Molecular imaging with contrast ultrasound and targeted microbubbles

Molecular imaging with contrast ultrasound and targeted microbubbles

FROM BENCH TO IMAGING Molecular imaging with contrast ultrasound and targeted microbubbles Jonathan R. Lindner, MD There is growing interest in the de...

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FROM BENCH TO IMAGING Molecular imaging with contrast ultrasound and targeted microbubbles Jonathan R. Lindner, MD There is growing interest in the development of methods for imaging cellular and molecular mediators of cardiovascular diseases. Techniques for imaging molecular and cellular alterations have been explored for essentially all noninvasive cardiac imaging modalities. Molecular imaging with contrast-enhanced ultrasound relies on the detection of novel site-targeted microbubble contrast agents. These microbubbles are retained within regions of a specific disease process, thereby allowing phenotypic characterization of tissue. As microbubbles are pure intravascular tracers, the disease processes assessed must be characterized by antigens that are expressed within the vascular compartment. Accordingly, the pathologic states that have been targeted include inflammation, neoplasms, angiogenesis, and thrombus formation, all of which are mediated in part by molecular events within the vascular space. This review describes (1) different strategies that have been used to target microbubbles to regions of disease, (2) the unique challenges for imaging targeted ultrasound contrast agents, and (3) some of the early experience imaging molecular events in animal models of disease. (J Nucl Cardiol 2004;11: 215-21.) A noninvasive imaging technique capable of accurately assessing molecular events that occur in cardiovascular disease could potentially have a large impact on clinical practice. Imaging pathophysiologic processes at their cellular or molecular level could be used to detect disease very early in its course and to determine spatial extent and severity of disease. It could also be used to guide treatment in patients and should facilitate the development and testing of new therapies in the laboratory setting. Methods for molecular imaging have been explored with essentially all noninvasive cardiovascular imaging techniques. For the specific purposes of imaging molecular phenotype, it is necessary that the imaging technique be sensitive, be selective for the molecular event targeted, and provide good spatial resolution. Imaging of cellular and molecular events with contrast-enhanced ultrasound (CEU) has recently been achieved by use of novel “site-targeted” microbubble contrast agents that are retained within diseased organs. As microbubbles are pure intravascular tracers, molecuFrom the Cardiovascular Division, University of Virginia Medical Center, Charlottesville, Va. Supported by grants from the National Institutes of Health (R01DK063508), Bethesda, Md, and the American Heart Association Mid-Atlantic Affiliate, Baltimore, Md. Reprint requests: Jonathan R. Lindner, MD, Box 800158, Cardiovascular Division, University of Virginia Medical Center, Charlottesville, VA 22908; [email protected] Copyright © 2004 by the American Society of Nuclear Cardiology. 1071-3581/2004/$30.00 ⫹ 0 doi:10.1016/j.nuclcard.2004.01.003

lar imaging with CEU has focused on diseases such as inflammation, thrombus formation, and angiogenesis, which are mediated by pathophysiologic events that occur within the vasculature. The relative advantage of using CEU is that it is well balanced in terms of its sensitivity and spatial resolution. In comparison to radionuclide imaging, CEU is slightly less sensitive, mostly as a result of the influence of background tissue signal, but has superior spatial resolution. Other potential advantages of CEU include low cost, high temporal resolution (10-15 minutes), and rapid data acquisition, which is necessary for high-throughput applications. TARGETING STRATEGIES AND IMAGING PROTOCOLS Several strategies have been used to target ultrasound contrast agents to regions of disease (Figure 1). The first strategy simply takes advantage of inherent chemical or electrostatic properties of the microbubble shell that promote retention of microbubbles within diseased organs. For example, certain disease states involve upregulation of receptors that are capable of binding either albumin or lipid components of the microbubble shell.1 A second and more selective strategy relies on the attachment of antibodies, peptides, or other ligands to the microbubble surface that recognize disease-related antigens. For this purpose, chemical spacers such as polyethylene glycol can be used to project the ligand from the microbubble surface, thereby increasing 215

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influenced by the kinetics of each specific tracer and the detectors. With targeted CEU, freely circulating microbubbles are cleared from the blood pool by 5 to 10 minutes, thereby allowing rapid data acquisition. To further reduce background signal, image processing techniques have been developed that null signal from residual free tracer.4 IMAGING INFLAMMATORY RESPONSES

Figure 1. Strategies used to target microbubbles to regions of disease, which rely on either nonspecific interactions between microbubbles and cells (A) or surface conjugation of monoclonal antibodies or other targeting ligands (B). See text for details. PEG, polyethyleneglycol.

microbubble affinity and decreasing steric hindrance.2 Another ligand-directed strategy using non-bubble lipid emulsion ultrasound contrast agents relies on administration of biotinylated monoclonal antibodies, followed by injection of streptavidin, to which biotinylated emulsion contrast agents attach. Examples of these targeting strategies will be discussed in the reviews of specific applications for molecular imaging with CEU. There are unique challenges when imaging targeted microbubbles with ultrasound. Two of the most important issues are (1) whether microbubbles that are attached to cells can generate a strong enough acoustic signal for clinical imaging and (2) how to differentiate the signal generated by retained microbubbles from that generated by freely circulating microbubbles. In regard to the first issue, in vitro studies suggest that the sensitivity for detecting adhered microbubbles is quite good despite the potential for damping from bubble-cell interactions.3 Hence, the primary objective has been to develop microbubbles with high affinity for the targeted molecule that can withstand physiologic vascular shear stresses. To selectively detect signal from microbubbles that have accumulated at their target, an imaging protocol common to most forms of targeted imaging has been used. As schematically illustrated in Figure 2, the majority of the signal in an organ early (interval A) after single administration of a targeted agent will be from free tracer. Tracer accumulation at the target occurs when the concentration is high (interval B). Late signal enhancement (interval C) that occurs after clearance of freely circulating or nonbound tracer should represent targeted signal. The relative heights and widths of the curves are

Acute and chronic inflammatory responses play an important role in many cardiovascular diseases such as atherosclerosis, ischemia-reperfusion injury, myocarditis, and transplant rejection. A critical component of inflammation is the activation and recruitment of freely circulating leukocytes in the blood pool, which relies upon a series of cellular and molecular events that can be targeted (Figure 3).5,6 Initial leukocyte capture and rolling are mediated primarily by the selectin family of endothelial cell adhesion molecules expressed on venular endothelial cells (P- and E-selectin) or on leukocytes (L-selectin) in response to proinflammatory cytokines. As the leukocyte rolls, it becomes progressively activated as a result of continued exposure to local chemokines on or around the endothelial surface.7 This results in expression and activation of integrins on the leukocyte surface, which interact with ligands expressed by activated endothelial cells such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). As a consequence, leukocytes become progressively more activated and eventually adhere firmly to the endothelial surface. Adhered leukocytes then exit the vascular space via endothelial clefts in response to chemoattractant signals. One technique for assessing inflammation with CEU is to image microbubbles that are targeted to activated leukocytes adherent to the vessel wall. This strategy takes advantage of the nonspecific interactions between activated leukocytes and either albumin or lipid-shelled microbubbles (Figure 4A). Attachment of albumin microbubbles to leukocytes is mediated primarily by the ␤2-integrin Mac-1, whereas lipid microbubble attachment is due to opsonization with serum complement.1,8 Although the mechanisms of leukocyte attachment for albumin and lipid microbubbles are different, they both require expression of adhesion molecules that occurs upon leukocyte activation in regions of inflammation. Soon after their attachment, microbubbles are often phagocytosed intact by leukocytes (Figure 4A).8 Despite their intracellular location, phagocytosed microbubbles retain their acoustic activity.9 Microbubble-leukocyte interactions that occur in vivo have been demonstrated by intravital microscopy (Figure 4B).1,4 The extent of microbubble attachment is proportional to the degree of

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Figure 2. Strategy for molecular imaging used with many site-targeted contrast agents. After a single-dose administration, total signal in an organ typical rises rapidly and is largely attributable to free tracer (A). The rate of targeted tracer attachment and accumulation in diseased tissue is greatest when the free tracer concentration is high (B). Late imaging (C) after clearance of most of the free tracer represents signal primarily from targeted retention.

Figure 3. The leukocyte adhesion cascade in regions of inflammation and potential processes that can be targeted with intravascular microbubble contrast agents. ECAM, Endothelial cell adhesion molecule. See text for details.

leukocyte recruitment that occurs in response to ischemic injury or exogenous cytokine exposure1,4. Improved microbubble targeting to inflammation and injury has been achieved by altering the shell constituents for lipid microbubbles to increase the avidity of microbubbles for activated leukocytes. The addition of a small amount of phosphatidylserine to the lipid shell increases complement deposition, thereby increasing microbubble attachment to leukocytes.4 Targeted CEU with phosphatidylserine-containing microbubbles has recently been used to noninvasively assess the spatial extent and severity of the myocardial inflammatory

responses to ischemia-reperfusion in both the infarct and risk areas (Figure 5).10 Binding of ultrasound contrast agents to activated endothelial cells in regions of inflammation has recently been accomplished by conjugating ligands against endothelial cell adhesion molecules to the surface of microbubbles or other acoustically active microparticles. Thus far, these strategies have involved attachment of monoclonal antibodies against either ICAM-1, P-selectin, or VCAM-1 to the surface of the contrast agent. For the microbubble-based preparations, an average of greater than 60,000 antibodies can be conjugated to the shell

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Figure 4. A, Light and transmission electron microscopy (EM) images of lipid microbubbles interacting with activated human neutrophils demonstrating attachment (at 3 minutes) and phagocytosis (at 15 minutes). Phagocytosis is confirmed on the transmission EM by the intracellular lucency. B, Intravital microscopic images of a venule treated with tumor necrosis factor-␣ demonstrating leukocyte adhesion (arrowhead) to the venular endothelium under transillumination (left) and a fluorescein-labeled microbubble adhering to an activated leukocyte during fluorescent epi-illumination (right). (Reproduced with permission from Lindner JR, Dayton PA, Coggins MP, et al. Non-invasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles. Circulation 2000;102:531-8.)

Figure 5. Myocardial short-axis images obtained in vivo with CEU and leukocyte-targeted microbubbles (MB-PS) and ex vivo by use of radionuclide imaging with a technetium-labeled neutrophil-avid tracer (Tc-RP517) after ischemia-reperfusion injury of the left circumflex artery territory. The location of inflammation is similar by the two techniques and extends outside of the triphenyltetrazoliumchloride (TTC)-defined infarct region into the noninfarcted risk area. Arrows denote a region of early microvascular no-reflow. (Reproduced with permission from Christiansen JP, Leong-Poi H, Xu F, et al. Non-invasive imaging of myocardial reperfusion injury using leukocyte-targeted contrast echocardiography. Circulation 2002;105:1764-7.)

surface.11 Perfluorocarbon gas–filled microbubbles targeted with monoclonal antibodies against ICAM-1 and VCAM-1 have been shown in flow chambers to attach to activated cultured endothelial cells at physiologic shear rates.12,13

The feasibility of detecting early inflammatory responses in vivo with CEU and intravenous injection of microbubbles targeted to the activated endothelium was first demonstrated with microbubbles targeted to Pselectin. Intravital microscopy was used to confirm

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preferential attachment of P-selectin–targeted microbubbles in inflamed postcapillary venules in mice.11 CEU of the kidneys with P-selectin–targeted and control microbubbles was then performed after ischemia-reperfusion injury. Marked signal enhancement with P-selectin–targeted microbubbles was seen only in postischemic kidneys in which P-selectin expression was seen on immunohistology.11 Enhancement in inflamed kidneys was reduced to control levels in gene-mutated P-selectin– deficient mice. There continues to be interest in selectin-targeted contrast agents for assessing early myocardial reperfusion injury and for detecting other inflammatory cardiovascular conditions. This strategy is, however, contingent on the development of adequate nonantibody selectin ligands. It is now well recognized that the inflammatory response plays an important role in the initiation and progression of atherosclerotic disease and in plaque instability. Targeted CEU imaging of inflammatory phenotype in large-vessel atherosclerotic disease has been explored with agents targeted against ICAM-1 and VCAM-1, which participate in monocyte and T-lymphocyte recruitment to atherosclerotic lesions.14 Atherosclerosis imaging was first described with acoustically active submicron liposomes composed of multilamellar lipid vesicles with a small amount of entrapped air.15 These liposomes were targeted by conjugation of antibody against ICAM-1 to the membrane. The in vivo binding capacity of this agent has been evaluated in an inflammatory atherosclerotic model in the carotid arteries of pigs.16 After their direct intra-arterial injection, anti– ICAM-1 liposomes were observed to attach to the endothelium overlying the atherosclerotic plaque, evident by focal regions of acoustic enhancement with high-frequency ultrasound imaging. It has recently become possible to target ultrasound contrast agents to inflamed plaque with intravenous contrast administration. Perfluorocarbon-filled lipid microbubbles targeted against VCAM-1 were found to adhere preferentially to inflamed atherosclerotic plaques in the aortas of apolipoprotein E– deficient mice, whereas control microbubble binding was essentially absent.13 These techniques are now being used in animal models to investigate the utility of imaging vascular inflammation for early risk stratification, for assessing response to drug therapy, and for identifying vulnerable lesions. The ability to image myocardial microvascular endothelial activation may also have an impact on the detection and management of myocarditis and cardiac transplant rejection. CEU with microbubbles targeted to ICAM-1 has been used to detect transplant rejection in a rat model of heterotopic cardiac transplantation.17 In these studies signal intensity from ICAM-1–targeted microbubbles was greater in allografts from strain-mismatched donors than from matched donors (Figure 6).

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Figure 6. Short-axis images of rat cardiac allografts obtained with CEU and ICAM-1–targeted microbubbles demonstrating strong signal enhancement in the setting of rejection (top) and low signal enhancement in the absence of rejection (bottom). (Reproduced with permission from Weller GER, Lu E, Csikari MM, et al. Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation 2003;108:218-24.)

These studies, as well as the numerous studies using radionuclide imaging for detecting transplantation rejection, represent an effort to develop a more sensitive and less invasive method for assessing the adequacy of immunosuppressive therapy than routine biopsy. MOLECULAR IMAGING OF ANGIOGENESIS There has been a recent surge of interest in techniques to image angiogenesis for the purpose of tumor imaging. To meet their metabolic demand, tumors promote their own vascular supply by creating an imbalance between angiogenic stimulators and inhibitors.18 The ability to image angiogenesis may be useful for diagnosing neoplasms, for detecting metastases, and for assessing susceptibility or response to novel antiangiogenic tumoricidal therapies. For cardiovascular disease, assessment of endogenous or therapeutic angiogenesis may be useful for treating patients with severe coronary or peripheral vascular disease, as well as for the development of new proangiogenic therapies. There is also evidence that imaging angiogenesis in adventitial and plaque neovessels may provide a means by which to detect unfavorable atherosclerotic phenotype.19 For molecular imaging of these processes, there are many

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Figure 7. Imaging of tumors with angiogenesis-targeted microbubbles. A, Confocal microscopic image demonstrating retention of a fluorescently labeled ␣v␤3-targeted microbubble (arrow) in a glioma tumor neovessel. B, Short-axis CEU images of a rat brain with a glioblastoma tumor. Top, The parametric flow image obtained with nontargeted perfusion imaging illustrates flow abnormalities in the region of the primary tumor (T). M, Metastasis. V, ventricles. Bottom, CEU imaging of angiogenesis with ␣v␤3-targeted microbubbles demonstrates enhancement in the region of the primary tumor, as well as in a very small periventricular metastasis. (Reproduced with permission from Ellegala DB, Leong-Poi H, Carpenter JE, et al. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to ␣v␤3. Circulation 2003;108:336-41.)

potential endothelial antigens that can be targeted, including growth factor receptors and endothelial cell surface integrins. Imaging angiogenesis with targeted CEU has recently been performed by conjugating to the microbubble surface either antibodies or peptides that recognize ␣v-integrins expressed by the neovascular endothelium.20 Microbubbles targeted to ␣v␤3 have been shown to preferentially bind to growth factor– stimulated neovessels, and the degree of their CEU signal enhancement correlates well with the extent of neovascularization in matrigel models of angiogenesis.20 In animal models of malignant glioma, contrast ultrasound imaging with microbubbles targeted to ␣v␤3 has been shown to provide information on the spatial distribution and extent of tumor angiogenesis and even to detect micrometastases (Figure 7).21 These microbubbles are now being applied in chronic ischemia models to determine whether targeted CEU imaging can provide information on fibroblast growth factor-2–mediated angiogenesis and arteriogenesis even before physiologic increases in blood flow. DETECTION OF INTRAVASCULAR THROMBUS The need to improve the diagnostic accuracy of ultrasound for detecting vascular or intracardiac thrombi

has led to the development of thrombus-targeted microbubbles. The ability to bind a high concentration of microbubbles to the surface of clots may also have therapeutic potential. Destruction of microbubbles in proximity to blood clots by exposure to high acoustic power ultrasound has been shown to improve the efficacy of chemical thrombolytic therapy and can even result in clot lysis in the absence of thrombolytic therapy.22,23 One strategy for targeting thrombus has been to create ultrasound contrast agents that bind the platelet glycoprotein IIb/IIIa receptor. This integrin is expressed at a very high density on the surface of platelets upon activation and is necessary for platelet aggregation. The ultrasound contrast agent MRX-408 (ImaRx Pharmaceutical Corp, Tucson, Ariz) is composed of lipid-shelled microbubbles bearing an oligopeptide sequence that is recognized by the active binding site of platelet IIb/IIIa receptors.24 Preliminary studies have demonstrated binding of these microbubbles to human blood clots in vitro.24 An alternate method for binding microbubbles to thrombus has been developed whereby an avidin-biotin bridge is used to bind a lipid microemulsion compound to fibrin.25 The initial step of this process is the administration of a biotinylated monoclonal antibody against fibrin, followed by administration of avidin. Finally, a lipid-encapsulated perfluorocarbon emulsion that contains a biotinylated phospholipid attaches to the thrombus by means of

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avidin cross-linking. Although such a technique is somewhat limited by the complexity of administration, there are certain advantages including long circulation times and the potential to amplify the contrast effect by cross-linking the microparticles to each other. SUMMARY AND FUTURE DIRECTIONS The relatively new field of molecular imaging will likely play an important role in both the clinical and research settings. Preliminary studies have demonstrated that CEU can be used for this purpose and that ultrasound contrast agents can be effectively targeted to inflammation, angiogenesis, and thrombus. There are ongoing efforts to improve both the targeting efficiency of microbubbles and the ultrasound detection methods. It is quite likely that targeted agents can also be used for therapeutic applications. Acoustic destruction of gene-laden microbubbles has been shown to enhance tissue deposition of plasmid and viral vector deoxyribonucleic acid and to enhance transfection.26-28 The destruction of microbubbles has also been shown to produce thrombolysis.22,23 Both of these applications are likely to be facilitated by targeting strategies that result in direct apposition of microbubbles to the vessel wall or to thrombus. Acknowledgment The author has indicated he has no financial conflicts of interest.

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