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. 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.
Methods
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.
Results
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.
Conclusions
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.
There are well-recognized limitations of the diagnostic approaches currently used to evaluate patients with possible acute coronary syndrome (ACS). The initial electrocardiogram and cardiac enzyme levels are often nondiagnostic in patients with ACS, resulting in delayed initiation of appropriate therapy. These tests also lack positive predictive value and can show abnormal results from conditions other than ACS. In response, new technologies are being developed to more accurately diagnose ACS or to exclude ischemia in the almost 6 million individuals who present each year to emergency departments in the United States with chest pain, the majority of whom do not have ACS but nonetheless require significant resources in their diagnostic evaluation.
Molecular imaging of ischemia has emerged as a promising technology to address these challenges and relies on targeted imaging probes to noninvasively detect molecular events that occur with the onset of ischemia and persist for hours after resolution, a strategy termed ischemic memory imaging. Myocardial contrast echocardiographic (MCE) molecular imaging of selectins has been used to detect recent ischemia in small animal models of disease. Selectins are long adhesion molecules expressed upon vascular endothelial activation. Endothelial P-selectin and E-selectin mediate leukocyte rolling by interacting with carbohydrate counterligands such as P-selectin glycoprotein ligand–1 (PSGL-1) found on the leukocyte membrane. By using PSGL-1 as a targeting ligand for ultrasound contrast agents, it has been possible to detect both early (P-selectin) and late (P-selectin and E-selectin) phases of myocardial ischemia-reperfusion injury.
The aim of this study was to evaluate both the safety and efficacy of an ultrasound microbubble contrast agent bearing a dimeric recombinant human PSGL-1 for ischemic memory imaging in nonhuman primates undergoing brief closed-chest myocardial ischemia produced by temporary coronary occlusion. These studies were performed as part of the development of a rapid bedside method for rapidly diagnosing ACS in patients.
Methods
Animal Preparation and Study Protocol
The study was approved by the animal care and use committees at Oregon Health & Science University and the Oregon National Primate Research Center. Nine adult male rhesus macaques ( Macaca mulatta ) 10 to 15 years of age were studied. Animals were randomized to ischemic ( n = 6) and nonischemic control ( n = 3) groups. Animals were sedated with ketamine HCl (10 mg/kg intramuscularly), followed by endotracheal intubation and maintenance of anesthesia with inhaled isoflurane (1.0%–1.5%) during spontaneous respiration. Continuous electrocardiographic (ECG) monitoring and oxygen saturation measurement was performed. The study protocols are schematically depicted in Figure 1 . After an equilibration period of 20 min, a full baseline echocardiographic study and complete laboratory blood testing were performed. For the ischemic group, coronary ischemia was induced by percutaneous balloon catheter occlusion for 5 to 10 min. Myocardial perfusion imaging was performed during ischemia to define the risk area and 100 to 110 min after reflow to exclude infarction. Molecular imaging of P-selectin was performed 30 and 90 min after reflow or after similar postinduction anesthesia time in controls. Blood pressure (BP) measurement, echocardiography, and blood sampling were performed immediately before and 5 to 10 min after each targeted microbubble injection.
Percutaneous Coronary Ischemia
A femoral artery sheath was placed, after which animals received intravenous heparin (70 U/kg) and aspirin (100 mg) per rectum. A 5-F left guide catheter was then used to selectively engage the left main coronary artery. A 2-mm balloon catheter was advanced into the proximal portion of either the left anterior descending or left circumflex coronary artery. The balloon catheter was inflated for 5 min ( n = 4) or 10 min ( n = 2) depending on whether hemodynamic instability necessitated early deflation. Ischemia was confirmed by ST-segment elevation on ECG monitoring and a new regional wall motion abnormality on two-dimensional echocardiography, and the ischemic risk area was spatially defined during occlusion by MCE perfusion imaging.
Perfusion Imaging
MCE imaging (Sonos 5500, S5-1 transducer; Philips Medical Systems, Andover, MA) was performed at baseline, during coronary occlusion, and after completion of the final targeted imaging protocol (100–110 min after reperfusion). Harmonic power Doppler imaging was performed at a transmission frequency of 1.3 MHz and a mechanical index of 1.2. Lipid-shelled octafluoropropane microbubbles (Definity; Lantheus Medical Imaging, North Billerica, MA) were diluted with saline and infused intravenously at 4 × 10 8 min −1 . End-systolic images were acquired in apical two-chamber, three-chamber, and four-chamber views with incremental pulsing intervals from every one to five cardiac cycles. The spatial extent of the perfusion defect (risk area) was defined as the region lacking opacification, defined by a reader blinded to treatment assignment and stage, and was expressed as a ratio to total myocardial end-systolic area. The final MCE study was used to exclude infarction or residual hypoperfusion. For quantitative analysis, regions of interest were placed over the risk area and a remote myocardial territory. Acoustic intensity within the regions was fit to the function y = A (1 − e −β t ), where y is acoustic intensity at time t , A is the plateau intensity reflecting relative blood volume, and the rate constant β is the microvascular flux rate. The myocardial blood flow was calculated as the product of A and β.
MCE Molecular Imaging
For selectin targeting, lipid-shelled decafluorobutane microbubbles containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyerthylene glycol)2000] (5% molar concentration) were prepared, and PSGL-1 synthesized as a dimeric fusion protein with human immunoglobulin G1 (YSPSL; Y’s Therapeutics, San Bruno, CA) was conjugated to the surface of microbubbles by maleimide coupling to form a stable thioether bond with the immunoglobulin G portion of the YSPSL (synthesized in a good manufacturing process laboratory and kindly provided by Thierry Bettinger, Bracco Research, Geneva, Switzerland). Molecular imaging was performed at 30 and 90 min after coronary reflow or at corresponding times in nonischemic animals by intravenous injection of 2 × 10 8 targeted microbubbles (MB-YSPSL). Imaging was performed 8 min after injection to allow microbubble attachment and clearance of freely circulating agent. End-systolic images were obtained using harmonic power Doppler imaging (1.3 MHz, mechanical index 1.2) in the apical two-chamber, three-chamber, and four-chamber views. Several frames were obtained at a pulsing interval of one and then every five cardiac cycles. Signal enhancement from retained targeted contrast agent alone was determined by signal from the initial frame acquired after digitally subtracting the signal from several averaged frames obtained at a pulsing interval of five cycles to remove signal from any freely circulating contrast, as previously described. Signal was measured from regions of interest placed over the risk area and the remote nonischemic territory in postischemic subjects and over the anterior wall in nonischemic controls. The spatial extent of enhancement on molecular imaging was measured independently by a reader blinded to animal identity and stage.
Echocardiography
Comprehensive two-dimensional and Doppler transthoracic echocardiographic studies were performed at baseline and at completion of the study protocol. Two-dimensional imaging was performed with tissue harmonic settings at a transmission frequency of 1.6 MHz. Left ventricular (LV) ejection fraction and LV end-systolic and end-diastolic volumes were measured using a modified biplane method. Stroke volume was calculated using the product of the LV outflow tract area and the time-velocity integral measured by pulsed-wave spectral Doppler. Cardiac output (CO) was determined by the product of heart rate (HR) and stroke volume. Radial wall thickening in the ischemic group was measured in the center of the risk area and in a remote territory defined by perfusion data during coronary occlusion or averaged for the anterior and lateral wall for the nonischemic group. Pulmonary artery systolic pressure was estimated from the peak pressure gradient between the right ventricle calculated using the tricuspid regurgitant velocity and the modified Bernoulli equation, and right atrial pressure was estimated from inferior vena cava dimensions. An index of pulmonary vascular resistance was calculated by dividing pulmonary arter systolic pressure by CO. Mitral valve inflow velocities in early (E) and late (A) diastole were evaluated using pulsed-wave Doppler at the mitral leaflet tips. The peak systolic (S′) and early diastolic (E′) longitudinal velocities were measured by tissue Doppler at the medial and lateral mitral annulus from the apical four-chamber view and averaged. The ratio of E to E′ was used as an index of left atrial pressure, and a second index of pulmonary vascular resistance was calculated as (pulmonary artery systolic pressure − [E/E′])/CO.
A focused echocardiographic examination was performed immediately before and 5 to 10 min after each of the two targeted imaging acquisitions. For these examinations, only LV ejection fraction, regional fractional thickening, stroke volume, CO, right ventricular systolic pressure, pulmonary vascular resistance, mitral inflow, and mitral annular longitudinal velocities were measured.
Laboratory Blood Testing
Peripheral venous blood samples were obtained for laboratory safety testing. The frequency and type of blood laboratory testing were limited by animal care and use committee guidelines that limit daily phlebotomy volume in nonhuman primates. In the nonischemic group, samples were obtained before and after each of the two targeted imaging acquisitions. In the ischemic group, in which there was additional procedure-related blood loss, samples were obtained at baseline and at the completion of the protocol. Blood tests included serum chemistry, a complete liver panel, blood coagulation studies (partial thromboplastin time, international normalized ratio), a complete blood count, C3 complement, lactate dehydrogenase, and troponin I.
Statistical Analysis
Data are expressed as mean ± SD unless otherwise noted. Comparisons of echocardiographic data, vital signs, ECG measurements, and laboratory blood testing data between time periods were performed using nonpaired Student’s t tests. Radial wall thickening and coronary flow data were compared using nonpaired Student’s t tests. For multiple comparisons of the molecular imaging data, Kruskal-Wallis tests with Dunn post hoc tests were used. Differences were considered significant at P < .05.
Results
ECG and Safety Studies
Data could not be obtained in two macaques in the ischemia group because of severe global LV dysfunction and arrhythmias after reperfusion or because of technical issues relating to vascular access. Results therefore represent ischemic ( n = 4) and nonischemic control ( n = 3) subjects that completed the entire protocol. In the ischemic group, HR decreased during ischemia and then stabilized after reperfusion ( Figure 2 ). In both the ischemic and nonischemic control groups, HR was unaffected by the injection of MB-YSPSL at 30 and 90 min. In nonischemic animals, BP decreased between baseline and the 30-min time interval, likely as a result of deeper anesthesia, and remained constant thereafter. In ischemic animals, BP was low during ischemia and was lower than in nonischemic animals only during the early postreflow period. In both groups, injection of MB-YSPSL did not alter systolic or diastolic BP. Oxygen saturation and body temperature were constant for both groups and were not altered by MB-YSPSL injection. On ECG monitoring, there were frequent ventricular extrasystoles during ischemia and in the first 10 min of reperfusion in several animals. However, administration of MB-YSPSL did not produce any arrhythmias, extrasystoles, ST-segment changes, or changes in ECG intervals, including the corrected QT interval ( Supplemental Tables 1 and 2 ; available at www.onlinejase.com ).
