Author + information
- Received September 20, 2012
- Accepted September 25, 2012
- Published online December 18, 2012.
- Kenji Fukushima, MD, PhD⁎,
- Paco E. Bravo, MD⁎,
- Takahiro Higuchi, MD, PhD⁎,
- Karl H. Schuleri, MD†,
- Xiaoping Lin, BS†,
- M. Roselle Abraham, MD†,
- Jinsong Xia, MD⁎,
- William B. Mathews, PhD⁎,
- Robert F. Dannals, PhD⁎,
- Albert C. Lardo, PhD†,
- Zsolt Szabo, MD⁎ and
- Frank M. Bengel, MD⁎,‡,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Frank M. Bengel, Department of Nuclear Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany
Objectives The goal of this study was to explore the feasibility of targeted imaging of the angiotensin II type 1 receptor (AT1R) in cardiac tissue, using clinical hybrid positron emission tomography/computed tomography (PET/CT).
Background AT1R is an attractive imaging target due to its key role in various cardiac pathologies, including post-infarct left ventricular remodeling.
Methods Using the novel AT1R ligand [11C]-KR31173, dynamic PET/CT was performed in young farm pigs under healthy conditions (n = 4) and 3 to 4 weeks after experimental myocardial infarction (n = 5). Ex vivo validation was carried out by immunohistochemistry and polymerase chain reaction. First-in-man application was performed in 4 healthy volunteers at baseline and under AT1R blocking.
Results In healthy pigs, myocardial KR31173 retention was detectable, regionally homogeneous, and specific for AT1R, as confirmed by blocking experiments. Metabolism in plasma was low (85 ± 2% of intact tracer after 60 min). After myocardial infarction, KR31173 retention, corrected for regional perfusion, revealed AT1R up-regulation in the infarct area relative to remote myocardium, whereas retention was elevated in both regions when compared with myocardium of healthy controls (8.7 ± 0.8% and 7.1 ± 0.3%/min vs. 5.8 ± 0.4%/min for infarct and remote, respectively, vs. healthy controls; p < 0.01 each). Postmortem analysis confirmed AT1R up-regulation in remote and infarct tissue. First-in-man application was safe, and showed detectable and specific myocardial KR31173 retention, albeit at a lower level than pigs (left ventricular average retention: 1.2 ± 0.1%/min vs. 4.4 ± 1.2%/min for humans vs. pigs; p = 0.04).
Conclusions Noninvasive imaging of cardiac AT1R expression is feasible using clinical PET/CT technology. Results provide a rationale for broader clinical testing of AT1R-targeted molecular imaging.
- angiotensin receptor
- molecular imaging
- myocardial infarction
- positron emission tomography
- renin-angiotensin system
Scientific work in recent years has highlighted the role of the renin-angiotensin system (RAS) in cardiac pathology (1–3). In addition to the circulating RAS, which contributes to the systemic regulation of global cardiovascular homeostasis, the heart has an intrinsic RAS that mediates locoregional mechanisms such as interstitial fibrosis, myocyte hypertrophy, and apoptosis (4). Activation of the intrinsic myocardial RAS may contribute to changes of geometry, structure, and function, which are hallmarks of heart failure progression.
Interest in targeted imaging of maladaptive mechanisms contributing to heart failure and left ventricular remodeling is increasing (5). Novel molecular imaging techniques may not only improve pathophysiological understanding. They may also provide prognostic value and refine therapy. In this context, the myocardial RAS appears to be an attractive target. Using ligands for the primary RAS mediator in myocardium, the angiotensin II type 1 receptor (AT1R), studies showed that regional AT1R up-regulation can be visualized noninvasively in rodents after myocardial infarction (6,7). But interspecies differences of RAS have been reported (8,9), and the usefulness of cardiac AT1R imaging in large mammals and humans remains to be demonstrated.
Accordingly, we sought to explore the translational potential of myocardial AT1R imaging, by using clinical hybrid positron emission tomography/computed tomography (PET/CT).
The study protocol was approved by the Johns Hopkins Institutional Animal Care and Use Committee. Animals were maintained according to the principles of the American Physiological Society. Nine young female farm pigs (20 to 30 kg) were enrolled. For experimental procedures, animals were held under general anesthesia (induction with ketamine hydrochloride 200 to 400 mg, maintenance with 1.2% to 2.0% isoflurane). For myocardial infarction, coronary catheterization was performed as previously described (10) in 5 animals. Balloon occlusion of the mid left anterior descending coronary artery was performed for 120 min. Post-operative treatment included narcotics and nonsteroidal anti-inflammatory drugs. PET/CT was performed 3 to 4 weeks later. Four animals underwent PET/CT under healthy conditions.
Synthesis of the AT1R-ligand [11C]-KR31173 was performed as described (11–13).
PET/CT was conducted using a GE Discovery Rx VCT scanner (GE Healthcare, Waukesha, Wisconsin). Pigs were anesthetized and positioned supine in a cradle. A CT scout scan was followed by low-dose CT for attenuation correction. [11C]-KR31173 was administered intravenously via ear vein (300 to 500 MBq). List-mode acquisition (60 min) was started simultaneously. Venous blood was taken at 20, 40, and 60 min to determine plasma metabolites by column-switch high-performance liquid chromatography (11). After waiting for radioactivity decay (100 min after injection), another low-dose CT was obtained, followed by intravenous [13N]-ammonia (NH3) infusion (370 to 555 MBq) and 20-min list-mode acquisition. Next, a delayed contrast-enhancement CT was performed as described (14), using 70 ml of Visipaque (GE Healthcare) and helical, retrospectively gated acquisition 10 min after injection.
In 3 healthy pigs, PET/CT was repeated under blocking conditions (SK-1080, 2 mg/kg intravenously, 30 min before [11C]-KR31173 injection) (12).
List-mode data were resampled to attenuation-corrected, iteratively reconstructed, tomographic images. Alignment of CT and PET was checked using fusion software (15). Static images were obtained for KR31173 (30 to 60 min) and NH3 (10 to 20 min). Electrocardiogram-gated images were obtained for NH3 (8 bins), and dynamic images were obtained for both agents (27 frames for KR31173: 12 × 10, 6 × 60, 4 × 180, 2 × 300, 3 × 600 s; 21 frames for NH3: 12 × 10, 6 × 30, 3 × 300 s).
Static PET data were volumetrically sampled, and myocardial polar maps were generated. A threshold of 60% of the individual maximum was used to define perfusion defect (16).
For quantitative analysis, myocardial segments defined from static NH3 images were applied to dynamic series, and time-activity curves were obtained. Additionally, the arterial input function was defined by a small region of interest in the left ventricular cavity. Absolute myocardial blood flow at rest was quantified using a 3-compartment model for NH3 (17). For KR31173, a myocardial retention index (KR-ret) was determined according to the following formula:
In this formula, t is the time after injection at which KR-ret is measured, Ctissue is the tracer concentration in myocardial tissue at the given time t, and the denominator describes the integral of the tracer concentration in arterial blood (Cblood) at all time points (θ) between injection and t.From gated NH3 datasets, resting left ventricular ejection fraction (LVEF) and end-diastolic volume were measured as previously reported (18).
Plasma levels of RAS components
Venous blood samples were obtained in animals before infarction and again on the day of the PET/CT scan. Plasma levels of renin, angiotensin II, and aldosterone were measured using commercial services (Quest Diagnostics, Madison, New Jersey).
After imaging, anesthetized pigs were euthanized (4 mmol/l potassium chloride intravenously). Hearts were removed, and gross left ventricular short-axis slices were created (10). Under guidance by TTC (triphenyltetrazolium chloride, 12.5 ml/kg, 2%) stain, samples were collected from the infarct, remote region, and right ventricle. Samples were frozen at −80°C for polymerase chain reaction (PCR), or fixed in 10% formaldehyde for histology/immunohistochemistry. Thin slices were stained by hematoxylin and eosin (HE) or Masson trichrome. AT1R immunohistochemistry was performed using goat polyclonal AT1R antibody (Santa Cruz Biotechnology, Santa Cruz, California).
For quantitative PCR, total RNA was extracted from homogenized tissue (RNAeasy, Qiagen, Valencia, California). Reverse-transcription PCR (1 μg of RNA) was performed using qScript cDNA Kit (Quanta BioSciences, Gaithersburg, Maryland). Real-time quantitative PCR was performed with StepOnePlus (Applied Biosystems, Foster City, California), using PerfeCTa SYBR Green FastMix, ROX (Quanta BioSciences). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) served as internal control. The sequences of the primers for porcine AT1R and GAPDH are specified in Table 1 (19). According to the delta CT method, expression of AT1R mRNA was normalized to GAPDH mRNA and related to sample measurements from noninfarcted myocardium. Specific amplification was confirmed by melting curve analysis. Experiments were performed in 3 samples and repeated 3 times for each sample.
First-in-man application of [11C]-KR31173
Approval for human application of KR31173 was obtained by investigational new drug application from the Food and Drug Administration, and from the Johns Hopkins Institutional Review Board. Safety was the primary focus. Accordingly, 4 healthy volunteers (male, age 24 ± 2 years) were included. Cardiovascular or other disease was ruled out by clinical examination, history, blood testing, and electrocardiogram. After written informed consent, subjects underwent KR31173 PET/CT. Injected activity was 650 ± 54 (579 to 709) MBq, specific activity 139.3 ± 66.9 (75 to 230.4) GBq/μmol, and injected cold mass 2.96 ± 1.18 (1.53 to 4.22) μg of KR31173. Based on animal data, which suggested an effective dose of 9.05 μSv/MBq, the individual effective dose was calculated to be 5.9 ± 0.5 mSv. Blood pressure, heart rate, and subjective symptoms were assessed before tracer injection, during imaging, and at the end of the scan. No NH3 or CT contrast agent was given. Otherwise, PET/CT acquisition and data analysis were performed as described in the previous text for animal studies. In 2 subjects, imaging was repeated on the subsequent day under AT1R blocking conditions, 3 h after an oral dose of 40-mg olmesartan.
All results are expressed as mean ± SD. Results within the same group were compared by paired t test. Results between different groups were compared by unpaired t test. Due to possible heterogeneity of variance, KR31173 retention in humans versus animals was compared by Welch t test. All testing was performed using SPSS version 13 (SPSS, Chicago, Illinois) and PRISM ver.5 (GraphPad Software, La Jolla, California). p < 0.05 was considered statistically significant.
Kinetics of [11C]-KR31173 in healthy porcine myocardium
Myocardial KR31173 was clearly visualized and regionally homogeneous, similar to NH3 uptake (Fig. 1A). Strong liver uptake of KR31173 was present, but did not interfere with myocardial visualization. Kinetic analysis showed stable myocardial retention over time (Fig. 1B). Global KR-ret was 5.5 ± 1.58%/min, 4.10 ± 0.76%/min, 3.62 ± 0.51%/min at 10, 30, and 60 min. Plasma metabolite analysis revealed tracer stability (89 ± 1% of tracer intact at 10 min after injection, 88 ± 3% at 30 min, and 85 ± 2% at 60 min). Blocking resulted in complete disappearance of myocardial KR31173 uptake (Fig. 1C). Quantitatively, KR-ret was reduced by >90% (0.39 ± 0.10%/min at 30 min; p < 0.001).
Pig model of myocardial infarction
Quantitatively, perfusion defect size comprised 26 ± 6% of the left ventricle. CT delayed enhancement matched the perfusion defect (Fig. 2), showing mostly transmural tissue damage associated with wall thinning, and nontransmural enhancement in a small border zone. The LVEF was 32 ± 5%. Compared with healthy normal myocardium (0.40 ± 0.14 ml/min/g), global myocardial blood flow was significantly reduced in the infarct region (0.18 ± 0.04 ml/min/g; p < 0.001), but not in the remote region (0.39 ± 0.08 ml/min/g; p = 0.89) of infarcted animals. Probably due to the general cardio-suppressive effect of anesthesia (20), overall blood flow was low.
Systemic renin, angiotensin II, and aldosterone were unchanged between pre- and post-infarct states. Plasma concentrations were 0.18 ± 0.2 μg/l versus 0.15 ± 0.12 μg/l, 15.0 ± 2.0 ng/ml versus 13.0 ± 3.6 ng/ml, and 2.9 ± 1.4 ng/ml versus 2.5 ± 1.9 ng/ml for renin, angiotensin II, and aldosterone, respectively (all p = NS).
Regional [11C]-KR31173 retention after myocardial infarction
Visual comparison of KR31173 and NH3 images suggests that KR31173 uptake in the hypoperfused infarct region exceeds residual regional perfusion (Fig. 2). Notably, right ventricular uptake appears enhanced, too.
Comparison of absolute KR-ret with healthy controls suggests a global up-regulation in infarcted animals (Fig. 3A). Uncorrected KR-ret at 30 min was significantly elevated in the remote area of infarcted animals (6.10 ± 0.64%/min) when compared with the infarct region (2.78 ± 0.82%/min; p = 0.002) and normal myocardium of healthy controls (4.10 ± 0.25%/min; p < 0.001). When KR-ret was normalized to regional perfusion in order to correct for partial volume effects and reduced flow in the infarct region (Fig. 3B), the elevation of KR-ret in remote regions persisted, but regional KR-ret was most strongly up-regulated in the infarct region (0.087 ± 0.008%/min vs. 0.071 ± 0.003%/min vs. 0.058 ± 0.004%/min for infarct, remote, and control, respectively; p = 0.001 for control vs. infarct, p = 0.015 for infarct vs. remote, and p = 0.005 for remote vs. control).
Ex vivo tissue analysis
Comparison of tissue samples (Fig. 4A) confirmed in vivo findings. Quantitative PCR revealed significantly increased AT1R expression in the infarct and remote regions compared with healthy controls (p < 0.001 and p = 0.016, respectively). There was also a trend toward higher expression in the infarct versus the remote region (p = 0.057). Notably, AT1R expression in the right ventricle of infarcted animals was highest (p < 0.001 vs. all others) (Fig. 4B).
Histological analysis showed necrosis and fibrosis on HE and Masson trichrome staining in the infarct area, whereas both were absent in remote myocardium. Immunohistochemistry showed anti-AT1R antibody binding to spindle-shaped cells, presumably myofibroblasts, in the infarct region, whereas there was also diffuse binding to cardiomyocytes in remote areas (Fig. 4C).
None of the human subjects reported any symptoms after KR31173 administration. Blood pressure (mean arterial pressure: 77 ± 9 mm Hg before vs. 77 ± 9 mm Hg during imaging) and heart rate (68 ± 9 beats/min vs. 65 ± 7 beats/min) remained stable. No adverse events were recorded.
At baseline, myocardial KR31173 retention was visually detectable and regionally homogeneous (Fig. 5). Similar to pigs, retention was stable over time, and strong liver uptake was present. Absolute left ventricular retention was 1.2 ± 0.1%/min at 40 min, which was significantly lower compared with healthy pigs (4.4 ± 1.2%/min; p = 0.04).
After blocking with olmesartan, myocardial retention disappeared, and blood pool activity increased. KR31173 retention dropped from 1.3 ± 0.1%/min to 0.7 ± 0.1%/min. Residual retention after blockade was mostly attributable to myocardial spillover from the elevated blood pool.
To our knowledge, this is the first report of noninvasive in vivo visualization of myocardial AT1R expression in large mammals and humans. Proof of safety and feasibility provides a rationale for further clinical testing.
On the myocardial tissue level, RAS activation stimulates cell growth, inflammation, fibrosis, and apoptosis through AT1R (21). Modest activation is thought to be part of the adaptive response to tissue damage and contributes to repair (4). But an excessive, maladaptive response may contribute to tissue remodeling and loss of function. A diagnostic method that identifies AT1R up-regulation on the myocardial tissue level may be of clinical value in several ways: First, if quantified early after acute myocardial infarction, the severity of up-regulation may predict risk for subsequent heart failure development. Second, myocardial AT1R imaging may be useful in other situations, such as left ventricular hypertrophy and hypertension, and it may further elucidate the link between renal and cardiovascular disease. And third, molecular imaging may be used for individual optimization of AT1R blockade, based on the level of receptor occupancy in target tissue. The present study provides a foundation for future testing of these hypotheses.
Of note, species differences of RAS/AT1R need to be considered. Prior work in rats (6) and mice (7) showed strong up-regulation of AT1R in the infarct area, without imaging signal from remote or healthy myocardium. This is in contrast to pigs, where remote myocardium showed significant AT1R binding, and where regional up-regulation in the infarct region was less pronounced. Prior experimental work confirms these differences between species (8,9,22). Notably, humans, like pigs, showed detectable levels of myocardial AT1R at baseline, although absolute retention was significantly lower. Whether this is due to further species differences, or due to an effect of anesthesia in animals cannot be clarified.
The presence of up-regulation of myocardial AT1R in the absence of changes of systemic RAS components emphasizes the value of noninvasive imaging of myocardial tissue. The existence of an intrinsic RAS in myocardial tissue is now well established (4,9,23). Work by other imaging groups has recently focused on imaging of angiotensin-converting enzyme as another component of myocardial tissue RAS, which is upstream of AT1R (24). This may add an additional perspective to RAS imaging, because multiple tracers may be combined for dissecting molecular mechanisms. In addition to developing tracers for other molecular components of RAS, probes for AT1R may also be optimized, for example, by focusing on labeling with the more broadly available isotope fluorine-18, or by focusing on a reduction of high nonspecific liver uptake.
It is noteworthy that our study also showed significant elevation of AT1R in the right ventricle. This is along the lines of prior work, where right ventricular angiotensin binding was also found to be enhanced after infarction (9), but the finding was not discussed in detail. Differential alterations of loading conditions during anesthesia may be a contributor (20), but the underlying mechanisms for this observation cannot be clarified from the present study. Although PET imaging of the right ventricle is not reliable due to partial volume effects resulting from the thin wall, the role of the low-pressure circulation in RAS-related cardiac pathology deserves some attention in future studies.
First, sacrifice of animals after imaging was necessary to obtain ex vivo validation. Our study thus lacks information about the time course of AT1R after myocardial infarction. Prior work in rats suggests a peak after 3 weeks (6), which supports the timing of imaging in the present study. Second, ex vivo tissue workup shows a general limitation of in vivo imaging at relatively low spatial resolution—the imaging signal is not specific for cell type. As suggested by immunostaining in this and other studies (7), the AT1R signal in the infarct region originates mostly from myofibroblasts (25), whereas the signal from remote and healthy myocardium originates from myocytes. It is not clear if and how the cell-specific origin of AT1R up-regulation is relevant for healing and remodeling, but integrated imaging may help to overcome this limitation. Combination with CT delayed enhancement localizes the AT1R signal to scar or intact tissue. Of note, specificity of the tracer for AT1R has been shown previously, not only in healthy myocardium, but also in infarcted myocardium or extracardiac tissue (6,13). Third, the employed pig model is not entirely representative of clinical post-infarct remodeling. General depressive effects of anesthesia may explain low LVEF and low blood flow (20), the observation period is short, and there is absence of coronary atherosclerotic disease. Nevertheless, this pig model is established to study the biological effects of tissue damage (20), and our results provide a rationale for further clinical testing. Fourth, we employed a retention index for [11C]-KR31173 to quantify AT1R expression. Although this approach corrects for the amount of available tracer, it is dependent on partial volume and does not provide truly quantitative receptor density. More sophisticated approaches are established for other tracers (26), but have not yet been validated for AT1R. Finally, our results in humans must be seen as a first step. With a focus on safety, healthy volunteers were studied as in a clinical phase 1 trial. Given the significant regulatory burden for human use of a new imaging compound, this represents an important transitional step and provides the foundation for subsequent disease-focused clinical studies.
Targeted molecular imaging of cardiac AT1R expression is feasible using clinical PET/CT technology. In the future, this technique may provide unique insights into regional myocardial RAS activation in cardiac disease.
The authors thank the staff of the PET/CT center of Johns Hopkins Hospital for their excellent technical assistance.
This work was supported in part by National Institutes of Health grant R01HL092985. Dr. Fukushima was supported by a SNM Wagner-Torizuka fellowship. [11C]-KR31173 precursor was courtesy of Dr. Sung-Eun Yoo from the Center for Biological Modulators/KRICT, Taejeon, Korea. The authors have stated that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- angiotensin II type 1 receptor
- glyceraldehyde-3-phosphate dehydrogenase
- hematoxylin and eosin
- retention index of KR31173
- left ventricular ejection fraction
- polymerase chain reaction
- hybrid positron emission tomography/computed tomography
- renin angiotensin system
- Received September 20, 2012.
- Accepted September 25, 2012.
- American College of Cardiology Foundation
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