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BACKGROUND: Non-invasive molecular imaging of recent ischemia can potentially be used to diagnose acute coronary syndrome (ACS) with high accuracy. OBJECTIVES: We hypothesized that bedside myocardial contrast echocardiography (MCE) ischemic memory imaging could be achieved with phosphatidylserine microbubbles (MB(PS)) that are retained in the microcirculation via ischemia-associated endothelial activation. METHODS: A dose-finding study was performed in healthy volunteers (n=17) to establish optimal MB(PS) dosing. Stable patients with ACS (n=30) and confirmed antecedent but resolved myocardial ischemia were studied within two hours of coronary angiography and PCI when indicated. MCE molecular imaging was performed 8 minutes after intravenous administration of MB(PS.) MCE perfusion imaging was used to assess status of the post-ischemic microcirculation. RESULTS. Based on dose-finding studies, 0.10 or 0.15 mL of MB(PS) based on body mass was selected. In patients with ACS, all but two underwent primary PCI. MCE molecular imaging signal intensity was greater in the post-ischemic risk area vs. remote territory (median [95% CI]: 56 [33–66] vs 8 [2–17] IU, p

Coronary microvascular dysfunction (MVD) is a syndrome of abnormal regulation of vascular tone, particularly during increased metabolic demand. While there are several risk factors for MVD, some of which are similar to those for coronary artery disease (CAD), the cause of MVD is not understood. We hypothesized that MVD in symptomatic non-elderly subjects would be characterized by specific lipidomic profiles. Subjects (n = 20) aged 35–60 years and referred for computed tomography coronary angiography (CTA) for chest pain but who lacked obstructive CAD (&gt, 50% stenosis), underwent quantitative regadenoson stress-rest myocardial contrast echocardiography (MCE) perfusion imaging for MVD assessment. The presence of MVD defined by kinetic analysis of MCE data was correlated with lipidomic profiles in plasma measured by liquid chromatography and high-resolution mass spectrometry. Nine of twenty subjects had evidence of MVD, defined by reduced hyperemic perfusion versus other subjects (beta-value 1.62 ± 0.44 vs. 2.63 ± 0.99 s−1, p = 0.009). Neither the presence of high-risk but non-obstructive CAD on CTA, nor CAD risk factors were different for those with versus without MVD. Lipidomic analysis revealed that patients with MVD had lower concentrations of long-carbon chain triacylglycerols and diacylglycerols, and higher concentrations of short-chain triacylglycerols. The diacylglycerol containing stearic and linoleic acid classified all participants correctly. We conclude that specific lipidomic plasma profiles occur in MVD involving saturated long-chain fatty acid-containing acylglycerols that are distinctly different from those in non-obstructive CAD. These patterns could be used to better characterize the pathobiology and potential treatments for this condition.

Background Microvascular dysfunction (MVD) is a potential cause of chest pain in younger individuals. The authors hypothesized that nonelderly patients referred for computed tomographic angiography (CTA) but without significant stenosis would have a high prevalence of MVD by myocardial contrast echocardiography (MCE). Secondary aims were to test whether the presence of nonobstructive coronary artery disease (CAD) or reduced brachial flow-mediated dilation (FMD) predicted MVD. Methods Subjects ≤60 years of age undergoing CTA were recruited if they had either no evidence of coronary plaque or evidence of mild CAD ( Results Of the 29 patients in whom MCE could be performed, 12 (41%) had MVD. These subjects, compared with those with normal microvascular function, had lower hyperemic perfusion (mean, 236 ± 68 vs 354 ± 161 intensity units/sec; P = .02) and microvascular flux rate (mean, 1.6 ± 0.4 vs 2.5 ± 0.9 sec−1; P = .002) on quantitative MCE. The degree of FMD was not significantly different in those with or without MVD (mean, 11 ± 4% vs 9 ± 4%; P = .32), and there was a poor correlation between results on stress MCE and FMD. Only eight of the 29 subjects were classified as having nonobstructive CAD. There were no groupwise differences in the prevalence of MVD function in those with versus without CAD (43% vs 38% for negative and positive findings on CTA, respectively, P = .79). Conclusions MVD is a common finding in the nonelderly population referred for CTA for evaluation of possible CAD but without obstructive stenosis. Neither the presence of noncritical atherosclerotic disease nor abnormal FMD increases the likelihood for detecting MVD in this population.

The ability to accurately evaluate skeletal muscle microvascular blood flow has broad clinical applications for understanding the regulation of skeletal muscle perfusion in health and disease states. Contrast-enhanced ultrasound (CEU) perfusion imaging, a technique originally developed to evaluate myocardial perfusion, is one of many techniques that have been applied to evaluate skeletal muscle perfusion. Among the advantages of CEU perfusion imaging of skeletal muscle is that it is rapid, safe and performed with equipment already present in most vascular medicine laboratories. The aim of this review is to discuss the use of CEU perfusion imaging in skeletal muscle. This article provides details of the protocols for CEU imaging in skeletal muscle, including two predominant methods for bolus and continuous infusion destruction-replenishment techniques. The importance of stress perfusion imaging will be highlighted, including a discussion of the methods used to produce hyperemic skeletal muscle blood flow. A broad overview of the disease states that have been studied in humans using CEU perfusion imaging of skeletal muscle will be presented including: (1) peripheral arterial disease; (2) sickle cell disease; (3) diabetes; and (4) heart failure. Finally, future applications of CEU imaging in skeletal muscle including therapeutic CEU imaging will be discussed along with technological developments needed to advance the field.

BACKGROUND: Ultrasound-mediated cavitation of microbubble contrast agents produces high intravascular shear. We hypothesized that microbubble cavitation increases myocardial microvascular perfusion through shear-dependent purinergic pathways downstream from ATP release that is immediate and sustained through cellular ATP channels such as Pannexin-1. METHODS: Quantitative myocardial contrast echocardiography (MCE) perfusion imaging and in vivo optical imaging of ATP was performed in wild-type and Pannexin-1-deficient (Panx1(−/−)) mice before, 5 and 30 minutes after 10 minutes of ultrasound-mediated (1.3 MHz, mechanical index 1.3) myocardial microbubble cavitation. Flow augmentation in a pre-clinical model closer to humans was evaluated in rhesus macaques undergoing MCE perfusion imaging after high-power cavitation in the apical 4-chamber plane for 10 minutes. RESULTS: Microbubble cavitation in wild-type mice (n=7) increased myocardial perfusion by 64±25% at 5 minutes and 95±55% at 30 minutes compared to baseline (p

Purpose Our aim was to determine whether pharmacologic vasodilation is an alternative to exercise stress during limb perfusion imaging for peripheral artery disease (PAD). Methods Quantitative contrast-enhanced ultrasound (CEU) perfusion imaging of the bilateral anterior thigh and calf was performed in nine control subjects and nine patients with moderate to severe PAD at rest and during vasodilator stress with dipyridamole. For those who were able, CEU of the calf was then performed during modest plantar flexion exercise (20 watts). CEU time-intensity data were analyzed to quantify microvascular blood flow (MBF) and its parametric components of microvascular blood volume and flux rate. Results Thigh and calf skeletal muscle MBF at rest was similar between control and PAD patients. During dipyridamole, MBF increased minimally (

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 signaling 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 10 minutes after intravenous injection of 2×10 8 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 signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or K ATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase 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. Results: Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈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 adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide 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 signaling 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: URL: http://clinicaltrials.gov . Unique identifier: NCT01566890.

Quantitative contrast-enhanced ultrasound (CEU) perfusion imaging, a technique originally developed to evaluate myocardial perfusion, has also been applied to evaluate limb perfusion. CEU relies on the ultrasound detection of encapsulated microbubble contrast agents that remain entirely within the vascular compartment. Advantages of CEU for assessing limb perfusion in PAD are, that is, rapid and safe and can be performed with equipment that is already present in almost all vascular medicine laboratories. It is uniquely suited for evaluating the physiologic impact of PAD because it directly assesses nutritive microvascular flow in muscle that can originate from multiple sources, including major conduit artery inflow, collateral vessel networks, or redistribution from other limb tissues and nonnutritive pathways. Moreover, by virtue of its speed, the technique is extremely well suited to imaging during or within seconds of completing exercise stress. In this chapter, the application of contrast-enhanced ultrasound (CEU) perfusion imaging in peripheral arterial disease will be discussed including details of the imaging technique and both clinical and preclinical studies.

Objectives The authors investigated ideal acoustic conditions on a clinical scanner custom-programmed for ultrasound (US) cavitation-mediated flow augmentation in preclinical models. We then applied these conditions in a first-in-human study to test the hypothesis that contrast US can increase limb perfusion in normal subjects and patients with peripheral artery disease (PAD). Background US-induced cavitation of microbubble contrast agents augments tissue perfusion by convective shear and secondary purinergic signalling that mediates release of endogenous vasodilators. Methods In mice, unilateral exposure of the proximal hindlimb to therapeutic US (1.3 MHz, mechanical index 1.3) was performed for 10 min after intravenous injection of lipid microbubbles. US varied according to line density (17, 37, 65 lines) and pulse duration. Microvascular perfusion was evaluated by US perfusion imaging, and in vivo adenosine triphosphate (ATP) release was assessed using in vivo optical imaging. Optimal parameters were then used in healthy volunteers and patients with PAD where calf US alone or in combination with intravenous microbubble contrast infusion was performed for 10 min. Results In mice, flow was augmented in the US-exposed limb for all acoustic conditions. Only at the lowest line density was there a stepwise increase in perfusion for longer (40-cycle) versus shorter (5-cycle) pulse duration. For higher line densities, blood flow consistently increased by 3-fold to 4-fold in the US-exposed limb irrespective of pulse duration. High line density and long pulse duration resulted in the greatest release of ATP in the cavitation zone. Application of these optimized conditions in humans together with intravenous contrast increased calf muscle blood flow by >2-fold in both healthy subjects and patients with PAD, whereas US alone had no effect. Conclusions US of microbubbles when using optimized acoustic environments can increase perfusion in limb skeletal muscle, raising the possibility of a therapy for patients with PAD. (Augmentation of Limb Perfusion With Contrast Ultrasound; NCT03195556)

This study hypothesized that microvascular retention of phosphatidylserine-containing microbubbles (MB-PS) would allow detection of recent but resolved myocardial ischemia with myocardial contrast echocardiographic (MCE) molecular imaging.Techniques for ischemic memory imaging which can detect and spatially assess resolved myocardial ischemia are being developed for rapid evaluation of patients with chest pain.MCE molecular imaging with MB-PS was performed 1.5 h, 3.0 h, and 6.0 h after brief (10 min) myocardial ischemia in mice; data were compared to selectin-targeted microbubbles. MCE molecular imaging with Sonazoid (GE Healthcare, Amersham, United Kingdom), a commercially produced phosphatidylserine (PS) - containing agent, was performed in separate mice at 1.5 h and 3.0 h after ischemia-reperfusion; and in dogs undergoing 135 min of ischemia and 60 min of reflow as well as in closed-chest nonischemic control dogs. The mechanism for MB-PS attachment was assessed by intravital microscopy of post-ischemic muscle and by flow cytometry analysis of cell-MB interactions.In mice undergoing ischemia-reperfusion without infarction, signal enhancement in the risk area for MB-PS and p-selectin glycoprotein ligand-1-targeted microbubbles was similar at reflow times of 1.5 h (23.3 ± 7.3 IU vs. 30.7 ± 4.1 IU), 3.0 h (42.2 ± 6.2 IU vs. 33.9 ± 7.4 IU), and 6.0 h (24.1 ± 4.3 IU vs. 25.5 ± 4.7 IU). For both agents, signal in the risk area was significantly (p 0.05) higher than remote region at all reflow times. Sonazoid also produced strong risk area enhancement at 1.5 h (34.7 ± 5.0 IU) and 3.0 h (52.5 ± 4.5 IU) which was approximately 3-fold greater than in the control region, and which correlated spatially with the microsphere-derived risk area. In dogs, Sonazoid signal in the risk area was5-fold higher than in closed-chest control myocardium (42.2 ± 8.1 IU vs. 7.9 ± 3.3 IU; p 0.001). Mechanistic studies indicated that MB-PS attached directly to venular endothelium and adherent leukocytes which was dependent on serum complement components C1q and C3.Ischemic memory imaging with MCE is possible using MB-PS which may obviate the need for ligand-directed targeting.

Key points In fetuses, chronic anaemia stimulates cardiac growth; simultaneously, blood flow to the heart muscle itself is increased, and reserve blood flow capacity of the coronary vascular bed is preserved. Here we examined functional adaptations of the capillaries and small blood vessels responsible for delivering oxygen to the anaemic fetal heart muscle using contrast-enhanced echocardiography. We demonstrate that coronary microvascular flux rate doubled in anaemic fetuses compared to control fetuses, both at rest and during maximal flow, suggesting reduced microvascular resistance consistent with capillary widening. Cardiac fractional microvascular blood volume was not greater in anaemic fetuses, suggesting that growth of new microvascular vessels does not contribute to the increased flow per volume of myocardium. These unusual changes in microvascular function during anaemia may indicate novel adaptive strategies in the fetal heart. Abstract Fetal anaemia causes cardiac adaptations that have immediate and life-long repercussions on heart function and health. It is known that resting and maximal coronary conductance both increase during chronic fetal anaemia, but the coronary microvascular changes responsible for the adaptive response are unknown. Until recently, technical limitations have prevented quantifying functional capillary-level adaptations in the in vivo fetal heart. Our objective was to characterise functional microvascular adaptations in chronically anaemic fetal sheep. Chronically instrumented fetuses were randomized to a control group (n = 11) or were made anaemic by isovolumetric haemorrhage (n = 12) for 1 week prior to myocardial contrast echocardiography at 85% of gestation. Anaemia augmented cardiac mass by 23% without changing body weight. In anaemic fetuses, microvascular blood flow per volume of myocardium was twice that of control fetuses at rest, during vasodilatory hyperaemia, and during hyperaemia plus increased aortic pressure. The elevated blood flow was attributable almost entirely to an increase in microvascular blood flux rate whereas microvascular blood volumes were not different between groups at baseline, during hyperaemia, or with hyperaemia plus increased aortic pressure. Increased coronary microvascular flux rate in response to chronic fetal anaemia is consistent with expected reductions in capillary resistance from capillary diameter widening detected in earlier histological studies.

Focal junctional tourniquets (JTs) have been developed to control hemorrhage from proximal limb injuries. These devices may permit greater collateral perfusion than circumferential tourniquets. We hypothesized that JTs eliminate large-vessel pulse pressure yet allow a small amount of residual limb perfusion that could be useful for maintaining tissue viability.Ten healthy control subjects were studied. Transthoracic echocardiography, Doppler ultrasound of the femoral artery (FA) and posterior tibial artery, and contrast-enhanced ultrasound (CEU) perfusion imaging of the anterior thigh extensor and calf plantar flexor muscles were performed at baseline and during application of a JT over the common FA. Intramuscular arterial pulsatility index was also measured from CEU intensity variation during the cardiac cycle.FA flow was eliminated by JTs in all subjects; posterior tibial flow was eliminated in all but one. Perfusion measured in the thigh and calf muscles was similar at baseline (0.33 ± 0.29 vs 0.29 ± 0.22 mL/min/g). Application of the JT resulted in a reduction of perfusion (P.05) that was similar for the thigh and calf (0.08 ± 0.07 and 0.10 ± 0.03 mL/min/g). On CEU, microvascular flux rate was reduced by ≈55%, and functional microvascular blood volume was reduced by ≈35%. Arterial pulsatility index was reduced by ≈90% in the calf. JT inflation did not alter left ventricle dimensions, fractional shortening, cardiac output, or arterial elastance as a measure of total systolic load.Application of a JT eliminates conduit arterial pulse and markedly reduces intramuscular pulse pressure, but thigh and calf skeletal muscle perfusion is maintained at 25% to 35% of basal levels. These data suggest that JTs that are used to control limb hemorrhage allow residual tissue perfusion even when pulse pressure is absent.

This study evaluated whether lipoprotein apheresis produces immediate changes in resting perfusion in subjects with severe hypercholesterolemia, and whether there is a difference in the response between peripheral and coronary microcirculations.Lipoprotein apheresis is used in patients with severe hypercholesterolemia to reduce plasma levels of low-density lipoprotein cholesterol.Quantitative contrast-enhanced ultrasound perfusion imaging of the myocardium at rest and skeletal muscle at rest and during calibrated contractile exercise was performed before and immediately after lipoprotein apheresis in 8 subjects with severe hypercholesterolemia, 7 of whom had a diagnosis of familial hypercholesterolemia. Myocardial perfusion imaging was also performed in 14 normal control subjects. Changes in myocardial work and left ventricular function were assessed by echocardiography. Ex vivo ovine coronary and femoral artery ring tension assays were assessed in the presence of pre- and post-apheresis plasma.Apheresis acutely decreased low-density lipoprotein cholesterol (234.9 ± 103.2 mg/dl vs. 67.1 ± 49.5 mg/dl; p 0.01) and oxidized phospholipid on apolipoprotein B-100 (60.2 ± 55.2 nmol/l vs. 47.0 ± 24.5 nmol/l; p = 0.01), and acutely increased resting myocardial perfusion (55.1 [95% confidence interval: 77.2 to 73.1] IU/s vs. 135 [95% confidence interval: 81.2 to 189.6] IU/s; p = 0.01), without changes in myocardial work. Myocardial longitudinal strain improved in those subjects with reduced pre-apheresis function. Skeletal muscle perfusion at rest and during contractile exercise was unchanged by apheresis. Acetylcholine-mediated dilation of ex vivo ovine coronary but not femoral arteries was impaired in pre-apheresis plasma and was completely reversed in post-apheresis plasma.Lipoprotein apheresis produces an immediate improvement in coronary microvascular function, which increases myocardial perfusion and normalizes endothelial-dependent vasodilation. These changes are not observed in the periphery. (Acute Microvascular Changes With LDL Apheresis; NCT02388633).

Small animal models of ischemic left ventricular (LV) dysfunction are important for the preclinical optimization of stem cell therapy. The aim of this study was to test the hypothesis that temporal changes in LV function and regional perfusion after cell therapy can be assessed in mice using echocardiographic imaging.Wild-type mice (n = 25) were studied 7 and 28 days after permanent ligation of the left anterior descending coronary artery. Animals were randomized to receive closed-chest ultrasound-guided intramyocardial delivery of saline (n = 13) or 5 × 10(5) multipotential adult progenitor cells (MAPCs; n = 12) on day 7. LV end-diastolic and end-systolic volumes, LV ejection fraction, and stroke volume were measured using high-frequency echocardiography. Multiplanar assessments of perfusion and defect area size were made using myocardial contrast echocardiography.Between days 7 and 28, MAPC-treated animals had 40% to 50% reductions in defect size (P .001) and 20% to 30% increases in total perfusion (P .01). Perfusion did not change in nontreated controls. Both LV end-diastolic and end-systolic volumes increased between days 7 and 28 in both groups, but LV end-systolic volume increased to a lesser degree in MAPC-treated compared with control mice (+4.2 ± 7.9 vs +19.2 ± 22.0 μL, P .05). LV ejection fraction increased in the MAPC-treated mice and decreased in control mice (+3.0 ± 4.3% vs -5.6 ± 5.9%, P .01). There was a significant linear relation between the change in LV ejection fraction and the change in either defect area size or total perfusion.High-frequency echocardiography and myocardial contrast echocardiography in murine models of ischemic LV dysfunction can be used to assess the response to stem cell therapy and to characterize the relationship among spatial flow, ventricular function, and ventricular remodeling.

Background In patients with peripheral artery disease (PAD), the severity of symptoms correlates poorly with ankle-brachial index (ABI). The aim of this study was to test the hypothesis that limb perfusion assessed using contrast-enhanced ultrasound (CEU) during contractile exercise varies according to functional class in patients with PAD, particularly those with ABIs in the 0.4 to 0.6 range whose symptoms vary widely. Methods Bilateral quantitative CEU perfusion imaging of the calf was performed in normal control subjects (n = 10) and patients with PAD who had at least one limb with a moderately reduced ABI (0.4–0.6; n = 17). Imaging was performed at rest and immediately after 30 sec of modest periodic (0.3-Hz) plantar flexion (10 W). Results In patients with PAD, Rutherford symptom classification for each limb varied widely, including in limbs with ABIs of 0.4 to 0.6 (n = 6 with mild or no symptoms, n = 14 with moderate to severe symptoms). CEU perfusion imaging parameters at rest were similar between control subjects and patients with PAD irrespective of ABI. In normal control subjects, limb flow increased on average by > 20-fold after only 30 sec of moderate exercise. In patients with PAD, muscle exercise perfusion for all limbs was reduced compared with control subjects and decreased according to the severity of ABI reduction, primarily from reduced microvascular flux rate. Even limbs with ABIs > 0.9 in patients with PAD had lower exercise perfusion than in control subjects (P = .03). In subjects with PAD, exercise perfusion was lower in those with moderate to severe versus mild symptoms when analyzed for all limbs (median, 30 IU/sec [interquartile range (IQR), 21–52 IU/sec] vs 84 IU/sec [IQR, 36–177 IU/sec]; P = .01) and limbs with ABIs of 0.4 to 0.6 (median, 26 IU/sec [IQR, 14–41 IU/sec] vs 54 IU/sec [IQR, 31–105 IU/sec]; P = .05). Conclusions In patients with PAD, CEU exercise perfusion imaging detects differences in limb muscle perfusion that are likely to be responsible for differences in symptom severity and can detect the flow abnormalities from microvascular dysfunction even in limbs with normal ABIs.

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/cm 2 ). 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.

In fetuses, chronic anaemia stimulates cardiac growth; simultaneously, blood flow to the heart muscle itself is increased, and reserve blood flow capacity of the coronary vascular bed is preserved. Here we examined functional adaptations of the capillaries and small blood vessels responsible for delivering oxygen to the anaemic fetal heart muscle using contrast-enhanced echocardiography. We demonstrate that coronary microvascular flux rate doubled in anaemic fetuses compared to control fetuses, both at rest and during maximal flow, suggesting reduced microvascular resistance consistent with capillary widening. Cardiac fractional microvascular blood volume was not greater in anaemic fetuses, suggesting that growth of new microvascular vessels does not contribute to the increased flow per volume of myocardium. These unusual changes in microvascular function during anaemia may indicate novel adaptive strategies in the fetal heart.Fetal anaemia causes cardiac adaptations that have immediate and life-long repercussions on heart function and health. It is known that resting and maximal coronary conductance both increase during chronic fetal anaemia, but the coronary microvascular changes responsible for the adaptive response are unknown. Until recently, technical limitations have prevented quantifying functional capillary-level adaptations in the in vivo fetal heart. Our objective was to characterise functional microvascular adaptations in chronically anaemic fetal sheep. Chronically instrumented fetuses were randomized to a control group (n = 11) or were made anaemic by isovolumetric haemorrhage (n = 12) for 1 week prior to myocardial contrast echocardiography at 85% of gestation. Anaemia augmented cardiac mass by 23% without changing body weight. In anaemic fetuses, microvascular blood flow per volume of myocardium was twice that of control fetuses at rest, during vasodilatory hyperaemia, and during hyperaemia plus increased aortic pressure. The elevated blood flow was attributable almost entirely to an increase in microvascular blood flux rate whereas microvascular blood volumes were not different between groups at baseline, during hyperaemia, or with hyperaemia plus increased aortic pressure. Increased coronary microvascular flux rate in response to chronic fetal anaemia is consistent with expected reductions in capillary resistance from capillary diameter widening detected in earlier histological studies.

Objectives 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.

With each heart beat, the left ventricle moves a volume of blood into the systemic arterial vasculature for distribution to the organs of the body. This interaction between the left ventricle and the systemic arterial vasculature (ventricular-arterial coupling) is an important determinant of left ventricular function. Study of ventricular-arterial coupling once required invasive techniques. However, advances in echocardiography now make it possible to noninvasively assess ventricular-arterial coupling in humans. In this issue of JASE, Kuznetsova et al use echocardiographic techniques to better understand ventricular-arterial coupling and its effects on left ventricular performance in patients with hypertension. 1 Such studies remind us of the core physiologic concepts pioneering cardiovascular scientists defined many years ago. As we enter an era of increasingly sophisticated noninvasive evaluation of cardiac performance, careful application of these core concepts is essential. These physiologic concepts form the basis for our understanding of the how the heart adapts to hemodynamic challenges during health and disease. Earlyresearchersworkedwithbothisolatedcardiacmuscleandthe intactheart,usingtheattributesofeachpreparationtotheiradvantage. Study of isolated cardiac muscle allowed more strict control of experimental variables to clearly determine the effects of load on muscle function. Researchers found that adding increasing weight to isolated cardiac muscle before contraction results in greater force generation during contraction. 2 These observations lead to the development of

Contrast echocardiography encompasses a broad variety of different applications whereby cardiovascular ultrasound is used to detect microbubble contrast agents that produce a strong acoustic signal. Ultrasound contrast agents are currently approved for use in cardiology for the purposes of opacifying the left ventricular chamber in order to aid interpretation of echocardiography, yet their off-label use has been applied for myocardial perfusion imaging in ischaemic heart disease for over a decade. This article provides a broad overview of some of the future uses of contrast ultrasound in patients with cardiovascular disease that are on the horizon, including: (1) evaluating vascular anatomy and plaque neovascularisation; (2) detecting abnormal microvascular function; (3) assessing peripheral vascular disease with stress/rest limb perfusion imaging; (4) molecular imaging of cardiovascular disease; (5) site targeted delivery of therapeutic genes or drugs; and (6) ultrasound facilitated thrombolysis. Signal enhancement during contrast enhanced ultrasound (CEU) relies on the detection of the acoustic energy produced by encapsulated microbubbles or other acoustically active particles. The basis for signal generation from these contrast agents is their compressibility.w1 w2 Ultrasound contrast agents contain gases or, less commonly, emulsions with a low vapour pressure. Hence, they are several orders of magnitude more compressible than water or tissue and, because they are also smaller than the wavelength of diagnostic ultrasound, undergo volumetric oscillation in the pressure fluctuations of an ultrasound field.w1 The ‘vibration’ of microbubbles produces, among other forms of energy (light, heat), strong backscattered acoustic pressure signals that produce contrast enhancement on ultrasound imaging. As predicted by the Rayleigh–Plesset principles, the magnitude of bubble oscillation and, hence, signal generation is influenced by the compressibility and density of the gas, the frequency and power of …

ObjectivesWe sought to determine whether contrast-enhanced ultrasound (CEU) microangiography with maximum intensity projection (MIP) processing could temporally evaluate proliferation of the vasa vasorum (VV) in a model of mural hemorrhage.BackgroundExpansion of the VV and plaque neovascularization contributes to plaque growth and instability and may be triggered by a variety of stimuli, including vascular hemorrhage. However, quantitative in vivo methods for temporal assessment of VV remodeling are lacking.MethodsIn 24 rabbits fed a high-fat diet, either autologous whole blood or saline was percutaneously injected into the media-adventitia of the femoral artery using ultrahigh-frequency ultrasound guidance. Functional VV density at the injection site and contralateral control artery was assessed 1, 2, and 6 weeks after injection with CEU imaging with MIP processing. In vitro studies with renathane microtubes were also performed to validate linear density measurement with CEU and MIP processing.ResultsIn vitro studies demonstrated that MIP processing of CEU data reflected the relative linear density of vessels in a manner that was relatively independent of contrast concentration or microtube flow rate. On CEU with MIP, there was a 3-fold increase in femoral artery VV microvascular density at 1 and 2 weeks after blood injection (p < 0.01 vs. contralateral control), whereas VV density increased minimally after saline injection. At 6 weeks, VV vascular density decreased in blood-treated vessels and was not different from saline-injected or contralateral control vessels.ConclusionsCEU with MIP processing can provide quantitative data on temporal changes in the functional density of the VV. This method may be useful for evaluating high-risk features of plaque neovascularization or response to therapies aimed at plaque neovessels.

BACKGROUND. We used ultrasound molecular imaging to evaluate the therapeutic effects of anti-oxidant therapy with EUK-207, which has superoxide dismutase and catalase activities, on suppressing high risk atherosclerotic features. METHODS. Mice with age-dependent atherosclerosis produced by deletion of the LDL-receptor and Apobec-1 were studied at 20 and 40 weeks of age. EUK-207 or vehicle was administered for the preceding 8 weeks. Therapy for 28 weeks was also studied for 40 week-old mice. Ultrasound molecular imaging of the thoracic aorta was performed with contrast agents targeted to endothelial P-selectin, von Willebrand factor (VWF) A1-domain, and platelet GPIbα; or control agent. Aortic plaque area and macrophage content were assessed by histology. RESULTS. In 20 week-old DKO mice, EUK-207 compared to sham therapy produced only non-significant trends for reduction in molecular imaging signal for endothelial P-selectin, VWF A1-domain and platelet adhesion. At 40 weeks, EUK-207 given for 8 or 28 weeks significantly (p

Background— Nonthrombotic platelet–endothelial interactions may contribute to atherosclerotic plaque development, although in vivo studies examining mechanism without platelet preactivation 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 low-density lipoprotein receptor and Apobec-1 were studied as an age-dependent model of atherosclerosis at 10, 20, 30, and 40 weeks of age, which provided progressive increase in stage from early fatty streak (10 weeks) to large complex plaques without rupture (40 weeks). 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, analysis of variance P Conclusions— Platelet–endothelial interactions occur in early atherosclerosis. These interactions are in part caused by endothelial von Willebrand factor large multimers, which can be reversed with exogenous ADAMTS13.

Background— In diabetes mellitus, reduced perfusion and capillary surface area in skeletal muscle, which is a major glucose storage site, contribute to abnormal glucose homeostasis. Using contrast-enhanced ultrasound, we investigated whether abdominal adipose tissue perfusion is abnormal in insulin resistance and correlates with glycemic control. Methods and Results— Contrast-enhanced ultrasound perfusion imaging of abdominal adipose tissue and skeletal muscle was performed in obese insulin resistance (db/db) mice at 11 to 12 or 14 to 16 weeks of age and in control lean mice. Time–intensity data were analyzed to quantify microvascular blood flow (MBF) and capillary blood volume (CBV). Blood glucose response for 1 hour was measured after insulin challenge (1 U/kg, IP). Compared with control mice, db/db mice at 11 to 12 and 14 to 16 weeks had a higher glucose concentration area under the curve after insulin (11.8±2.8, 20.6±4.3, and 28.4±5.9 mg·min/dL [×1000], respectively; P =0.0002) and also had lower adipose MBF (0.094±0.038, 0.035±0.010, and 0.023±0.01 mL/min per gram; P =0.0002) and CBV (1.6±0.6, 1.0±0.3, and 0.5±0.1 mL/100 g; P =0.0017). The glucose area under the curve correlated in a nonlinear fashion with both adipose and skeletal muscle MBF and CBV. There were significant linear correlations between adipose and muscle MBF ( r =0.81) and CBV ( r =0.66). Adipocyte cell volume on histology was 25-fold higher in 14- to 16-week db/db versus control mice. Conclusions— Abnormal adipose MBF and CBV in insulin resistance can be detected by contrast-enhanced ultrasound and correlates with the degree of impairment in glucose storage. Abnormalities in adipose tissue and muscle seem to be coupled. Impaired adipose tissue perfusion is in part explained by an increase in adipocyte size without proportional vascular response.

Background Contrast-enhanced ultrasound (CEU) limb perfusion imaging is a promising approach for evaluating peripheral artery disease (PAD). However, low signal enhancement in skeletal muscle has necessitated high-power intermittent imaging algorithms, which are not clinically feasible. We hypothesized that CEU using a combination of intermediate power and a contrast agent resistant to inertial cavitation would allow real-time limb stress perfusion imaging. Methods In normal volunteers, CEU of the calf skeletal muscle was performed on separate days with Sonazoid, Optison, or Definity. Progressive reduction in the ultrasound pulsing interval was used to assess the balance between signal enhancement and agent destruction at escalating mechanical indices (MI, 0.1–0.4). Real-time perfusion imaging at MI 0.1–0.4 using postdestructive replenishment kinetics was performed at rest and during 25 W plantar flexion contractile exercise. Results For Optison, limb perfusion imaging was unreliable at rest due to very low signal enhancement generated at all MIs and was possible during exercise-induced hyperemia only at MI 0.1 due to agent destruction at higher MIs. For Definity, signal intensity progressively increased with MI but was offset by microbubble destruction, which resulted in modest signal enhancement during CEU perfusion imaging and distortion of replenishment curves at MI ≥ 0.2. For Sonazoid, there strong signal enhancement at MI ≥ 0.2, with little destruction detected only at MI 0.4. Accordingly, high signal intensity and nondistorted perfusion imaging was possible at MI 0.2–0.3 and detected an 8.0- ± 5.7-fold flow reserve. Conclusions Rest-stress limb perfusion imaging in humans with real-time CEU, which requires only seconds to perform, is possible using microbubbles with viscoelastic properties that produce strong nonlinear signal generation without destruction at intermediate acoustic pressures.

Skeletal muscle microvascular blood flow (MBF) increases in response to physiological hyperinsulinemia. This vascular action of insulin may facilitate glucose uptake. We hypothesized that epoxyeicosatrienoic acids (EETs), a family of arachadonic, acid-derived, endothelium-derived hyperpolarizing factors, are mediators of insulin's microvascular effects. Contrast-enhanced ultrasound (CEU) was performed to quantify skeletal muscle capillary blood volume (CBV) and MBF in wild-type and obese insulin-resistant ( db/db) mice after administration of vehicle or trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid ( t-AUCB), an inhibitor of soluble epoxide hydrolase that converts EETs to less active dihydroxyeicosatrienoic acids. Similar studies were performed in rats pretreated with l-NAME. CEU was also performed in rats undergoing a euglycemic hyperinsulinemic clamp, half of which were pretreated with the epoxygenase inhibitor MS-PPOH to inhibit EET synthesis. In both wild-type and db/db mice, intravenous t-AUCB produced an increase in CBV (65–100% increase at 30 min, P < 0.05) and in MBF. In db/db mice, t-AUCB also reduced plasma glucose by ∼15%. In rats pretreated with l-NAME, t-AUCB after produced a significant ≈20% increase in CBV, indicating a component of vascular response independent of nitric oxide (NO) production. Hyperinsulinemic clamp produced a time-dependent increase in MBF (19 ± 36 and 76 ± 49% at 90 min, P = 0.026) that was mediated in part by an increase in CBV. Insulin-mediated changes in both CBV and MBF during the clamp were blocked entirely by MS-PPOH. We conclude that EETs are a mediator of insulin-mediated augmentation in skeletal muscle perfusion and are involved in regulating changes in CBV during hyperinsulinemia.

The etiology of flank pain sometimes experienced during the administration of ultrasound contrast agents is unknown. The aim of this study was to investigate whether microbubble ultrasound contrast agents are retained within the renal microcirculation, which could lead to either flow disturbance or local release of vasoactive and pain mediators downstream from complement activation.Retention of lipid-shelled microbubbles in the renal microcirculation of mice was assessed by confocal fluorescent microscopy and contrast-enhanced ultrasound imaging with dose-escalating intravenous injection. Studies were performed with size-segregated microbubbles to investigate physical entrapment, after glycocalyx degradation and in wild-type and C3-deficient mice to investigate complement-mediated retention. Urinary bradykinin was measured before and after microbubble administrations. Renal contrast-enhanced ultrasound in human subjects (n = 13) was performed 7 to 10 min after the completion of lipid microbubble administration.In both mice and humans, microbubble retention was detected in the renal cortex by persistent contrast-enhanced ultrasound signal enhancement. Microbubble retention in mice was linearly related to dose and occurred almost exclusively in cortical glomerular microvessels. Microbubble retention did not affect microsphere-derived renal blood flow. Microbubble retention was not influenced by glycocalyx degradation or by microbubble size, thereby excluding lodging, but was reduced by 90% (P.01) in C3-deficient mice. Urinary bradykinin increased by 65% 5 min after microbubble injection.Lipid-shelled microbubbles are retained in the renal cortex because of complement-mediated interactions with glomerular microvascular endothelium. Microbubble retention does not adversely affect renal perfusion but does generate complement-related intermediates that are known to mediate nociception and could be responsible for flank pain.

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 (MAPCs) promotes recovery of blood flow through the recruitment of proangiogenic monocytes. Methods and Results— Hind-limb ischemia was produced in mice by iliac artery ligation, and MAPCs were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPCs indicated that cells survived for 1 week. Contrast-enhanced ultrasound on days 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 CX 3 CR-1-positive monocytes were significantly higher in MAPC-treated mice than in the control groups at days 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 tumor necrosis factor-α–treated cremaster muscle demonstrated that MAPCs migrate to perimicrovascular 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, MAPCs stimulate the recruitment of proangiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond the MAPC lifespan, suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype.

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 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. 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.

Objective— Diabetes mellitus (DM) is associated with impaired ischemia-related vascular remodeling and also dysregulation of the inflammatory response. We sought to determine whether impaired selectin-mediated monocyte recruitment in ischemic tissues contributes to blunted ischemia-mediated angiogenesis in DM. Methods and Results— Contrast-enhanced ultrasound perfusion imaging and molecular imaging of endothelial P-selectin expression in the proximal hindlimb were performed at 1, 3, and 21 days after arterial ligation in wild-type and db/db mice. Ligation reduced muscle blood flow to ≈0.05 mL/minute per gram in both strains. Significant recovery of flow occurred only in wild-type mice (60%–65% of baseline flow). On molecular imaging, baseline P-selectin signal was 4-fold higher in db/db compared with wild-type mice ( P P Conclusion— In DM, there is dysregulation of the selectin response to limb ischemia, which leads to impaired monocyte recruitment, which may be mechanistically related to reduced vascular remodeling in limb ischemia.

There is growing interest in limb contrast-enhanced ultrasound (CEU) perfusion imaging for the evaluation of peripheral artery disease. Because of low resting microvascular blood flow in skeletal muscle, signal enhancement during limb CEU is prohibitively low for real-time imaging. The aim of this study was to test the hypothesis that this obstacle can be overcome by intermediate- rather than low-power CEU when performed with an acoustically resilient microbubble agent.Viscoelastic properties of Definity and Sonazoid were assessed by measuring bulk modulus during incremental increases in ambient pressure to 200 mm Hg. Comparison of in vivo microbubble destruction and signal enhancement at a mechanical index (MI) of 0.1 to 0.4 was performed by sequential reduction in pulsing interval from 10 to 0.05 sec during limb CEU at 7 MHz in mice and 1.8 MHz in dogs. Destruction was also assessed by broadband signal generation during passive cavitation detection. Real-time CEU perfusion imaging with destruction-replenishment was then performed at 1.8 MHz in dogs using an MI of 0.1, 0.2, or 0.3.Sonazoid had a higher bulk modulus than Definity (66 ± 12 vs 29 ± 2 kPa, P = .02) and exhibited less inertial cavitation (destruction) at MIs ≥ 0.2. On in vivo CEU, maximal signal intensity increased incrementally with MI for both agents and was equivalent between agents except at an MI of 0.1 (60% and 85% lower for Sonazoid at 7 and 1.8 MHz, respectively, P .05). However, on progressive shortening of the pulsing interval, Definity was nearly completely destroyed at MIs ≥ 0.2 at 1.8 and 7 MHz, whereas Sonazoid was destroyed only at 1.8 MHz at MIs ≥ 0.3. As a result, real-time CEU perfusion imaging demonstrated approximately fourfold greater enhancement for Sonazoid at an MI of 0.3 to 0.4.Robust signal enhancement during real-time CEU perfusion imaging of the limb is possible when using intermediate-power imaging coupled with a durable microbubble contrast agent.

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. The aim of this study was to test the hypothesis that molecular imaging of selectins with myocardial contrast echocardiographic (MCE) molecular imaging could be used to detect recent brief ischemia in closed-chest nonhuman primates.Myocardial ischemia was produced in anesthetized adult rhesus macaques (n = 6) by percutaneous balloon catheter occlusion of the left anterior descending or circumflex coronary artery for 5 to 10 min. Three separate macaques served as nonischemic controls. MCE perfusion imaging was performed during coronary occlusion to measure risk area and at 100 to 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 safety data, electrocardiography, and echocardiography were performed at baseline and before and 10 min after each MB-YSPSL injection.Vital signs, oxygen saturation, electrocardiographic results, ventricular systolic function, pulmonary vascular resistance, and serum safety markers were unchanged by intravenous injection of MB-YSPSL. On echocardiography, left ventricular dysfunction in the risk area had resolved by 30 min, and there was no evidence of infarction on MCE perfusion imaging. On selectin-targeted MCE molecular imaging, signal enhancement was greater (P.05) in the risk area 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 nonischemic subjects.In primates, MCE molecular imaging of selectins using MB-YSPSL, 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.