IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control ,
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Role of Intracellular Calcium and Reactive Oxygen Species in Microbubble-Mediated Alterations of Endothelial Layer Permeability Klazina Kooiman, Member, IEEE, Antonius F. W. van der Steen, Fellow, IEEE, and Nico de Jong, Associate Member, IEEE Abstract—Drugs will be delivered to diseased tissue more efficiently if the vascular endothelial permeability is increased. Ultrasound in combination with an ultrasound contrast agent is known to increase the permeability of the endothelial layer, but the mechanism is not known. The goal of this study was to elucidate whether intracellular calcium ions, [Ca2+]i, and reactive oxygen species (ROS) are part of the mechanism that leads to an increased endothelial layer permeability following ultrasound and microbubble treatment. Human umbilical vein endothelial cells (HUVECs) treated for 2 min with ultrasoundactivated microbubbles (1 MHz, 210 kPa, 10 000 cycles, 20 Hz repetition rate) had an increased permeability that lasted up to 12 h. Recovery of permeability after 2 h was only found when HUVECs were preincubated with the [Ca2+]i chelator BAPTAAM or the antioxidant butylated hydroxytoluene (BHT). This suggests that both [Ca2+]i and ROS play an important role in the mechanism of increased permeability following ultrasound in combination with microbubble treatment.
I. Introduction
B
lood vessels are lined by endothelium, which is composed of a single layer of endothelial cells. Endothelium regulates several functions, including vascular smooth muscle tone, host-defense reactions, and tissue fluid homeostasis. Homeostasis is kept by actively controlling the extravasation of fluids, solutes, and cells into the extravascular tissue. However, the endothelial layer forms a major barrier for drug delivery into the extravascular tissue because drugs do not readily cross healthy endothelial cells. To enhance drug delivery, a controlled, temporary, and local increase in endothelial layer permeability is needed. At the same time, transient opening of the vascular barrier should also make it possible to reduce drug doses and side effects at distant sites [1]–[3]. It is known that the permeability of the endothelial layer can be increased through the following pathway of events: 1) activation of specific receptors; 2) generation of intracellular signals through second messengers and protein phosphorylations; and 3) alteration of cell–cell junctions [1], [4]. These pathways
Manuscript received December 10, 2012; accepted May 22, 2012. This project is supported by innovation subsidies collaborative projects by the Dutch Ministry of Economic Affairs under number IS042035. The authors are with the Department of Biomedical Engineering, Erasmus MC, Rotterdam, The Netherlands (e-mail: k.kooiman@ erasmusmc.nl). A. F. W. van der Steen and N. de Jong are also with the Interuniversity Cardiology Institute of The Netherlands, Utrecht, The Netherlands. DOI http://dx.doi.org/10.1109/TUFFC.2013.2767 0885–3010/$25.00
can be activated either biochemically by, for example, histamine or thrombin [1], [5], or mechanically by, for example, mechanical poking or cyclic stretching [6]. We previously reported that ultrasound, when combined with a contrast agent, increased the permeability of an endothelial layer while overall cell layer integrity remained [7]. Because ultrasound can be locally applied [8], a local increase in permeability can be achieved, thus enhancing drug delivery only locally. However, the mechanism by which ultrasound in combination with a contrast agent increases endothelial layer permeability is not known. In addition to an increase in endothelial layer permeability, several other bioeffects have also been reported by others, such as a rise in intracellular calcium ions [9], [10], [Ca2+]i, and reactive oxygen species (ROS) formation [11]. Interestingly, all of these responses have also been reported in endothelial cells following mechanotransduction [6], [12]–[14]. Because mechanotransduction can lead to an increase in [Ca2+]i and/or ROS, and subsequently to an increase in endothelial layer permeability, we determined whether this was also involved in microbubble-mediated increases in endothelial layer permeability. Permeability of treated endothelial monolayers was therefore determined in the absence and presence of the [Ca2+]i chelator BAPTA-AM [15] and the general antioxidant butylated hydroxytoluene (BHT) [16], [17]. II. Materials and Methods A. Endothelial Monolayers Primary human umbilical vein endothelial cells (HUVECs; passages 4–7) were cultured as previously described [7]. For experiments, HUVECs were replated onto ultrasound-transparent membranes of cell culture inserts (23.1 mm diameter, PET membrane; 353090, BD Falcon, BD Biosciences, San Jose, CA) at a density of 1.25 × 106 cells per insert. Before plating, the membranes were treated for 30 min with 1000 µL of 1% gelatin in MilliQ (1.04070.0500; Merck and Co. Inc., Whitehouse Station, NJ) at 37°C in the humidified cell incubator, followed by incubation for 30 min with 1273 µL of 5 µg/mL of fibronectin (10 838 039 001; Roche Diagnostics Corp., Indianapolis, IN) at 37°C in the humidified cell incubator. After removal of the fibronectin, EGM-2 cell culture medium was added to both inserts (i.e., apical; 2 mL) and wells
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(i.e., basolateral; 3 mL). Fluid volumes were selected to yield no hydrostatic pressure gradient across the cells. Experiments were performed 3 d post-plating to allow monolayers to develop well [7]. B. Treatment of Endothelial Monolayer Endothelial layer permeability was assessed by measuring transendothelial electrical resistance (TEER) in an Endohm-SNAP chamber (World Precision Instruments, Berlin, Germany) as previously described by Kooiman et al. [7]. TEER was measured before and after preincubation for 30 min in EGM-2 with or without BAPTAAM [10 µM from a 10 mM stock in dimethyl sulfoxide (DMSO); B6769, Invitrogen Corp., Carlsbad, CA; the highest dose that by itself did not alter the TEER] or BHT (50 µM from a 50 mM stock in ethanol; DI0315, Scharlab S.L., Barcelona, Spain). Monolayers were treated with ultrasound and BR14 microbubbles (Bracco Suisse SA, Geneva, Switzerland) as previously described [7]. Briefly, inserts were placed upside-down in a custom-made micropositioner in front of the ultrasound beam at a standoff distance of 60 mm. Microbubbles were added in a microbubble-cell ratio of 1:1 using a 1-mL syringe and customcurved blunt 19-gauge needle. To determine the number of microbubbles needed, cells of three inserts were counted in a haemacytometer before each experiment. Microbubbles and cells were insonified for 2 min with a 10 000-cycle sine wave burst at a repetition rate of 20 Hz at 1.0 MHz (unfocused single-element transducer 1.27 cm in diameter; V303SU, Panametrics, Waltham, MA) at an applied peak negative acoustic pressure (P−) of 210 kPa (MI = 0.21). The −6-dB lateral beam profile of the 1-MHz transducer at the standoff distance of 60 mm, i.e., where the endothelial monolayer was positioned, was 10 mm wide (see also [7]). Sham was used as control treatment. After treatment, the inserts were taken out of the tank and 2 mL of EGM-2 [plus 50 µM BHT for cells preincubated with BHT; BAPTA-AM preincubated cells were placed back on EGM-2 only, because a 1 h incubation with BAPTA-AM decreased TEER (data not shown)]. TEER was measured before, immediately after ultrasound and BR14 treatment, and up to 12 h after treatment.
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after treatment. Ultrasound alone or BR14 alone did not decrease TEER values [7]. In our current study, TEER was followed for up to 12 h after treatment. TEER values before treatment were 24.0 ± 0.7 Ω·cm2, which is in line with what others have previously reported [18], [19]. Fig. 1 shows that TEER significantly decreased from 24.0 ± 0.7 Ω·cm2 before treatment to 17.3 ± 1.0 Ω·cm2 (p 240 min time point. At all time points, TEER values were significantly different from sham-treated monolayers. TEER for sham-treated monolayers did not significantly change from before sham-treatment to up to 120 min after sham treatment (see Fig. 1). To explore the mechanism of decreased TEER after ultrasound in combination with microbubble treatment, we determined whether preincubating the monolayers with the [Ca2+]i chelator BAPTA-AM or the antioxidant BHT had an effect on TEER. Preincubation with either BAPTA-AM or BHT only did not have an effect on TEER (data not shown). Also, TEER for sham-treated monolayers preincubated with BAPTA-AM or BHT did not significantly differ from sham-treated monolayers preincubated with only EGM-2 culture medium for up to 120 min after sham treatment (data not shown). For monolayers treated with ultrasound in combination with BR14, BAPTA-AM preincubation did not have an effect on the decrease in TEER directly after and up to 60 min after treatment (see Fig. 2). Interestingly, BAPTA-AM preincubation did have an effect 120 min after treatment; TEER was significantly higher than for monolayers treated with ultrasound in
C. Statistical Analysis Results were expressed as mean ± SEM. Comparisons among multiple groups were performed using a two-way analysis of variance (ANOVA) followed by Bonferroni’s test. Differences were considered significant if p < 0.05. All tests were performed using GraphPad InStat verion 5.04 (GraphPad Software, San Diego, CA). III. Results Previously, we reported that ultrasound in combination with BR14 microbubbles decreased TEER directly
Fig. 1. Transendothelial electrical resistance (TEER) before (t = −2 min) and after (t ≥ 0 min) sham treatment (circle) or ultrasound in combination with BR14 treatment (square). Points = means; bars = ±SEM; n = 4–12 inserts.
kooiman et al.: microbubble-mediated alterations of endothelial layer permeability
Fig. 2. Effect of BAPTA-AM preincubation on transendothelial electrical resistance (TEER) before treatment (before), directly after treatment (0 min), and 60 and 120 min after treatment (60 min, 120 min) for sham-treated monolayers (white columns), ultrasound in combination with BR14 treated monolayers (gray columns), or monolayers preincubated with 10 µM BAPTA-AM and treated with ultrasound in combination with BR14 (checkered columns). Columns = means; bars = ±SEM; n = 4–12 inserts; * = significantly lower than sham; # = significantly higher than ultrasound in combination with BR14 treatment.
combination with BR14 that were not preincubated with BAPTA-AM. In fact, TEER was no longer significantly different from sham-treated monolayers. Preincubation of monolayers with BHT followed by treatment with ultrasound in combination with BR14 gave similar results as for BAPTA-AM preincubated monolayers, albeit that 60 min after ultrasound in combination with BR14 treatment, TEER for BHT preincubated monolayers was not significantly different from sham-treated or ultrasound in combination with BR14 treated monolayers (see Fig. 3). IV. Discussion This study shows that ultrasound in combination with the contrast agent BR14 results in a prolonged decrease of TEER. Previously, we reported that overall cell layer integrity remained and cell viability was not altered when
Fig. 3. Effect of BHT preincubation on transendothelial electrical resistance (TEER) before treatment (before), directly after treatment (0 min), and 60 and 120 min after treatment (60 min, 120 min) for sham-treated monolayers (white columns), ultrasound in combination with BR14 treated monolayers (gray columns), or monolayers preincubated with 50 µM BHT and treated with ultrasound in combination with BR14 (striped columns). Columns = means; bars = ±SEM; n = 4–12 inserts; * = significantly lower than sham; # = significantly higher than ultrasound in combination with BR14 treatment.
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TEER decreased [7]. Because TEER reflects changes in endothelial permeability that are associated with increased passage across a monolayer [20], our results indicate a prolonged increase in permeability following microbubble and ultrasound treatment. A prolonged increase in permeability was also reported by Böhmer et al. [21], who reported that local ultrasound in combination with microbubble treatment locally increased extravasation of Evans Blue in mice. The increased extravasation was reported when Evans Blue was injected 5 min before the ultrasound in combination with microbubble treatment, but also when Evans Blue was injected 1 h after ultrasound in combination with microbubble treatment. In this study, we set out to explore the mechanism of increased endothelial layer permeability through ultrasound-activated microbubbles. In endothelial cells, TEER is also known to immediately decrease upon addition of histamine or thrombin. Both histamine and thrombin induce intracellular signaling that leads to an increased permeability [5]. A rise in [Ca2+]i is part of this signaling mechanism and BAPTA-AM, which chelates [Ca2+]i, completely inhibits the histamine-induced increase in permeability while only partly inhibiting the thrombin-induced increase in permeability. Although histamine and thrombin induce a biochemical rise in [Ca2+]i in endothelial cells, a mechanically induced [Ca2+]i rise has also been reported, namely upon mechanical stimulation by a micropipette or microprobe [22]–[25]. Our previous study [26] showed that ultrasound-activated microbubbles are capable of poking endothelial cells. This mechanical poking by ultrasoundactivated microbubbles is known to induce a [Ca2+]i rise [9], [10] which may increase endothelial layer permeability through signaling [6]. In our current study, BAPTA-AM did not inhibit the ultrasound and microbubble-mediated decrease in TEER in the initial phase (0 to 60 min) after treatment. This suggests that [Ca2+]i does not play a role in the ultrasound-induced decrease in TEER in the initial phase. On the other hand, BAPTA-AM was able to facilitate the return of TEER to initial values 2 h after treatment. This suggests that [Ca2+]i plays a role in the late phase after treatment and could also mean that the microbubble-mediated rise in [Ca2+]i in the initial phase was too high and therefore exceeded the chelating capacity of BAPTA-AM, resulting in no inhibiting effect of BAPTA-AM in the initial phase. During this initial phase, BAPTA-AM preincubated endothelial cells would be able to decrease the remaining excess [Ca2+]i from their cytoplasm by pumping it out of the cell or into intracellular stores, resulting in the return of TEER to initial values in the late phase after treatment. In addition to a rise in [Ca2+]i, increased H2O2 production has also been reported following ultrasound in combination with microbubble treatment [11]. H2O2 is an ROS and is known to be involved in an increase in endothelial layer permeability [12], [13]. In our study, we inhibited ROS by preincubating the cells with BHT. Although BHT did not inhibit the decrease in TEER in the initial phase
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following ultrasound in combination with microbubble treatment, it did induce a recovery in TEER to initial values within 2 h. This, therefore, suggests that ROS play a role in delaying the recovery of endothelial layer permeability following ultrasound in combination with microbubble treatment. It is remarkable, as shown in Fig. 1, that TEER first decreased after ultrasound in combination with microbubble treatment, then increased between 30 and 60 min and decreased again after 60 min. It is our hypothesis that the mechanism by which TEER decreases is not a single, but a multifactorial, process that takes place at different time scales. This will be subject of our future studies. Although HUVECs are widely used to study endothelial layer permeability [15], [20], they remain an in vitro model system. We used this model system to simulate microbubble interactions with the vessel wall and to study effects of ultrasound-activated microbubbles on endothelial layer permeability. However, the disadvantage of this model is that flow is not taken into consideration. This will be subject of our future studies. Our study shows that both [Ca2+]i and ROS are suggested to be involved in the prolonged decrease in TEER of an endothelial layer following ultrasound and microbubble treatment. The mechanism could therefore involve the activation of signaling mechanisms that induce endothelial layer permeability. Further studies are needed to fully elucidate the mechanism, including the microbubble dynamics, microstreaming, jetting, clustering, etc. Fully elucidating the mechanism would mean that in vitro and in vivo effects of ultrasound in combination with microbubble treatment could be more controlled. This is important for drugs that have difficulty crossing the endothelial layer. Our future studies will also focus on determining which drugs will have an increased transport across the endothelial layer following ultrasound in combination with contrast agent treatment and whether this is due to increased transcellular or paracellular passage. In addition, our current study suggests that [Ca2+]i chelators or antioxidants can be applied when a prolonged increase in endothelial layer permeability is not needed following ultrasound and microbubble treatment.
Acknowledgments The authors are grateful to Bracco Suisse SA, Geneva, Switzerland, for kindly providing the BR14 samples. The authors thank M. Manten (Dept. of Biomedical Engineering, Erasmus MC, The Netherlands) for his technical assistance and are grateful to Dr. G. P. van Nieuw Ame rongen, Prof. V. W. M. van Hinsbergh (both Dept. of Physiology, VU University Medical Center, The Netherlands), and Dr. L. J. M. Juffermans (Dept. of Physiology and Dept. of Cardiology, VU University Medical Center, The Netherlands) for discussions about the results.
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References [1] D. Mehta and A. B. Malik, “Signaling mechanisms regulating endothelial permeability,” Physiol. Rev., vol. 86, pp. 279–367, Jan. 2006. [2] P. Telo, S. Lostaglio, and E. Dejana, “Structure of intercellular junctions in the endothelium,” Therapie, vol. 52, pp. 395–398, Sep.–Oct. 1997. [3] G. P. van Nieuw Amerongen and V. W. van Hinsbergh, “Targets for pharmacological intervention of endothelial hyperpermeability and barrier function,” Vascul. Pharmacol., vol. 39, pp. 257–272, Nov. 2002. [4] F. R. Haselton, J. S. Alexander, S. N. Mueller, and A. P. Fishman, Modulation of Endothelial Paracellular Permeability. New York: Plenum Press, 1992. [5] V. W. van Hinsbergh and G. P. van Nieuw Amerongen, “Intracellular signalling involved in modulating human endothelial barrier function,” J. Anat., vol. 200, pp. 549–560, Jun. 2002. [6] P. F. Davies, “Flow-mediated endothelial mechanotransduction,” Physiol. Rev., vol. 75, pp. 519–560, Jul. 1995. [7] K. Kooiman, M. Emmer, M. Foppen-Harteveld, A. van Wamel, and N. de Jong, “Increasing the endothelial layer permeability through ultrasound-activated microbubbles,” IEEE Trans. Biomed. Eng., vol. 57, pp. 29–32, Jan. 2010. [8] M. R. Böhmer, A. L. Klibanov, K. Tiemann, C. S. Hall, H. Gruell, and O. C. Steinbach, “Ultrasound triggered image-guided drug delivery,” Eur. J. Radiol., vol. 70, pp. 242–253, May 2009. [9] Z. Fan, R. E. Kumon, J. Park, and C. X. Deng, “Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles,” J. Control. Release, vol. 142, pp. 31–39, Feb. 25, 2010. [10] L. J. Juffermans, A. van Dijk, C. A. Jongenelen, B. Drukarch, A. Reijerkerk, H. E. de Vries, O. Kamp, and R. J. Musters, “Ultrasound and microbubble-induced intra- and intercellular bioeffects in primary endothelial cells,” Ultrasound Med. Biol., vol. 35, pp. 1917–1927, Nov. 2009. [11] L. J. Juffermans, P. A. Dijkmans, R. J. Musters, C. A. Visser, and O. Kamp, “Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide,” Am. J. Physiol. Heart Circ. Physiol., vol. 291, pp. H1595– H1601, Oct. 2006. [12] K. G. Birukov, “Cyclic stretch, reactive oxygen species, and vascular remodeling,” Antioxid. Redox Signal., vol. 11, pp. 1651–1667, Jul. 2009. [13] M. Ushio-Fukai, R. S. Frey, T. Fukai, and A. B. Malik, “Reactive oxygen species and endothelial permeability,” in Free Radical Effects on Membranes. vol. 61, San Diego, CA: Elsevier Academic, 2008, pp. 147–189. [14] P. Sobolewski, J. Kandel, A. L. Klinger, and D. M. Eckmann, “Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores,” Am. J. Physiol. Cell Physiol., vol. 301, pp. C679–C686, Sep. 2011. [15] G. P. van Nieuw Amerongen, R. Draijer, M. A. Vermeer, and V. W. van Hinsbergh, “Transient and prolonged increase in endothelial permeability induced by histamine and thrombin: Role of protein kinases, calcium, and RhoA,” Circ. Res., vol. 83, pp. 1115–1123, Nov. 30, 1998. [16] T. Kaneko, K. Kaji, and M. Matsuo, “Protection of linoleic acid hydroperoxide-induced cytotoxicity by phenolic antioxidants,” Free Radic. Biol. Med., vol. 16, pp. 405–409, Mar. 1994. [17] J. M. Choi, B. S. Yoon, S. K. Lee, J. K. Hwang, and R. Ryang, “Antioxidant properties of neohesperidin dihydrochalcone: Inhibition of hypochlorous acid-induced DNA strand breakage, protein degradation, and cell death,” Biol. Pharm. Bull., vol. 30, pp. 324–330, Feb. 2007. [18] P. G. Bannon, M. J. Kim, R. T. Dean, and J. Dawes, “Augmentation of vascular endothelial barrier function by heparin and low molecular weight heparin,” Thromb. Haemost., vol. 73, pp. 706–712, Apr. 1995. [19] W. Tschugguel, Z. Zhegu, L. Gajdzik, M. Maier, B. R. Binder, and J. Graf, “High precision measurement of electrical resistance across endothelial cell monolayers,” Pflugers Arch., vol. 430, pp. 145–147, May 1995. [20] N. Gautam, P. Hedqvist, and L. Lindbom, “Kinetics of leukocyteinduced changes in endothelial barrier function,” Br. J. Pharmacol., vol. 125, pp. 1109–1114, Nov. 1998.
kooiman et al.: microbubble-mediated alterations of endothelial layer permeability [21] M. R. Böhmer, C. H. T. Chlon, B. I. Raju, C. T. Chin, T. Shevchenko, and A. L. Klibanov, “Focused ultrasound and microbubbles for enhanced extravasation,” J. Control. Release, vol. 148, no. 1, pp. 18–24, 2010. [22] M. S. Goligorsky, “Mechanical stimulation induces Ca2+i transients and membrane depolarization in cultured endothelial cells. Effects on Ca2+i in co-perfused smooth muscle cells,” FEBS Lett., vol. 240, pp. 59–64, Nov. 21, 1988. [23] M. Sano, K. Imura, T. Ushida, and T. Tateishi, “Simultaneous measurement of [Ca2+](i) and membrane potential under mechanical or biochemical stimulation,” JSME Int. J. C, vol. 45, pp. 889–896, Dec. 2002. [24] M. Moerenhout, B. Himpens, and J. Vereecke, “Intercellular communication upon mechanical stimulation of CPAE-endothelial cells is mediated by nucleotides,” Cell Calcium, vol. 29, pp. 125–136, Feb. 2001. [25] L. L. Demer, C. M. Wortham, E. R. Dirksen, and M. J. Sanderson, “Mechanical stimulation induces intercellular calcium signaling in bovine aortic endothelial cells,” Am. J. Physiol., vol. 264, pp. H2094–H2102, Jun. 1993. [26] A. van Wamel, K. Kooiman, M. Harteveld, M. Emmer, F. J. ten Cate, M. Versluis, and N. de Jong, “Vibrating microbubbles poking individual cells: Drug transfer into cells via sonoporation,” J. Control. Release, vol. 112, pp. 149–155, May 15, 2006.
Klazina Kooiman (M’12) studied bio-pharmaceutical sciences at Leiden University, The Netherlands, and obtained her M.Sc. degree cum laude, specializing in pharmaceutical technology. From 2005 to 2010, she was a Ph.D. student in the Department of Biomedical Engineering of the Thoraxcentre, Erasmus MC, The Netherlands. On January 19, 2011, she obtained her Ph.D. degree on the topic of ultrasound contrast agents for therapy. She currently holds a postdoctoral position in the Department of Biomedical Engineering of the Thoraxcentre, Erasmus MC, focusing on using ultrasound contrast agents for drug delivery and molecular imaging. At EFUSMB 2011 in Vienna, she won the young investigator award. She was awarded the ICIN Fellowship 2012 and is currently performing research at the lab of Professor F. S. Villanueva, director of the Center for Ultrasound Molecular Imaging and Therapy at UPMC, Pittsburgh, PA.
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A. F. W. (Ton) van der Steen (M’94–SM’03– F’13) is Professor in Biomedical Engineering in Cardiology. He has an M.Sc. degree in applied physics (1989, Technical University Delft) and a Ph.D. degree in medical science (1994, Catholic University Nijmegen). Since 1994, he has been connected to the Thoraxcentre and the Interuniversity Cardiology Institute of the Netherlands. He is head of Biomedical Engineering of the Thoraxcentre, Erasmus MC. His expertise is mainly in diagnostic cardiologic imaging devices, with emphasis on echography. His current research interests are focused on vulnerable atherosclerotic plaque detection, ultrasound contrast agents, ultrasound transducers, and vascular biomechanics. His research on vulnerable plaque detection has resulted in many publications and several patents on IVUS flow, IVUS palpography, harmonic IVUS, and vasa vasorum detection. He was the IEEE UFFC Distinguished lecturer for 2011–2012.
Nico de Jong was born in 1954. He graduated from Delft University of Technology, The Netherlands, in 1978. He received his M.Sc. degree in physics, specialized in the field of pattern recognition. Since 1980, he has been a staff member of the Thoraxcentre of the Erasmus Medical Center, Rotterdam. In 1993, he received his Ph.D. degree for work on the acoustic properties of ultrasound contrast agents. In 2003, he became part-time professor at the University of Twente in the group Physics of Fluids, headed by Detlef Lohse (Spinoza winner, 2005). He is organizer of the annual European Symposium on Ultrasound Contrast Imaging, held in Rotterdam and attended by approximately 175 scientists from universities and industries all over the world. He is on the safety committee of the WFUMB (World Federation of Ultrasound in Medicine and Biology), associate editor of Ultrasound in Medicine and Biology, and has been guest editor for special issues of different journals. Over the last 5 years, he has given more than 30 invited lectures and has given numerous scientific presentations for international industries. He teaches at Technical Universities and the Erasmus MC. He has been Promotor of 15 Ph.D. students and is currently supervising 9 Ph.D. students. Since October 1, 2011, he has been professor in Molecular Ultrasonic Imaging and Therapy at the Erasmus MC and the Technical University of Delft.
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Role of Intracellular Calcium and Reactive Oxygen Species in Microbubble-Mediated Alterations of Endothelial Layer Permeability Klazina Kooiman, Member, IEEE, Antonius F. W. van der Steen, Fellow, IEEE, and Nico de Jong, Associate Member, IEEE Abstract—Drugs will be delivered to diseased tissue more efficiently if the vascular endothelial permeability is increased. Ultrasound in combination with an ultrasound contrast agent is known to increase the permeability of the endothelial layer, but the mechanism is not known. The goal of this study was to elucidate whether intracellular calcium ions, [Ca2+]i, and reactive oxygen species (ROS) are part of the mechanism that leads to an increased endothelial layer permeability following ultrasound and microbubble treatment. Human umbilical vein endothelial cells (HUVECs) treated for 2 min with ultrasoundactivated microbubbles (1 MHz, 210 kPa, 10 000 cycles, 20 Hz repetition rate) had an increased permeability that lasted up to 12 h. Recovery of permeability after 2 h was only found when HUVECs were preincubated with the [Ca2+]i chelator BAPTAAM or the antioxidant butylated hydroxytoluene (BHT). This suggests that both [Ca2+]i and ROS play an important role in the mechanism of increased permeability following ultrasound in combination with microbubble treatment.
I. Introduction
B
lood vessels are lined by endothelium, which is composed of a single layer of endothelial cells. Endothelium regulates several functions, including vascular smooth muscle tone, host-defense reactions, and tissue fluid homeostasis. Homeostasis is kept by actively controlling the extravasation of fluids, solutes, and cells into the extravascular tissue. However, the endothelial layer forms a major barrier for drug delivery into the extravascular tissue because drugs do not readily cross healthy endothelial cells. To enhance drug delivery, a controlled, temporary, and local increase in endothelial layer permeability is needed. At the same time, transient opening of the vascular barrier should also make it possible to reduce drug doses and side effects at distant sites [1]–[3]. It is known that the permeability of the endothelial layer can be increased through the following pathway of events: 1) activation of specific receptors; 2) generation of intracellular signals through second messengers and protein phosphorylations; and 3) alteration of cell–cell junctions [1], [4]. These pathways
Manuscript received December 10, 2012; accepted May 22, 2012. This project is supported by innovation subsidies collaborative projects by the Dutch Ministry of Economic Affairs under number IS042035. The authors are with the Department of Biomedical Engineering, Erasmus MC, Rotterdam, The Netherlands (e-mail: k.kooiman@ erasmusmc.nl). A. F. W. van der Steen and N. de Jong are also with the Interuniversity Cardiology Institute of The Netherlands, Utrecht, The Netherlands. DOI http://dx.doi.org/10.1109/TUFFC.2013.2767 0885–3010/$25.00
can be activated either biochemically by, for example, histamine or thrombin [1], [5], or mechanically by, for example, mechanical poking or cyclic stretching [6]. We previously reported that ultrasound, when combined with a contrast agent, increased the permeability of an endothelial layer while overall cell layer integrity remained [7]. Because ultrasound can be locally applied [8], a local increase in permeability can be achieved, thus enhancing drug delivery only locally. However, the mechanism by which ultrasound in combination with a contrast agent increases endothelial layer permeability is not known. In addition to an increase in endothelial layer permeability, several other bioeffects have also been reported by others, such as a rise in intracellular calcium ions [9], [10], [Ca2+]i, and reactive oxygen species (ROS) formation [11]. Interestingly, all of these responses have also been reported in endothelial cells following mechanotransduction [6], [12]–[14]. Because mechanotransduction can lead to an increase in [Ca2+]i and/or ROS, and subsequently to an increase in endothelial layer permeability, we determined whether this was also involved in microbubble-mediated increases in endothelial layer permeability. Permeability of treated endothelial monolayers was therefore determined in the absence and presence of the [Ca2+]i chelator BAPTA-AM [15] and the general antioxidant butylated hydroxytoluene (BHT) [16], [17]. II. Materials and Methods A. Endothelial Monolayers Primary human umbilical vein endothelial cells (HUVECs; passages 4–7) were cultured as previously described [7]. For experiments, HUVECs were replated onto ultrasound-transparent membranes of cell culture inserts (23.1 mm diameter, PET membrane; 353090, BD Falcon, BD Biosciences, San Jose, CA) at a density of 1.25 × 106 cells per insert. Before plating, the membranes were treated for 30 min with 1000 µL of 1% gelatin in MilliQ (1.04070.0500; Merck and Co. Inc., Whitehouse Station, NJ) at 37°C in the humidified cell incubator, followed by incubation for 30 min with 1273 µL of 5 µg/mL of fibronectin (10 838 039 001; Roche Diagnostics Corp., Indianapolis, IN) at 37°C in the humidified cell incubator. After removal of the fibronectin, EGM-2 cell culture medium was added to both inserts (i.e., apical; 2 mL) and wells
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(i.e., basolateral; 3 mL). Fluid volumes were selected to yield no hydrostatic pressure gradient across the cells. Experiments were performed 3 d post-plating to allow monolayers to develop well [7]. B. Treatment of Endothelial Monolayer Endothelial layer permeability was assessed by measuring transendothelial electrical resistance (TEER) in an Endohm-SNAP chamber (World Precision Instruments, Berlin, Germany) as previously described by Kooiman et al. [7]. TEER was measured before and after preincubation for 30 min in EGM-2 with or without BAPTAAM [10 µM from a 10 mM stock in dimethyl sulfoxide (DMSO); B6769, Invitrogen Corp., Carlsbad, CA; the highest dose that by itself did not alter the TEER] or BHT (50 µM from a 50 mM stock in ethanol; DI0315, Scharlab S.L., Barcelona, Spain). Monolayers were treated with ultrasound and BR14 microbubbles (Bracco Suisse SA, Geneva, Switzerland) as previously described [7]. Briefly, inserts were placed upside-down in a custom-made micropositioner in front of the ultrasound beam at a standoff distance of 60 mm. Microbubbles were added in a microbubble-cell ratio of 1:1 using a 1-mL syringe and customcurved blunt 19-gauge needle. To determine the number of microbubbles needed, cells of three inserts were counted in a haemacytometer before each experiment. Microbubbles and cells were insonified for 2 min with a 10 000-cycle sine wave burst at a repetition rate of 20 Hz at 1.0 MHz (unfocused single-element transducer 1.27 cm in diameter; V303SU, Panametrics, Waltham, MA) at an applied peak negative acoustic pressure (P−) of 210 kPa (MI = 0.21). The −6-dB lateral beam profile of the 1-MHz transducer at the standoff distance of 60 mm, i.e., where the endothelial monolayer was positioned, was 10 mm wide (see also [7]). Sham was used as control treatment. After treatment, the inserts were taken out of the tank and 2 mL of EGM-2 [plus 50 µM BHT for cells preincubated with BHT; BAPTA-AM preincubated cells were placed back on EGM-2 only, because a 1 h incubation with BAPTA-AM decreased TEER (data not shown)]. TEER was measured before, immediately after ultrasound and BR14 treatment, and up to 12 h after treatment.
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after treatment. Ultrasound alone or BR14 alone did not decrease TEER values [7]. In our current study, TEER was followed for up to 12 h after treatment. TEER values before treatment were 24.0 ± 0.7 Ω·cm2, which is in line with what others have previously reported [18], [19]. Fig. 1 shows that TEER significantly decreased from 24.0 ± 0.7 Ω·cm2 before treatment to 17.3 ± 1.0 Ω·cm2 (p 240 min time point. At all time points, TEER values were significantly different from sham-treated monolayers. TEER for sham-treated monolayers did not significantly change from before sham-treatment to up to 120 min after sham treatment (see Fig. 1). To explore the mechanism of decreased TEER after ultrasound in combination with microbubble treatment, we determined whether preincubating the monolayers with the [Ca2+]i chelator BAPTA-AM or the antioxidant BHT had an effect on TEER. Preincubation with either BAPTA-AM or BHT only did not have an effect on TEER (data not shown). Also, TEER for sham-treated monolayers preincubated with BAPTA-AM or BHT did not significantly differ from sham-treated monolayers preincubated with only EGM-2 culture medium for up to 120 min after sham treatment (data not shown). For monolayers treated with ultrasound in combination with BR14, BAPTA-AM preincubation did not have an effect on the decrease in TEER directly after and up to 60 min after treatment (see Fig. 2). Interestingly, BAPTA-AM preincubation did have an effect 120 min after treatment; TEER was significantly higher than for monolayers treated with ultrasound in
C. Statistical Analysis Results were expressed as mean ± SEM. Comparisons among multiple groups were performed using a two-way analysis of variance (ANOVA) followed by Bonferroni’s test. Differences were considered significant if p < 0.05. All tests were performed using GraphPad InStat verion 5.04 (GraphPad Software, San Diego, CA). III. Results Previously, we reported that ultrasound in combination with BR14 microbubbles decreased TEER directly
Fig. 1. Transendothelial electrical resistance (TEER) before (t = −2 min) and after (t ≥ 0 min) sham treatment (circle) or ultrasound in combination with BR14 treatment (square). Points = means; bars = ±SEM; n = 4–12 inserts.
kooiman et al.: microbubble-mediated alterations of endothelial layer permeability
Fig. 2. Effect of BAPTA-AM preincubation on transendothelial electrical resistance (TEER) before treatment (before), directly after treatment (0 min), and 60 and 120 min after treatment (60 min, 120 min) for sham-treated monolayers (white columns), ultrasound in combination with BR14 treated monolayers (gray columns), or monolayers preincubated with 10 µM BAPTA-AM and treated with ultrasound in combination with BR14 (checkered columns). Columns = means; bars = ±SEM; n = 4–12 inserts; * = significantly lower than sham; # = significantly higher than ultrasound in combination with BR14 treatment.
combination with BR14 that were not preincubated with BAPTA-AM. In fact, TEER was no longer significantly different from sham-treated monolayers. Preincubation of monolayers with BHT followed by treatment with ultrasound in combination with BR14 gave similar results as for BAPTA-AM preincubated monolayers, albeit that 60 min after ultrasound in combination with BR14 treatment, TEER for BHT preincubated monolayers was not significantly different from sham-treated or ultrasound in combination with BR14 treated monolayers (see Fig. 3). IV. Discussion This study shows that ultrasound in combination with the contrast agent BR14 results in a prolonged decrease of TEER. Previously, we reported that overall cell layer integrity remained and cell viability was not altered when
Fig. 3. Effect of BHT preincubation on transendothelial electrical resistance (TEER) before treatment (before), directly after treatment (0 min), and 60 and 120 min after treatment (60 min, 120 min) for sham-treated monolayers (white columns), ultrasound in combination with BR14 treated monolayers (gray columns), or monolayers preincubated with 50 µM BHT and treated with ultrasound in combination with BR14 (striped columns). Columns = means; bars = ±SEM; n = 4–12 inserts; * = significantly lower than sham; # = significantly higher than ultrasound in combination with BR14 treatment.
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TEER decreased [7]. Because TEER reflects changes in endothelial permeability that are associated with increased passage across a monolayer [20], our results indicate a prolonged increase in permeability following microbubble and ultrasound treatment. A prolonged increase in permeability was also reported by Böhmer et al. [21], who reported that local ultrasound in combination with microbubble treatment locally increased extravasation of Evans Blue in mice. The increased extravasation was reported when Evans Blue was injected 5 min before the ultrasound in combination with microbubble treatment, but also when Evans Blue was injected 1 h after ultrasound in combination with microbubble treatment. In this study, we set out to explore the mechanism of increased endothelial layer permeability through ultrasound-activated microbubbles. In endothelial cells, TEER is also known to immediately decrease upon addition of histamine or thrombin. Both histamine and thrombin induce intracellular signaling that leads to an increased permeability [5]. A rise in [Ca2+]i is part of this signaling mechanism and BAPTA-AM, which chelates [Ca2+]i, completely inhibits the histamine-induced increase in permeability while only partly inhibiting the thrombin-induced increase in permeability. Although histamine and thrombin induce a biochemical rise in [Ca2+]i in endothelial cells, a mechanically induced [Ca2+]i rise has also been reported, namely upon mechanical stimulation by a micropipette or microprobe [22]–[25]. Our previous study [26] showed that ultrasound-activated microbubbles are capable of poking endothelial cells. This mechanical poking by ultrasoundactivated microbubbles is known to induce a [Ca2+]i rise [9], [10] which may increase endothelial layer permeability through signaling [6]. In our current study, BAPTA-AM did not inhibit the ultrasound and microbubble-mediated decrease in TEER in the initial phase (0 to 60 min) after treatment. This suggests that [Ca2+]i does not play a role in the ultrasound-induced decrease in TEER in the initial phase. On the other hand, BAPTA-AM was able to facilitate the return of TEER to initial values 2 h after treatment. This suggests that [Ca2+]i plays a role in the late phase after treatment and could also mean that the microbubble-mediated rise in [Ca2+]i in the initial phase was too high and therefore exceeded the chelating capacity of BAPTA-AM, resulting in no inhibiting effect of BAPTA-AM in the initial phase. During this initial phase, BAPTA-AM preincubated endothelial cells would be able to decrease the remaining excess [Ca2+]i from their cytoplasm by pumping it out of the cell or into intracellular stores, resulting in the return of TEER to initial values in the late phase after treatment. In addition to a rise in [Ca2+]i, increased H2O2 production has also been reported following ultrasound in combination with microbubble treatment [11]. H2O2 is an ROS and is known to be involved in an increase in endothelial layer permeability [12], [13]. In our study, we inhibited ROS by preincubating the cells with BHT. Although BHT did not inhibit the decrease in TEER in the initial phase
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following ultrasound in combination with microbubble treatment, it did induce a recovery in TEER to initial values within 2 h. This, therefore, suggests that ROS play a role in delaying the recovery of endothelial layer permeability following ultrasound in combination with microbubble treatment. It is remarkable, as shown in Fig. 1, that TEER first decreased after ultrasound in combination with microbubble treatment, then increased between 30 and 60 min and decreased again after 60 min. It is our hypothesis that the mechanism by which TEER decreases is not a single, but a multifactorial, process that takes place at different time scales. This will be subject of our future studies. Although HUVECs are widely used to study endothelial layer permeability [15], [20], they remain an in vitro model system. We used this model system to simulate microbubble interactions with the vessel wall and to study effects of ultrasound-activated microbubbles on endothelial layer permeability. However, the disadvantage of this model is that flow is not taken into consideration. This will be subject of our future studies. Our study shows that both [Ca2+]i and ROS are suggested to be involved in the prolonged decrease in TEER of an endothelial layer following ultrasound and microbubble treatment. The mechanism could therefore involve the activation of signaling mechanisms that induce endothelial layer permeability. Further studies are needed to fully elucidate the mechanism, including the microbubble dynamics, microstreaming, jetting, clustering, etc. Fully elucidating the mechanism would mean that in vitro and in vivo effects of ultrasound in combination with microbubble treatment could be more controlled. This is important for drugs that have difficulty crossing the endothelial layer. Our future studies will also focus on determining which drugs will have an increased transport across the endothelial layer following ultrasound in combination with contrast agent treatment and whether this is due to increased transcellular or paracellular passage. In addition, our current study suggests that [Ca2+]i chelators or antioxidants can be applied when a prolonged increase in endothelial layer permeability is not needed following ultrasound and microbubble treatment.
Acknowledgments The authors are grateful to Bracco Suisse SA, Geneva, Switzerland, for kindly providing the BR14 samples. The authors thank M. Manten (Dept. of Biomedical Engineering, Erasmus MC, The Netherlands) for his technical assistance and are grateful to Dr. G. P. van Nieuw Ame rongen, Prof. V. W. M. van Hinsbergh (both Dept. of Physiology, VU University Medical Center, The Netherlands), and Dr. L. J. M. Juffermans (Dept. of Physiology and Dept. of Cardiology, VU University Medical Center, The Netherlands) for discussions about the results.
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References [1] D. Mehta and A. B. Malik, “Signaling mechanisms regulating endothelial permeability,” Physiol. Rev., vol. 86, pp. 279–367, Jan. 2006. [2] P. Telo, S. Lostaglio, and E. Dejana, “Structure of intercellular junctions in the endothelium,” Therapie, vol. 52, pp. 395–398, Sep.–Oct. 1997. [3] G. P. van Nieuw Amerongen and V. W. van Hinsbergh, “Targets for pharmacological intervention of endothelial hyperpermeability and barrier function,” Vascul. Pharmacol., vol. 39, pp. 257–272, Nov. 2002. [4] F. R. Haselton, J. S. Alexander, S. N. Mueller, and A. P. Fishman, Modulation of Endothelial Paracellular Permeability. New York: Plenum Press, 1992. [5] V. W. van Hinsbergh and G. P. van Nieuw Amerongen, “Intracellular signalling involved in modulating human endothelial barrier function,” J. Anat., vol. 200, pp. 549–560, Jun. 2002. [6] P. F. Davies, “Flow-mediated endothelial mechanotransduction,” Physiol. Rev., vol. 75, pp. 519–560, Jul. 1995. [7] K. Kooiman, M. Emmer, M. Foppen-Harteveld, A. van Wamel, and N. de Jong, “Increasing the endothelial layer permeability through ultrasound-activated microbubbles,” IEEE Trans. Biomed. Eng., vol. 57, pp. 29–32, Jan. 2010. [8] M. R. Böhmer, A. L. Klibanov, K. Tiemann, C. S. Hall, H. Gruell, and O. C. Steinbach, “Ultrasound triggered image-guided drug delivery,” Eur. J. Radiol., vol. 70, pp. 242–253, May 2009. [9] Z. Fan, R. E. Kumon, J. Park, and C. X. Deng, “Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles,” J. Control. Release, vol. 142, pp. 31–39, Feb. 25, 2010. [10] L. J. Juffermans, A. van Dijk, C. A. Jongenelen, B. Drukarch, A. Reijerkerk, H. E. de Vries, O. Kamp, and R. J. Musters, “Ultrasound and microbubble-induced intra- and intercellular bioeffects in primary endothelial cells,” Ultrasound Med. Biol., vol. 35, pp. 1917–1927, Nov. 2009. [11] L. J. Juffermans, P. A. Dijkmans, R. J. Musters, C. A. Visser, and O. Kamp, “Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide,” Am. J. Physiol. Heart Circ. Physiol., vol. 291, pp. H1595– H1601, Oct. 2006. [12] K. G. Birukov, “Cyclic stretch, reactive oxygen species, and vascular remodeling,” Antioxid. Redox Signal., vol. 11, pp. 1651–1667, Jul. 2009. [13] M. Ushio-Fukai, R. S. Frey, T. Fukai, and A. B. Malik, “Reactive oxygen species and endothelial permeability,” in Free Radical Effects on Membranes. vol. 61, San Diego, CA: Elsevier Academic, 2008, pp. 147–189. [14] P. Sobolewski, J. Kandel, A. L. Klinger, and D. M. Eckmann, “Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores,” Am. J. Physiol. Cell Physiol., vol. 301, pp. C679–C686, Sep. 2011. [15] G. P. van Nieuw Amerongen, R. Draijer, M. A. Vermeer, and V. W. van Hinsbergh, “Transient and prolonged increase in endothelial permeability induced by histamine and thrombin: Role of protein kinases, calcium, and RhoA,” Circ. Res., vol. 83, pp. 1115–1123, Nov. 30, 1998. [16] T. Kaneko, K. Kaji, and M. Matsuo, “Protection of linoleic acid hydroperoxide-induced cytotoxicity by phenolic antioxidants,” Free Radic. Biol. Med., vol. 16, pp. 405–409, Mar. 1994. [17] J. M. Choi, B. S. Yoon, S. K. Lee, J. K. Hwang, and R. Ryang, “Antioxidant properties of neohesperidin dihydrochalcone: Inhibition of hypochlorous acid-induced DNA strand breakage, protein degradation, and cell death,” Biol. Pharm. Bull., vol. 30, pp. 324–330, Feb. 2007. [18] P. G. Bannon, M. J. Kim, R. T. Dean, and J. Dawes, “Augmentation of vascular endothelial barrier function by heparin and low molecular weight heparin,” Thromb. Haemost., vol. 73, pp. 706–712, Apr. 1995. [19] W. Tschugguel, Z. Zhegu, L. Gajdzik, M. Maier, B. R. Binder, and J. Graf, “High precision measurement of electrical resistance across endothelial cell monolayers,” Pflugers Arch., vol. 430, pp. 145–147, May 1995. [20] N. Gautam, P. Hedqvist, and L. Lindbom, “Kinetics of leukocyteinduced changes in endothelial barrier function,” Br. J. Pharmacol., vol. 125, pp. 1109–1114, Nov. 1998.
kooiman et al.: microbubble-mediated alterations of endothelial layer permeability [21] M. R. Böhmer, C. H. T. Chlon, B. I. Raju, C. T. Chin, T. Shevchenko, and A. L. Klibanov, “Focused ultrasound and microbubbles for enhanced extravasation,” J. Control. Release, vol. 148, no. 1, pp. 18–24, 2010. [22] M. S. Goligorsky, “Mechanical stimulation induces Ca2+i transients and membrane depolarization in cultured endothelial cells. Effects on Ca2+i in co-perfused smooth muscle cells,” FEBS Lett., vol. 240, pp. 59–64, Nov. 21, 1988. [23] M. Sano, K. Imura, T. Ushida, and T. Tateishi, “Simultaneous measurement of [Ca2+](i) and membrane potential under mechanical or biochemical stimulation,” JSME Int. J. C, vol. 45, pp. 889–896, Dec. 2002. [24] M. Moerenhout, B. Himpens, and J. Vereecke, “Intercellular communication upon mechanical stimulation of CPAE-endothelial cells is mediated by nucleotides,” Cell Calcium, vol. 29, pp. 125–136, Feb. 2001. [25] L. L. Demer, C. M. Wortham, E. R. Dirksen, and M. J. Sanderson, “Mechanical stimulation induces intercellular calcium signaling in bovine aortic endothelial cells,” Am. J. Physiol., vol. 264, pp. H2094–H2102, Jun. 1993. [26] A. van Wamel, K. Kooiman, M. Harteveld, M. Emmer, F. J. ten Cate, M. Versluis, and N. de Jong, “Vibrating microbubbles poking individual cells: Drug transfer into cells via sonoporation,” J. Control. Release, vol. 112, pp. 149–155, May 15, 2006.
Klazina Kooiman (M’12) studied bio-pharmaceutical sciences at Leiden University, The Netherlands, and obtained her M.Sc. degree cum laude, specializing in pharmaceutical technology. From 2005 to 2010, she was a Ph.D. student in the Department of Biomedical Engineering of the Thoraxcentre, Erasmus MC, The Netherlands. On January 19, 2011, she obtained her Ph.D. degree on the topic of ultrasound contrast agents for therapy. She currently holds a postdoctoral position in the Department of Biomedical Engineering of the Thoraxcentre, Erasmus MC, focusing on using ultrasound contrast agents for drug delivery and molecular imaging. At EFUSMB 2011 in Vienna, she won the young investigator award. She was awarded the ICIN Fellowship 2012 and is currently performing research at the lab of Professor F. S. Villanueva, director of the Center for Ultrasound Molecular Imaging and Therapy at UPMC, Pittsburgh, PA.
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A. F. W. (Ton) van der Steen (M’94–SM’03– F’13) is Professor in Biomedical Engineering in Cardiology. He has an M.Sc. degree in applied physics (1989, Technical University Delft) and a Ph.D. degree in medical science (1994, Catholic University Nijmegen). Since 1994, he has been connected to the Thoraxcentre and the Interuniversity Cardiology Institute of the Netherlands. He is head of Biomedical Engineering of the Thoraxcentre, Erasmus MC. His expertise is mainly in diagnostic cardiologic imaging devices, with emphasis on echography. His current research interests are focused on vulnerable atherosclerotic plaque detection, ultrasound contrast agents, ultrasound transducers, and vascular biomechanics. His research on vulnerable plaque detection has resulted in many publications and several patents on IVUS flow, IVUS palpography, harmonic IVUS, and vasa vasorum detection. He was the IEEE UFFC Distinguished lecturer for 2011–2012.
Nico de Jong was born in 1954. He graduated from Delft University of Technology, The Netherlands, in 1978. He received his M.Sc. degree in physics, specialized in the field of pattern recognition. Since 1980, he has been a staff member of the Thoraxcentre of the Erasmus Medical Center, Rotterdam. In 1993, he received his Ph.D. degree for work on the acoustic properties of ultrasound contrast agents. In 2003, he became part-time professor at the University of Twente in the group Physics of Fluids, headed by Detlef Lohse (Spinoza winner, 2005). He is organizer of the annual European Symposium on Ultrasound Contrast Imaging, held in Rotterdam and attended by approximately 175 scientists from universities and industries all over the world. He is on the safety committee of the WFUMB (World Federation of Ultrasound in Medicine and Biology), associate editor of Ultrasound in Medicine and Biology, and has been guest editor for special issues of different journals. Over the last 5 years, he has given more than 30 invited lectures and has given numerous scientific presentations for international industries. He teaches at Technical Universities and the Erasmus MC. He has been Promotor of 15 Ph.D. students and is currently supervising 9 Ph.D. students. Since October 1, 2011, he has been professor in Molecular Ultrasonic Imaging and Therapy at the Erasmus MC and the Technical University of Delft.
