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Seeing the Invisible—Ultrasound Molecular Imaging

      Abstract

      Ultrasound molecular imaging has been developed in the past two decades with the goal of non-invasively imaging disease phenotypes on a cellular level not depicted on anatomic imaging. Such techniques already play a role in pre-clinical research for the assessment of disease mechanisms and drug effects, and are thought to in the future contribute to earlier diagnosis of disease, assessment of therapeutic effects and patient-tailored therapy in the clinical field. In this review, we first describe the chemical composition and structure as well as the in vivo behavior of the ultrasound contrast agents that have been developed for molecular imaging. We then discuss the strategies that are used for targeting of contrast agents to specific cellular targets and protocols used for imaging. Next we describe pre-clinical data on imaging of thrombosis, atherosclerosis and microvascular inflammation and in oncology, including the pathophysiological principles underlying the selection of targets in each area. Where applicable, we also discuss efforts that are currently underway for translation of this technique into the clinical arena.

      Key Words

      Introduction

      Tremendous advancements have been made in medical imaging in the past decade. Taking advantage of improvements in hardware and computing power, the human body is imaged with ever-increasing spatial and temporal resolution. However, early disease processes are often characterized by phenotypical changes at the cellular level and will remain out of range for conventional imaging. To overcome this limitation, molecular imaging that uses contrast media to image biological processes at a cellular level has been developed for all major medical imaging modalities. It is thought that molecular imaging will, in the future, allow the earlier detection of disease processes and will lead to a better understanding of pathophysiology. Already, molecular imaging is being used for drug discovery and development, and this increases the likelihood that it will be an integral part of personalized medicine. Thus, nuclear tracers have been functionalized to allow molecular imaging with single-photon-emission computed tomography and positron emission tomography. For magnetic resonance imaging, magnetic nanoparticles such as ultrasmall particles of iron oxide have been used, while macrophage uptake of iodinated nanoparticles has been used for imaging of inflammation with computed tomography. For ultrasound molecular imaging, microbubbles (MBs) and other contrast agents have been functionalized for molecular imaging. Compared with other imaging modalities, ultrasound molecular imaging is characterized by a favorable balance between resolution and sensitivity for the tracer employed. In addition, a distinct advantage of ultrasound is its wide availability and low cost. Thus, from a technical and economical point of view, ultrasound is well suited for screening of large populations for a particular disease phenotype. In this review, we will first describe the contrast agents that have been developed for ultrasound molecular imaging, including the targeting strategies and imaging strategies used. We then describe how ultrasound molecular imaging has been used in pre-clinical studies, and, where applicable, in clinical studies, for imaging of thrombosis, atherosclerosis and microvascular inflammation and in oncology. All studies cited in this review describing research involving humans were approved by an ethics committee or institutional review board, and all studies describing research in animals were approved by an institutional animal care and use committee.

      Ultrasound contrast agents

      A prerequisite for ultrasound molecular imaging is the availability of a contrast agent that efficiently backscatters ultrasound waves and thus can be detected and differentiated from surrounding tissue. For this purpose, several ultrasound contrast agents (UCAs) have been developed such as MBs, nanobubbles, phase change contrast agents and echogenic liposomes (ELIPs) (Fig. 1, Table 1).
      Fig 1
      Fig. 1Contrast agents for contrast-enhanced ultrasound molecular imaging. (a) Microbubbles consist of a lipid monolayer shell and a gas core. The shell is functionalized with an antibody bound to the surface through linker molecules (here, biotin–streptavidin). (b) Echogenic liposomes (ELIP) consist of a bilayer lipid shell and an aqueous core. Entrapped gas pockets provide enhancement of the ultrasound signal. (c) Nanobubbles are used for imaging the extravascular space, as they can extravasate from the vessel. (d) Phase change contrast agents can be activated in a controlled manner, which allows for spatial and temporal control of contrast generation. PEG = polyethylene glycol.
      Table 1Main properties of ultrasound contrast agents
      AgentSizeCompositionCharacteristicsApplications/potential applications
      ShellCore
      MBs1–8 µmPhospholipid

      Polymer

      Albumin
      Gas (air, perfluorocarbons, hexafluoride)Restriction to vascular compartmentBlood pool opacification, molecular imaging

      Ultrasound-mediated drug delivery
      ELIPs20 nm–10 µmPhospholipid bilayer (with air/perfluorocarbon gas pockets)Aqueous core (with air/perfluorocarbon gas pockets)Encapsulation of hydrophilic (aqueous core) drugsMolecular imaging

      Ultrasound-mediated drug delivery
      NBs100 nm–1 µmPhospholipid or polymerGas (air, perfluorocarbon, hexafluoride)Passive extravasation into tissueMolecular imaging,

      Molecular imaging of non-vascular surface markers
      PCCAs100–400 nmPhospholipid polymer or solidLiquid

      perfluorocarbon
      Passive extravasation into tissue, spatial and temporal control of contrast activationMolecular imaging

      Molecular imaging of non-vascular surface markers
      MBs = microbubbles; ELIPs = echogenic liposomes; NBs = nanobubbles; PCCAs = phase change contrast agents.
      The most commonly used UCAs are MBs. MBs are composed of an amphiphilic lipid, biopolymer or albumin shell and a gas core (
      • Kaufmann B.A.
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      Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography.
      ,
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      • Davis C.
      • Xie A.
      • Aldred P.
      • Sarembock I.J.
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      Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1.
      ,
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      • Wei K.
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      Contrast echocardiography.
      ). Biologically inert heavy-weight gases such as perfluorocarbons and sulfur hexafluoride with low diffusion constants and low solubility in blood are typically used to improve MB stability. Such MB formulations are polydisperse with a size range from 1–8 µm. Microfluidic chips have been used to produce monodisperse MBs with a much smaller size distribution (
      • Talu E.
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      • Zhao S.
      • Powell R.L.
      • Lee A.P.
      • Longo M.L.
      • Dayton P.A.
      Tailoring the size distribution of ultrasound contrast agents: Possible method for improving sensitivity in molecular imaging.
      ). Monodisperse MBs can potentially be of advantage for molecular imaging by allowing detection of a majority of attached monodisperse MBs versus only a fraction of polydisperse MBs with ultrasound systems operating at a specific frequency bandwidth. However, this will also make necessary hardware adjustments for tuning of ultrasound frequency and bandwidth to a specific MB size. Insertion of a polyethylene glycol (PEG) brush into the shell limits MB coalescence, increases half-life in circulation (
      • Klibanov A.L.
      • Maruyama K.
      • Torchilin V.P.
      • Huang L.
      Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes.
      ) and decreases interactions with endothelial cells or leukocytes by limiting deposition of proteins such as complement on the MB surface (
      • Fisher N.G.
      • Christiansen J.P.
      • Klibanov A.
      • Taylor R.P.
      • Kaul S.
      • Lindner J.R.
      Influence of microbubble surface charge on capillary transit and myocardial contrast enhancement.
      ;
      • Chen C.C.
      • Borden M.A.
      Ligand conjugation to bimodal poly(ethylene glycol) brush layers on microbubbles.
      ).
      Because of their size, MBs transit unimpeded through the microcirculation. They do not aggregate or cause microvascular obstruction and have been reported to behave similar to red blood cells in terms of flow velocity (
      • Jayaweera A.R.
      • Edwards N.
      • Glasheen W.P.
      • Villanueva F.S.
      • Abbott R.D.
      • Kaul S.
      In vivo myocardial kinetics of air-filled albumin microbubbles during myocardial contrast echocardiography. Comparison with radiolabeled red blood cells.
      ;
      • Lindner J.R.
      • Song J.
      • Jayaweera A.R.
      • Sklenar J.
      • Kaul S.
      Microvascular rheology of Definity microbubbles after intra-arterial and intravenous administration.
      ). However, MBs are far too large to leave the vascular space, which has implications for molecular imaging as described below. After several minutes of circulation (
      • Landmark K.E.
      • Johansen P.W.
      • Johnson J.A.
      • Johansen B.
      • Uran S.
      • Skotland T.
      Pharmacokinetics of perfluorobutane following intravenous bolus injection and continuous infusion of sonazoid in healthy volunteers and in patients with reduced pulmonary diffusing capacity.
      ), MBs are taken up by the hepatic and splenic reticuloendothelial system and phagocytosed by Kupffer cells (
      • Yanagisawa K.
      • Moriyasu F.
      • Miyahara T.
      • Yuki M.
      • Iijima H.
      Phagocytosis of ultrasound contrast agent microbubbles by Kupffer cells.
      ;
      • Willmann J.K.
      • Cheng Z.
      • Davis C.
      • Lutz A.M.
      • Schipper M.L.
      • Nielsen C.H.
      • Gambhir S.S.
      Targeted microbubbles for imaging tumor angiogenesis: Assessment of whole-body biodistribution with dynamic micro-PET in mice.
      ), the gas component is eliminated via the lungs.
      Signal enhancement of MBs in an ultrasound field relies on their ability to undergo volumetric oscillation when exposed to ultrasound (
      • Dayton P.
      • Klibanov A.
      • Brandenburger G.
      • Ferrara K.
      Acoustic radiation force in vivo: a mechanism to assist targeting of microbubbles.
      ). This oscillation becomes most efficient when the frequency of incident ultrasound waves is in the range of the resonance frequency of the MBs. At sufficient ultrasound pressure near the resonant frequency, MBs start to oscillate in a non-linear manner termed stable cavitation, resulting in a strong backscattered signal not only at the incident frequency, but also at its multiples (harmonic frequencies) (
      • Burns P.N.
      Harmonic imaging with ultrasound contrast agents.
      ). As the surrounding tissue is less compressible than the MBs, it undergoes a weaker non-linear oscillation and backscatters a weaker signal at harmonic frequencies, while still backscattering a strong signal at the incident frequency. By filtering the harmonic signals only, it is possible to improve contrast-to-tissue signal ratio. Insonication of MBs at high pulse pressures leads to exaggerated MB oscillation, also termed inertial cavitation, and results in MB destruction (
      • Shi W.T.
      • Forsberg F.
      • Tornes A.
      • Ostensen J.
      • Goldberg B.B.
      Destruction of contrast microbubbles and the association with inertial cavitation.
      ).
      In clinical ultrasound, MBs are routinely used to opacify the blood pool for better delineation of the left ventricular (LV) cavity to assess LV function and structure or to assess myocardial perfusion (
      • Lindner J.R.
      • Firschke C.
      • Wei K.
      • Goodman N.C.
      • Skyba D.M.
      • Kaul S.
      Myocardial perfusion characteristics and hemodynamic profile of MRX-115, a venous echocardiographic contrast agent, during acute myocardial infarction.
      ;
      • Wei K.
      • Jayaweera A.R.
      • Firoozan S.
      • Linka A.
      • Skyba D.M.
      • Kaul S.
      Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion.
      ;
      • Senior R.
      • Khattar R.S.
      Cardiac investigation for prognosis in coronary artery disease: Where negative is positive.
      ). Also, MBs are routinely used for the detection of hepatocellular carcinoma and the differentiation of focal liver lesions (
      • Wilson S.R.
      • Greenbaum L.D.
      • Goldberg B.B.
      Contrast-enhanced ultrasound: What is the evidence and what are the obstacles?.
      ).
      MBs are far too large to leave the vascular space and, thus, only allow for molecular imaging of events taking place on the endothelial lining of the vasculature. The microvasculature in tumor tissue lacks tight junctions and has open pores at inter-endothelial junctions that may be as large as 800 nm (
      • Hobbs S.K.
      • Monsky W.L.
      • Yuan F.
      • Roberts W.G.
      • Griffith L.
      • Torchilin V.P.
      • Jain R.K.
      Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment.
      ). Thus, possible extravasation of small-sized contrast agents in tumor tissue has resulted in development of nanobubbles (NBs) capable of reaching beyond the endothelial barrier in tumors. The chemical composition of NBs is similar to that of MBs with a polymer or phospholipid shell and a heavy-weight gas core. They are obtained through post-formulation modification of polydisperse MB/NB emulsions by flotation–centrifugation or physical filtration. Dependent on the modification protocols applied, the produced NBs range from 100–1000 nm in diameter (
      • Yin T.
      • Wang P.
      • Zheng R.
      • Zheng B.
      • Cheng D.
      • Zhang X.
      • Shuai X.
      Nanobubbles for enhanced ultrasound imaging of tumors.
      ). Alternatively, the addition of tensides such as poloxamers to the emulsion of lipid shell constituents has been used for size control and production of NBs with a 200-nm mean diameter (
      • Krupka T.M.
      • Solorio L.
      • Wilson R.E.
      • Wu H.
      • Azar N.
      • Exner A.A.
      Formulation and characterization of echogenic lipid-Pluronic nanobubbles.
      ). NBs are detectable using clinical ultrasound frequency ranges (
      • Wu H.
      • Rognin N.G.
      • Krupka T.M.
      • Solorio L.
      • Yoshiara H.
      • Guenette G.
      • Sanders C.
      • Kamiyama N.
      • Exner A.A.
      Acoustic characterization and pharmacokinetic analyses of new nanobubble ultrasound contrast agents.
      ,
      • Wu W.
      • Zhang Z.
      • Zhuo L.
      • Zhou L.
      • Liu P.
      • He Y.
      • Gao Y.
      • Li R.
      • Chen Q.
      • Hua X.
      Ultrasound molecular imaging of acute cellular cardiac allograft rejection in rat with T-cell-specific nanobubbles.
      ), but head-to-head comparisons of backscattered signal in in vivo settings and based on contrast agents’ number or gas volume have not been performed. NBs can extravasate into tumor tissue, and targeting of cancer cell surface molecules such as CA-125 in ovarian cancer has been reported (
      • Gao Y.
      • Hernandez C.
      • Yuan H.X.
      • Lilly J.
      • Kota P.
      • Zhou H.
      • Wu H.
      • Exner A.A.
      Ultrasound molecular imaging of ovarian cancer with CA-125 targeted nanobubble contrast agents.
      ). After extravasation into tumor tissue, temperature- or ultrasound-induced coalescence of NBs has been hypothesized to further increase echogenicity within tissue (
      • Rapoport N.
      • Gao Z.
      • Kennedy A.
      Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy.
      ).
      Another type of contrast agent that may overcome the size limitations of MBs are phase-change contrast agents (PCCAs) (
      • Rojas J.D.
      • Dayton P.A.
      In vivo molecular imaging using low-boiling-point phase-change contrast agents: A proof of concept study.
      ). Cooling and application of mechanical pressure are used to change low-boiling-point gases such as octafluorobutane and decafluorobutane within MBs to the liquid phase and thus obtain nanodroplets in the submicron size range. Of note, nanodroplets can also be produced through low-temperature and high-pressure exposure from commercially available MBs such as Definity (
      • Choudhury S.A.
      • Xie F.
      • Dayton P.A.
      • Porter T.R.
      Acoustic behavior of a reactivated, commercially available ultrasound contrast agent.
      ). On injection into the circulation, PCCAs become superheated and easily vaporize when exposed to pulsed ultrasound at diagnostic pressures and frequencies (
      • Sheeran P.S.
      • Luois S.
      • Dayton P.A.
      • Matsunaga T.O.
      Formulation and acoustic studies of a new phase-shift agent for diagnostic and therapeutic ultrasound.
      ;
      • Wilson K.
      • Homan K.
      • Emelianov S.
      Biomedical photoacoustics beyond thermal expansion using triggered nanodroplet vaporization for contrast-enhanced imaging.
      ). The resulting MBs can be detected only after vaporization, which allows for spatial and temporal control of contrast generation. PCCAs have been used for targeting of the integrin αvß3 involved in tumor angiogenesis in animal models (
      • Rojas J.D.
      • Dayton P.A.
      In vivo molecular imaging using low-boiling-point phase-change contrast agents: A proof of concept study.
      ). The longer circulation time of PCCAs (
      • Sheeran P.S.
      • Rojas J.D.
      • Puett C.
      • Hjelmquist J.
      • Arena C.B.
      • Dayton P.A.
      Contrast-enhanced ultrasound imaging and in vivo circulatory kinetics with low-boiling-point nanoscale phase-change perfluorocarbon agents.
      ) may improve targeting efficiency, and the specific acoustic signature that results from PCCA vaporization (
      • Sheeran P.S.
      • Matsunaga T.O.
      • Dayton P.A.
      Phase change events of volatile liquid perfluorocarbon contrast agents produce unique acoustic signatures.
      ) could improve signal-to-noise ratio for molecular imaging compared with MBs; however, this has so far not specifically been assessed. It should also be noted that it remains to be investigated whether the inability to control the size of MBs generated after vaporization may lead to physical entrapment and, possibly, tissue damage.
      Entrapment of gas (usually air) either within the phospholipid bilayer or within the core during the rehydration process after lyophilization results in the generation of ELIPs (
      • Huang S.L.
      Liposomes in ultrasonic drug and gene delivery.
      ;
      • Paul S.
      • Nahire R.
      • Mallik S.
      • Sarkar K.
      Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery.
      ). ELIPs have a size distribution of 20 nm to 10 µm (
      • Huang S.L.
      • Hamilton A.J.
      • Nagaraj A.
      • Tiukinhoy S.D.
      • Klegerman M.E.
      • McPherson D.D.
      • Macdonald R.C.
      Improving ultrasound reflectivity and stability of echogenic liposomal dispersions for use as targeted ultrasound contrast agents.
      ). The echogenicity of ELIPs has been studied in comparison to the commercially available MB contrast agent Optison. Although larger concentrations of ELIPs relative to MBs are required to achieve similar echogenicity (
      • Coussios C.C.
      • Holland C.K.
      • Jakubowska L.
      • Huang S.L.
      • MacDonald R.C.
      • Nagaraj A.
      • McPherson D.D.
      In vitro characterization of liposomes and Optison by acoustic scattering at 3.5 MHz.
      ), such high concentrations have been well tolerated in animal studies (
      • Hamilton A.J.
      • Huang S.L.
      • Warnick D.
      • Rabbat M.
      • Kane B.
      • Nagaraj A.
      • Klegerman M.
      • McPherson D.D.
      Intravascular ultrasound molecular imaging of atheroma components in vivo.
      ). The surface of ELIPs can be loaded with targeting agents such as antibodies, and ELIPs have been used to target endothelial surface markers of atherosclerosis (
      • Hamilton A.J.
      • Huang S.L.
      • Warnick D.
      • Rabbat M.
      • Kane B.
      • Nagaraj A.
      • Klegerman M.
      • McPherson D.D.
      Intravascular ultrasound molecular imaging of atheroma components in vivo.
      ). In addition, ELIPs can carry hydrophilic drugs in the aqueous compartment or hydrophobic drugs within the lipid bilayer and are thus attractive for drug delivery (
      • Tiukinhoy S.D.
      • Khan A.A.
      • Huang S.
      • Klegerman M.E.
      • MacDonald R.C.
      • McPherson D.D.
      Novel echogenic drug—Immunoliposomes for drug delivery.
      ).

      Targeting strategies for ultrasound molecular imaging

      Ultrasound molecular imaging relies on delivering the contrast agent to a specific site in the vasculature where a disease process has resulted in the expression of cell surface markers. There are two general strategies for targeting of ultrasound contrast agents: (i) modification of the shell surface, or (ii) conjugation of a targeting ligand on the contrast agent shell surface.
      Incorporation of phosphatidylserine into the MB shell results in a highly negative surface charge and in activation and surface attachment of complement fragments (
      • Fisher N.G.
      • Christiansen J.P.
      • Klibanov A.
      • Taylor R.P.
      • Kaul S.
      • Lindner J.R.
      Influence of microbubble surface charge on capillary transit and myocardial contrast enhancement.
      ), which in turn mediate attachment of MBs to activated leukocytes within inflamed tissue (
      • Lindner J.R.
      • Coggins M.P.
      • Kaul S.
      • Klibanov A.L.
      • Brandenburger G.H.
      • Ley K.
      Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes.
      ,
      • Lindner J.R.
      • Dayton P.A.
      • Coggins M.P.
      • Ley K.
      • Song J.
      • Ferrara K.
      • Kaul S.
      Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles.
      ,
      • Lindner J.R.
      • Song J.
      • Xu F.
      • Klibanov A.L.
      • Singbartl K.
      • Ley K.
      • Kaul S.
      Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes.
      ). Similarly, albumin-shelled MBs have been reported to bind to activated leukocytes via β2-integrins on leukocytes (
      • Lindner J.R.
      • Coggins M.P.
      • Kaul S.
      • Klibanov A.L.
      • Brandenburger G.H.
      • Ley K.
      Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes.
      ,
      • Lindner J.R.
      • Dayton P.A.
      • Coggins M.P.
      • Ley K.
      • Song J.
      • Ferrara K.
      • Kaul S.
      Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles.
      ,
      • Lindner J.R.
      • Song J.
      • Xu F.
      • Klibanov A.L.
      • Singbartl K.
      • Ley K.
      • Kaul S.
      Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes.
      ) and via complement fragments (
      • Anderson D.R.
      • Tsutsui J.M.
      • Xie F.
      • Radio S.J.
      • Porter T.R.
      The role of complement in the adherence of microbubbles to dysfunctional arterial endothelium and atherosclerotic plaque.
      ). After being phagocytized by the leukocytes, MBs can still be detected in vivo by ultrasound and used for imaging of inflammation (
      • Lindner J.R.
      • Coggins M.P.
      • Kaul S.
      • Klibanov A.L.
      • Brandenburger G.H.
      • Ley K.
      Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes.
      ,
      • Lindner J.R.
      • Dayton P.A.
      • Coggins M.P.
      • Ley K.
      • Song J.
      • Ferrara K.
      • Kaul S.
      Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles.
      ,
      • Lindner J.R.
      • Song J.
      • Xu F.
      • Klibanov A.L.
      • Singbartl K.
      • Ley K.
      • Kaul S.
      Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes.
      ).
      In a more specific approach to targeting, the conjugation of ligands such as antibodies, peptides, glycoproteins and oligosaccharides to the MB surface is used. Antibodies are widely used in pre-clinical ultrasound molecular imaging studies, because they offer several advantages such as commercial availability of a wide range of antibodies to endothelial target molecules and binding with high affinity and specificity. However, while being suitable for pre-clinical studies, antibodies as targeting ligands are unlikely to be clinically translated, as they are potentially immunogenic and expensive. Therefore, low-molecular-weight ligands have been developed to overcome the limitations of antibodies. Small peptide binders are an emerging class of targeting ligands with high potential for clinical application. Such high-affinity binders can be selected from synthetic libraries using ribosome display and screened against a given target (
      • Binz H.K.
      • Amstutz P.
      • Pluckthun A.
      Engineering novel binding proteins from nonimmunoglobulin domains.
      ;
      • Hosse R.J.
      • Rothe A.
      • Power B.E.
      A new generation of protein display scaffolds for molecular recognition.
      ;
      • Skerra A.
      Alternative non-antibody scaffolds for molecular recognition.
      ;
      • Zahnd C.
      • Amstutz P.
      • Pluckthun A.
      Ribosome display: Selecting and evolving proteins in vitro that specifically bind to a target.
      ). Production of small peptide binders is easily accomplished in bacteria, offering an additional advantage over the expensive production of monoclonal antibodies in hybridoma cell cultures. It has recently been reported that MBs bearing small peptide ligands can be used for imaging of inflammatory cells and platelets in vivo (
      • Moccetti F.
      • Brown E.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ruggeri Z.
      • Shentu W.
      • Chen J.
      • Lopez J.A.
      • Lindner J.R.
      Myocardial infarction produces sustained proinflammatory endothelial activation in remote arteries.
      ,
      • Moccetti F.
      • Weinkauf C.C.
      • Davidson B.P.
      • Belcik J.T.
      • Marinelli E.R.
      • Unger E.
      • Lindner J.R.
      Ultrasound molecular imaging of atherosclerosis using small-peptide targeting ligands against endothelial markers of inflammation and oxidative stress.
      ). In addition, Lewis oligosaccharide derivatives, such as sialyl Lewis X (sLex), have been successfully used for targeting of the endothelial cell adhesion molecules (ECAMs) P-selectin and E-selectin (
      • Klibanov A.L.
      Ligand-carrying gas-filled microbubbles: Ultrasound contrast agents for targeted molecular imaging.
      ). Multitargeting using sLex and antibodies displayed on the MB shell surface may offer improved adhesion characteristics (
      • Weller G.E.
      • Villanueva F.S.
      • Tom E.M.
      • Wagner W.R.
      Targeted ultrasound contrast agents: In vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx.
      ). Also, acoustic radiation force has been used to improve targeting efficiency. This principle relies on absorption of the momentum of an incident sound wave and, therefore, displacement of MBs toward the vessel wall. As a result, the targeting efficiency is improved as receptor–ligand interactions are promoted (
      • Dayton P.
      • Klibanov A.
      • Brandenburger G.
      • Ferrara K.
      Acoustic radiation force in vivo: a mechanism to assist targeting of microbubbles.
      ;
      • Zhao S.
      • Borden M.
      • Bloch S.H.
      • Kruse D.
      • Ferrara K.W.
      • Dayton P.A.
      Radiation-force assisted targeting facilitates ultrasonic molecular imaging.
      ;
      • Rychak J.J.
      • Klibanov A.L.
      • Ley K.F.
      • Hossack J.A.
      Enhanced targeting of ultrasound contrast agents using acoustic radiation force.
      ).
      Different strategies have been used for conjugation of targeting ligands to the MB surface Coupling can be accomplished either directly to the MB shell or more often to a PEG spacer arm to project the binder away from the MB surface and, therefore, enhance ligand–target interaction. The use of longer PEG spacer arms can improve targeting efficiency because of the better accessibility of the conjugated ligand (
      • Khanicheh E.
      • Mitterhuber M.
      • Kinslechner K.
      • Xu L.
      • Lindner J.R.
      • Kaufmann B.A.
      Factors affecting the endothelial retention of targeted microbubbles: Influence of microbubble shell design and cell surface projection of the endothelial target molecule.
      ). However, being exposed on the PEG spacer, targeting ligands are more likely to be recognized by the immune system and cause an unwanted immune response. For this reason, MBs with buried-ligand architecture have been developed, where the targeting ligands are coupled to relatively short PEG spacer arms and are surrounded by longer PEG chains reducing recognition by the immune system. For the ligand–target interaction necessary for molecular imaging, such buried-ligand architecture MBs need to be brought into proximity with the target molecule using ultrasound radiation force (
      • Borden M.A.
      • Streeter J.E.
      • Sirsi S.R.
      • Dayton P.A.
      In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.
      ). Microbubble adherence can be improved by adjusting the acoustic setting of an imaging protocol, for example, by applying acoustic radiation force (
      • Dayton P.
      • Klibanov A.
      • Brandenburger G.
      • Ferrara K.
      Acoustic radiation force in vivo: a mechanism to assist targeting of microbubbles.
      ;
      • Zhao S.
      • Borden M.
      • Bloch S.H.
      • Kruse D.
      • Ferrara K.W.
      • Dayton P.A.
      Radiation-force assisted targeting facilitates ultrasonic molecular imaging.
      ;
      • Rychak J.J.
      • Klibanov A.L.
      • Ley K.F.
      • Hossack J.A.
      Enhanced targeting of ultrasound contrast agents using acoustic radiation force.
      ). When radiation force is applied in the direction of ultrasound wave propagation, MBs can be displaced in the same direction over distances in the millimeter range and, thus, be concentrated near the vessel wall. As a result, the targeting efficiency is improved as receptor–ligand interactions are promoted.
      For the conjugation of ligands to the PEG spacer, a biotin–streptavidin linking has been commonly used for proof of concept in pre-clinical studies. However, the major concern of this method is the immunogenicity of streptavidin in humans, which makes this method difficult to translate into clinics. To overcome this limitation, other conjugation strategies were developed that rely on the formation of covalent bonds between the targeting agent and the PEG spacer. A maleimide on the end of the PEG spacer facilitates covalent conjugation to a thiol group on the targeting agent, (e.g., a cysteine residue) (
      • Kirpotin D.
      • Park J.W.
      • Hong K.
      • Zalipsky S.
      • Li W.L.
      • Carter P.
      • Benz C.C.
      • Papahadjopoulos D.
      Sterically stabilized anti-HER2 immunoliposomes: Design and targeting to human breast cancer cells in vitro.
      ). This strategy has the advantage of allowing site-specific conjugation of molecular probes, which do not contain cysteine in their structure, for instance, small peptide binders, by incorporating an additional cysteine at the C- or N-terminus of the peptide. Another type of covalent conjugation is N-hydroxysuccinimide (NHS) binding. NHS ester reagent binds primary amines on the proteins or peptides and, thus, provides an undirected binding compared with maleimide (
      • Klibanov A.L.
      Ligand-carrying gas-filled microbubbles: Ultrasound contrast agents for targeted molecular imaging.
      ).
      To achieve a successful ultrasound molecular imaging, a number of requirements for the targeting ligand and the target itself should be met. For MBs and ELIPs, the target molecule should be present on the endothelial surface. The target molecule expression should be specific for a particular disease with low constitutive expression in the absence of disease. It is desirable, that the expression of the target molecule correlates with disease severity. Moreover, off-target binding of the ligand should be as low as possible for a high signal-to-noise ratio. The targeting ligand should be attached in high density to the MBs for high attachment efficiency (
      • Weller G.E.
      • Villanueva F.S.
      • Klibanov A.L.
      • Wagner W.R.
      Modulating targeted adhesion of an ultrasound contrast agent to dysfunctional endothelium.
      ). Fifty thousand to 100,000 ligands can be attached to a 2- to 3-µm MB, which allows for firm adhesion under flow conditions (
      • Takalkar A.M.
      • Klibanov A.L.
      • Rychak J.J.
      • Lindner J.R.
      • Ley K.
      Binding and detachment dynamics of microbubbles targeted to P-selectin under controlled shear flow.
      ). The ligand should be specific for its target, bind it quickly, thus having a high on-rate constant kon, and stay attached for a long time, having a low off-rate constant koff. These factors are crucial particularly for targeting at high shear stresses, such as in large arteries (Fig. 2).
      Fig 2
      Fig. 2Illustration of imaging of targeted signal. Ultrasound molecular imaging using microbubbles targeted to vascular cell adhesion molecule 1 in a mouse hindlimb inflammation model. See text for details. Adapted with permission from
      • Lindner J.R.
      Molecular imaging with contrast ultrasound and targeted microbubbles.
      .

      Imaging of targeted signal

      A common approach to molecular imaging of a target molecule on the endothelium is to inject a bolus of one to several million MBs and let them circulate for 5–10 min. During this time a fraction of the MBs bind to their target within the tissue while the majority of non-bound freely circulating MBs are cleared from the blood pool because of uptake by reticuloendothelial system, gas loss and shell disintegration. After circulation time, image frames containing both attached and circulating MBs are acquired, followed by destruction of MBs in the tissue of interest by application of high-power ultrasound impulses. The total number of circulating MBs is not affected, and the tissue of interest will be replenished by circulating MBs. At this time point, images of the circulating MBs only are acquired. Digital subtraction of pre- and post-destruction frames will then yield signal from attached MBs only (Fig. 3). This molecular imaging protocol is sufficiently short to allow the assessment of several targets within one imaging session (
      • Lindner J.R.
      • Song J.
      • Christiansen J.
      • Klibanov A.L.
      • Xu F.
      • Ley K.
      Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin.
      ). It has also been reported that repeated injections of MBs targeted to the same molecule do not result in a signal decay, as targeting efficiency is relatively low (
      • Streeter J.E.
      • Dayton P.A.
      An in vivo evaluation of the effect of repeated administration and clearance of targeted contrast agents on molecular imaging signal enhancement.
      ). Both low-mechanical-index and high-mechanical-index protocols have been used for imaging of targeted signal, and head-to-head comparisons have revealed that although high-mechanical-index imaging leads to a more robust signal, relative signal enhancement is comparable for both imaging techniques (
      • Kaufmann B.A.
      • Carr C.L.
      • Belcik J.T.
      • Xie A.
      • Yue Q.
      • Chadderdon S.
      • Caplan E.S.
      • Khangura J.
      • Bullens S.
      • Bunting S.
      • Lindner J.R.
      Molecular imaging of the initial inflammatory response in atherosclerosis: Implications for early detection of disease.
      ,
      • Kaufmann B.A.
      • Carr C.L.
      • Belcik T.
      • Xie A.
      • Kron B.
      • Yue Q.
      • Lindner J.R.
      Effect of acoustic power on in vivo molecular imaging with targeted microbubbles: Implications for low-mechanical index real-time imaging.
      ).
      Fig 3
      Fig. 3Determinants of targeted microbubble retention. See text for details. Adapted, with permission, from
      • Lindner J.R.
      Molecular imaging of vascular phenotype in cardiovascular disease: new diagnostic opportunities on the horizon.
      .
      The retention fraction of MBs within a given tissue is influenced not only by the expression of the target molecule and MB characteristics, but also by the perfusion of the tissue. To account for this, mathematical modeling using deconvolution of time–intensity curves has been used to derive a retention fraction of MBs that controls for tissue perfusion. Such techniques have been used, for example, to assess inflammatory responses to ischemia in animal models with diabetes mellitus-induced vascular dysregulation (
      • Carr C.L.
      • Qi Y.
      • Davidson B.
      • Chadderdon S.
      • Jayaweera A.R.
      • Belcik J.T.
      • Benner C.
      • Xie A.
      • Lindner J.R.
      Dysregulated selectin expression and monocyte recruitment during ischemia-related vascular remodeling in diabetes mellitus.
      ).

      Molecular imaging of thrombosis

      Thrombosis plays a major role in many disease states. There are many different anti-thrombotic and anti-platelet pharmacologic strategies that are designed to inhibit thrombus formation or to accelerate dissolution of clot (
      • Blann A.D.
      • Landray M.J.
      • Lip G.Y.
      ABC of antithrombotic therapy: An overview of antithrombotic therapy.
      ). Because these anti-thrombotic therapies are all associated with the risk of bleeding, there is often a need for the clinician to have a high level of confidence that a patient will benefit from their use. Accordingly, methods for non-invasively imaging thrombus in the cardiac chamber, in large vessels or in the microcirculation will likely have a role in selecting the most appropriate therapy in patients based on refining risk-to-benefit ratio. In cardiovascular disease, some of the most common scenarios where thrombus imaging would affect management include the diagnosis of acute venous or peripheral arterial thrombus, detection of high-risk carotid or coronary arterial plaque, detection of left atrial or LV thrombus, diagnosis of prosthetic valve thrombosis and detection of microvascular thrombosis in post-myocardial infarction no-reflow phenomenon. The diagnosis of systemic disorders that involve microvascular thrombosis could also be refined, including the diagnosis of thrombotic microangiopathies (thrombotic thrombocytopenic purpura [TTP], hemolytic–uremic syndrome, paroxysmal nocturnal hemoglobinuria), sickle cell disease and even forms of placental insufficiency (
      • Moake J.L.
      Thrombotic microangiopathies.
      ;
      • Sparkenbaugh E.
      • Pawlinski R.
      Interplay between coagulation and vascular inflammation in sickle cell disease.
      ;
      • Sibley C.P.
      Treating the dysfunctional placenta.
      ). The ability to image thrombus may also be important for monitoring the efficacy of antithrombotic therapies. It has been used in pre-clinical studies to evaluate new strategies aimed at inhibiting platelet-mediated thrombosis in atherosclerotic disease and in thrombotic complications of anti-cancer drugs (
      • Moccetti F.
      • Brown E.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ruggeri Z.
      • Shentu W.
      • Chen J.
      • Lopez J.A.
      • Lindner J.R.
      Myocardial infarction produces sustained proinflammatory endothelial activation in remote arteries.
      ,
      • Moccetti F.
      • Weinkauf C.C.
      • Davidson B.P.
      • Belcik J.T.
      • Marinelli E.R.
      • Unger E.
      • Lindner J.R.
      Ultrasound molecular imaging of atherosclerosis using small-peptide targeting ligands against endothelial markers of inflammation and oxidative stress.
      ;
      • Ozawa K.
      • Packwood W.
      • Varlamov O.
      • Qi Y.
      • Xie A.
      • Wu M.D.
      • Ruggeri Z.
      • Lopez J.A.
      • Lindner J.R.
      Molecular imaging of VWF (von Willebrand factor) and platelet adhesion in postischemic impaired microvascular reflow.
      ;
      • Latifi Y.
      • Moccetti F.
      • Wu M.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ozawa K.
      • Shentu W.
      • Brown E.
      • Shirai T.
      • McCarty O.J.
      • Ruggeri Z.
      • Moslehi J.
      • Chen J.
      • Druker B.J.
      • Lopez J.A.
      • Lindner J.R.
      Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib.
      ). Finally, the targeting of MBs to a specific disease process could also be important in increasing their use in ultrasound-mediated therapy such as delivery of fibrinolytics or sonothrombolysis (
      • Marsh J.N.
      • Senpan A.
      • Hu G.
      • Scott M.J.
      • Gaffney P.J.
      • Wickline S.A.
      • Lanza G.M.
      Fibrin-targeted perfluorocarbon nanoparticles for targeted thrombolysis.
      ;
      • Xie F.
      • Lof J.
      • Matsunaga T.
      • Zutshi R.
      • Porter T.R.
      Diagnostic ultrasound combined with glycoprotein IIb/IIIa-targeted microbubbles improves microvascular recovery after acute coronary thrombotic occlusions.
      ;
      • Kawata H.
      • Uesugi Y.
      • Soeda T.
      • Takemoto Y.
      • Sung J.H.
      • Umaki K.
      • Kato K.
      • Ogiwara K.
      • Nogami K.
      • Ishigami K.
      • Horii M.
      • Uemura S.
      • Shima M.
      • Tabata Y.
      • Saito Y.
      A new drug delivery system for intravenous coronary thrombolysis with thrombus targeting and stealth activity recoverable by ultrasound.
      ).
      The notion that CEU is a promising approach for imaging of thrombosis is based on the knowledge that most pathologic thrombotic complications occur within the vascular space where MBs are confined. The adoption of any thrombus-targeted non-invasive imaging technologies into any clinical or research paradigm requires two separate considerations regarding the target molecule or cell. The first consideration is whether target fibrin-rich thrombus should be targeted versus platelet adhesion and aggregation. For example, activation of the clotting cascade with eventual activation of thrombin and conversion of fibrinogen to fibrin are critical processes in venous thrombosis and valve thrombosis. On the other hand, platelet activation, adhesion and aggregation are recognized to be primary thrombotic mechanisms in complications of plaque rupture or erosion (
      • Sato Y.
      • Hatakeyama K.
      • Yamashita A.
      • Marutsuka K.
      • Sumiyoshi A.
      • Asada Y.
      Proportion of fibrin and platelets differs in thrombi on ruptured and eroded coronary atherosclerotic plaques in humans.
      ), early plaque activation (
      • Wu M.D.
      • Atkinson T.M.
      • Lindner J.R.
      Platelets and von Willebrand factor in atherogenesis.
      ) and TTP (
      • Levy G.G.
      • Nichols W.C.
      • Lian E.C.
      • Foroud T.
      • McClintick J.N.
      • McGee B.M.
      • Yang A.Y.
      • Siemieniak D.R.
      • Stark K.R.
      • Gruppo R.
      • Sarode R.
      • Shurin S.B.
      • Chandrasekaran V.
      • Stabler S.P.
      • Sabio H.
      • Bouhassira E.E.
      • Upshaw Jr, J.D.
      • Ginsburg D.
      • Tsai H.M.
      Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.
      ). A second consideration is whether it is desirable to detect thrombus itself (platelets, fibrin, etc.) or to simply detect a pro-thrombotic milieu (vascular tissue factor, von Willebrand factor [VWF] and exposed collagen).
      Because of the importance of selecting the right targeting moiety vis-à-vis the intended clinical role of molecular imaging, molecular imaging of thrombus has been performed with a broad variety of targeted UCAs (
      • Lindner J.R.
      Molecular imaging of thrombus: Technology in evolution.
      ). An additional consideration has been whether or not MBs need to outcompete endogenous ligands. For example, when targeting GPIIb/IIIa, platelet-targeted MBs need to outcompete fibrinogen; however, little endogenous competition exists when targeting platelet GPIbα.
      For imaging the fibrin component of thrombus, ultrasound contrast agents, mostly in the form of ELIPs or other nano-emulsions, have been targeted directly to fibrin (
      • Lanza G.M.
      • Wallace K.D.
      • Scott M.J.
      • Cacheris W.P.
      • Abendschein D.R.
      • Christy D.H.
      • Sharkey A.M.
      • Miller J.G.
      • Gaffney P.J.
      • Wickline S.A.
      A novel site-targeted ultrasonic contrast agent with broad biomedical application.
      ;
      • Hamilton A.J.
      • Huang S.L.
      • Warnick D.
      • Rabbat M.
      • Kane B.
      • Nagaraj A.
      • Klegerman M.
      • McPherson D.D.
      Intravascular ultrasound molecular imaging of atheroma components in vivo.
      ). These agents were developed to enhance the areas of mechanical injury in animal models of atherosclerosis and to detect ventricular thrombi in large animal models (
      • Hamilton A.
      • Huang S.L.
      • Warnick D.
      • Stein A.
      • Rabbat M.
      • Madhav T.
      • Kane B.
      • Nagaraj A.
      • Klegerman M.
      • MacDonald R.
      • McPherson D.
      Left ventricular thrombus enhancement after intravenous injection of echogenic immunoliposomes: Studies in a new experimental model.
      ,
      • Hamilton A.
      • Rabbat M.
      • Jain P.
      • Belkind N.
      • Huang S.L.
      • Nagaraj A.
      • Klegerman M.
      • Macdonald R.
      • McPherson D.D.
      A physiologic flow chamber model to define intravascular ultrasound enhancement of fibrin using echogenic liposomes.
      ,
      • Hamilton A.J.
      • Huang S.L.
      • Warnick D.
      • Rabbat M.
      • Kane B.
      • Nagaraj A.
      • Klegerman M.
      • McPherson D.D.
      Intravascular ultrasound molecular imaging of atheroma components in vivo.
      ). This fibrin-targeting approach has not advanced rapidly because ligands that recognize only fibrin and do not bind plasma fibrinogen have only recently been developed and conjugated to UCAs (
      • Gormley C.A.
      • Keenan B.J.
      • Buczek-Thomas J.A.
      • Pessoa A.
      • Xu J.
      • Monti F.
      • Tabeling P.
      • Holt R.G.
      • Nagy J.O.
      • Wong J.Y.
      Fibrin-targeted polymerized shell microbubbles as potential theranostic agents for surgical adhesions.
      ). MBs have also been targeted to tissue factor in regions of vascular injury (
      • Lanza G.M.
      • Abendschein D.R.
      • Hall C.S.
      • Scott M.J.
      • Scherrer D.E.
      • Houseman A.
      • Miller J.G.
      • Wickline S.A.
      In vivo molecular imaging of stretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles.
      ;
      • Hamilton A.J.
      • Huang S.L.
      • Warnick D.
      • Rabbat M.
      • Kane B.
      • Nagaraj A.
      • Klegerman M.
      • McPherson D.D.
      Intravascular ultrasound molecular imaging of atheroma components in vivo.
      ), which may provide a useful target for evaluating subclinical plaque rupture. This approach is likely to be quite specific as tissue factor in inflamed plaques is ordinarily not available to binding by intravascular agents.
      The targeting of platelets has been of substantial interest based on the critical role of platelet adhesion and aggregation in microvascular angiopathies and complications of atherosclerotic disease. A common approach has been to image the platelet GPIIb/IIIa receptor, which is expressed in an activated state on activated platelets and plays a role in platelet aggregation (
      • Willoughby S.
      • Holmes A.
      • Loscalzo J.
      Platelets and cardiovascular disease.
      ). This targeting can be achieved through surface conjugation of antibodies, antibody fragments or peptides that have the RGD motif (Arg–Gly–Asp) that is found on fibronectin and many other matrix proteins (
      • Schumann P.A.
      • Christiansen J.P.
      • Quigley R.M.
      • McCreery T.P.
      • Sweitzer R.H.
      • Unger E.C.
      • Lindner J.R.
      • Matsunaga T.O.
      Targeted-microbubble binding selectively to GPIIb IIIa receptors of platelet thrombi.
      ;
      • Alonso A.
      • Della Martina A.
      • Stroick M.
      • Fatar M.
      • Griebe M.
      • Pochon S.
      • Schneider M.
      • Hennerici M.
      • Allemann E.
      • Meairs S.
      Molecular imaging of human thrombus with novel abciximab immunobubbles and ultrasound.
      ;
      • Xie F.
      • Lof J.
      • Matsunaga T.
      • Zutshi R.
      • Porter T.R.
      Diagnostic ultrasound combined with glycoprotein IIb/IIIa-targeted microbubbles improves microvascular recovery after acute coronary thrombotic occlusions.
      ;
      • Wang X.
      • Hagemeyer C.E.
      • Hohmann J.D.
      • Leitner E.
      • Armstrong P.C.
      • Jia F.
      • Olschewski M.
      • Needles A.
      • Peter K.
      • Ahrens I.
      Novel single-chain antibody-targeted microbubbles for molecular ultrasound imaging of thrombosis: Validation of a unique noninvasive method for rapid and sensitive detection of thrombi and monitoring of success or failure of thrombolysis in mice.
      ). By targeting GPIIb/IIIa it has been possible to use contrast-enhanced ultrasound (CEU) molecular imaging to detect thrombus in the carotid artery in animal models of carotid thrombosis and to monitor clot lysis (
      • Alonso A.
      • Della Martina A.
      • Stroick M.
      • Fatar M.
      • Griebe M.
      • Pochon S.
      • Schneider M.
      • Hennerici M.
      • Allemann E.
      • Meairs S.
      Molecular imaging of human thrombus with novel abciximab immunobubbles and ultrasound.
      ;
      • Wang X.
      • Hagemeyer C.E.
      • Hohmann J.D.
      • Leitner E.
      • Armstrong P.C.
      • Jia F.
      • Olschewski M.
      • Needles A.
      • Peter K.
      • Ahrens I.
      Novel single-chain antibody-targeted microbubbles for molecular ultrasound imaging of thrombosis: Validation of a unique noninvasive method for rapid and sensitive detection of thrombi and monitoring of success or failure of thrombolysis in mice.
      ). In vitro studies have suggested these MBs may also enhance the ability to detect microembolization by transcutaneous Doppler (
      • Martin M.J.
      • Chung E.M.
      • Goodall A.H.
      • Della Martina A.
      • Ramnarine K.V.
      • Fan L.
      • Hainsworth S.V.
      • Naylor A.R.
      • Evans D.H.
      Enhanced detection of thromboemboli with the use of targeted microbubbles.
      ). The targeting of MBs to GPIIb/IIIa has also been used to modestly improve the efficacy of ultrasound-mediated thrombolysis using energy produced by inertial cavitation (
      • Xie F.
      • Lof J.
      • Matsunaga T.
      • Zutshi R.
      • Porter T.R.
      Diagnostic ultrasound combined with glycoprotein IIb/IIIa-targeted microbubbles improves microvascular recovery after acute coronary thrombotic occlusions.
      ;
      • Zhou X.B.
      • Qin H.
      • Li J.
      • Wang B.
      • Wang C.B.
      • Liu Y.M.
      • Jia X.D.
      • Shi N.
      Platelet-targeted microbubbles inhibit re-occlusion after thrombolysis with transcutaneous ultrasound and microbubbles.
      ).
      One of the earliest events in platelet-mediated thrombosis is the interaction between the GPIbα component of the GPIb-IX-V complex with VWF (
      • Ruggeri Z.M.
      Von Willebrand factor, platelets and endothelial cell interactions.
      ;
      • Wu M.D.
      • Atkinson T.M.
      • Lindner J.R.
      Platelets and von Willebrand factor in atherogenesis.
      ). In areas of vascular compromise or plaque rupture, VWF multimers bind to collagen and undergo conformational change with exposure of the A1 binding domain for GPIbα (
      • Ruggeri Z.M.
      Von Willebrand factor, platelets and endothelial cell interactions.
      ). However, excess VWF can also occur in conditions such as TTP, atherosclerosis and ischemia–reperfusion injury. Ultrasound molecular imaging of constitutively expressed GPIbα on platelets and VWF A1 domain has been accomplished with targeted MBs. In animal models of atherosclerosis, these agents have been useful for early detection of disease and for understanding the role of VWF-mediated platelet adhesion in atherosclerosis development and progression (
      • McCarty O.J.
      • Conley R.B.
      • Shentu W.
      • Tormoen G.W.
      • Zha D.
      • Xie A.
      • Qi Y.
      • Zhao Y.
      • Carr C.
      • Belcik T.
      • Keene D.R.
      • de Groot P.G.
      • Lindner J.R.
      Molecular imaging of activated von Willebrand factor to detect high-risk atherosclerotic phenotype.
      ;
      • Shim C.Y.
      • Liu Y.N.
      • Atkinson T.
      • Xie A.
      • Foster T.
      • Davidson B.P.
      • Treible M.
      • Qi Y.
      • Lopez J.A.
      • Munday A.
      • Ruggeri Z.
      • Lindner J.R.
      Molecular Imaging of Platelet-Endothelial Interactions and Endothelial von Willebrand factor in early and mid-stage atherosclerosis.
      ). They have also been used to detect plaque vulnerability to the accelerated progression that occurs after myocardial infarction or with certain chemotherapeutic agents (
      • Moccetti F.
      • Brown E.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ruggeri Z.
      • Shentu W.
      • Chen J.
      • Lopez J.A.
      • Lindner J.R.
      Myocardial infarction produces sustained proinflammatory endothelial activation in remote arteries.
      ,
      • Moccetti F.
      • Weinkauf C.C.
      • Davidson B.P.
      • Belcik J.T.
      • Marinelli E.R.
      • Unger E.
      • Lindner J.R.
      Ultrasound molecular imaging of atherosclerosis using small-peptide targeting ligands against endothelial markers of inflammation and oxidative stress.
      ;
      • Latifi Y.
      • Moccetti F.
      • Wu M.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ozawa K.
      • Shentu W.
      • Brown E.
      • Shirai T.
      • McCarty O.J.
      • Ruggeri Z.
      • Moslehi J.
      • Chen J.
      • Druker B.J.
      • Lopez J.A.
      • Lindner J.R.
      Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib.
      ). More recently, these agents have been used not only to detect microvascular thrombosis in post-infarct microvascular no-reflow, but also to evaluate the role of new therapies for this condition (Fig. 4) (
      • Ozawa K.
      • Packwood W.
      • Varlamov O.
      • Qi Y.
      • Xie A.
      • Wu M.D.
      • Ruggeri Z.
      • Lopez J.A.
      • Lindner J.R.
      Molecular imaging of VWF (von Willebrand factor) and platelet adhesion in postischemic impaired microvascular reflow.
      ). They have also been used to detect renal platelet adhesion from VWF (
      • Latifi Y.
      • Moccetti F.
      • Wu M.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ozawa K.
      • Shentu W.
      • Brown E.
      • Shirai T.
      • McCarty O.J.
      • Ruggeri Z.
      • Moslehi J.
      • Chen J.
      • Druker B.J.
      • Lopez J.A.
      • Lindner J.R.
      Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib.
      ), giving hope that they could be eventually applied for more accurate diagnosis of thrombotic microangiopathies.
      Fig 4
      Fig. 4Contrast echocardiography molecular imaging of microvascular platelet GPIbα in ischemia–reperfusion injury after acute molecular imaging. Images show background-subtracted images of the left ventricle with control or platelet-targeted microbubbles after production of brief anterior ischemia. The microsphere-derived risk area is shown. Molecular imaging data indicate microvascular platelet signal in the risk area. Platelet adhesion was completely eliminated by therapy with the protease (ADAMTS13) that degrades von Willebrand factor, whereas ADAMTS13-deficient animals had much greater platelet adhesion. In this study, the degree of platelet adhesion was inversely correlated with adequacy of microvascular reflow. Reprinted with permission from
      • Ozawa K.
      • Packwood W.
      • Varlamov O.
      • Qi Y.
      • Xie A.
      • Wu M.D.
      • Ruggeri Z.
      • Lopez J.A.
      • Lindner J.R.
      Molecular imaging of VWF (von Willebrand factor) and platelet adhesion in postischemic impaired microvascular reflow.
      . MB = microbubbles.

      Molecular imaging of atherosclerosis

      Complications of atherosclerotic disease such as myocardial infarction and stroke are responsible for a large proportion of morbidity and mortality worldwide and lead to enormous economic losses (
      • Laslett L.J.
      • Alagona Jr, P.
      • Clark III, B.A.
      • Drozda Jr, J.P.
      • Saldivar F.
      • Wilson S.R.
      • Poe C.
      • Hart M.
      The worldwide environment of cardiovascular disease: Prevalence, diagnosis, therapy, and policy issues: A report from the American College of Cardiology.
      ). In recent years, better and more timely treatment of acute complications of atherosclerosis has led to a reduction in mortality, particularly in patients having a myocardial infarction in high-income countries. However, assessment of an individual's risk for future atherosclerotic disease complications continues to be challenging, with currently available risk models placing a large proportion of adults in high-income countries in an intermediate risk category (
      • Assmann G.
      • Cullen P.
      • Schulte H.
      Simple scoring scheme for calculating the risk of acute coronary events based on the 10-year follow-up of the prospective cardiovascular Munster (PROCAM) study.
      ;
      • Ginghina C.
      • Bejan I.
      • Ceck C.D.
      Modern risk stratification in coronary heart disease.
      ). In this group, methods such as non-invasive imaging tools that detect early atherosclerotic changes and therefore refine the risk assessment and guide prevention strategies are a clinical need. Also, diagnostic modalities that detect early stages of atherosclerosis may be essential for selecting patients for emerging therapies that slow down or even reverse atherosclerotic plaque formation.
      The development of atherosclerosis is initiated by the accumulation of low-density lipoprotein (LDL) cholesterol particles in the subendothelial space, predominantly at the sites of disturbed laminar flow such as arterial bifurcations. Oxidative modification of LDL cholesterol to oxidized LDL (oxLDL) then results in inflammatory activation of the vascular endothelium with expression of cell adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1] and P-selectin), secretion of chemokines and recruitment of monocytes from the bloodstream into the nascent atherosclerotic plaque. Scavenging of oxLDL by monocytes/macrophages leads to the formation of foam cells, amplification of the inflammatory process and plaque growth within the arterial wall. A growing plaque encroaching on the vessel lumen can lead to reduced blood flow, causing symptoms such as angina pectoris. An imbalance between factors that stabilize or destabilize plaques can result in acute thrombotic vessel occlusion and, thus, myocardial infarction or stroke. Plaque-stabilizing factors include smooth muscle cell infiltration and collagen deposition, while plaques are destabilized by inflammatory cell infiltration, neovascularization with microbleeding, foam cell apoptosis with necrotic core formation and endothelial dysfunction or erosion with formation of a pro-thrombotic milieu. With respect to the latter, interaction of platelet GPIb with VWF on dysfunctional endothelium or in the subendothelial space can lead to atherothrombotic events, but there is also evidence that platelet adhesion to the endothelium occurs throughout all stages of plaque development and may in fact accelerate the disease process (
      • Massberg S.
      • Brand K.
      • Gruner S.
      • Page S.
      • Muller E.
      • Muller I.
      • Bergmeier W.
      • Richter T.
      • Lorenz M.
      • Konrad I.
      • Nieswandt B.
      • Gawaz M.
      A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation.
      ).
      Developments in molecular imaging have been geared both toward detection of a pro-atherogenic phenotype and toward non-invasive imaging of features that destabilize plaques, also termed vulnerable plaques. However, it should be noted that pathologic studies and studies using intracoronary ultrasound have indicated that the presence of vulnerable plaques in the coronary arteries is frequent in patients without acute myocardial infarction (
      • Mann J.
      • Davies M.J.
      Mechanisms of progression in native coronary artery disease: Role of healed plaque disruption.
      ) and that a high proportion of plaques identified as vulnerable on intravascular ultrasound stabilize over time and do not result in clinical events (
      • Kubo T.
      • Maehara A.
      • Mintz G.S.
      • Doi H.
      • Tsujita K.
      • Choi S.Y.
      • Katoh O.
      • Nasu K.
      • Koenig A.
      • Pieper M.
      • Rogers J.H.
      • Wijns W.
      • Bose D.
      • Margolis M.P.
      • Moses J.W.
      • Stone G.W.
      • Leon M.B.
      The dynamic nature of coronary artery lesion morphology assessed by serial virtual histology intravascular ultrasound tissue characterization.
      ;
      • Stone G.W.
      • Maehara A.
      • Lansky A.J.
      • de Bruyne B.
      • Cristea E.
      • Mintz G.S.
      • Mehran R.
      • McPherson J.
      • Farhat N.
      • Marso S.P.
      • Parise H.
      • Templin B.
      • White R.
      • Zhang Z.
      • Serruys P.W.
      • Investigators P.
      A prospective natural-history study of coronary atherosclerosis.
      ). Thus, research efforts have been shifting toward a global vascular risk assessment rather than the identification of individual plaque characteristics.
      Several target events occurring on the vascular endothelium have been explored in animal models of atherosclerosis for ultrasound molecular imaging.
      • Anderson D.R.
      • Tsutsui J.M.
      • Xie F.
      • Radio S.J.
      • Porter T.R.
      The role of complement in the adherence of microbubbles to dysfunctional arterial endothelium and atherosclerotic plaque.
      have used albumin-shelled MBs for imaging of endothelial dysfunction in rodent models of early and established atherosclerosis. They found that the attachment of albumin-shelled MBs is dependent on serum complement C3; however, the exact mechanism of attachment to dysfunctional vascular endothelium was not examined (
      • Anderson D.R.
      • Tsutsui J.M.
      • Xie F.
      • Radio S.J.
      • Porter T.R.
      The role of complement in the adherence of microbubbles to dysfunctional arterial endothelium and atherosclerotic plaque.
      ). Targeting of ECAMs has been examined extensively. ECAMs such as VCAM-1 and P-selectin promote recruitment of monocytes to activated endothelium by interaction with their monocyte ligands VLA-4 and P-selectin glycoprotein ligand 1 (PSGL-1). Antibody-mediated targeting of MBs to VCAM-1 has been reported to result in a specific signal enhancement that quantitatively depends on the degree of endothelial activation (
      • Kaufmann B.A.
      • Lewis C.
      • Xie A.
      • Mirza-Mohd A.
      • Lindner J.R.
      Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography.
      ,
      • Kaufmann B.A.
      • Sanders J.M.
      • Davis C.
      • Xie A.
      • Aldred P.
      • Sarembock I.J.
      • Lindner J.R.
      Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1.
      ,
      • Kaufmann B.A.
      • Wei K.
      • Lindner J.R.
      Contrast echocardiography.
      ). The fact that both VCAM-1 and P-selectin are expressed at very early stages of atherosclerotic plaque development has successfully been exploited for molecular imaging in murine models at the initial fatty streak stage (
      • Kaufmann B.A.
      • Carr C.L.
      • Belcik J.T.
      • Xie A.
      • Yue Q.
      • Chadderdon S.
      • Caplan E.S.
      • Khangura J.
      • Bullens S.
      • Bunting S.
      • Lindner J.R.
      Molecular imaging of the initial inflammatory response in atherosclerosis: Implications for early detection of disease.
      ,
      • Kaufmann B.A.
      • Carr C.L.
      • Belcik T.
      • Xie A.
      • Kron B.
      • Yue Q.
      • Lindner J.R.
      Effect of acoustic power on in vivo molecular imaging with targeted microbubbles: Implications for low-mechanical index real-time imaging.
      ). In contrast to targets in the microvasculature, high shear forces in arteries decrease firm attachment of MBs and thus negatively affect specific signal enhancement. Therefore, several strategies to increase targeting efficiency have been described. Magnetic field guidance of MBs targeted to VCAM-1 containing a magnetic streptavidin bridge has been used to increase targeting efficiency via approximation of MBs to the endothelial target (
      • Wu J.
      • Leong-Poi H.
      • Bin J.
      • Yang L.
      • Liao Y.
      • Liu Y.
      • Cai J.
      • Xie J.
      • Liu Y.
      Efficacy of contrast-enhanced US and magnetic microbubbles targeted to vascular cell adhesion molecule-1 for molecular imaging of atherosclerosis.
      ). Pre-treatment with ELIPs delivering nitric oxide (NO) to the target tissue and subsequent imaging with ELIPs targeted to intercellular cell adhesion molecule 1 (ICAM-1), which is expressed not only on the endothelium but also within plaques, lead to increased signal. This effect is presumably due to NO-induced increases in vascular permeability and, thus, accumulation of ICAM-1-targeted ELIPs within plaques (
      • Kee P.H.
      • Kim H.
      • Huang S.
      • Laing S.T.
      • Moody M.R.
      • Vela D.
      • Klegerman M.E.
      • McPherson D.D.
      Nitric oxide pretreatment enhances atheroma component highlighting in vivo with intercellular adhesion molecule-1-targeted echogenic liposomes.
      ). In addition, dual targeting of MBs to P-selectin and VCAM-1 has been found to increase attachment efficiency compared with single targeting in flow chamber studies (
      • Ferrante E.A.
      • Pickard J.E.
      • Rychak J.
      • Klibanov A.
      • Ley K.
      Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow.
      ). Although all of the aforementioned studies were performed in rodent models of atherosclerosis, ultrasound molecular imaging with MBs targeted to VCAM-1 and P-selectin has also been reported to detect early endothelial activation in non-human primate models of diet-induced obesity (
      • Chadderdon S.M.
      • Belcik J.T.
      • Bader L.
      • Kirigiti M.A.
      • Peters D.M.
      • Kievit P.
      • Grove K.L.
      • Lindner J.R.
      Proinflammatory endothelial activation detected by molecular imaging in obese nonhuman primates coincides with onset of insulin resistance and progressively increases with duration of insulin resistance.
      ). With clinical translation in mind, targeting ligands that are smaller than full-size antibodies have recently been explored and found to be feasible for targeting of ECAMs in murine models of atherosclerosis and in human thrombo-endarterectomy specimens (
      • Moccetti F.
      • Brown E.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ruggeri Z.
      • Shentu W.
      • Chen J.
      • Lopez J.A.
      • Lindner J.R.
      Myocardial infarction produces sustained proinflammatory endothelial activation in remote arteries.
      ,
      • Moccetti F.
      • Weinkauf C.C.
      • Davidson B.P.
      • Belcik J.T.
      • Marinelli E.R.
      • Unger E.
      • Lindner J.R.
      Ultrasound molecular imaging of atherosclerosis using small-peptide targeting ligands against endothelial markers of inflammation and oxidative stress.
      ;
      • Punjabi M.
      • Xu L.
      • Ochoa-Espinosa A.
      • Kosareva A.
      • Wolff T.
      • Murtaja A.
      • Broisat A.
      • Devoogdt N.
      • Kaufmann B.A.
      Ultrasound molecular imaging of atherosclerosis with nanobodies: translatable microbubble targeting murine and human VCAM (vascular cell adhesion molecule) 1.
      ). Besides assessment of vascular inflammation in atherosclerosis, targeting of VCAM-1 with MBs has recently been used to illustrate that myocardial infarction leads to inflammatory endothelial activation in remote arteries (
      • Moccetti F.
      • Brown E.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ruggeri Z.
      • Shentu W.
      • Chen J.
      • Lopez J.A.
      • Lindner J.R.
      Myocardial infarction produces sustained proinflammatory endothelial activation in remote arteries.
      ,
      • Moccetti F.
      • Weinkauf C.C.
      • Davidson B.P.
      • Belcik J.T.
      • Marinelli E.R.
      • Unger E.
      • Lindner J.R.
      Ultrasound molecular imaging of atherosclerosis using small-peptide targeting ligands against endothelial markers of inflammation and oxidative stress.
      ), which could explain the increased risk for vascular events in non-infarct-related arteries in the month after an acute coronary syndrome.
      Based on the importance of platelet–endothelial interactions in the progression of atherosclerosis and thrombotic events as a cause for clinical events, ultrasound molecular imaging of both activated VWF and platelets on the vascular endothelium has been developed. Using MBs targeted toward either the A1 fragment of VWF or GPIb on platelets, endothelial thrombogenicity and platelet–endothelium interactions have been imaged in both early and late stages of atherosclerosis (
      • McCarty O.J.
      • Conley R.B.
      • Shentu W.
      • Tormoen G.W.
      • Zha D.
      • Xie A.
      • Qi Y.
      • Zhao Y.
      • Carr C.
      • Belcik T.
      • Keene D.R.
      • de Groot P.G.
      • Lindner J.R.
      Molecular imaging of activated von Willebrand factor to detect high-risk atherosclerotic phenotype.
      ;
      • Shim C.Y.
      • Liu Y.N.
      • Atkinson T.
      • Xie A.
      • Foster T.
      • Davidson B.P.
      • Treible M.
      • Qi Y.
      • Lopez J.A.
      • Munday A.
      • Ruggeri Z.
      • Lindner J.R.
      Molecular Imaging of Platelet-Endothelial Interactions and Endothelial von Willebrand factor in early and mid-stage atherosclerosis.
      ).
      Apart from its diagnostic use, ultrasound molecular imaging has also been reported to be useful for assessing the effects of drugs on vascular inflammation (
      • Khanicheh E.
      • Mitterhuber M.
      • Xu L.
      • Haeuselmann S.P.
      • Kuster G.M.
      • Kaufmann B.A.
      Noninvasive ultrasound molecular imaging of the effect of statins on endothelial inflammatory phenotype in early atherosclerosis.
      ,
      • Khanicheh E.
      • Qi Y.
      • Xie A.
      • Mitterhuber M.
      • Xu L.
      • Mochizuki M.
      • Daali Y.
      • Jaquet V.
      • Krause K.H.
      • Ruggeri Z.M.
      • Kuster G.M.
      • Lindner J.R.
      • Kaufmann B.A.
      Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties.
      ) and thrombogenicity (
      • Liu Y.
      • Davidson B.P.
      • Yue Q.
      • Belcik T.
      • Xie A.
      • Inaba Y.
      • McCarty O.J.
      • Tormoen G.W.
      • Zhao Y.
      • Ruggeri Z.M.
      • Kaufmann B.A.
      • Lindner J.R.
      Molecular imaging of inflammation and platelet adhesion in advanced atherosclerosis: Effects of antioxidant therapy with NADPH oxidase inhibition.
      ;
      • Atkinson T.
      • Packwood W.
      • Xie A.
      • Liang S.
      • Qi Y.
      • Ruggeri Z.
      • Lopez J.
      • Davidson B.P.
      • Lindner J.R.
      Assessment of novel antioxidant therapy in atherosclerosis by contrast ultrasound molecular imaging.
      ). The ability to assess therapeutic effects will not only be useful for clinical purposes, but also for the pre-clinical assessment of novel drug candidates.

      Molecular imaging of microvascular inflammation

      Inflammation is a ubiquitous process in health and disease and is critical to the body's response to infection or injury. Although there are many different components of the inflammatory response, in mammals and other vertebrates inflammatory responses can be broadly classified as being part of either the innate or adaptive immune processes. The innate immune response relies on the recruitment of specialized immune cells (leukocyte subtypes) and involves the production of cytokines and chemokines, activation of the complement system and cell–cell interactions that participate directly in leukocyte recruitment and migration. The adaptive immune response, sometimes called acquired immunity, often involves the processing of pathogen-specific, non-self molecules by antigen-presenting cells, and culminates in the subsequent production of antibodies that act through complement activation and effector T cells.
      With respect to inflammatory processes that are amenable to imaging with conventional contrast ultrasound molecular probes, endothelial activation and leukocyte adhesion to the activated endothelium are hallmarks of innate immunity and, occasionally, adaptive immunity. The canonical pathway for how leukocyte recruitment is orchestrated is illustrated in Figure 5. Leukocyte recruitment is regulated by local generation of cytokines and chemokines and the subsequent expression of ECAMs, which interact with leukocyte counterligands (
      • Springer T.A.
      Adhesion receptors of the immune system.
      ;
      • Ley K.
      Molecular mechanisms of leukocyte recruitment in the inflammatory process.
      ). Initial leukocyte capture is attributed largely to the interaction between endothelial selectins (P-selectin, E-selectin), a family of long adhesion molecules that extend beyond the endothelial glycocalyx, and their sialylated glycoproteins (e.g., PSGL-1) that are constitutively expressed on leukocytes (
      • Ley K.
      The role of selectins in inflammation and disease.
      ). The selectin bond formation and similar types of adhesive interactions, such as those mediated by fractalkines, occur even in high-shear vessels, but are transient, resulting in leukocyte rolling. Rolling allows cells to be exposed to cytokines, which produce activation of integrins, which are heterodimeric molecules that interact with ECAMs such as ICAMs and VCAM-1 for firm adhesion (
      • Springer T.A.
      Adhesion receptors of the immune system.
      ;
      • Ley K.
      Molecular mechanisms of leukocyte recruitment in the inflammatory process.
      ). An array of other molecules are involved in transmigration through either leukocyte adhesion (platelet and endothelial cell adhesion molecule 1 [PECAM-1]) or in coordinating the opening of gap junctions (junctional adhesion molecules and vascular endothelial-cadherin) (
      • Muller W.A.
      Mechanisms of leukocyte transendothelial migration.
      ).
      Fig 5
      Fig. 5Classic pathways for recruitment of inflammatory cells. Initial leukocyte recruitment and rolling are mediated largely through interaction between selectins and sialylated glycoprotein counter-receptors such as P-selectin glycoprotein ligand 1 (PSGL-1). Rolling not only permits exposure of leukocytes to pro-inflammatory cytokines, but also the formation of tight interactions between integrins (many of which require cellular activation) and endothelial cell adhesion molecules: vascular cell adhesion molecule 1 (VCAM-1), intercellular cell adhesion molecules (ICAMs), mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Adhesion allows leukocytes to follow chemokine signals that coax migration, which in part involves changes in other endothelial cell adhesion molecules (ECAMs), for example, platelet and endothelial cell adhesion molecule 1 (PECAM-1), junctional adhesion molecules (JAMs) and vascular endothelial cadherin (VE-cadherin), at cell–cell junctions.
      The wide array of adhesion molecules involved in leukocyte recruitment and the large library of ligands that have been created to detect or inhibit them have enabled many forms of CEU molecular imaging of inflammation. MBs bearing targeting ligands have been used for molecular imaging of ECAMs including selectins, VCAM-1, ICAM-1 and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) (
      • Villanueva F.S.
      • Jankowski R.J.
      • Klibanov S.
      • Pina M.L.
      • Alber S.M.
      • Watkins S.C.
      • Brandenburger G.H.
      • Wagner W.R.
      Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells.
      ;
      • Hamilton A.J.
      • Huang S.L.
      • Warnick D.
      • Rabbat M.
      • Kane B.
      • Nagaraj A.
      • Klegerman M.
      • McPherson D.D.
      Intravascular ultrasound molecular imaging of atheroma components in vivo.
      ;
      • Bachmann C.
      • Klibanov A.L.
      • Olson T.S.
      • Sonnenschein J.R.
      • Rivera-Nieves J.
      • Cominelli F.
      • Ley K.F.
      • Lindner J.R.
      • Pizarro T.T.
      Targeting mucosal addressin cellular adhesion molecule (MAdCAM)-1 to noninvasively image experimental Crohn's disease.
      ;
      • Kaufmann B.A.
      • Lewis C.
      • Xie A.
      • Mirza-Mohd A.
      • Lindner J.R.
      Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography.
      ,
      • Kaufmann B.A.
      • Sanders J.M.
      • Davis C.
      • Xie A.
      • Aldred P.
      • Sarembock I.J.
      • Lindner J.R.
      Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1.
      ;
      • Chadderdon S.M.
      • Belcik J.T.
      • Bader L.
      • Kirigiti M.A.
      • Peters D.M.
      • Kievit P.
      • Grove K.L.
      • Lindner J.R.
      Proinflammatory endothelial activation detected by molecular imaging in obese nonhuman primates coincides with onset of insulin resistance and progressively increases with duration of insulin resistance.
      ;
      • Davidson B.P.
      • Chadderdon S.M.
      • Belcik J.T.
      • Gupta S.
      • Lindner J.R.
      Ischemic memory imaging in nonhuman primates with echocardiographic molecular imaging of selectin expression.
      ). It is also important to realize that a non-canonical pathway of inflammation has been defined whereby VWF-mediated platelet adhesion to the endothelial cell surface can participate not only in endothelial activation, but directly in leukocyte adhesion (
      • Jackson S.P.
      • Darbousset R.
      • Schoenwaelder S.M.
      Thromboinflammation: Challenges of therapeutically targeting coagulation and other host defense mechanisms.
      ). Knowledge of these processes, referred to as thrombo-inflammation, has been leveraged in molecular imaging to use platelet adhesion and VWF signal as markers of endothelial inflammatory activation (
      • Shim C.Y.
      • Liu Y.N.
      • Atkinson T.
      • Xie A.
      • Foster T.
      • Davidson B.P.
      • Treible M.
      • Qi Y.
      • Lopez J.A.
      • Munday A.
      • Ruggeri Z.
      • Lindner J.R.
      Molecular Imaging of Platelet-Endothelial Interactions and Endothelial von Willebrand factor in early and mid-stage atherosclerosis.
      ;
      • Moccetti F.
      • Brown E.
      • Xie A.
      • Packwood W.
      • Qi Y.
      • Ruggeri Z.
      • Shentu W.
      • Chen J.
      • Lopez J.A.
      • Lindner J.R.
      Myocardial infarction produces sustained proinflammatory endothelial activation in remote arteries.
      ,
      • Moccetti F.
      • Weinkauf C.C.
      • Davidson B.P.
      • Belcik J.T.
      • Marinelli E.R.
      • Unger E.
      • Lindner J.R.
      Ultrasound molecular imaging of atherosclerosis using small-peptide targeting ligands against endothelial markers of inflammation and oxidative stress.
      ). Leukocyte cell surface molecules not involved in adhesion can also be targeted for inflammation imaging. This strategy can be useful for detecting specific leukocyte populations of interest that are in the process of intravascular recruitment. Examples of this strategy include the targeting of specific reparative monocyte subpopulations using CX3 CR-1 (
      • Ryu J.C.
      • Davidson B.P.
      • Xie A.
      • Qi Y.
      • Zha D.
      • Belcik J.T.
      • Caplan E.S.
      • Woda J.M.
      • Hedrick C.C.
      • Hanna R.N.
      • Lehman N.
      • Zhao Y.
      • Ting A.
      • Lindner J.R.
      Molecular imaging of the paracrine proangiogenic effects of progenitor cell therapy in limb ischemia.
      ), of granulocytes through targeting complement receptors (
      • Lindner J.R.
      Molecular imaging with contrast ultrasound and targeted microbubbles.
      ;
      • Mott B.
      • Packwood W.
      • Xie A.
      • Belcik J.T.
      • Taylor R.P.
      • Zhao Y.
      • Davidson B.P.
      • Lindner J.R.
      Echocardiographic ischemic memory imaging through complement-mediated vascular adhesion of phosphatidylserine-containing microbubbles.
      ) and of certain T-lymphocyte populations by targeting CD3 or CD4 (
      • Steinl D.C.
      • Xu L.
      • Khanicheh E.
      • Ellertsdottir E.
      • Ochoa-Espinosa A.
      • Mitterhuber M.
      • Glatz K.
      • Kuster G.M.
      • Kaufmann B.A.
      Noninvasive contrast-enhanced ultrasound molecular imaging detects myocardial inflammatory response in autoimmune myocarditis.
      ;
      • Liu J.
      • Chen Y.
      • Wang G.
      • Lv Q.
      • Yang Y.
      • Wang J.
      • Zhang P.
      • Liu J.
      • Xie Y.
      • Zhang L.
      • Xie M.
      Ultrasound molecular imaging of acute cardiac transplantation rejection using nanobubbles targeted to T lymphocytes.
      ).
      Contrast-enhanced ultrasound imaging of inflammation has been proven to be feasible in a wide array of inflammatory conditions involving almost every tissue of the body with the possible exceptions of bone and lung. Because it is impossible to cover all studies in a brief review, our comments focus on several topics where imaging of inflammation may provide unique information that may revolutionize clinical care. One of these topical areas, inflammation imaging for early detection of atherosclerotic disease, has been discussed earlier.
      The diagnosis of acute coronary syndrome (ACS) in symptomatic patients relies on clinical history, laboratory evaluation and electrocardiogram, which, except in the case of ST elevation myocardial infarction, are often non-diagnostic on initial evaluation (
      • Mehta R.H.
      • Eagle K.A.
      Missed diagnoses of acute coronary syndromes in the emergency room—Continuing challenges.
      ;
      • Brieger D.
      • Eagle K.A.
      • Goodman S.G.
      • Steg P.G.
      • Budaj A.
      • White K.
      • Montalescot G.
      Acute coronary syndromes without chest pain, an underdiagnosed and undertreated high-risk group: Insights from the Global Registry of Acute Coronary Events.
      ). To address this limitation, ultrasound molecular imaging techniques for detecting ischemia-related inflammation have been developed and may be useful for diagnosing ACS when initial tests are negative or when ischemia has resolved and not resulted in much myocardial necrosis (
      • Taegtmeyer H.
      • Dilsizian V.
      Imaging myocardial metabolism and ischemic memory.
      ). They could potentially also identify high-risk individuals based on the spatial extent of the area at risk. Molecular imaging of resolved ischemia without infarction (ischemic memory imaging) has been accomplished with myocardial contrast echocardiography in rodent and non-human primate models using MBs targeted to the selectin family of ECAMs that are rapidly expressed in response to ischemia (
      • Kaufmann B.A.
      • Lewis C.
      • Xie A.
      • Mirza-Mohd A.
      • Lindner J.R.
      Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography.
      ,
      • Kaufmann B.A.
      • Sanders J.M.
      • Davis C.
      • Xie A.
      • Aldred P.
      • Sarembock I.J.
      • Lindner J.R.
      Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1.
      ,
      • Kaufmann B.A.
      • Wei K.
      • Lindner J.R.
      Contrast echocardiography.
      ;
      • Villanueva F.S.
      • Lu E.
      • Bowry S.
      • Kilic S.
      • Tom E.
      • Wang J.
      • Gretton J.
      • Pacella J.J.
      • Wagner W.R.
      Myocardial ischemic memory imaging with molecular echocardiography.
      ;
      • Davidson B.P.
      • Kaufmann B.A.
      • Belcik J.T.
      • Xie A.
      • Qi Y.
      • Lindner J.R.
      Detection of antecedent myocardial ischemia with multiselectin molecular imaging.
      ,
      • Davidson B.P.
      • Chadderdon S.M.
      • Belcik J.T.
      • Gupta S.
      • Lindner J.R.
      Ischemic memory imaging in nonhuman primates with echocardiographic molecular imaging of selectin expression.
      ). These studies relied on the use of a variety of ligands against selectins including monoclonal antibodies, recombinant forms of the endogenous selectin ligand PSGL-1 or the carbohydrate moieties (sialyl Lewis-x) that are on PSGL-1. The data indicate an accurate depiction of the recently ischemic region for more than 6 h after resolution. More recently, it was discovered that ischemic memory is possible simply using MBs that contain phosphatidylserine in their shell (
      • Mott B.
      • Packwood W.
      • Xie A.
      • Belcik J.T.
      • Taylor R.P.
      • Zhao Y.
      • Davidson B.P.
      • Lindner J.R.
      Echocardiographic ischemic memory imaging through complement-mediated vascular adhesion of phosphatidylserine-containing microbubbles.
      ). This approach, which was as effective as selectin targeting, relies on the amplification of complement-mediated adhesion to leukocytes and the activated endothelium (
      • Lindner J.R.
      • Coggins M.P.
      • Kaul S.
      • Klibanov A.L.
      • Brandenburger G.H.
      • Ley K.
      Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes.
      ,
      • Lindner J.R.
      • Dayton P.A.
      • Coggins M.P.
      • Ley K.
      • Song J.
      • Ferrara K.
      • Kaul S.
      Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles.
      ,
      • Lindner J.R.
      • Song J.
      • Xu F.
      • Klibanov A.L.
      • Singbartl K.
      • Ley K.
      • Kaul S.
      Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes.
      ;
      • Christiansen J.P.
      • Leong-Poi H.
      • Klibanov A.L.
      • Kaul S.
      • Lindner J.R.
      Noninvasive imaging of myocardial reperfusion injury using leukocyte-targeted contrast echocardiography.
      ). A clinical phase 1 dose-ranging trial (ClinicalTrials.gov identifier: NCT03009266) using commercially available MBs containing phosphatidylserine (Sonazoid) is currently recruiting normal controls.
      There is no more precious resource in the practice of medicine than a transplanted allograft. Transplant networks have been designed to ensure that a structured approach is taken that balances the urgency of need for each individual patient and the adequacy immunologic match to maximize the survival of the transplanted tissue and, in certain cases, minimize risk of graft-versus-host disease. Once a transplant has been performed, the ability to monitor the innate and adaptive immune processes that compromise graft health is critical. Biopsy still remains the cornerstone for assessment for rejection. Non-invasive methods for accurately detecting rejection or associated processes such as transplant vasculopathy would be valuable for early detection and treatment and for assessing the response to immunosuppressive therapies that often need to be adjusted based on inflammatory status. Contrast ultrasound molecular imaging of host responses to cardiac and renal allografts have been in rodent models of allograft rejection. Approaches have included the use of MBs or nano-scale UCAs targeted to endothelial ICAM-1 (
      • Weller G.E.
      • Lu E.
      • Csikari M.M.
      • Klibanov A.L.
      • Fischer D.
      • Wagner W.R.
      • Villanueva F.S.
      Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1.
      ) or to T-cell antigens such as CD4, CD3 and CD25 (
      • Wu H.
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      • Sanders C.
      • Kamiyama N.
      • Exner A.A.
      Acoustic characterization and pharmacokinetic analyses of new nanobubble ultrasound contrast agents.
      ,
      • Wu W.
      • Zhang Z.
      • Zhuo L.
      • Zhou L.
      • Liu P.
      • He Y.
      • Gao Y.
      • Li R.
      • Chen Q.
      • Hua X.
      Ultrasound molecular imaging of acute cellular cardiac allograft rejection in rat with T-cell-specific nanobubbles.
      ;
      • Liu J.
      • Chen Y.
      • Wang G.
      • Lv Q.
      • Yang Y.
      • Wang J.
      • Zhang P.
      • Liu J.
      • Xie Y.
      • Zhang L.
      • Xie M.
      Ultrasound molecular imaging of acute cardiac transplantation rejection using nanobubbles targeted to T lymphocytes.
      ;
      • Liao T.
      • Zhang Y.
      • Ren J.
      • Zheng H.
      • Zhang H.
      • Li X.
      • Liu X.
      • Yin T.
      • Sun Q.
      Noninvasive quantification of intrarenal allograft C4d deposition with targeted ultrasound imaging.
      ;
      • Xie Y.
      • Chen Y.
      • Zhang L.
      • Wu M.
      • Deng Z.
      • Yang Y.
      • Wang J.
      • Lv Q.
      • Zheng H.
      • Xie M.
      • Yan F.
      Ultrasound molecular imaging of lymphocyte-endothelium adhesion cascade in acute cellular rejection of cardiac allografts.
      ). Studies have also suggested that non-specific interactions between non-targeted lipid MBs with an anionic charge and leukocytes can also be used to detect rejection (
      • Kondo I.
      • Ohmori K.
      • Oshita A.
      • Takeuchi H.
      • Yoshida J.
      • Shinomiya K.
      • Fuke S.
      • Suzuki T.
      • Mizushige K.
      • Kohno M.
      Leukocyte-targeted myocardial contrast echocardiography can assess the degree of acute allograft rejection in a rat cardiac transplantation model.
      ).
      Inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis are debilitating diseases that affect several million people worldwide and lead to severe symptoms and chronic morbidity. The disease is characterized by periodic cycles of inflammatory disruption of gastrointestinal function and even bowel necrosis. A major challenge of the disease is that patient symptoms often lag behind the beginning of IBD activity flares. Methods for monitoring disease activity could lead to earlier treatment of disease flares with immunosuppressive therapies. The first description of IBD activity molecular imaging was made in a mouse model of IBD (SAMP1 YitFC) using MBs targeted against MAdCAM-1 (mucosal vascular addressin cell adhesion molecule 1), an ECAM involved in gut lymphocyte adhesion and trafficking (
      • Bachmann C.
      • Klibanov A.L.
      • Olson T.S.
      • Sonnenschein J.R.
      • Rivera-Nieves J.
      • Cominelli F.
      • Ley K.F.
      • Lindner J.R.
      • Pizarro T.T.
      Targeting mucosal addressin cellular adhesion molecule (MAdCAM)-1 to noninvasively image experimental Crohn's disease.
      ). More recently, selectin-targeted agents have been used in mice with chemically induced IBD (
      • Wang H.
      • Machtaler S.
      • Bettinger T.
      • Lutz A.M.
      • Luong R.
      • Bussat P.
      • Gambhir S.S.
      • Tranquart F.
      • Tian L.
      • Willmann J.K.
      Molecular imaging of inflammation in inflammatory bowel disease with a clinically translatable dual-selectin-targeted US contrast agent: Comparison with FDG PET/CT in a mouse model.
      ).

      Molecular imaging in oncology

      Ultrasound is a suitable diagnostic technology for use in primary care and is widely accepted in the clinical setting; however, it suffers from low specificity (
      • Deshpande N.
      • Needles A.
      • Willmann J.K.
      Molecular ultrasound imaging: current status and future directions.
      ,
      • Deshpande N.
      • Pysz M.A.
      • Willmann J.K.
      Molecular ultrasound assessment of tumor angiogenesis.
      ;
      • Wilson S.R.
      • Burns P.N.
      Microbubble-enhanced US in body imaging: What role?.
      ;
      • Pysz M.A.
      • Willmann J.K.
      Targeted contrast-enhanced ultrasound: An emerging technology in abdominal and pelvic imaging.
      ). Improvement of the diagnostic accuracy of ultrasound imaging is critical. Ultrasound molecular imaging is an emerging imaging modality that can substantially increase diagnostic accuracy, and has been reported to play a major role in the field of cancer imaging (
      • Deshpande N.
      • Needles A.
      • Willmann J.K.
      Molecular ultrasound imaging: current status and future directions.
      ,
      • Deshpande N.
      • Pysz M.A.
      • Willmann J.K.
      Molecular ultrasound assessment of tumor angiogenesis.
      ). The introduction of molecularly targeted MBs and the evolving molecular imaging technology is a promising new modality for oncological imaging and may pave the way for earlier cancer detection, molecular profiling of cancer, improved characterization of focal lesions and treatment monitoring (
      • Pysz M.A.
      • Willmann J.K.
      Targeted contrast-enhanced ultrasound: An emerging technology in abdominal and pelvic imaging.
      ;
      • Kiessling F.
      • Fokong S.
      • Bzyl J.
      • Lederle W.
      • Palmowski M.
      • Lammers T.
      Recent advances in molecular, multimodal and theranostic ultrasound imaging.
      ). In cancer, the formation and recruitment of new blood vessels from the host surrounding tissue, also known as neo-angiogenesis, is one of the foremost hallmarks of early tumor development (
      • Deshpande N.
      • Needles A.
      • Willmann J.K.
      Molecular ultrasound imaging: current status and future directions.
      ,
      • Deshpande N.
      • Pysz M.A.
      • Willmann J.K.
      Molecular ultrasound assessment of tumor angiogenesis.
      ;
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: The next generation.
      ). Many tumors depend on an adequate supply of oxygen and nutrients, and rely on increased neovascularization once they grow beyond 1–2 mm in size (
      • Folkman J.
      • Long Jr, D.M.
      • Becker F.F.
      Growth and metastasis of tumor in organ culture.
      ;
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: The next generation.
      ). In the last decade, with the deeper understanding of cancer biology and the discovery and validation of new receptors differentially expressed on the cancer neovasculature, several biomarkers have been studied for tumor detection at early stages (
      • Pysz M.A.
      • Willmann J.K.
      Targeted contrast-enhanced ultrasound: An emerging technology in abdominal and pelvic imaging.
      ). Ultrasound molecular imaging with molecularly targeted contrast agents enabled the visualization of disease progression at the molecular level. Among those receptors expressed on the neovasculature of various cancers are vascular endothelial growth factor receptor type 2 (VEGFR2), ⍺vβ3 integrin, endoglin (CD105) and prostate-specific membrane antigen, to mention a few (
      • Deshpande N.
      • Ren Y.
      • Foygel K.
      • Rosenberg J.
      • Willmann J.K.
      Tumor angiogenic marker expression levels during tumor growth: Longitudinal assessment with molecularly targeted microbubbles and US imaging.
      ;
      • Bachawal S.V.
      • Jensen K.C.
      • Lutz A.M.
      • Gambhir S.S.
      • Tranquart F.
      • Tian L.
      • Willmann J.K.
      Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model.
      ). Several groups have found that molecularly targeted MBs against the vascular marker VEGFR2, αvβ3 integrin or endoglin could be used successfully to detect neo-angiogenesis with high sensitivity. Recently,
      • Bachawal S.V.
      • Jensen K.C.
      • Lutz A.M.
      • Gambhir S.S.
      • Tranquart F.
      • Tian L.
      • Willmann J.K.
      Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model.
      reported that ultrasound molecular imaging signal significantly increased when normal mammary tissue transformed from early precursor lesions to invasive breast cancer using a clinical-grade VEGFR2-targeted MB, also called BR55 (Bracco, Italy), in a transgenic mouse model of breast cancer development (FVB/N-Tg(MMTVPyMT)634 Mul) (
      • Bachawal S.V.
      • Jensen K.C.
      • Lutz A.M.
      • Gambhir S.S.
      • Tranquart F.
      • Tian L.
      • Willmann J.K.
      Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model.
      ). BR55, the first clinical-grade molecular ultrasound contrast agent, consists of a gas core (mixture of perfluorobutane and nitrogen), surrounded by a phospholipid shell with a mean diameter of 1.5 µm. The heterodimeric peptide-binding ligand is targeted at the kinase insert domain receptor (the human analogue of VEGFR2) (
      • Pochon S.
      • Tardy I.
      • Bussat P.
      • Bettinger T.
      • Brochot J.
      • von Wronski M.
      • Passantino L.
      • Schneider M.
      BR55: A lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.
      ). Numerous pre-clinical trials have revealed that after intravenous injection, BR55 MBs are rapidly cleared from the circulation primarily through the reticuloendothelial system (
      • Walday P.
      • Tolleshaug H.
      • Gjoen T.
      • Kindberg G.M.
      • Berg T.
      • Skotland T.
      • Holtz E.
      Biodistributions of air-filled albumin microspheres in rats and pigs.
      ;
      • Willmann J.K.
      • Cheng Z.
      • Davis C.
      • Lutz A.M.
      • Schipper M.L.
      • Nielsen C.H.
      • Gambhir S.S.
      Targeted microbubbles for imaging tumor angiogenesis: Assessment of whole-body biodistribution with dynamic micro-PET in mice.
      ). Remarkably, the rapid clearance allows for repetitive injections of molecularly targeted MBs with analysis of several targets within the same imaging session. Similarly,
      • Pysz M.A.
      • Machtaler S.B.
      • Seeley E.S.
      • Lee J.J.
      • Brentnall T.A.
      • Rosenberg J.
      • Tranquart F.
      • Willmann J.K.
      Vascular endothelial growth factor receptor type 2-targeted contrast-enhanced US of pancreatic cancer neovasculature in a genetically engineered mouse model: potential for earlier detection.
      also reported that ultrasound molecular imaging with BR55 can visualize up to submillimeter-sized foci of pancreatic ductal adenocarcinoma (PDAC) in a transgenic mouse model (Pdx1-Cre, KRasG12-D, Ink4 a−/−). Both proof-of-concept studies highlight the power of ultrasonic molecular imaging for detecting early breast and pancreatic cancer. Various types of cancer typically produce no symptoms when the tumor is small and relatively easy to treat; therefore, patient survival and successful treatment depend on early cancer screenings in populations at risk. However, early cancer detection with high specificity and sensitivity still remains one of the most desired goals of oncological imaging. Hence, in addition to the more general neo-angiogenesis markers such as VEGFR2, cancer-specific vascular molecular markers differentially expressed on the neovasculature of various cancer types are crucially needed. Advances in the field of cancer-specific vascular target discovery and CEU imaging revealed that molecular targeting of novel designed molecularly targeted MB contrast agents may further improve the diagnostic accuracy of ultrasound imaging in early cancer detection (
      • Pysz M.A.
      • Willmann J.K.
      Targeted contrast-enhanced ultrasound: An emerging technology in abdominal and pelvic imaging.
      ). Recently, several vascular markers have been discovered through proteomic analysis and validated with immunohistochemical staining in human tissues. Thymocyte differentiating antigen 1 (Thy1) and B7-H3 (CD276, a member of B7 family of immunomodulators) are two such novel markers that have recently been identified and validated as specific, differentially expressed vascular targets for ultrasound molecular imaging of pancreatic, ovarian and breast cancer (
      • Foygel K.
      • Wang H.
      • Machtaler S.
      • Lutz A.M.
      • Chen R.
      • Pysz M.
      • Lowe A.W.
      • Tian L.
      • Carrigan T.
      • Brentnall T.A.
      • Willmann J.K.
      Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1.
      ;
      • Lutz A.M.
      • Bachawal S.V.
      • Drescher C.W.
      • Pysz M.A.
      • Willmann J.K.
      • Gambhir S.S.
      Ultrasound molecular imaging in a human CD276 expression-modulated murine ovarian cancer model.
      ;
      • Bachawal S.V.
      • Jensen K.C.
      • Wilson K.E.
      • Tian L.
      • Lutz A.M.
      • Willmann J.K.
      Breast cancer detection by B7-H3-targeted ultrasound molecular imaging.
      ). Our group has validated a novel neovascular molecular marker of breast cancer, B7-H3, which has recently been found to be differentially expressed with very high accuracy (up to area under the curve = 0.96) in all four major subtypes of human breast cancer compared with a broad spectrum of benign breast lesions. In a pilot experiment in a transgenic mouse model of breast cancer, we found that the ultrasound molecular imaging signal using B7-H3-targeted MBs was substantially higher in breast cancer (Fig. 6a) than in normal breast tissue (
      • Bachawal S.V.
      • Jensen K.C.
      • Wilson K.E.
      • Tian L.
      • Lutz A.M.
      • Willmann J.K.
      Breast cancer detection by B7-H3-targeted ultrasound molecular imaging.
      ). In addition, our group has identified and validated Thy1 as a novel, highly specific neo-angiogenesis marker in patients with PDAC (
      • Foygel K.
      • Wang H.
      • Machtaler S.
      • Lutz A.M.
      • Chen R.
      • Pysz M.
      • Lowe A.W.
      • Tian L.
      • Carrigan T.
      • Brentnall T.A.
      • Willmann J.K.
      Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1.
      ). This has been confirmed by a subsequent independent study that reported significant overexpression of Thy1 in patients with PDAC compared with those with a normal pancreas and chronic pancreatitis (
      • Zhu J.
      • Thakolwiboon S.
      • Liu X.
      • Zhang M.
      • Lubman D.M.
      Overexpression of CD90 (Thy-1) in pancreatic adenocarcinoma present in the tumor microenvironment.
      ;
      • Pei X.
      • Zhu J.
      • Yang R.
      • Tan Z.
      • An M.
      • Shi J.
      • Lubman D.M.
      CD90 and CD24 co-expression is associated with pancreatic intraepithelial neoplasias.
      ).
      • Abou-Elkacem L.
      • Wang H.
      • Chowdhury S.M.
      • Kimura R.H.
      • Bachawal S.V.
      • Gambhir S.S.
      • Tian L.
      • Willmann J.K.
      Thy1-targeted microbubbles for ultrasound molecular imaging of pancreatic ductal adenocarcinoma.
      also used a clinically translatable dual human and murine Thy1-binding single-chain variable fragment (scFv) conjugated to MBs to illustrate the feasibility of in vivo Thy1-targeted ultrasound molecular imaging of PDAC in two animal models, a human PDAC xenograft and a transgenic mouse model (Fig. 6b).
      Fig 6
      Fig. 6Examples of pre-clinical and clinical ultrasound molecular imaging of targets relevant in oncology. (a) Ultrasound molecular imaging of B7-H3 in normal breast tissue (left panels) versus breast cancer tissue; top panels indicate region of interest, and bottom panels, color-coded molecular imaging signals. (b) Molecular imaging of tumor neo-angiogenesis using Thy-1 as a target in pancreatic ductal adenocarcinoma. Left panels represent transgenic mouse model; right panels, human xenograft model; top panels, targeted signals; and bottom panels, control signals. (c, d) Examples of imaging studies using BR-55 for targeting of vascular endothelial growth receptor type 2 (VEGFR2) expression in ovarian cancer (c) and breast cancer (d). Yellow arrows denote strong stationary contrast signal within tumor tissue. Blue arrows denote specular reflection artifacts within tissue.
      In pre-clinical research, ultrasound molecular imaging with molecularly targeted MBs has successfully been applied to investigate early neo-angiogenesis in cancer. However, in almost all pre-clinical studies, non-translatable ligands are coupled to MBs via (strept)avidin–biotin, which will not be translatable into humans (
      • Marshall D.
      • Pedley R.B.
      • Boden J.A.
      • Boden R.
      • Melton R.G.
      • Begent R.H.
      Polyethylene glycol modification of a galactosylated streptavidin clearing agent: Effects on immunogenicity and clearance of a biotinylated anti-tumour antibody.
      ).
      In the recent past, however, significant strides have been made toward developing a clinical-grade MB suitable for molecular imaging of cancer. As mentioned earlier, the first formulation that has made good progress toward clinical use is BR55, a lipoprotein-based VEGFR-2-targeted ultrasound contrast agent developed by Bracco Diagnostics (
      • Pochon S.
      • Tardy I.
      • Bussat P.
      • Bettinger T.
      • Brochot J.
      • von Wronski M.
      • Passantino L.
      • Schneider M.
      BR55: A lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.
      ), which has gone into clinical trials in both the United States and Europe after demonstrating high efficacy and a good tolerability profile in multiple pre-clinical studies (
      • Pysz M.A.
      • Foygel K.
      • Rosenberg J.
      • Gambhir S.S.
      • Schneider M.
      • Willmann J.K.
      Antiangiogenic cancer therapy: Monitoring with molecular US and a clinically translatable contrast agent (BR55).
      ;
      • Bachawal S.V.
      • Jensen K.C.
      • Lutz A.M.
      • Gambhir S.S.
      • Tranquart F.
      • Tian L.
      • Willmann J.K.
      Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model.
      ). Notably, initial results from separate phase 0 and I clinical trials for BR55 have already underscored the potential of this contrast agent in cancer diagnostics.
      In a phase 0 exploratory study, (

      Smeenge M, Tranquart F, Mannaerts CK, de Reijke TM, van de Vijver MJ, Laguna MP, Pochon S, de la Rosette JJMCH, Wijkstra H. Invest Radiol. 2017;52:419–427.

      ) (University Hospital Amsterdam) evaluated the tolerability and efficacy of BR55 in the detection of prostate cancer (ClinicalTrials.gov identifier: NCT01253213). Twenty-four patients with biopsy-proven pancreatic cancer scheduled for radical prostatectomy were intravenously injected with BR55 (0.03–0.05 mL/kg weight), and ultrasound molecular imaging was performed (ClinicalTrials.gov identifier: NCT02142608). The first 12 patients were used to fine-tune the dosing and imaging protocol, while the next 12 patients were imaged with the improved protocol. The results indicated that at the doses used, BR55 did not produce any significant adverse effects in patients. Furthermore, BR55-aided ultrasound molecular imaging could successfully identify 68% of malignant lesions, whereas 32 % of malignant lesions could not be identified (malignant lesions were confirmed post-surgery from the resected prostate). This was the first study to find that BR55 can indeed bind to VEGFR-2 in humans to produce contrast enhancement and aid cancer detection. Overall, these results validated the tolerability profile of BR55 in humans while also highlighting the future potential for this contrast agent in the detection of prostate cancer. In a similar study from Stanford University, clinical trials exploring the efficacy of BR55-enhanced volumetric ultrasound molecular imaging in detecting prostate cancer before radical prostatectomy have recently been completed. Results from this study will shed further light on how BR55 can be utilized in the early detection of prostate cancer.
      A recently published clinical trial from Stanford University has explored the tolerability of BR55 in humans while also assessing the efficacy of this contrast agent in detecting VEGFR2 expression (immunohistochemistry used as gold standard) in ovarian and breast cancer (
      • Willmann J.K.
      • Bonomo L.
      • Carla Testa A.
      • Rinaldi P.
      • Rindi G.
      • Valluru K.S.
      • Petrone G.
      • Martini M.
      • Lutz A.M.
      • Gambhir S.S.
      Ultrasound molecular imaging with BR55 in patients with breast and ovarian lesions: First-in-human results.
      ). In the study, 24 women with ovarian cancer (aged 48–79 y) and 21 women with breast cancer (aged 34–66 y) were intravenously injected with BR55 (0.03–0.08 mL/kg weight) and ultrasound molecular imaging was performed starting from 5 min post-injection to up to 29 min. Results revealed evidence of high VEGFR2 expression (high targeted signal) in 77% of malignant ovarian lesions (Fig. 6c) and 93% of malignant breast lesions (Fig. 6d). In comparison, 78% of benign ovarian lesions and 67% of benign breast lesions manifested no targeted signal. Overall, 85% of ovarian lesions and 93% of breast lesions had good correlation with VEGFR2 expression obtained from histology of resected tumor tissue. This study was instrumental in highlighting BR55 as a prime candidate for use in detection and staging of breast and ovarian cancer. As a continuation of this study, a phase 2 trial further exploring the potential role of BR55 in ovarian cancer detection is currently underway at Stanford University (
      • Willmann J.K.
      • Bonomo L.
      • Carla Testa A.
      • Rinaldi P.
      • Rindi G.
      • Valluru K.S.
      • Petrone G.
      • Martini M.
      • Lutz A.M.
      • Gambhir S.S.
      Ultrasound molecular imaging with BR55 in patients with breast and ovarian lesions: First-in-human results.
      ). In addition to these published studies, a trial assessing the potential role of BR55 in the detection and staging of pancreatic cancer is also currently being completed at Stanford University. This study specifically explores the potential use of BR55 along with transabdominal ultrasound imaging toward the detection of pancreatic ductal adenocarcinoma.

      Future perspectives

      The feasibility of ultrasound molecular imaging studies has been proven in a large number of pre-clinical studies and in a vast array of disease models. As a consequence, ultrasound molecular imaging is already being used in pre-clinical research for in vivo assessment of pathophysiology and thus contributes to advances in our understanding of disease mechanisms. Another area where ultrasound molecular imaging is already being applied is pre-clinical testing of the effect of novel drugs. This area can be expected to expand if better standardization of the targeted contrast agents and ultrasound imaging protocols is achieved. As described in the preceding section of this review, first clinical trials have not raised any tolerability concerns and have shown potential for tumor diagnosis and phenotypic characterization. The ability to better characterize a particular disease phenotype will become crucial as an increasing number of drugs with specific molecular targets are developed. However, widespread use of ultrasound molecular imaging in the clinical field will depend on additional, extensive tolerability testing and larger clinical trials demonstrating incremental diagnostic value over existing techniques. Also, integration of the additional information gained from molecular imaging will need to be integrated into diagnostic and therapeutic pathways, and the impact on patient prognosis will have to be assessed.

      Acknowledgments

      This work was supported by grants from the Swiss National Science Foundation ( SNF 310030_169905 ), the Swiss Heart Foundation and the Kardiovaskuläre Stiftung Basel to Dr. Kaufmann, and Grants R01-HL078610 , R01-HL130046 , and P51-OD011092 from the National Institutes of Health (NIH) to Dr. Lindner.

      Conflict of interest disclosure

      The authors declare no competing interests.

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