David K. Glover

Comparison Between 201Tl and 99mTc Sestamibi Uptake During Adenosine-Induced Vasodilation as a Function of Coronary Stenosis Severity

BACKGROUND Myocardial uptake of either 201Tl or 99mTc-sestamibi (sestamibi) is known to plateau at high coronary flow rates. However, few direct comparisons have been made between these tracers to determine what effect differences in the uptake plateau for the two tracers may have on the detection of coronary stenoses of various severities. METHODS AND RESULTS Twenty-two dogs were instrumented with flow transducers on the left anterior descending (LAD) and circumflex (LCx) arteries. In 6 nonstenotic dogs, adenosine was infused directly into the LAD, whereas 16 dogs with either critical (n = 7) or mild (n = 9) LAD stenoses received an intravenous infusion. At peak flow, 201Tl (0.5 mCi), sestamibi (5 to 8 mCi), and radiolabeled microspheres were injected simultaneously. Five minutes later, dogs were killed, and ex vivo imaging of heart slices and gamma-well counting of multiple myocardial samples was performed. Neither 201Tl nor sestamibi uptake increased in direct proportion to flow. In the 6 nonstenotic dogs, a fivefold increase in LAD flow increased 201Tl and sestamibi uptake by only 202 +/- 6% and 138 +/- 4%, respectively (P < .0001). In the dogs with critical stenosis, the ratios of stenotic to normal activity by well counting for 201Tl (0.37 +/- 0.05) and sestamibi (0.53 +/- 0.06) underestimated the microsphere-determined flow disparity (0.17 +/- 0.03) (P < .005), but the degree of underestimation was greater for sestamibi (P = .001). Similarly, in the dogs with mild stenosis, the stenotic-to-normal ratio for 201Tl (0.62 +/- 0.04) approximated the flow ratio (0.43 +/- 0.04) better than sestamibi (0.79 +/- 0.03) (P < .0001). Sestamibi defects, however, were visually identifiable on the images of the myocardial slices. By image quantification, sestamibi defect magnitude (LAD-to-LCx count ratio) in the critical stenosis group (0.62 +/- 0.05) was significantly less than in the mild stenosis group (0.80 +/- 0.02) (P < .01). CONCLUSIONS Thus, with adenosine-induced hyperemic flow, both 201Tl and sestamibi significantly underestimated the magnitude of the flow disparity between stenotic and normal perfusion beds. The degree of underestimation was greater for sestamibi. The clinical implication of these experimental findings for vasodilator perfusion imaging remains to be determined, since factors such as greater redistribution and scatter with 201Tl could offset its advantages.

Molecular Imaging of Atherosclerotic Plaques Targeted to Oxidized LDL Receptor LOX-1 by SPECT/CT and Magnetic Resonance

Background—The oxidized low-density lipoprotein receptor (LDLR) LOX-1 plays a crucial role in atherosclerosis. We sought to detect and assess atherosclerotic plaque in vivo by using single-photon emission computed tomography/computed tomography and magnetic resonance imaging and a molecular probe targeted at LOX-1. Methods and Results—Apolipoprotein E−/− mice fed a Western diet and LDLR−/− and LDLR−/−/LOX-1−/− mice fed an atherogenic diet were used. Imaging probes consisted of liposomes decorated with anti–LOX-1 antibodies or nonspecific immunoglobulin G, 111indium or gadolinium, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine fluorescence markers. In vivo imaging was performed 24 hours after intravenous injection (150 &mgr;L) of LOX-1 or nonspecific immunoglobulin G probes labeled with either 111indium (600 &mgr;Ci) or gadolinium (0.075 mmol/kg), followed by aortic excision for phosphor imaging and Sudan IV staining, or fluorescence imaging and hematoxylin/eosin staining. The LOX-1 probe also colocalized with specific cell types, apoptosis, and matrix metalloproteinase-9 expression in frozen aortic sections. Single-photon emission computed tomography/computed tomography imaging of the LOX-1 probe showed aortic arch “hot spots” in apolipoprotein E−/− mice (n=8), confirmed by phosphor imaging. Magnetic resonance imaging showed significant Gd enhancement in atherosclerotic plaques in LDLR−/− mice with the LOX-1 (n=7) but not with the nonspecific immunoglobulin G (n=5) probe. No signal enhancement was observed in LDLR−/−/LOX-1−/− mice injected with the LOX-1 probe (n=5). These results were confirmed by ex vivo fluorescence imaging. The LOX-1 probe bound preferentially to the plaque shoulder, a region with vulnerable plaque features, including extensive LOX-1 expression, macrophage accumulation, apoptosis, and matrix metalloproteinase-9 expression. Conclusions—LOX-1 can be used as a target for molecular imaging of atherosclerotic plaque in vivo. Furthermore, the LOX-1 imaging signal is associated with markers of rupture-prone atherosclerotic plaque.

Pharmacological Stress Thallium Scintigraphy With 2-Cyclohexylmethylidenehydrazinoadenosine (WRC-0470) A Novel, Short-Acting Adenosine A2A Receptor Agonist

BACKGROUND Pharmacological stress imaging with adenosine or dipyridamole is associated with a high incidence of side effects, including hypotension, chest pain, AV conduction abnormalities, and bronchospasm. Although the desired coronary vasodilatory response is mediated primarily by the adenosine A2A receptors, these side effects result from stimulation of the A1, A2B, or A3 adenosine receptors. We hypothesized that a selective adenosine A2A receptor agonist would induce coronary vasodilatation appropriate for pharmacological stress imaging, without evoking adenosine receptor-mediated side effects. METHODS AND RESULTS Infusions of a potent and selective A2A adenosine receptor agonist, WRC-0470 (0.1 to 3 micrograms kg-1. min-1 for 10 minutes), to five open-chest dogs produced dose-related left anterior descending (LAD) and left circumflex (LCx) coronary artery vasodilatation without altering mean arterial pressure, heart rate, left atrial pressure, or left ventricular dP/dt. In the same dogs, adenosine (300 micrograms . kg-1. min-1 for 4 minutes) produced coronary vasodilatation that was limited by significant hypotension. To determine the utility of WRC-0470 for pharmacological stress imaging, the hemodynamic responses to WRC-0470 (0.6 microgram.kg-1.min-1 for 10 minutes) and adenosine (250 micrograms.kg-1.min-1 for 4 minutes) were compared in dogs with critical LAD stenoses. 201T1 was injected at the peak WRC-0470 stress response. WRC-0470 increased LCx flow nearly fivefold but did not significantly lower mean arterial pressure. Anteroseptal defects were readily apparent in slice images from all dogs. The mean defect ratio (LAD/LCx) was 0.59 +/- 0.06. CONCLUSIONS The potent A2A-selective adenosine receptor agonist WRC-0470 is a short-acting coronary vasodilator with potential utility for pharmacological stress perfusion imaging.

Imaging Atherosclerosis and Vulnerable Plaque

Identifying patients at high risk for an acute cardiovascular event such as myocardial infarction or stroke and assessing the total atherosclerotic burden are clinically important. Currently available imaging modalities can delineate vascular wall anatomy and, with novel probes, target biologic processes important in plaque evolution and plaque stability. Expansion of the vessel wall involving remodeling of the extracellular matrix can be imaged, as can angiogenesis of the vasa vasorum, plaque inflammation, and fibrin deposits on early nonocclusive vascular thrombosis. Several imaging platforms are available for targeted vascular imaging to acquire information on both anatomy and pathobiology in the same imaging session using either hybrid technology (nuclear combined with CT) or MRI combined with novel probes targeting processes identified by molecular biology to be of importance. This article will discuss the current state of the art of these modalities and challenges to clinical translation.

Myocardial 99mTc-Tetrofosmin Uptake During Adenosine-Induced Vasodilatation With Either a Critical or Mild Coronary Stenosis Comparison With 201Tl and Regional Myocardial Blood Flow

BACKGROUND Clinical studies have shown a comparable coronary stenosis detection rate between 99mTc-tetrofosmin and 201Tl but with smaller defect magnitudes for 99mTc-tetrofosmin. The goals of this study were to measure the first-pass extraction fraction (EF) of 99mTc-tetrofosmin in canine myocardium and to compare 99mTc-tetrofosmin with 201Tl uptake during adenosine-induced vasodilatation in dogs with various degrees of coronary stenosis. METHODS AND RESULTS EF was calculated in 4 anesthetized, open-chest dogs after intracoronary administration of 125I-labeled albumin and 99mTc-tetrofosmin. In another 16 dogs with either critical (n=6) or mild (n= 10) left anterior descending coronary artery (LAD) stenoses, 201Tl and 99mTc-tetrofosmin were administered during adenosine infusion (250 microg x kg(-1) x min(-1)). Dogs were killed 5 minutes later, and tracer activities were determined by ex vivo imaging of heart slices and by well counting. Mean 99mTc-tetrofosmin EF was 54.0+/-3.7%. In the 6 critical-stenosis dogs, the LAD-to-left circumflex artery (LCx) microsphere flow ratio was 0.22+/-0.02 with adenosine. The LAD-to-LCx activity ratios were 0.37+/-0.04 for 201Tl and 0.67+/-0.05 for 99mTc-tetrofosmin (P<.01). For the 10 mild-stenosis dogs, the LAD-to-LCx flow ratio was 0.44+/-0.05. The 201Tl activity ratio was 0.58+/-0.04, compared with 0.81+/-0.04 for 99mTc-tetrofosmin (P<.01). Thus, in both groups, 99mTc-tetrofosmin uptake underestimated the flow disparity more than 201Tl. Similarly, magnitudes of ex vivo image defects were significantly greater for 201Tl than for 99mTc-tetrofosmin in both groups. CONCLUSIONS In this canine model, relative underperfusion with adenosine stress is better resolved with 201Tl than with 99mTc-tetrofosmin and may be explained by the lower EF for 99mTc tetrofosmin. With clinical imaging, greater 201Tl attenuation and redistribution may lessen this advantage.

Glucose regulation of load-induced mTOR signaling and ER stress in mammalian heart.

Background Changes in energy substrate metabolism are first responders to hemodynamic stress in the heart. We have previously shown that hexose‐6‐phosphate levels regulate mammalian target of rapamycin (mTOR) activation in response to insulin. We now tested the hypothesis that inotropic stimulation and increased afterload also regulate mTOR activation via glucose 6‐phosphate (G6P) accumulation. Methods and Results We subjected the working rat heart ex vivo to a high workload in the presence of different energy‐providing substrates including glucose, glucose analogues, and noncarbohydrate substrates. We observed an association between G6P accumulation, mTOR activation, endoplasmic reticulum (ER) stress, and impaired contractile function, all of which were prevented by pretreating animals with rapamycin (mTOR inhibition) or metformin (AMPK activation). The histone deacetylase inhibitor 4‐phenylbutyrate, which relieves ER stress, also improved contractile function. In contrast, adding the glucose analogue 2‐deoxy‐d‐glucose, which is phosphorylated but not further metabolized, to the perfusate resulted in mTOR activation and contractile dysfunction. Next we tested our hypothesis in vivo by transverse aortic constriction in mice. Using a micro‐PET system, we observed enhanced glucose tracer analog uptake and contractile dysfunction preceding dilatation of the left ventricle. In contrast, in hearts overexpressing SERCA2a, ER stress was reduced and contractile function was preserved with hypertrophy. Finally, we examined failing human hearts and found that mechanical unloading decreased G6P levels and ER stress markers. Conclusions We propose that glucose metabolic changes precede and regulate functional (and possibly also structural) remodeling of the heart. We implicate a critical role for G6P in load‐induced mTOR activation and ER stress.

Comparison of Thallium-201 Resting Redistribution With Technetium-99m–Sestamibi Uptake and Functional Response to Dobutamine for Assessment of Myocardial Viability

BACKGROUND 201Tl scintigraphy is useful for determination of viability in patients with coronary artery disease and depressed left ventricular function. Whether 99mTc sestamibi is adequate for viability detection remains controversial. The primary goal of this study was to compare 99mTc-sestamibi uptake with 201Tl uptake in canine models of sustained low flow and regional asynergy for determination of viability. A secondary objective was to compare myocardial uptake of these tracers with the functional response to low-dose dobutamine. METHODS AND RESULTS In protocol 1, 14 open-chested, anesthetized dogs with a 50% reduction in resting left anterior descending coronary artery (LAD) flow underwent 1 hour of transient LAD occlusion followed by reperfusion through the severe stenosis. Then 1.0 mCi of 201Tl was injected, and serial imaging was performed 5 minutes and 2 hours later. After acquisition of the delayed 201Tl image, 10 mCi of 99mTc sestamibi was injected, and imaging was repeated 45 minutes later. No significant difference was seen between the 201Tl defect ratio (LAD/left circumflex coronary artery [LCx]) on redistribution images (0.62 +/- 0.02) and 99mTc-sestamibi defect ratio (0.60 +/- 0.02). Similarly, LAD/LCx activity ratios by gamma-well counting were comparable (0.62 +/- 0.02 versus 0.59 +/- 0.04) and reflected the flow decrement. Systolic thickening was -11 +/- 3% at the time of tracer injection. In protocol 2, 16 dogs underwent serial 201Tl and 99mTc-sestamibi imaging during a 50% reduction in LAD flow with no superimposed transient LAD occlusion. In this model, the 99mTc-sestamibi LAD/LCx image defect ratio (0.61 +/- 0.03) was significantly less than the 201Tl redistribution image defect ratio (0.66 +/- 0.03, P < .01). In 10 dogs, the stenosis was released, resulting in a significant increase in systolic thickening (P = .003), which increased further in response to 5 micrograms.kg-1.min-1 of dobutamine (P = .02). In contrast, thickening increased only from -7 +/- 3% to 2 +/- 4% (P = .004) in response to dobutamine infusion in the remaining 6 dogs with persistent severe LAD stenoses. In protocol 3, 5 dogs received both 201Tl and 99mTc sestamibi to compare the degree of delayed redistribution between tracers at 2 hours. The LAD/LCx microsphere flow ratios when 201Tl and 99mTc sestamibi were injected were 0.44 +/- 0.06 and 0.43 +/- 0.05 (P = NS), respectively. The LAD/LCx activity ratio by gamma-well counting was greater for 201Tl (0.56 +/- 0.08) than 99mTc sestamibi (0.50 +/- 0.07) at 2 hours of redistribution (P < .05), indicating greater redistribution for 201Tl. The LAD/LCx 99mTc-sestamibi defect ratios on serial imaging improved from 0.49 +/- 0.07 to 0.52 +/- 0.07 (P = .0005), consistent with a slight amount of 99mTc-sestamibi redistribution. In protocol 4, no difference between 201Tl and 99mTc-sestamibi defect magnitudes was seen in 4 dogs undergoing 3 hours of total LAD occlusion and ligation of visible coronary collaterals. Infarct size was 68 +/- 19% of the risk area. CONCLUSIONS Although 99mTc-sestamibi and 201Tl defect magnitudes and regional activities were comparable in dogs with sustained low coronary flows and superimposed subendocardial infarctions and in dogs with large infarctions, approximately 5% more 201Tl than 99mTc-sestamibi uptake was observed in dogs with chronic low flow and severe systolic dysfunction. Substantial 99mTc-sestamibi uptake in asynergic zones was observed in this low-flow model, with some slight resting 99mTc-sestamibi redistribution observed on serial images. Systolic thickening was negligibly enhanced during dobutamine infusion in dogs with sustained low flow, whereas 201Tl uptake was only mildly reduced.

99mTc-sestamibi uptake and retention during myocardial ischemia and reperfusion.

Background99mTc-methoxyisobutyl isonitrile (Sestamibi) is a new perfusion agent that has shown promise for the noninvasive detection of myocardial salvage after coronary reperfusion in acute myocardial infarction. The objective of this study was to further validate that myocardial uptake and retention of Sestamibi after reperfusion in a canine myocardial infarction model are markers of tissue viability. The hypotheses tested were that if Sestamibi is given early after reperfusion and myocardial uptake is quantitated soon afterward, the degree of ultimate myocardial salvage will be overestimated, and that there will be continued loss of myocardial Sestamibi from ischemic tissue during 3 hours of reperfusion due to accelerated release of Sestamibi from cells already irreversibly injured during the phase of coronary occlusion, reperfusion injury to myocytes still viable early after reflow, or a combination of both mechanisms Methods and ResultsIn protocol 1, 8.0 mCi Sestamibi was injected intravenously in anesthetized dogs 2-5 minutes after reperfusion preceded by 3 hours of left anterior descending coronary artery ([AD) occlusion. Animals were killed either 5 minutes (n=7) or 3 hours (n=9) after Sestamibi administration. Mean endocardial Sestamibi activity was 74±3% of nonischemic activity in dogs killed early and 31±2% of nonischemic activity in dogs killed late after Sestamibi administration, indicating myocardial loss of Sestamibi during 3 hours of reflow. Regional flow (percent nonischemic) at the time of Sestamibi administration (2–5 minutes after reperfusion) was comparable in dogs killed early (144±23%) and dogs killed late (118±4%, p=NS). In protocol 2, Sestamibi was given intravenously at baseline under normal conditions followed by 3 hours of LAD occlusion and either 4 (n=6), 30 (n=9), or 180 minutes (n=10) of reperfusion. At postmortem, myocardial slices were imaged for quantification of defect magnitude and regional flow (radiolabeled microspheres), and tissue Sestamibi activities were determined by gamma well counting. Coronary sinus Sestamibi activity was serially measured. In these dogs, which were preloaded with Sestamibi at baseline, 3 hours of LAD occlusion followed by 3 hours of reperfusion resulted in a loss of Sestamibi in the endocardial zone of the ischemic region to 40±6% of nonischemic levels (p<0.0001). This loss corresponded to a sustained elevation of coronary sinus activity throughout the reflow period. The loss of myocardial Sestamibi was significantly greater than that observed in dogs killed 4 or 30 minutes after reflow. Defect magnitude also worsened over 3 hours of reperfusion as assessed by gamma camera imaging of slices of the excised hearts. ConclusionThese experimental data suggest that Sestamibi uptake and retention are dependent on myocardial viability as well as regional flow. If Sestamibi is administered early after reperfusion and imaging is performed soon afterward, the degree of myocardial salvage could be significantly overestimated.

Myocardial Uptake and Redistribution of 99mTc-N-NOET in Dogs With Either Sustained Coronary Low Flow or Transient Coronary Occlusion Comparison With 201Tl and Myocardial Blood Flow

BACKGROUND 99mTc-N-NOET (NOET) is a new myocardial perfusion imaging agent that redistributes over time. We sought to better define the redistribution kinetics of NOET using open-chest canine models of sustained low coronary flow (protocol 1) and transient coronary occlusion followed by reflow (protocol 2). METHODS AND RESULTS In protocol 1 (n=10), NOET and 201Tl were injected during low flow in the left anterior descending coronary artery (LAD) that was sustained for 2 hours. Protocol 2 dogs (n=6) were injected with NOET during 20 minutes of LAD occlusion followed by 2 hours of reflow. In both protocols, serial NOET planar images were acquired, and myocardial flow and 2-hour tracer activities were determined by gamma-well counting. Defect resolution was observed on images in both protocols. Initial defect count ratios, reflecting flow disparity at injection (0.66+/-0.03 and 0.57+/-0.04, respectively), increased over 2 hours (0.73+/-0.02 and 0.75+/-0.04, respectively; P<.001 versus initial). Quantitative imaging showed that NOET redistribution resulted from greater clearance from normal areas versus low-flow or transiently occluded areas. In protocol 1, 2-hour NOET and 201Tl stenotic-to-normal tissue activity ratios were similar (0.76+/-0.06 versus 0.73+/-0.04, P=NS) and higher than injection flow ratios (0.52+/-0.06 and 0.56+/-0.07, respectively, P<.001), consistent with tracer redistribution. In protocol 2, NOET redistributed to an even greater extent (injection flow ratio, 0.27+/-0.04; 2-hour tissue activity ratio, 0.84+/-0.03, P<.001). CONCLUSIONS NOET is the first 99mTc-labeled myocardial imaging agent with kinetics similar to 201Tl in experimental models, permitting redistribution imaging. NOET appears to be a promising agent for assessing patients with coronary artery disease.

Intravenously Delivered Mesenchymal Stem Cells

Rationale: Virtually all mesenchymal stem cell (MSC) studies assume that therapeutic effects accrue from local myocardial effects of engrafted MSCs. Because few intravenously administered MSCs engraft in the myocardium, studies have mainly utilized direct myocardial delivery. We adopted a different paradigm. Objective: To test whether intravenously administered MSCs reduce left ventricular (LV) dysfunction both post–acute myocardial infarction and in ischemic cardiomyopathy and that these effects are caused, at least partly, by systemic anti-inflammatory activities. Methods and Results: Mice underwent 45 minutes of left anterior descending artery occlusion. Human MSCs, grown chronically at 5% O2, were administered intravenously. LV function was assessed by serial echocardiography, 2,3,5-triphenyltetrazolium chloride staining determined infarct size, and fluorescence-activated cell sorting assessed cell composition. Fluorescent and radiolabeled MSCs (1×106) were injected 24 hours post–myocardial infarction and homed to regions of myocardial injury; however, the myocardium contained only a small proportion of total MSCs. Mice received 2×106 MSCs or saline intravenously 24 hours post–myocardial infarction (n=16 per group). At day 21, we harvested blood and spleens for fluorescence-activated cell sorting and hearts for 2,3,5-triphenyltetrazolium chloride staining. Adverse LV remodeling and deteriorating LV ejection fraction occurred in control mice with large infarcts (≥25% LV). Intravenous MSCs eliminated the progressive deterioration in LV end-diastolic volume and LV end-systolic volume. MSCs significantly decreased natural killer cells in the heart and spleen and neutrophils in the heart. Specific natural killer cell depletion 24 hours pre–acute myocardial infarction significantly improved infarct size, LV ejection fraction, and adverse LV remodeling, changes associated with decreased neutrophils in the heart. In an ischemic cardiomyopathy model, mice 4 weeks post–myocardial infarction were randomized to tail-vein injection of 2×106 MSCs, with injection repeated at week 3 (n=16) versus PBS control (n=16). MSCs significantly increased LV ejection fraction and decreased LV end-systolic volume. Conclusions: Intravenously administered MSCs for acute myocardial infarction attenuate the progressive deterioration in LV function and adverse remodeling in mice with large infarcts, and in ischemic cardiomyopathy, they improve LV function, effects apparently modulated in part by systemic anti-inflammatory activities.