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Am J Physiol Heart Circ Physiol 291: H1595-H1601, 2006. First published April 21, 2006; doi:10.1152/ajpheart.01120.2005
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Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide

L. J. M. Juffermans,1,2 P. A. Dijkmans,1 R. J. P. Musters,1 C. A. Visser,1,2 and O. Kamp1,2

1Institute for Cardiovascular Research, VU University Medical Center, Amsterdam; and 2Interuniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands

Submitted 24 October 2005 ; accepted in final form 12 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we addressed the interactions among ultrasound, microbubbles, and living cells as well as consequent arising bioeffects. We specifically investigated whether hydrogen peroxide (H2O2) is involved in transient permeabilization of cell membranes in vitro after ultrasound exposure at low diagnostic power, in the presence of stable oscillating microbubbles, by measuring the generation of H2O2 and Ca2+ influx. Ultrasound, in the absence or presence of SonoVue microbubbles, was applied to H9c2 cells at 1.8 MHz with a mechanical index (MI) of 0.1 or 0.5 during 10 s. This was repeated every minute, for a total of five times. The production of H2O2 was measured intracellularly with CM-H2DCFDA. Cell membrane permeability was assessed by measuring real-time changes in intracellular Ca2+ concentration with fluo-4 using live-cell fluorescence microscopy. Ultrasound, in the presence of microbubbles, caused a significant increase in intracellular H2O2 at MI 0.1 of 50% and MI 0.5 of 110% compared with control (P < 0.001). Furthermore, we found increases in intracellular Ca2+ levels at both MI 0.1 and MI 0.5 in the presence of microbubbles, which was not detected in the absence of extracellular Ca2+. In addition, in the presence of catalase, Ca2+ influx immediately following ultrasound exposure was completely blocked at MI 0.1 (P < 0.01) and reduced by 50% at MI 0.5 (P < 0.001). Finally, cell viability was not significantly affected, not even 24 h later. These results implicate a role for H2O2 in transient permeabilization of cell membranes induced by ultrasound-exposed microbubbles.

membrane permeability; calcium; ultrasound contrast agents

SEVERAL YEARS AGO, contrast-enhanced echocardiography was introduced in the clinic. Ultrasound contrast agents consist of microbubbles that reflect ultrasound more effectively than the surrounding tissue and are used for contrast enhancement of ultrasound images and improvement of diagnosis (7, 8). However, interactions among ultrasound, microbubbles, and living cells as well as consequent, The Netherlands arising bioeffects have not yet been fully explored. In addition, in light of the fact that ultrasound and microbubbles were recently shown to enhance transgene uptake (16, 17, 19, 22), it is especially important to understand the different bioeffects caused by ultrasound and microbubbles.

Different experimental studies have indicated several subtle bioeffects as a consequence of the interaction among ultrasound, microbubbles, and cell membranes (3, 15, 20, 23, 25). These bioeffects can be divided into three groups: 1) thermal effect: there is a local rise in temperature due to the absorption and dissipation of ultrasound energy (25); 2) chemical effect: the generation of reactive oxygen species (ROS) (2, 3, 15); and 3) mechanical effect: due to the oscillations of the microbubble, the surrounding fluid is set in motion, causing microstreaming along the cell membrane (23). These effects together may result in permeability changes of the cell membrane, or even in the formation of physical pores in the cell membrane (20), and an increased uptake of transgenes (2). Deng et al. (6) used a Ca2+ influx to demonstrate these permeability changes in the presence of microbubbles exposed to continuous wave ultrasound (6). Being an important second messenger, Ca2+ influx is not only an indicator for membrane permeability changes but also may have several consequences for intracellular Ca2+ homeostasis and signal transduction. For instance, it has been shown that patients can have extrasystoles during contrast-echocardiography (5, 21), which might be explained by triggered Ca2+ influxes.

However, it remains to be elucidated whether extracellular ROS production, in particular H2O2, by ultrasound exposure in the presence of microbubbles provokes Ca2+ influx. In the present study, therefore, we focused on the possible role of H2O2 in transient permeabilization of cell membranes. We are specifically interested in these effects at low diagnostic ultrasound power in the presence of stable oscillating microbubbles. We investigated 1) whether more H2O2 is generated in the presence of microbubbles than when ultrasound alone is applied; 2) whether H2O2 affects cell membrane permeability for Ca2+ by measuring Ca2+ influx; and 3) whether cell viability is affected.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ultrasound exposure. Ultrasound was applied using two different settings: a diagnostic apparatus for off-line measurements and a single-element transducer for online measurements. In the first setting, ultrasound was applied in the second harmonic mode with the Sonos 5500 (Philips, Eindhoven, The Netherlands) equipped with a S4 transducer emitting at 1.8 MHz and receiving at 3.6 MHz, with a mechanical index (MI) of 0.1 or 0.5 for a 10-s duration. This was repeated every minute for a total of five times. The tip of the transducer was positioned in a 37°C water bath at a 3-cm distance from the cell surface. In the second ultrasound setting, a 1-MHz unfocused single-element transducer (Panametrics, Waltham, MA) was mounted in a 37°C water bath on the microscope (Fig. 1A). The ultrasonic wave was generated by an arbitrary waveform generator (Agilent 33220A; Agilent Technology, Palo Alto, CA) and amplified (150A100B; EMV Benelux, Nieuwkoop, The Netherlands). Peak-to-peak acoustic pressure generated at the region of interest (ROI) was MI 0.1 or MI 0.5, with a duty cycle of 0.2% and pulse repetition frequency of 20 Hz.


Figure 1
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  		Fig. 1. Experimental design. A: schematic drawing of the ultrasound setting on the microscope used for online measurements. B: schematic overview of experiments with 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA). C: schematic overview of experiments with fluo-4.

 
Ultrasound contrast agent. SonoVue (Bracco Research) is a second-generation contrast agent. It contains sulfur hexafluoride gas and has a phospholipid monolayer shell. The suspension contains 2 x 108 microbubbles/ml with a mean diameter of 2.5 µm (18).

Cell culture. H9c2 rat cardiomyoblast cells (LGC Prochem, Teddington, UK) were grown in cell culture flasks (Micronic, Lelystad, The Netherlands) in 12 ml of Dulbecco's modified Eagle's medium (GIBCO, Breda, The Netherlands) supplemented with 10% fetal bovine serum (GIBCO) and 1% penicillin/streptomycin (GIBCO) in a humidified incubator at 37°C with 5% CO2. Two days before the day of the experiment, the cells were plated in an OptiCell growth chamber (50-cm2 growth surface) (Sanyo Gallenkamp, Etten-Leur, The Netherlands), reaching ~70% confluence on the day of the experiment.

Generation of H2O2. The generation of H2O2 was measured intracellular with 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) (Molecular Probes, Leiden, The Netherlands), which is a carboxy derivative of 2',7'-dichlorofluorescein diacetate acetyl ester (DCF-DA) that exhibits much better retention in living cells than DCF-DA. CM-H2DCFDA, being nonpolar, diffuses passively into cells, where its acetate groups are cleaved by intracellular esterases, and is trapped within the cell. In this status it provides a substrate for oxidation by H2O2, resulting in the production of a highly fluorescent intracellular product emitting fluorescence with intensity proportional to the level of intracellular H2O2 (1). H9c2 cells were grown in an OptiCell growth chamber, loaded with CM-H2DCFDA (5 µM) in ADS buffer (in mM: 116 NaCl, 5.3 KCl, 1.2 MgSO4·7H2O, 1.13 NaH2PO4·H2O, 20 HEPES, and 1 CaCl2, pH 7.4), and incubated for 15 min at 37°C (Fig. 1B). Next, 0.1 ml of SonoVue microbubbles or 0.1 ml of ADS buffer (control) was added, followed by ultrasound exposure with the Sonos 5500. After ultrasound exposure, cells were incubated 60 min at 37°C, allowing the oxidized CM-H2DCFDA to accumulate in the cells (Fig. 1B). Fluorescence microscopy was performed to qualitatively and quantitatively assess the generation of H2O2, using a wide-field digital imaging fluorescence microscope (Marianas; I.I.I., Denver, CO) with a x10 objective. All images were digitally processed using custom-made software (SlideBook version 4.0.8.3 [EC] ; I.I.I.) to quantify the intensity of fluorescence. Fluorescence was corrected for background and expressed in arbitrary units as mean intensity of fluorescence (MIF). Necrotic cells were not included in the evaluation. Changes in CM-H2DCFDA were also measured in the presence of the H2O2 scavenger catalase (1,250 U/ml) (Sigma). Finally, to quantify the increase in MIF induced by ultrasound and microbubbles, cells were loaded with CM-H2DCFDA as described above. After 15 min, different concentrations of exogenous H2O2 (0, 1, 2.5, 5, 7.5, and 10 µM) were added to the cells.

Ca2+ permeability. Changes in intracellular Ca2+ concentration were measured with the fluorescent probe fluo-4 (C51H50F2N2O23), a cell-permeant acetoxymethyl ester (AM) sensitive to free cytosolic Ca2+ (Molecular Probes). Cells were grown in the OptiCell growth chamber, loaded with fluo-4 (3 µM) in ADS buffer, and incubated for 30 min at 37°C; the cells were washed in ADS and incubated for another 30 min at 37°C to allow the fluorescence to stabilize (Fig. 1C). SonoVue microbubbles (0.1 ml) were added, followed by ultrasound exposure. Intensity of fluorescence was measured in real time, i.e., during ultrasound exposure, with the use of live-cell fluorescence microscopy. Changes in intracellular Ca2+ level were also measured in the presence of the H2O2 scavenger catalase (1,250 U/ml) and the L-type Ca2+ channel blocker verapamil (10 µM) (Sigma). To quantify the increase in MIF induced by ultrasound and microbubbles, we loaded cells with fluo-4 as described above. After incubation with fluo-4, different concentrations of exogenous H2O2 (0, 1, 2.5, 5, 7.5, and 10 µM) were added to the cells.

Cell viability. Cell viability was assessed by flow cytometry (FACS Calibur). After ultrasound exposure, cells were harvested from the OptiCell membrane with trypsin (0.25%; GIBCO). The cell suspension (200 µl) was placed in 5 ml of polystyrene round-bottom tubes. To assess viability, we added 10 µl of annexin-V (Molecular Probes). Annexin-V has a high affinity for phosphatidylserine, which is translocated from the internal to the external surface of early apoptotic cells. Necrotic cells also stain for annexin-V due to loss of membrane integrity; they are discriminated from apoptotic cells with propidium iodide (PI) (Molecular Probes), which is excluded from viable and apoptotic cells. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay (Promega, Leiden, The Netherlands) was performed to detect DNA fragmentation by endonucleases in late apoptosis using fluorescence microscopy. Furthermore, cells were observed using differential interference contrast (DIC) microscopy to detect possible morphological changes after ultrasound and microbubble exposure.

Statistical analysis. Data are expressed as means ± SE. Differences between different groups were evaluated using one-way ANOVA followed by Bonferroni's posttest if values passed a normality test. If values did not pass a normality test (P < 0.05), the one-way ANOVA was replaced with a nonparametric Kruskal-Wallis test followed by Dunn's posttest. Results were considered significant when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of H2O2. Ultrasound at low diagnostic power caused a significant increase in intracellular H2O2 in the presence of stable oscillating microbubbles, i.e., 50% increase at MI 0.1 and 110% increase at MI 0.5, respectively (P < 0.001 compared with control cells) (see Fig. 2). Ultrasound applied in the absence of microbubbles at both MI 0.1 and 0.5, however, did not increase H2O2 production compared with control cells. Representative images of cells displaying the oxidized fluorescent product of CM-H2DCFDA are shown in Fig. 3. To quantify the amount of H2O2 generated by ultrasound exposure and microbubbles, we added different concentrations of exogenous H2O2 to the cells. The increase in intensity of fluorescence induced by ultrasound and microbubbles corresponded to adding ~2.5 µM H2O2 for MI 0.1 and ~7.5 µM H2O2 for MI 0.5, respectively (see Fig. 2C).


Figure 2
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  		Fig. 2. Generation of H2O2 measured 60 min after ultrasound exposure is shown as mean intensity of fluorescence (MIF) expressed as a percentage of control cells. A: cells exposed to ultrasound (US) at mechanical index (MI) 0.1, demonstrating a 50% increase in H2O2 in the presence of microbubbles (US + microbubbles) ({zeta}P < 0.001 compared with control cells). Catalase completely blocked the increase in cells exposed to ultrasound and microbubbles at MI 0.1 ({zeta}P < 0.001). Control, 398 cells; US, 283 cells; US + microbubbles, 378 cells; US + microbubbles + catalase, 884 cells. B: cells exposed to US at MI 0.5, demonstrating a 110% increase in the presence of microbubbles ({zeta}P < 0.001 compared with control cells). Catalase significantly reduced the H2O2 by ~80% ({zeta}P < 0.001) in cells exposed to ultrasound and microbubbles at MI 0.5. Control, 365 cells; US, 268 cells; US + microbubbles, 217 cells; US + microbubbles + catalase, 548 cells. C: cells exposed to exogenous H2O2. Control, 688 cells; 1 µM, 308 cells; 2.5 µM, 387 cells; 5 µM, 725 cells; 7.5 µM, 317 cells; 10 µM, 305 cells. Data are shown as means ± SE.

 

Figure 3
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  		Fig. 3. Representative images of H9c2 cells displaying the oxidized fluorescent product of CM-H2DCFDA. A: control cells. B: cells exposed to ultrasound at MI 0.1 in the presence of microbubbles. C: cells exposed to ultrasound at MI 0.5 in the presence of microbubbles. Bars represent 10 µm.

 
Ca2+ permeability. Ultrasound, in the presence of microbubbles, caused a transient increase in the permeability of the cell membrane for Ca2+ at both MI 0.1 and MI 0.5 (Fig. 4, A and C). There was no detectable Ca2+ influx in the absence of microbubbles. Furthermore, in the presence of microbubbles, more Ca2+ entered the cells at MI 0.5 compared with MI 0.1. This finding is in line with the levels of H2O2 production: there was no increase in H2O2 production in the absence of microbubbles, and in the presence of microbubbles, significantly more H2O2 was detected at MI 0.5 compared with MI 0.1. Control cells not exposed to ultrasound or microbubbles showed a minor decrease in intracellular Ca2+ level over time (Fig. 4A), probably because of bleaching of fluo-4. In addition, cells exposed to ultrasound and microbubbles with only 1 µM Ca2+ in the buffer showed an even more pronounced decrease in intracellular Ca2+ concentration compared with control cells (P < 0.001, from 60 s onwards), indicating that there is a net loss of intracellular Ca2+ from the cell. However, under this condition, the increase in intracellular H2O2 was still observed (data not shown). The increase in intracellular Ca2+ level corresponded with the addition of ~2.5 µM exogenous H2O2 for MI 0.1 and ~7.5 µM for MI 0.5, respectively, as shown in Fig. 4E. This was also in line with the levels of H2O2 measured with CM-H2DCFDA.


Figure 4
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  		Fig. 4. Changes in intracellular Ca2+ concentration over time in H9c2 cells loaded with fluo-4. Data are shown as MIF (±SE), expressed as %MIF at time t = 0 s. Dotted lines represent moments of ultrasound on and off switch, respectively. A: cells exposed to ultrasound at MI 0.1 (control cells received no US). Cells exposed to ultrasound alone did not display an increase in intracellular Ca2+ level. Cells exposed to ultrasound in the presence of microbubbles displayed a significant increase in intracellular Ca2+ level directly after ultrasound exposure (#P < 0.01; *P < 0.05). B: cells exposed to MI 0.1 and microbubbles compared with cells exposed to MI 0.1 and microbubbles in the presence of catalase. Catalase completely blocked the increase in intracellular Ca2+ level (*P < 0.05 at t = 56 s; #P < 0.01 at t = 64 s; {zeta}P < 0.001 from t = 72 s, indicated by arrow). C: cells exposed to US at MI 0.5 (control cells received no ultrasound). Cells exposed to ultrasound alone did not display increases in intracellular Ca2+ level, whereas cells exposed to ultrasound in the presence of microbubbles show a pronounced increase in intracellular Ca2+ level ({zeta}P < 0.001 from t = 56 s). Cells exposed to MI 0.5 and microbubbles in the presence of only 1 µM Ca2+ in the buffer did not show increases in intracellular Ca2+ level. D: cells exposed to MI 0.5 and microbubbles compared with cells exposed to MI 0.5 and microbubbles in the presence of catalase. Catalase significantly reduced the increase in intracellular Ca2+ level ({zeta}P < 0.001 from t = 56 s). Note the different y-axis scales at MI 0.1 and MI 0.5. E: peak fluorescent intensities of cells exposed to exogenous H2O2. Control, 147 cells; 1 µM, 118 cells; 2.5 µM, 104 cells; 5 µM, 136 cells; 7.5 µM, 103 cells; 10 µM, 111 cells.

 
In the presence of catalase, the Ca2+ influx following ultrasound exposure was completely blocked at MI 0.1 (P < 0.01) and reduced by 50% at MI 0.5 (P < 0.001) (Fig. 4, B and D). In contrast, L-type Ca2+ channel blocker verapamil was not able to prevent Ca2+ influx (Fig. 4B). Representative images of cells loaded with Fluo-4 before and during ultrasound exposure in the presence of microbubbles are shown in Fig. 5.


Figure 5
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  		Fig. 5. H9c2 cells loaded with the Ca2+ fluorescence indicator fluo-4 in the presence of microbubbles. A: before the onset of ultrasound exposure. B: during ultrasound exposure (MI 0.1): increases in intracellular Ca2+ concentration. Images are an overlay of fluorescence (dark field) with bright field. Bars represent 10 µm.

 
Cell viability. Ultrasound exposure at both MI 0.1 and MI 0.5 in the absence or presence of microbubbles did not cause any alterations in overall cell morphology or excessive detachment of cells. In addition, all cells were still viable 24 h later. This was confirmed by flow cytometry: there was only a small, insignificant increase in the percentage of annexin-V (+6.4%, P > 0.05)- and annexin-V/PI (+1.4%, P > 0.05)-positive cells exposed to ultrasound and microbubbles compared with controls (Fig. 6). Likewise, apoptosis could not be detected with the TUNEL assay.


Figure 6
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  		Fig. 6. Propidium iodide (PI) and annexin-V positivity in control cells and in cells exposed to MI 0.5 in the presence of microbubbles. There is no significant increase in necrosis (PI positivity) or early apoptosis (annexin-V positivity) after exposure to ultrasound and microbubbles.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that ultrasound at low diagnostic power, causing stable oscillations of the microbubbles, results in a transient increase in membrane permeability for Ca2+, as reflected by a Ca2+ influx, which is partially caused by generation of H2O2. In the presence of microbubbles, we detected a significant increase in H2O2 production at both MI 0.1 and MI 0.5 (50 and 110%, respectively; P < 0.001 compared with controls) (Fig. 2). Basta et al. (3) previously reported a 50% increase after ultrasound exposure at MI 1.3 after 30 min of exposure time in the absence of microbubbles. Interestingly, we had already detected a similar increase after five 10-s periods of ultrasound exposure at MI 0.1 in the presence of microbubbles, indicating that the presence of stable oscillating microbubbles has significant effects on surrounding cells. Furthermore, our findings are supported by a recent study by Marmottant et al. (11), who demonstrated that gentle (linear) microbubble oscillations induce pronounced effects on phospholipid bilayers.

Role of H2O2 in Ca2+ permeability. The presence of ultrasound-exposed microbubbles in close proximity to cells has an effect on the permeability of cells. This permeability change is a result of a number of as yet undefined physical, chemical, and mechanical forces acting on microdomains of the cell membrane. Because of the difficulty of investigating ultrarapid alterations in cell membrane phospholipid bilayer organization, specific mechanisms of pore formation have not yet been definitely proven. Microbubble oscillation, cavitation, collapse, and the resulting shear forces are all suggested to play a role in the increase in permeability of the cell (15, 23). We demonstrated increased permeability of the cell membrane by revealing Ca2+ influxes at both MI 0.1 and MI 0.5 in the presence of microbubbles, already with pulsed ultrasound with a duty cycle of 0.2% and pulse repetition frequency of 20 Hz. This is ultrasound with low energy compared with ultrasound used by other groups (6, 13, 20). This is an important finding, because it indicates that ultrasound exposed microbubbles can indeed provoke alterations in cell membrane permeability, which may have several consequences for intracellular ion homeostasis. Especially relevant to this, it has been shown that patients can have extrasystoles during contrast echocardiography (5, 21), which may be explained by triggered Ca2+ influxes. Furthermore, our findings are in correspondence with a voltage-clamp study performed by Deng et al. (6). In the presence of microbubbles, an inward Ca2+ current was already demonstrated after 1 s of continuous wave ultrasound exposure. They suggested that the increased ion permeability resulted from pore formation in the cell membrane, rather than increased opening of endogenous voltage- or ligand-gated ion channels. During ultrasound exposure, the membrane potential was clamped at a constant voltage and the solution bathing the cells contained no ligand known to activate endogenous ion channels. By comparing the amplitude of the current step increase (~0.1 µA) with the maximum current amplitude through a single K+ channel (~100 pA), and assuming that the current amplitude is only dependent on pore size, the ultrasound-induced pore can be as large as 0.1 µm (6). This corresponds with our results from the experiments with verapamil, an L-type Ca2+-channel blocker, where we still measured a Ca2+ influx. In addition, it also is in agreement with the results from a study from Honda et al. (9), who showed that the Ca2+ influx does not occur via the L-type Ca2+ channels. Furthermore, Tachibana et al. (20) showed such pores by electron microscopy in leukemia cells after insonation with continuous wave ultrasound.

To detect whether Ca2+ enters the cytoplasmic region via the cell membrane or by a release from intracellular Ca2+ stores, we exposed cells to ultrasound in buffer with only 1 µM Ca2+. In the absence of extracellular Ca2+, we measured a decrease in cytosolic Ca2+ concentration after ultrasound exposure, which also is in agreement with Deng et al. (6), who demonstrated that there was no resealing of the cell membrane in the absence of extracellular Ca2+ and that Ca2+ could leak out of the cells. Most importantly, in the presence of catalase, a H2O2 scavenger, the Ca2+ influx was completely blocked at MI 0.1 (P < 0.01) and significantly reduced by ~50% at MI 0.5 (P < 0.001), as shown in Fig. 4. It is now recognized that Ca2+ signaling and H2O2 production are two closely related aspects of cell functioning (4). We show an effect of H2O2 on the influx of Ca2+ in ultrasound-exposed cells in the presence of microbubbles.

Our present findings, i.e., no increase in intracellular Ca2+ concentration in the presence of only 1 µM extracellular Ca2+ and a reduced increase in cytosolic Ca2+ concentration when H2O2 is scavenged, implicate a role for H2O2 in transient permeabilization of cell membranes during ultrasound exposure in the presence of microbubbles. In addition, Marumo et al. (12) reported a VEGF-elicited, ROS-dependent increase in the macromolecule permeability of bovine microvascular endothelial cells. In contrast with these findings are the results of an earlier study by Lawrie et al. (10) indicating that ultrasound- enhanced transgene expression is not dependent on ROS. However, they used high-intensity ultrasound (MI 2.0), which causes inertial cavitation with high mechanical forces acting on cell membranes (10).

Transient permeabilization and implications for cell viability. There are several reports on ultrasound-exposed microbubble-induced apoptosis and necrosis (9, 14). Importantly, using our ultrasound parameters, we found that cell viability was not significantly affected, despite the transient permeabilization with concomitant Ca2+ influx. Only a small, nonsignificant increase in early-apoptotic and necrotic cells was detected using flow cytometry. In addition, no increase in late-apoptotic cells could be detected using TUNEL, and DIC microscopic observation of exposed cells showed no alteration in overall cell morphology or excessive cell detachment. Also, after 24 h, these cells were still viable. Thus H2O2-induced Ca2+ influxes are not necessarily a precursor for cell death. This finding is in agreement with a study of Volk et al. (24) in which low levels of H2O2 triggered subtoxic Ca2+ oscillations. The majority of cells recover fast from the primary wave of Ca2+ influx: directly after ultrasound exposure, fluo-4 fluorescence peaks and then rapidly falls back to baseline levels.

Transient permeabilization of cell membranes during and after ultrasound exposure in the presence of microbubbles is of great interest in relation to local gene delivery and enhanced transgene uptake. Increased cell membrane permeability has been demonstrated by several groups by an increase in ion fluxes (6) as well as enhanced uptake of different macromolecules (13) or transgenes (17, 19, 22). Although the present findings are in support of a role for H2O2 in transient permeabilization of cell membranes by ultrasound-exposed microbubbles, this is probably not the sole effect involved in permeabilization of cell membranes. Most likely, it is a combination of the production of H2O2 and the bioeffects mentioned in the Introduction, i.e., a local rise in temperature and microstreaming along the cell membrane that will finally lead to physical pores in the membrane, causing increased uptake of macromolecules and transgenes. At different intensities and energy levels of ultrasound, one bioeffect may play a greater role than the others. Further studies have to be directed toward investigating the combination of these effects of ultrasound-exposed microbubbles at different intensities and energy levels on cell membrane permeability and transgene uptake. Although enhanced transgene uptake is often accomplished by microbubble destruction with high-intensity ultrasound, studying bioeffects at lower intensity ultrasound is important for our understanding of increased permeability. If effects like H2O2 production and Ca2+ influx are already present at these low intensities in vitro, they are also likely to occur in vivo during contrast echocardiography, where imaging takes place for a longer period of time.

In conclusion, we observed a significant increase in the production of H2O2 after ultrasound-exposed microbubbles compared with ultrasound alone. This is a novel observation in the setting of low power ultrasound in the presence of stable oscillating microbubbles. Furthermore, increased H2O2 production was associated with increased cell membrane permeability, reflected by Ca2+ influxes, because scavenging H2O2 reduced this influx.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. J. M. Juffermans, VU Univ. Medical Center, Van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands (e-mail: ljm.juffermans{at}vumc.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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B. D.M. Meijering, L. J.M. Juffermans, A. van Wamel, R. H. Henning, I. S. Zuhorn, M. Emmer, A. M.G. Versteilen, W. J. Paulus, W. H. van Gilst, K. Kooiman, et al.
Ultrasound and Microbubble-Targeted Delivery of Macromolecules Is Regulated by Induction of Endocytosis and Pore Formation
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