six rats had a return of diabetes at 90 days, accompanied by ß-cell apoptosis on Tunel staining. Pretreatment with the JNK inhibitor SP600125 successfully blocked ß-cell apoptosis and resulted in restoration of ß-cell mass and normalization of blood glucose level for up to 90 days. This technique allows in vivo islet regeneration, restoration of ß-cell mass and normalization of blood sugar, insulin and C-peptide in rats without viruses.

In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.

AIMS/HYPOTHESIS: Delivery of the gene for human vascular endothelial growth factor (VEGF, also known as VEGFA) to both the transplanted islets and the surrounding tissue may promote islet revascularisation and survival. We previously showed the effective delivery of VEGF gene to rat myocardium by an ultrasound-mediated gene-transfer method named ultrasound-targeted microbubble destruction (UTMD). Here we examined the effect of non-viral VEGF delivery using UTMD on transplanted islets in vivo. METHODS: A marginal number of human islets were transplanted into livers of mice which were a model for diabetes. Then, non-viral plasmid vectors encoding VEGF (VEGF group, n = 11) or the gene for green fluorescent protein (GFP) (GFP group, n = 7) were introduced into the host liver by UTMD. Transplantation without gene delivery was performed as a control (no-UTMD group, n = 8). Blood glucose, serum human insulin, C-peptide levels and the revascularisation in graft islets were evaluated. RESULTS: Restoration of euglycaemia occurred in 13% in the no-UTMD group and 14% in the GFP group, whereas 73% mice in the VEGF group became euglycaemic at day 30 (p < 0.05 in no-UTMD vs VEGF). Serum human insulin and C-peptide were significantly higher in the VEGF group at day 32 (insulin: no-UTMD, 17 +/- 8; GFP, 37 +/- 17; VEGF, 109 +/- 26 pmol/l, respectively, p < 0.05; C-peptide: no-UTMD, 68 +/- 38; GFP, 115 +/- 58; VEGF, 791 +/- 230 pmol/l, respectively, p < 0.05). Vessel density in graft islets was significantly higher in the VEGF group (no-UTMD, 169 +/- 36; GFP, 227 +/- 39; VEGF, 649 +/- 51 counts/mm(2), respectively, p < 0.05). CONCLUSIONS/INTERPRETATION: Delivery of VEGF gene to host liver using UTMD promoted islet revascularisation after islet transplantation and improved the restoration of euglycaemia.

Efficient, glucose responsive and islet-specific transgene expression by a modified rat insulin promoter.

This study was done to improve efficiency and islet specificity of the rat insulin promoter (RIP). Various RIP lengths were prepared and tested in vitro to drive luciferase reporter gene expression in INS1-cells, alpha-cells, acinar cells, ductal cells and fibroblasts. The CMV promoter was used as a positive control. In addition, the DsRed reporter gene was administered in vivo to rat pancreas by ultrasound-targeted microbubble destruction (UTMD). Confocal microscopy was used to detect the presence and distribution of DsRed within the pancreas after UTMD. A modified RIP3.1 promoter, which includes portions of the insulin gene after its transcription start site is fivefold more active in INS-1 cells than the full-length RIP promoter or the CMV promoter. RIP3.1 is regulated by glucose level and various islet transcription factors in vitro, and exhibits activity in alpha-cells, but not in exocrine cells. In vivo delivery of RIP3.1-DsRed resulted in expression of DsRed protein in beta-cells, and to a lesser extent in alpha-cells under normal glucose conditions. No DsRed signal was present in exocrine pancreas under RIP3.1. A modified RIP, RIP3.1, efficiently and specifically directs gene expression to endocrine pancreas.

Reversal of streptozotocin-induced diabetes in rats by gene therapy with betacellulin and pancreatic duodenal homeobox-1.

Ultrasound-targeted microbubble destruction (UTMD) was used to direct betacellulin (BTC) and pancreatic duodenal homeobox-1 (PDX1) to rat pancreas 48 h after islet destruction by streptozotocin (STZ). Sprague-Dawley rats were rendered diabetic by STZ injection. Controls included normal rats, STZ only without UTMD, and UTMD with DsRed reporter gene. Blood glucose increased dramatically in all rats 48 h after STZ, and continued to rise after UTMD with BTC alone. Blood glucose declined from day 3 to day 10 after UTMD with PDX1, but remained elevated (261+/-8 mg/dl). However, in rats treated with both BTC and PDX1, blood glucose remained below 200 mg/dl throughout day 10. This was accompanied by normalization of blood insulin and C-peptide. Histology demonstrated islet-like clusters of glucagon-staining cells in the rats treated with BTC and PDX1, but these clusters disappeared by 30 days after UTMD treatment. Although regeneration of insulin-producing islets was not seen, diabetes was reversed for up to 15 days after a single UTMD treatment by ectopic insulin production by pancreatic acinar cells. These cells co-expressed amylase and insulin and demonstrated several beta-cell markers by reverse transcription-PCR. Gene therapy by UTMD can reverse diabetes in vivo in adult rats by restoring pancreatic insulin production.

Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles.

Myocardial angiogenesis mediated by human vascular endothelial growth factor 165 (hVEGF165) cDNA was promoted in rat myocardium using an in vivo-targeted gene delivery system known as ultrasound-targeted microbubble destruction (UTMD). Microbubbles carrying plasmids encoding hVEGF165, or control solutions were infused intravenously during ultrasonic destruction of the microbubbles within the myocardium. Biochemical and histological assessment of gene expression and angiogenesis were performed 5, 10, and 30 days after UTMD. UTMD-treated myocardium contained hVEGF165 protein and mRNA. The myocardium of UTMD-treated animals showed hypercellular foci associated with hVEGF165 expression and endothelial cell markers. Capillary density in UTMD-treated rats increased 18% at 5 days and 33% at 10 days, returning to control levels at 30 days (P<0.0001). Similarly, arteriolar density increased 22% at 5 days, 86% at 10 days, and 31% at 30 days (P<0.0001). Thus, noninvasive delivery of hVEGF165 to rat myocardium by UTMD resulted in significant increases in myocardial capillary and arteriolar density.

Myocardial contrast agents: recent advances and future directions.

The assessment of perfusion by myocardial contrast echocardiography has evolved from the early contrast agents, including agitated saline solutions and hydrogen peroxide, to the current second-generation contrast agents. Unlike the first-generation contrast agents, which are composed of air, the newer, second-generation agents contain gases with a higher molecular weight and less solubility and diffusivity, improving microbubble persistence. The newer contrast agents are capable of transpulmonary passage and opacification of the left-heart chambers and the myocardial microcirculation after intravenous administration. Also, innovative imaging techniques using harmonics and triggered imaging have minimized tissue signal and improved signal-to-noise ratio, making the assessment of myocardial perfusion possible. Currently, microbubbles are being designed for specific research or clinical use by exploiting certain characteristics of the microbubble such as the shell, surface characteristics, and/or gas content. Some novel applications of microbubble technology include tissue-targeted gene therapy, drug delivery, ultrasound-enhanced thrombolysis, and the assessment of endothelial function and integrity. This review focuses on the composition, physical properties, and acoustic characteristics of the currently available myocardial contrast agents and those under clinical investigation. In addition, the clinical trials involving these agents will also be discussed.

Assessment of aortic regurgitation by transesophageal color Doppler imaging of the vena contracta: validation against an intraoperative aortic flow probe.

OBJECTIVES: This study was performed to validate the accuracy of color flow vena contracta (VC) measurements of aortic regurgitation (AR) severity by comparing them to simultaneous intraoperative flow probe measurements of regurgitant fraction (RgF) and regurgitant volume (RgV). BACKGROUND: Color Doppler imaging of the vena contracta has emerged as a simple and reliable measure of the severity of valvular regurgitation. This study evaluated the accuracy of VC imaging of AR by transesophageal echocardiography (TEE). METHODS: A transit-time flow probe was placed on the ascending aorta during cardiac surgery in 24 patients with AR. The flow probe was used to measure RgF and RgV simultaneously during VC imaging by TEE. Flow probe and VC imaging were interpreted separately and in blinded fashion. RESULTS: A good correlation was found between VC width and RgF (r = 0.85) and RgV (r = 0.79). All six patients with VC width >6 mm had a RgF >0.50. All 18 patients with VC width <5 mm had a RgF <0.50. Vena contracta area also correlated well with both RgF (r = 0.81) and RgV (r = 0.84). All six patients with VC area >7.5 mm2 had a RgF >0.50, and all 18 patients with a VC area <7.5 mm2 had a RgF <0.50. In a subset of nine patients who underwent afterload manipulation to increase diastolic blood pressure, RgV increased significantly (34 +/- 26 ml to 41 +/- 27 ml, p = 0.042) while VC width remained unchanged (5.4 +/- 2.8 mm to 5.4 +/- 2.8 mm, p = 0.41). CONCLUSIONS: Vena contracta imaging by TEE color flow mapping is an accurate marker of AR severity. Vena contracta width and VC area correlate well with RgF and RgV obtained by intraoperative flow probe. Vena contracta width appears to be less afterload-dependent than RgV.

Vasodilator therapy for chronic aortic and mitral regurgitation.

The use of vasodilator therapy in chronic AR and MR may be beneficial in selected patients and harmful in others. The hemodynamics of the two conditions are different and must be taken into account. In AR, vasodilators reduce afterload mismatch and can preserve LV function and delay the need for surgery. However, if the patient has severely reduced diastolic blood pressure, vasodilators could potentially impair coronary perfusion. In MR, vasodilators may reduce regurgitant volume and LV preload depending on the mechanism of MR. In patients with MR caused by dilated cardiomyopathy, vasodilators reduce symptoms, and improve functional class. However, in mitral valve prolapse or hypertrophic cardiomyopathy, vasodilators may worsen the MR and should be avoided. In other primary causes of MR, vasodilators could potentially mask the development of LV dysfunction and lead to unnecessary and harmful delays in surgery.

Optimization of the size distribution and myocardial contrast effect of perfluorocarbon-filled albumin microbubbles by lyophilization under continuous negative pressure.

This study was undertaken to evaluate the effect of lyophilization under continuous negative pressure on perfluoropropane-filled albumin microbubble size distribution and myocardial contrast effect. Three different microbubble preparations were studied: (1) 1% albumin solution without a sugar (Optison), (2) 1% albumin and 5% dextrose (PESDA), and (3) 1% albumin and 5% fructose (PESFA). The 2 preparations containing sugar were also subjected to lyophilization under continuous negative pressure. Microbubble size distribution was measured with a Coulter Multisizer II (Beckman Coulter, Inc, Fullerton, Calif). The microbubbles were injected intravenously into a rat during intravital microscopy of the mesenteric microcirculation. Finally, the different albumin microbubbles were injected intravenously into 10 dogs, and myocardial contrast effect was assessed by videodensitometry. Results of the Coulter counter studies showed lyophilized PESFA to have a smaller size distribution with 99.9% + or - 0.1% of microbubbles <10 microm in diameter and 88.5% + or - 1.4% <4 microm in diameter (P <.05 compared with Optison or PESDA). On intravital microscopy, PESFA microbubbles behaved as intravascular tracers without microvascular plugging or coalescence. Finally, myocardial peak gray scale and area under the curve were significantly higher for PESFA than for PESDA or Optison, respectively. In conclusion, lyophilization of perfluoropropane-filled albumin microbubbles results in smaller microbubbles with a more uniform size distribution and brighter myocardial contrast. In addition, the substitution of fructose for dextrose improves size distribution and contrast effect. These findings have important implications regarding the use of novel imaging technologies that take advantage of microbubble destruction to image myocardial perfusion.

Esophageal Doppler monitor determinations of cardiac output and preload during cardiac operations.

BACKGROUND: Perioperative management of cardiac surgical patients frequently mandates measurements of cardiac output and left ventricular filling. This study compared cardiac output and left ventricular filling measured by pulmonary artery (PA) catheter and esophageal Doppler monitor (EDM). METHODS: Thirty-four patients undergoing coronary artery bypass grafting were prepared by implanting a PA catheter, an EDM, and a transit-time ultrasonic flow probe around the ascending aorta. In 20 patients, left ventricular end-diastolic short-axis area (EDA) was measured by transesophageal echocardiography. At five time points, cardiac output was measured from the flow probe, the EDM, and the PA catheter (by thermodilution), and left ventricular filling was assessed from the PA catheter (as PA diastolic pressure), the EDM (corrected flow time), and the EDA. For cardiac output, concordance correlations relating EDM to flow probe and PA catheter to flow probe were calculated, transformed (Fisher's z transformation), and compared by Student's t test. For left ventricular filling, regression coefficients were created between corrected flow time and EDA and between PA diastolic pressure and EDA. Spearman correlations were compared by Wilcoxon rank sum test. RESULTS: The EDM and the PA catheter exhibited similar relationships to the flow probe (concordance correlations, 0.55 +/- 0.35 [mean +/- standard deviation] and 0.49 +/- 0.34, respectively; p = 0.088). The correlation between corrected flow time and EDA was better than the correlation between PA diastolic pressure and EDA (concordance correlations, 0.49 +/- 0.55 versus 0.10 +/- 0.43, respectively; p < 0.01). CONCLUSIONS: These data suggest that the EDM may offer a less invasive technique for evaluating cardiac output and a more accurate estimate for preload compared with the PA catheter.

Assessment of myocardial perfusion by harmonic power Doppler imaging at rest and during adenosine stress: comparison with (99m)Tc-sestamibi SPECT imaging.

BACKGROUND: Harmonic power Doppler imaging (HPDI) is a novel technique for assessing myocardial perfusion by contrast echocardiography in humans. The purpose of this study was to compare myocardial perfusion by HPDI with that obtained by (99m)Tc-sestamibi single photon emission computed tomography (SPECT) during rest and pharmacological stress. METHODS AND RESULTS: HPDI was performed on 123 patients who were referred for SPECT imaging for known or suspected coronary artery disease. Images were obtained at baseline and during adenosine infusion (0.14 mg. kg(-)(1). min(-)(1)x6 minutes) in 3 apical views. Myocardial perfusion by HPDI was graded for each coronary territory as absent, patchy, or full. The persistence of absent or patchy myocardial perfusion by HPDI between rest and adenosine was interpreted as a fixed defect, whereas any decrease in perfusion grade was interpreted as a reversible defect. Overall concordance between HPDI and SPECT was 83 (81%) of 103 for normal versus abnormal perfusion. Agreement between the 2 methods for each of the 3 coronary territories was 81% (kappa=0.57) for the left anterior descending artery, 76% (kappa=0.52) for the right coronary artery, and 72% (kappa=0.40) for the left circumflex artery. Discrepancies between the 2 techniques were most notable in the circumflex territory, where fixed defects were observed in 33% by HPDI but in only 14% by SPECT (chi(2)=15.8, P=0.0001). CONCLUSIONS: This study demonstrates that HPDI can reliably detect myocardial perfusion during pharmacological stress, although there was a significantly higher number of falsely abnormal results in the circumflex territory.

Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium.

BACKGROUND: The noninvasive, tissue-specific delivery of therapeutic agents to the heart would be a valuable clinical tool. This study addressed the hypothesis that albumin-coated microbubbles could be used to effectively deliver an adenoviral transgene to rat myocardium by ultrasound-mediated microbubble destruction. METHODS AND RESULTS: Recombinant adenovirus containing beta-galactosidase and driven by a constitutive promoter was attached to the surface of albumin-coated, perfluoropropane-filled microbubbles. These bubbles were infused into the jugular vein of rats with or without simultaneous echocardiography. Additional controls included ultrasound of microbubbles that did not contain virus, virus alone, and virus plus ultrasound. One group underwent ultrasound-mediated destruction of microbubbles followed by adenovirus infusion. Rats were killed after 4 days and examined for beta-galactosidase expression. The hearts of all rats that underwent ultrasound-mediated destruction of microbubbles containing virus showed nuclear staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside substrate, indicating expression of the transgene. None of the control animals showed myocardial expression of the beta-galactosidase transgene. By quantitative analysis, beta-galactosidase activity was 10-fold higher in the treated group than in controls (P<0.0001). CONCLUSIONS: Ultrasound-mediated destruction of albumin-coated microbubbles is a promising method for the delivery of bioactive agents to the heart.