OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)–stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm2). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1–targeted microbubbles and by ultrasound molecular imaging of P-selectin–targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin–targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin–targeted microbubbles. CONCLUSIONS We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

Background 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. Methods and Results Mice deficient for both the LDL 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/day orally). In vivo molecular imaging of VCAM-1, P-selectin and platelet GPIbα in the thoracic aorta was performed with targeted contrast-enhanced ultrasound (CEU) molecular imaging. Arterial elastic modulus and pulse wave transit time were assessed using ultra-high frequency ultrasound and invasive hemodynamic measurements. Plaque size and composition were assessed by histology. Molecular imaging in non-treated mice detected a 2-fold increase in P-selectin expression, VCAM-1 expression, and platelet adhesion between 30 and 40 wks 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. Conclusions 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.

Background Non-thrombotic platelet-endothelial interactions may contribute to atherosclerotic plaque development, although in vivo studies examining mechanism without platelet pre-activation are lacking. Using in vivo molecular imaging at various stages of atherosclerosis, we quantified platelet-endothelial interactions and evaluated the contribution of major adhesion pathways. Methods and Results Mice deficient for the LDL-receptor and Apobec-1 were studied as an age-dependent model of atherosclerosis at 10, 20, 30, and 40 wks of age, which provided progressive increase in stage from very early fatty streak (10 wks) to large complex plaques without rupture (40 wks). Platelet-targeted contrast ultrasound molecular imaging of the thoracic aorta performed with microbubbles targeted to GPIbα demonstrated selective signal enhancement as early as 10 weeks of age. This signal increased progressively with age (almost 8-fold increase from 10 to 40 weeks, ANOVA p<0.001). Specificity for platelet targeting was confirmed by the reduction in platelet-targeted signal commensurate with the decrease in platelet count after immunodepletion with anti-GPIb or anti-CD41 antibody. Inhibition of P-selectin in 20 and 40 wk atherosclerotic mice resulted in a small (15-30%) reduction in platelet signal. Molecular imaging with microbubbles targeted to the A1 domain of von Willebrand factor (VWF) demonstrated selective signal enhancement at all time points which did not significantly increase with age. Treatment of 20 and 40 week mice with recombinant ADAMTS13 eliminated platelet and VWF molecular imaging signal. Conclusions Platelet-endothelial interactions occur in early atherosclerosis. These interactions are in part due to endothelial VWF large multimers which can be reversed with exogenous ADAMTS13.

Background Stem cells are thought to enhance vascular remodeling in ischemic tissue in part through paracrine effects. Using molecular imaging, we tested the hypothesis that treatment of limb ischemia with multipotential adult progenitor cells (MAPC) promotes recovery of blood flow through the recruitment of pro-angiogenic monocytes. Methods and Results Hindlimb ischemia was produced in mice by iliac artery ligation and MAPC were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPC indicated that cells survived for 1 week. Contrast-enhanced ultrasound on day 3, 7 and 21 showed a more complete recovery of blood flow and greater expansion of microvascular blood volume in MAPC-treated mice than in controls. Fluorescent microangiography demonstrated more complete distribution of flow to microvascular units in MAPC-treated mice. On ultrasound molecular imaging, expression of endothelial P-selectin and intravascular recruitment of CX3CR-1-positive monocytes was significantly higher in MAPC-treated than control groups at day 3 and 7 after arterial ligation. Muscle immunohistology showed a >10-fold greater infiltration of monocytes in MAPC-treated than control-treated ischemic limbs at all time points. Intravital microscopy of ischemic or TNF-α-treated cremaster muscle demonstrated that MAPC migrate to peri-microvascular locations and potentiate selectin-dependent leukocyte rolling. In vitro migration of human CD14+ monocytes was 10-fold greater in response to MAPC-conditioned than basal media. Conclusions In limb ischemia, MAPC stimulate the recruitment of pro-angiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond MAPC lifespan suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype.

Background Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signalling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. Methods Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for ten minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signalling pathways were assessed by studying interventions that either (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signalling pathways involving endothelial nitric oxide synthase (eNOS) or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease (SCD). Results Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hrs in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with SCD. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced a nearly 40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or through adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of eNOS abolished the effects of therapeutic ultrasound, indicating downstream signalling through both NO and prostaglandins. Conclusions Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP which can act through a diverse portfolio of purinergic signalling pathways. These events can reverse hindlimb ischemia in mice for >24 hours, and increase muscle blood flow in patients with sickle cell disease. Clinical Trial Registration NCT01566890 (https://clinicaltrials.gov/ct2/show/NCT01566890)

Objective Our aim was to develop an echocardiographic molecular imaging approach for detecting recent myocardial ischemia by using recombinant P-selectin glycoprotein ligand (PSGL)-1 as a targeting ligand, which is a feasible approach for human use. Background Ischemic memory imaging using human PSGL-1 as a targeting moiety may extend the time window for postischemic detection by targeting the early (P-selectin) and late (E-selectin) endothelial ischemic response. Methods Lipid microbubbles bearing recombinant human PSGL-1 (MBYSPSL) or P-selectin antibody (MBAb) were prepared. Targeted attachment was evaluated by using flow chamber and intravital microscopy. In vivo ultrasound molecular imaging was first performed in the hindlimb in wild-type and P-selectin–deficient (P−/−) mice 45 to 360 min after brief ischemia-reperfusion injury. Myocardial contrast echocardiography molecular imaging was performed 1.5, 3, 6, and 18 h after brief left anterior descending coronary artery ischemia-reperfusion. Results Microbubble attachment to P-selectin–immunoglobulin G fusion protein in flow chamber experiments (shear stress 0.5 to 8.0 dyne/cm2) and to activated venular endothelium on intravital microscopy were similar for MBAb and MBYSPSL. Intense enhancement was seen for MBAb and MBYSPSL in postischemic muscle and was more stable over time for MBYSPSL. On myocardial contrast echocardiography, both MBYSPSL and MBAb produced similar signal enhancement at 90 min and 3 h after ischemia, which spatially correlated with the postischemic risk area. Signal significantly decreased but was still present at 6 to 18 h. Conclusions Echocardiographic molecular imaging with a human multi-selectin–targeted contrast agent bearing recombinant human PSGL-1 can detect myocardial ischemia hours after resolution. This approach may potentially be used for rapid bedside evaluation of patients with recent chest pain.

Background Selectins are adhesion molecules that are expressed by the vascular endothelium upon activation and may be an imaging target for detecting myocardial ischemia long after resolution. We hypothesized that molecular imaging of selectins with myocardial contrast echocardiography (MCE) molecular imaging could be used to detect recent brief ischemia in closed-chest non-human primates. Methods Myocardial ischemia was produced in anesthetized adult rhesus macaques (n=6) by percutaneous balloon catheter occlusion of the LAD or circumflex coronary artery for 5–10 minutes. Three separate macaques served as non-ischemic controls. MCE perfusion imaging was performed during coronary occlusion to measure risk area (RA) and at 100–110 min to exclude infarction. MCE molecular imaging was performed at 30 and 90 min after reperfusion using a lipid microbubble bearing dimeric recombinant human P-selectin glycoprotein ligand-1 (MB-YSPSL). Collection of blood for a safety data, electrocardiography, and echocardiography were performed at baseline, and before and 10 min after each MB-YSPSL injection. Results Vital signs, O2 saturation, ECG, ventricular systolic function, pulmonary vascular resistance, and serum safety markers were unchanged by intravenous injection of MB-YSPSL. On echocardiography, LV dysfunction in the RA had resolved by 30 min and there was no evidence for infarction on MCE perfusion imaging. On selectin-targeted MCE molecular imaging, signal enhancement was greater (p<0.05) in the RA than remote territory at 30 min (25±11 vs 11±4 IU) and 90 min (13±3 vs 3±2 IU) after ischemia. There was no enhancement (<1 IU) in control non-ischemic subjects. Conclusion In primates, MCE molecular imaging of selectins using a recombinant ligand appropriate for humans is both safe and effective for imaging recent myocardial ischemia. This technique may be useful for detecting recent ischemia in patients with chest pain even in the absence of necrosis.

Coronary artery disease (CAD) is one of the major causes of mortality and morbidity worldwide, with a high socioeconomic impact.(1) Non-invasive imaging modalities play a fundamental role in the evaluation and management of patients with known or suspected CAD. Imaging end-points have served as surrogate markers in many observational studies and randomized clinical trials that evaluated the benefits of specific therapies for CAD.(2) A number of guidelines and recommendations have been published about coronary syndromes by cardiology societies and associations, but have not focused on the excellent opportunities with cardiac imaging. The recent European Society of Cardiology (ESC) 2019 guideline on chronic coronary syndromes (CCS) and 2020 guideline on acute coronary syndromes in patients presenting with non-ST-segment elevation (NSTE-ACS) highlight the importance of non-invasive imaging in the diagnosis, treatment, and risk assessment of the disease.(3)(4) The purpose of the current recommendations is to present the significant role of non-invasive imaging in coronary syndromes in more detail. These recommendations have been developed by the European Association of Cardiovascular Imaging (EACVI) and the American Society of Echocardiography (ASE), in collaboration with the American Society of Nuclear Cardiology, the Society of Cardiovascular Computed Tomography, and the Society for Cardiovascular Magnetic Resonance, all of which have approved the final document.