Contrast-enhanced Ultrasound Molecular Imaging Research Articles

Nanobodies as Diagnostic and Therapeutic Tools for Cardiovascular Diseases (CVDs).

The aim of this review is to summarize some of the most recent work in the field of cardiovascular disease (CVD) diagnosis and therapy, focusing mainly on the role of nanobodies in the development of non-invasive imaging methods, diagnostic devices, and advanced biotechnological therapy tools. In the context of the increased number of people suffering from CVDs due to a variety of factors such as sedentariness, poor nutrition, stress, and smoking, there is an urgent need for new and improved diagnostic and therapeutic methods. Nanobodies can be easily produced in prokaryotes, lower eukaryotes, and plant and mammalian cells, and offer great advantages. In the diagnosis domain, they are mainly used as labeled probes that bind to certain surface receptors or other target molecules and give important information on the severity and extent of atherosclerotic lesions, using imaging methods such as contrast-enhanced ultrasound molecular imaging (CEUMI), positron emission tomography (PET), single-photon emission computed tomography coupled with computed tomography (SPECT/CT), and PET/CT. As therapy tools, nanobodies have been used either for transporting drug-loaded vesicles to specific targets or as inhibitors for certain enzymes and receptors, demonstrated to be involved in various CVDs.

Liraglutide Lowers Endothelial Vascular Cell Adhesion Molecule-1 in Murine Atherosclerosis Independent of Glucose Levels

The authors determined the effect of the GLP-1 receptor agonist liraglutide on endothelial surface expression of vascular cell adhesion molecule (VCAM)-1 in murine apolipoprotein E knockout atherosclerosis. Contrast-enhanced ultrasound molecular imaging using microbubbles targeted to VCAM-1 and control microbubbles showed a 3-fold increase in endothelial surface VCAM-1 signal in vehicle-treated animals, whereas in the liraglutide-treated animals the signal ratio remained around 1 throughout the study. Liraglutide had no influence on low-density lipoprotein cholesterol or glycated hemoglobin, but reduced TNF-α, IL-1β, MCP-1, and OPN. Aortic plaque lesion area and luminal VCAM-1 expression on immunohistology were reduced under liraglutide treatment.

FN3 linked nanobubbles as a targeted contrast agent for US imaging of cancer-associated human PD-L1.

PD-L1 (programmed death-ligand 1) targeted therapies may be useful for several cancers. The use of non-invasive diagnostic and prognostic molecular imaging platforms could improve clinical assessment of PD-L1 tumor status during these therapies. Contrast enhanced ultrasound molecular imaging (CE-USMI) techniques may offer versatile and cost-effective ways to detect and quantify the expression levels of cellular targets in vivo. However, conventional use of microbubbles as a blood pool contrast agent for CE-USMI is limited to accessing intravascular biomarkers rather than reflecting the tumor molecular status. Using a microfluidic based reconstruction process we therefore developed ultra-stable nanobubbles (NBs) as a contrast agent for molecular imaging of vascular and extravascular cell surface markers. We then functionalized these NBs by covalently linking to nanobody (FN3hPD-L1) targeting human (h)PD-L1 to measure the expression of human PD-L1 in the tumor microenvironment (TME) in vivo. We showed the specific binding of hPD-L1 targeted NBs in cell culture, and in xenografted mouse models of hPD-L1 expressing CT26 tumors. CE-USMI of hPD-L1 in the TME in vivo showed ~3-fold increase in contrast signal compared to non-targeted NBs. Overall, in vivo use of CE-USMI with hPD-L1 targeted NBs has the potential for clinical translation and imaging of human cancers during immunotherapy, and for prognostic evaluation of patient response to PD-L1 targeted immunotherapy.

Contrast-Enhanced Ultrasound Molecular Imaging in Atherosclerosis Research.

The management of cardiovascular conditions will likely be improved by noninvasive in vivo molecular imaging technologies that can provide earlier or more accurate diagnosis. These techniques are already having a positive impact in preclinical research by providing insight into disease pathobiology or efficacy of new therapies. Contrast enhanced ultrasound (CEU) molecular imaging is a technique that relies on the ultrasound detection of targeted microbubble contrast agents to examine molecular or cellular events that occur at the blood pool-endothelial interface. For the most part, targeted contrast agents are composed of encapsulated gas microbubbles (MBs) that are 2-4μm in diameter, or other acoustically active micro- or nanoparticles. These agents bear several tens of thousands of binding molecules per particle. Because nonadhered agent is cleared rapidly, CEU molecular imaging can be performed in a matter of minutes. MBs are detected using contrast-specific techniques that generate and receive nonlinear signals produced by MB cavitation, thereby increasing signal-to-noise ratio. Dedicated kinetic models for molecular imaging have been generated that permit the elimination of signal from nonadherent agent.

Contrast-enhanced ultrasound molecular imaging of activated platelets in the progression of atherosclerosis using microbubbles bearing the von Willebrand factor A1 domain.

Platelet-endothelial interactions have been linked to increased inflammatory activation and a prothrombotic state in atherosclerosis. The interaction between von Willebrand factor (vWF)-A1 domain and platelet glycoprotein (GP) Ib/IX plays a significant role in mediating the adhesion of platelets to the injured endothelium. In the present study, contrast-enhanced ultrasound (CEU) molecular imaging with microbubbles bearing the vWF-A1 domain was performed to non-invasively monitor activated platelets on the vascular endothelium in the procession of atherosclerosis. A targeted CEU contrast agent was prepared by attaching the vWF-A1 domain to the shell of microbubbles (MbA1). Rat isotype control antibody was used to produce control (Mbctrl) microbubbles. The binding of MbA1 and Mbctrl to activated platelets was assessed in in vitro flow chamber experiments. Apolipoprotein E (ApoE-/-) deficient mice were studied as a model of atherosclerosis. At 8, 16 and 32 weeks of age, CEU molecular imaging of the proximal aorta with MbA1 and Mbctrl was performed and the imaging signals from microbubbles were quantified. Atherosclerotic lesion severity and platelets on the endothelial surface were assessed by histology and immunohistochemistry. In in vitro flow chamber studies, attachment of MbA1 to activated platelets on culture dishes was significantly greater than that of Mbctrl across a range of shear stresses (P<0.05). The attachment of Mbctrl was sparse and not related to the aggregated platelets. As lesion development progressed in the ApoE-/- mice, molecular imaging of activated platelets demonstrated selective signal enhancement of MbA1 (P<0.05 vs. Mbctrl) at all ages. Selective signal enhancement from MbA1 increased from 8 to 32 weeks of age. Immunohistochemistry for GPIIb revealed the presence of platelets on the endothelial cell surface in each group of ApoE-/- mice and that the degree of platelet deposits was age-dependent. The results of the present study indicated that non-invasive CEU molecular imaging with targeted microbubbles bearing the vWF-A1 domain could not only detect activated platelets on the vascular endothelium but also indicate lesion severity in atherosclerosis.

Therapeutic efficacy and cardioprotection of nucleolin-targeted doxorubicin-loaded ultrasound nanobubbles in treating triple-negative breast cancer

Targeted lipid nanobubbles as theranostic ultrasound molecular probes with both targeted contrast-enhanced ultrasound molecular imaging and synergistic treatment capabilities are expected to overcome severe challenges in the diagnosis and treatment of refractory triple-negative breast cancer (TNBC). In this study, AS1411 aptamer-functionalised nucleolin-targeted doxorubicin-loaded lipid nanobubbles (AS1411-DOX-NBs) were constructed, and their physicochemical properties as well as anti-tumour and cardioprotective efficacies were systematically tested and evaluated. The results showed that AS1411-DOX-NBs can carry and maintain the physicochemical and pharmacodynamic properties of doxorubicin (DOX) and show stronger tumour cell-killing ability in vitro by increasing the active uptake of drugs. AS1411-DOX-NBs also significantly inhibited the growth of TNBC xenografts while maintaining the weight and health of the mice. Echocardiography and pathological examination further confirmed that AS1411-DOX-NBs effectively caused tumour tissue apoptosis and necrosis while reducing DOX-induced cardiotoxicity. The AS1411-DOX-NBs constructed in this study enable both targeted contrast-enhanced ultrasound molecular imaging and synergistic therapeutic efficacy and can be used as safe and efficient theranostic ultrasound molecular probes for the diagnosis and treatment of TNBC.

Construction of Nucleolin-Targeted Lipid Nanobubbles and Contrast-Enhanced Ultrasound Molecular Imaging in Triple-Negative Breast Cancer.

To construct aptamer AS1411-functionalized targeted lipid nanobubbles that could simultaneously target abnormally highly expressed nucleolin (NCL) on tumor tissue and neovasculature. Additionally, the study of their contrast-enhanced ultrasound molecular imaging capabilities in vitro and in vivo to explore new methods and approaches for the early and accurate diagnosis of triple-negative breast cancer (TNBC). First, the targeted lipid-nucleic acid molecules were constructed by an amide reaction. Then, the targeted lipid nanobubbles (AS1411-NBs) and nontargeted lipid nanobubbles (NBs) were prepared by membrane hydration, mechanical vibration and centrifugal floatation. The physicochemical characteristics and contrast-enhanced ultrasound imaging capabilities of AS1411-NBs and NBs were compared and analyzed in vitro and in vivo. There were no significant differences between the AS1411-NBs and NBs in their concentration, average particle size or ultrasound imaging capabilities in vitro (P > 0.05). However, AS1411-NBs could simultaneously target NCL in tumor tissue and neovasculature to effectively prolong the duration of contrast-enhanced ultrasound imaging compared to NBs in vivo. The area under the time-intensity curve was significantly different between AS1411-NBs and NBs (P < 0.001), and the drug loading capacity of the AS1411-NBs was also significantly higher than that of the NBs (P < 0.05). Aptamer AS1411-functionalized targeted lipid nanobubbles could significantly prolong the duration of contrast-enhanced ultrasound imaging to achieve dual-targeted ultrasound molecular imaging of tumor tissue and neovasculature. AS1411-NBs also have higher drug loading and targeted drug delivery capabilities compared with NBs, which can provide new methods and approaches for the early accurate diagnosis and effective treatment of TNBC.

418 Ultrasound molecular imaging of the role of von willebrand factor-mediated platelet adhesion in atherogenesis

Abstract Funding Acknowledgements JSPS Overseas Research Fellowship Background Platelets are known to be both pro-inflammatory and pro-mitogenic. However, the role of platelet-endothelial interactions in the initiation and growth of atherosclerotic lesions is not well understood. Purpose We used contrast-enhanced ultrasound (CEU) molecular imaging of the arterial endothelium to test the hypothesis that platelet attachment to endothelial Von Willebrand Factor (VWF) promotes atherogenesis. Methods We studied wild-type mice (WT), low-density lipoprotein deficient mice fed western diet to produce atherosclerosis (LDLR-/-), and LDLR-/- mice also deficient for ADAMTS-13 (LDLR-/-ADAMTS13-/-) which is the enzyme responsible for proteolytic cleavage of endothelial-associated VWF. Mice were studied at 20 weeks and 30 weeks of age. A subset of LDLR-/- mice were treated with recombinant ADAMTS13 1 hr prior to study. Proximal aortic CEU molecular imaging of P-selectin, vascular cell adhesion molecule (VCAM)-1, von Willebrand factor (VWF), and platelet GPIbα was performed. Aortic distensibility was assessed using high-frequency (30 MHz) transthoracic echocardiography and tail cuff blood pressure systems. NF-κB of aorta was assessed by ELISA kit. Plaque size and composition were assessed by histology. Platelets and macrophage immunohistochemistry were also performed on confocal microscopy. Results Aortic molecular imaging signal for P-selectin, VCAM-1, VWF, and platelet adhesion was significantly higher in LDLR-/- than WT mice, and increased by 2-fold between 20 and 30 wks of age. Signal for VWF and platelet adhesion was abolished 1 h after administration of ADAMTS13, confirming that platelet adhesion was VWF-mediated. At 20 and 30 wks of age, molecular imaging signal for all targets was 2-fold higher (p &amp;lt; 0.01) in LDLR-/-ADAMTS13-/- versus LDLR-/- mice. The LDLR-/-ADAMTS13-/- mice also had lower aortic distensibility (p &amp;lt; 0.05), had a 2-fold higher NF-κB signal (p &amp;lt; 0.05), and had a 2-fold greater total plaque area (p &amp;lt; 0.01). Fluorescent immunohistochemistry confirmed that the LDLR-/-ADAMTS13-/- mice also had greater platelets (p &amp;lt; 0.05) and increased macrophage content (p &amp;lt; 0.05) than LDLR-/- mice in aortic plaque. Conclusion In early to mid-stage atherosclerosis, abnormal regulation of endothelial-associated VWF results in platelet adhesion and secondary up-regulation of endothelial inflammatory adhesion molecules, thereby promoting atherosclerotic plaque progression. These results indicate an important role of platelet-endothelial interactions in early atherogenesis. Abstract 418 Figure

Ultrasound Molecular Imaging of Atherosclerosis With Nanobodies: Translatable Microbubble Targeting Murine and Human VCAM (Vascular Cell Adhesion Molecule) 1.

Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The microbubble contrast agents developed for preclinical studies are not suitable for clinical translation. Our aim was to characterize and validate a microbubble contrast agent using a clinically translatable single-variable domain immunoglobulin (nanobody) ligand. Approach and Results: Microbubble with a nanobody targeting VCAM-1 (MBcAbVcam1-5) and microbubble with a control nanobody (MBVHH2E7) were prepared and characterized in vitro. Attachment efficiency to VCAM-1 under continuous and pulsatile flow was investigated using activated murine endothelial cells. In vivo CEUMI of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MBcAbVcam1-5 and MBVHH2E7. Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×105 per microbubble. Compared with MBVHH2E7, MBcAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm2 while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MBcAbVcam1-5 in early (P=0.0003 versus MBVHH2E7) and late atherosclerosis (P=0.007 versus MBVHH2E7); in wild-type mice, there were no differences between MBcAbVcam1-5 and MBVHH2E7. CEUMI in human endarterectomy specimens showed a 100% increase in signal for MBcAbVcam1-5versus MBVHH2E7 (20.6±27.7 versus 9.6±14.7, P=0.0156). CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody.

Ultrasound Molecular Imaging: Principles and Applications in Cardiovascular Medicine.

Non-invasive molecular imaging is currently used as a research technique to better understand disease pathophysiology. There are also many potential clinical applications where molecular imaging may provide unique information that allows either earlier or more definitive diagnosis, or can guide precision medicine-based decisions on therapy. Contrast-enhanced ultrasound (CEU) with targeted microbubble contrast agents is one such technique that has been developed that has the unique properties of providing rapid information and revealing information only on events that occur within the vascular space. CEU molecular probes have been developed for a wide variety of disease states including atherosclerosis, vascular inflammation, thrombosis, tumor neovascularization, and ischemic injury. While the technique has not yet been adapted to clinical use, it has been used to reveal pathological processes, to identify new therapeutic targets, and to test the efficacy of novel treatments. This review will explore the physical basis for CEU molecular imaging, its strengths and limitations compared to other molecular imaging modalities, and the pre-clinical translational research experience.

Abstract 044: Platelet-Endothelial Interactions in Atherosclerosis-Prone Arteries in a Non-Human Primate Model of Obesity and Insulin Resistance

Background: Platelet interactions with the vascular endothelium contribute to early atherogenesis, and occur in part from oxidative stress and secondary dysregulation of endothelial-associated von Willebrand factor (VWF). We used in vivo contrast enhanced ultrasound (CEU) molecular imaging of the carotid endothelium in a non-human primate model of diet-induced obesity to test whether platelet adhesion is present in atherosclerosis-susceptible arteries prior to plaque development, and whether it can be modified through NADPH-oxidase (Nox) inhibition. Methods: Six adult rhesus macaques fed a high-fat diet (HFD), with 30% of calories from fat, for &gt;2 years were studied at baseline and after 8 weeks of therapy with the Nox inhibitor apocynin (50 mg/kg/d). Six lean chow-fed controls were also studied. Intravenous glucose tolerance tests (IVGTT) and body composition were assessed at each interval. CEU molecular imaging of VCAM-1 and platelet GPIbα of the carotid arteries bilaterally was used to assess endothelial activation and platelet adhesion. Carotid intimal thickening (IMT) and brachial flow-mediated dilation (FMD) were assessed by ultrasound. Results: Before therapy, animals on HFD compared to controls were obese (16.0 vs 9.3 kg, p=0.003), had increased visceral adiposity (49% vs 25% truncal fat, p=0.002), and were insulin resistant (4-fold higher insulin AUC on IVGTT, p=0.002). FMD and IMT were similar between cohorts, although mild plaque was seen in the carotid bulb in 3 HFD macaques. HFD animals had greater (p&lt;0.01 vs. controls) carotid CEU signal for VCAM-1 (20.0 vs -2.7 IU) and GPIbα (12.7 vs -0.4 IU). There was a linear correlation between VCAM-1 and GP1ba signal (r 2 =0.50, p=0.0001). Apocynin significantly reduced (p&lt;0.01 vs pre-treatment) signal for VCAM-1 (3.96 IU) and GPIbα (0.19 IU); but did not alter IMT, FMD, visceral adiposity, or insulin resistance on IVGTT. Conclusions: Platelet- endothelial interactions occur early in atherosclerosis-prone carotid arteries of obese and insulin resistant non-human primates, and correlates with the degree of endothelial adhesion molecule expression. Inhibition of Nox suppresses platelet adhesion and reduces VCAM-1 expression independent of any effects on insulin resistance and obesity.

Highlights of This Issue

Preclinical experiments demonstrate that interleukin-15 (IL-15) can control homeostasis and stimulate natural killer (NK) and antigen-specific CD8+ T cell activity without causing activation induced cell death or promoting T regulatory (Treg) cell function. Miller and colleagues conducted the first-in-human outpatient phase I dose escalation of subcutaneous (SC) rhIL-15 in refractory solid tumor cancer patients. SC rhIL-15 treatment was well tolerated and resulted in substantial increases in circulating NK and CD8+ T cells. These results will allow the export of IL-15 immunotherapy to an outpatient setting and testing of combinatorial strategies to improve cancer treatment.Treatment of cancer by targeting VEGF-A with bevacizumab has been extensively validated in the clinic; however, not all tumors respond to VEGF-A blockade. Angiopoietin-2 (Ang-2) and VEGF-A have complementary roles in regulating tumor angiogenesis and metastasis, suggesting that dual pathway inhibition is necessary to improve treatment outcomes. Vanucizumab is an investigational, first-in-class, bi-specific IgG1-like monoclonal antibody that simultaneously blocks VEGF-A and Ang-2 from interacting with their receptors. Hidalgo and colleagues conducted a first-in-human study that demonstrated an acceptable safety and tolerability profile for vanucizumab in patients with advanced solid tumors. Vanucizumab also demonstrated consistent pharmacokinetic and pharmacodynamic effects, and showed encouraging signs of antitumor activity that support further evaluation of vanucizumab in clinical trials.Adjuvant sunitinib prolonged disease-free survival over placebo in patients with locoregional renal cell carcinoma at high risk of recurrence postnephrectomy in the randomized phase III STRAC trial. In a prospectively designed exploratory analysis of tissue samples from a subset of patients in this trial, George and colleagues identify predictive biomarkers that could facilitate future patient selection for adjuvant sunitinib and mechanisms of interaction that might explain the durable treatment effect, potentially leading to future combination approaches. Tumor tissue expression levels of CD4, CD8, CD68, and PD-L1 were compared with efficacy outcomes. The observed association between higher CD8+ T-cell density in tumor tissue with longer disease-free survival with sunitinib, but not placebo, suggested predictive potential of CD8+ T-cell density, which warrants further independent cohort validation studies. The prognostic value of PD-L1 expression in primary tumors in this setting also should be further explored.The thymocyte differentiation antigen-1 (Thy1) is differentially expressed on the neovasculature of human PDAC compared to chronic pancreatitis and normal pancreas. To engineer a human Thy1-binding single-chain-antibody-ligand (Thy1-scFv) conjugated to microbubbles (MBThy1-scFv), Abou-Elkacem and colleagues designed and validated a clinically translatable Thy1-scFv for contrast-enhanced ultrasound molecular imaging of PDAC. The MBThy1-scFv was highly specific to murine/human Thy1, and in vivo ultrasound molecular imaging of PDAC-bearing mice demonstrated specific binding to Thy1. Ongoing translational studies are targeted at developing clinical-grade MBThy1-scFv which may improve visualization of PDAC and enable diagnosing at earlier disease state to ultimately improve survival of PDAC patients.

Noninvasive Contrast-Enhanced Ultrasound Molecular Imaging Detects Myocardial Inflammatory Response in Autoimmune Myocarditis.

Cardiac tests for diagnosing myocarditis lack sensitivity or specificity. We hypothesized that contrast-enhanced ultrasound molecular imaging could detect myocardial inflammation and the recruitment of specific cellular subsets of the inflammatory response in murine myocarditis. Microbubbles (MB) bearing antibodies targeting lymphocyte CD4 (MBCD4), endothelial P-selectin (MBPSel), or isotype control antibody (MBIso) and MB with a negative electric charge for targeting of leukocytes (MBLc) were prepared. Attachment of MBCD4 was validated in vitro using murine spleen CD4+ T cells. Twenty-eight mice were studied after the induction of autoimmune myocarditis by immunization with α-myosin-peptide; 20 mice served as controls. Contrast-enhanced ultrasound molecular imaging of the heart was performed. Left ventricular function was assessed by conventional and deformation echocardiography, and myocarditis severity graded on histology. Animals were grouped into no myocarditis, moderate myocarditis, and severe myocarditis. In vitro, attachment of MBCD4 to CD4+ T cells was significantly greater than of MBIso. Of the left ventricular ejection fraction or strain and strain rate readouts, only longitudinal strain was significantly different from control animals in severe myocarditis. In contrast, contrast-enhanced ultrasound molecular imaging showed increased signals for all targeted MB versus MBIso both in moderate and severe myocarditis, and MBCD4 signal correlated with CD4+ T-lymphocyte infiltration in the myocardium. Contrast-enhanced ultrasound molecular imaging can detect endothelial inflammation and leukocyte infiltration in myocarditis in the absence of a detectable decline in left ventricular performance by functional imaging. In particular, imaging of CD4+ T cells involved in autoimmune responses could be helpful in diagnosing myocarditis.

High-frequency ultrasound-guided disruption of glycoprotein VI-targeted microbubbles targets atheroprogressison in mice

Targeted contrast-enhanced ultrasound (CEU) using microbubble agents is a promising non-invasive imaging technique to evaluate atherosclerotic lesions. In this study, we decipher the diagnostic and therapeutic potential of targeted-CEU with soluble glycoprotein (GP)-VI in vivo. Microbubbles were conjugated with the recombinant fusion protein GPVI-Fc (MBGPVI) that binds with high affinity to atherosclerotic lesions. MBGPVI or control microbubbles (MBC) were intravenously administered into ApoE−/− or wild type mice and binding of the microbubbles to the vessel wall was visualized by high-resolution CEU. CEU molecular imaging signals of MBGPVI were substantially enhanced in the aortic arch and in the truncus brachiocephalicus in ApoE−/− as compared to wild type mice. High-frequency ultrasound (HFU)-guided disruption of MBGPVI enhanced accumulation of GPVI in the atherosclerotic lesions, which may interfere with atheroprogression. Thus, we establish targeted-CEU with soluble GPVI as a novel non-invasive molecular imaging method for atherosclerosis. Further, HFU-guided disruption of GPVI-targeted microbubbles is an innovate therapeutic approach that potentially prevents progression of atherosclerotic disease.

Cardiovascular Molecular Imaging with Contrast Ultrasound: Principles and Applications

Methods for imaging the molecular or cellular profile of tissue are being developed for all forms of non-invasive cardiovascular imaging. It is thought that these technologies will potentially improve patient outcomes by allowing diagnosis of disease at an early-stage, monitoring disease progression, providing important information on patient risk, and for tailoring therapy to the molecular basis of disease. Molecular imaging is also already assuming an important role in science by providing a better understanding of the molecular basis of cardiovascular pathology, for assessing response to new therapies, and for rapidly optimizing new or established therapies. Ultrasound-based molecular imaging is one of these new approaches. Contrast-enhanced ultrasound molecular imaging relies on the detection of novel site-targeted microbubbles (MB) or other acoustically active particles which are administered by intravenous injection, circulate throughout the vascular compartment, and are then retained and imaged within regions of disease by ligand-directed binding. The technique is thought to be advantageous in practical terms of cost, time, and ease of use. The aim of this review is to discuss the molecular participants of cardiovascular disease that have been targeted for ultrasound imaging, general features of site-targeted MB, imaging protocols, and potential roles of ultrasound molecular imaging in cardiovascular research and clinical medicine.

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

Inflammation and insulin resistance (IR) are associated processes that potentiate risk for cardiovascular disease in obesity. The temporal relation between IR and inflammation is not completely characterized. We hypothesized that endothelial cell adhesion molecule expression in large arteries is an early event that coincides with diet-induced obesity and IR in primates. Ten adult male rhesus macaques were studied at baseline and every 4 to 6 months on a high-fat diet for 2 years. Truncal fat, carotid intima-media thickness, plasma inflammatory biomarkers, and carotid P-selectin and vascular cell adhesion molecule-1 expression by contrast-enhanced ultrasound molecular imaging were assessed. Intravenous glucose tolerance test was performed at baseline and at 4 and 18 months. A high-fat diet produced a rapid increase (P<0.01) in weight, truncal fat, and degree of IR indicated by the insulin area under the curve and glucose disappearance rate on intravenous glucose tolerance test, all of which worsened minimally thereafter. Molecular imaging detected a progressive increase in endothelial cell adhesion molecule expression over time (5- to 7-fold greater than control agent signal at 2 years; P<0.01). Changes in intima-media thickness were not detected until 2 years and, although there was a trend toward an increase in plasma markers of inflammation (monocyte chemotactic protein-1, C-reactive protein), the pattern of increase varied considerably over time. In primates with diet-induced obesity, endothelial inflammatory activation is an early event that occurs coincident with the development of IR and long before any measurable change in carotid intima-media thickness. Endothelial activation is related more to the duration rather than to the severity of IR and is not mirrored by changes in plasma biomarkers.

Molecular Imaging Reveals Rapid Reduction of Endothelial Activation in Early Atherosclerosis With Apocynin Independent of Antioxidative Properties

Antioxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an nicotinamide adenine dinucleotide phosphate oxidase inhibitor with anti-inflammatory properties. We used contrast-enhanced ultrasound molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation Genetically modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4 mg/kg per day IP) or saline. Contrast-enhanced ultrasound molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MB(V)), to platelet glycoprotein Ibα (MB(Pl)), and control microbubbles (MB(Ctr)). Aortic vascular cell adhesion molecule 1 was measured using Western blot. Aortic reactive oxygen species generation was measured using a lucigenin assay. Hydroethidine oxidation was used to assess aortic superoxide generation. Baseline signal for MBV (1.3 ± 0.3 AU) and MB(Pl )(1.5 ± 0.5 AU) was higher than for MBCtr (0.5 ± 0.2 AU; P<0.01). In saline-treated animals, signal did not significantly change for any microbubble agent, whereas short-term apocynin significantly (P<0.05) reduced vascular cell adhesion molecule 1 and platelet signal (MBV: 0.3 ± 0.1; MBPl: 0.4 ± 0.1; MBCtr: 0.3 ± 0.2 AU; P=0.6 between agents). Apocynin reduced aortic vascular cell adhesion molecule 1 expression by 50% (P<0.05). However, apocynin therapy did not reduce reactive oxygen species content, superoxide generation, or macrophage content. Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content and is not associated with a detectable change in oxidative burden.

Epithelial cell biocompatibility of silica nanospheres for contrast-enhanced ultrasound molecular imaging

Nanosized particles are receiving increasing attention as future contrast agents (CAs) for ultrasound (US) molecular imaging, possibly decorated on its surface with biological recognition agents for targeted delivery and deposition of therapeutics. In particular, silica nanospheres (SiNSs) have been demonstrated to be feasible in terms of contrast enhancement on conventional US systems. In this work, we evaluated the cytotoxicity of SiNSs on breast cancer (MCF-7) and HeLa (cervical cancer) cells employing NSs with sizes ranging from 160 to 330 nm and concentration range of 1.5–5 mg/mL. Cell viability was evaluated in terms of size, dose and time dependence, performing the MTT reduction assay with coated and uncoated SiNSs. Whereas uncoated SiNSs caused a variable significant decrease in cell viability on both cell lines mainly depending on size and exposure time, PEGylated SiNSs (SiNSs-PEG) exhibit a high level of biocompatibility. In fact, after 72-h incubation, viability of both cell types was above the cutoff value of 70 % at concentration up to 5 mg/mL. We also investigated the acoustical behavior of coated and uncoated SiNSs within conventional diagnostic US fields in order to determine a suitable configuration, in terms of particle size and concentration, for their employment as targetable CAs. Our results indicate that the employment of SiNSs with diameters around 240 nm assures the most effective contrast enhancement even at the lowest tested concentration, coupled with the possibility of targeting all tumor tissues, being the SiNSs still in a size range where reticuloendothelial system trapping effect is relatively low.

Noninvasive Ultrasound Molecular Imaging of the Effect of Statins on Endothelial Inflammatory Phenotype in Early Atherosclerosis

Background/ObjectivesInflammatory changes on the endothelium are responsible for leukocyte recruitment to plaques in atherosclerosis. Noninvasive assessment of treatment-effects on endothelial inflammation may be of use for managing medical therapy and developing novel therapies. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) with contrast enhanced ultrasound (CEU) could assess treatment effects on endothelial phenotype in early atherosclerosis.MethodsMice with atherosclerosis produced by gene deletion of the LDL-receptor and Apobec-1-editing protein were studied. At 12 weeks of age, mice received 8 weeks of regular chow or atorvastatin-enriched chow (10 mg/kg/day). At 20 weeks, CEU molecular imaging for aortic endothelial VCAM-1 expression was performed with VCAM-1-targeted (MBVCAM) and control microbubbles (MBCtr). Aortic wall thickness was assessed with high frequency ultrasound. Histology, immunohistology and Western blot were used to assess plaque burden and VCAM-1 expression.ResultsPlaque burden was reduced on histology, and VCAM-1 was reduced on Western blot by atorvastatin, which corresponded to less endothelial expression of VCAM-1 on immunohistology. High frequency ultrasound did not detect differences in aortic wall thickness between groups. In contrast, CEU molecular imaging demonstrated selective signal enhancement for MBVCAM in non-treated animals (MBVCAM 2±0.3 vs MBCtr 0.7±0.2, p<0.01), but not in statin-treated animals (MBVCAM 0.8±0.2 vs MBCtr 1.0±0.2, p = ns; p<0.01 for the effect of statin on MBVCAM signal).ConclusionsNon-invasive CEU molecular imaging detects the effects of anti-inflammatory treatment on endothelial inflammation in early atherosclerosis. This easily accessible, low-cost technique may be useful in assessing treatment effects in preclinical research and in patients.

Molecular Imaging of Inflammation and Platelet Adhesion in Advanced Atherosclerosis Effects of Antioxidant Therapy With NADPH Oxidase Inhibition

In atherosclerosis, local generation of reactive oxygen species amplifies the inflammatory response and contributes to plaque vulnerability. We used molecular imaging to test whether inhibition of NADPH oxidase with apocynin would reduce endothelial inflammatory activation and endothelial-platelet interactions, thereby interrupting progression to high-risk plaque phenotype. Mice deficient for both the low-density lipoprotein receptor and Apobec-1 were studied at 30 weeks of age and again after 10 weeks with or without apocynin treatment (10 or 50 mg/kg per day orally). In vivo molecular imaging of vascular cell adhesion molecule-1 (VCAM 1) P-selectin, and platelet glycoprotein-1bα (GPIbα) in the thoracic aorta was performed with targeted contrast-enhanced ultrasound molecular imaging. Arterial elastic modulus and pulse wave transit time were assessed using ultrahigh frequency ultrasound and invasive hemodynamic measurements. Plaque size and composition were assessed by histology. Molecular imaging in nontreated mice detected a 2-fold increase in P-selectin expression, VCAM-1 expression, and platelet adhesion between 30 and 40 weeks of age. Apocynin reduced all of these endothelial events in a dose-dependent fashion (25% and 50% reduction in signal at 40 weeks for low- and high-dose apocynin). Apocynin also decreased aortic elastic modulus and increased the pulse transit time. On histology, apocynin reduced total monocyte accumulation in a dose-dependent manner as well as platelet adhesion, although total plaque area was reduced in only the high-dose apocynin treatment group. Inhibition of NADPH oxidase in advanced atherosclerosis reduces endothelial activation and platelet adhesion, which are likely responsible for the arrest of plaque growth and improvement of vascular mechanical properties.