Open Access

Preliminary evaluation of 18F‑AlF‑NOTA‑MAL‑Cys40‑Exendin‑4 in rodent heart after myocardial ischemia and reperfusion

  • Authors:
    • Xietian Pan
    • Qing Xu
    • Jiangwei Chen
    • Tingting Wang
    • Mingming Zhang
    • Haichang Wang
    • Haokao Gao
  • View Affiliations

  • Published online on: June 27, 2019     https://doi.org/10.3892/mmr.2019.10432
  • Pages: 2276-2284
  • Copyright: © Pan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Glucagon‑like peptide‑1 (GLP‑1) and its receptor (GLP‑1R) exert cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in animal models and human clinical trials. Receptor imaging with positron emission tomography (PET) provides a non‑invasive method for monitoring GLP‑1R expression. In the present study, a fluorine‑18‑labeled aluminum fluoride exendin‑4 analog [18F‑AlF conjugated with 1,4,7‑triazacyclononanetriacetic acid (NOTA)‑maleimide (MAL)‑Cys40‑exendin‑4] was synthesized and evaluated in a rat MI/R model for GLP‑1R imaging. NOTA‑MAL‑Cys40‑exendin‑4 was synthesized by coupling Cys40‑exendin‑4 with NOTA‑MAL. NOTA‑MAL‑Cys40‑exendin‑4 was then conjugated with 18F‑AlF to obtain 18F‑AlF‑NOTA‑MAL‑Cys40‑exendin‑4. The yield of 18F‑AlF‑NOTA‑MAL‑Cys40‑exendin‑4 was 18.5±3.4% (not decay corrected). The process was completed within ~30 min. In rat MI/R models, the tracer exhibited specific binding to GLP‑1R and an appropriate signal‑to‑noise ratio. At 8 h post‑MI/R, tracer uptake reached its peak [0.35±0.053% of injected dose (%ID)/g; n=6] in ischemic myocardium. Localized tracer uptake decreased 1 day (0.20±0.032 %ID/g; n=6) and 3 days (0.16±0.017 %ID/g; n=6) post‑MI/R compared with 8 h post‑MI/R, but still remained higher compared with sham‑operated groups (0.06±0.012 %ID/g; n=6). Pre‑injected unlabeled exendin‑4 effectively blocked tracer accumulation (0.09±0.041 %ID/g; n=6). In conclusion, 18F‑AlF‑NOTA‑MAL‑Cys40‑exendin‑4 demonstrated favorable characteristics for GLP‑1R imaging following MI/R. PET imaging using 18F‑AlF‑NOTA‑MAL‑Cys40‑exendin‑4 in rodent hearts after MI/R revealed a dynamic pattern of GLP‑1R upregulation.

Introduction

Although mortality rates of coronary heart disease worldwide have declined over the past four decades, the disease remains responsible for approximately one-third of all deaths of individuals over the age of 35 (1). Upon acute myocardial ischemia (MI) due to coronary occlusion, restoring myocardial perfusion through either percutaneous coronary intervention or thrombolytic therapy is the most effective treatment. However, reperfusion causes ischemia/reperfusion (I/R) injury, which aggravates myocardial damage (2,3). Recent research suggests great interest in alleviating reperfusion injury following myocardial ischemia (4,5).

Glucagon-like peptide-1 (GLP-1) is a 30-amino-acid-long gut hormone secreted from intestinal endocrine L-cells (6,7). GLP-1 exerts physiological functions through binding to its receptor (GLP-1R), which is a class B heterotrimeric G-protein-coupled receptor (8). GLP-1R is expressed in a number of tissues, including pancreatic islets, heart, lung, kidney, stomach, intestine, pituitary, skin and nervous tissue. Previous studies have demonstrated that GLP-1 exerts cytoprotective effects in the heart after I/R injury (911). Thus, in addition to treating hyperglycemia, GLP-1 offers promising prospects in treating reperfusion injury of the myocardium. However, the cardioprotective effects of GLP-1 after MI/R injury are temporary (9). Therefore, in vivo imaging of GLP-1R may be a useful tool to further study the role of GLP-1 and GLP-1R in treating reperfusion injury.

GLP-1 cannot be used as an imaging tracer as it is rapidly degraded by dipeptidyl-peptidase-IV (DPP-IV) upon being released into the blood (12). Exendin-4, an analog of GLP-1, has a 53% amino acid homology with GLP-1, but is resistant to DPP-IV, thus prolonging its half-life (T1/2) in plasma (13). Numerous attempts have been made to attach exendin-4 to radioisotopes, such as 111In, 99mTc for single-photon emission computed tomography (SPECT) and 68Ga, 64Cu for positron emission tomography (PET) (1416). However, these combinations have certain limits; SPECT has a lower density resolution compared with PET, and 68Ga and 64Cu are not commonly used in the clinic. Fluorine-18 (18F), however, has suitable imaging properties (β+, 0.635 MeV 97%; T1/2, 110 min). Previously, 18F-4-fluorobenzamido-N-ethylamino-maleimide (FBEM)-Cys40-exendin-4 was developed for GLP-1R imaging by PET; 18F-FBEM-Cys40-exendin-4 exhibited high affinity with GLP-1R in an INS-1 rat insulinoma xenograft model and a rat MI/R model (17,18). However, the radiosynthesis of 18F-FBEM-Cys40-exendin-4 is time-consuming, which complicates the application of the tracer.

In the present study, a radiotracer of GLP-1R was synthesized by conjugating 18F-labled aluminum fluoride (18F-AlF) with 1,4,7-triazacyclononanetriacetic acid (NOTA)-maleimide (MAL)-Cys40-exendin-4 in a one-step procedure. Subsequently, 18F-AlF-NOTA-MAL-Cys40-Exendin-4 was evaluated in a rat MI/R model for longitudinal PET imaging.

Materials and methods

Materials and high-performance liquid chromatography (HPLC)

Cys40-exendin-4 was a generous gift from Professor Min Yang (Jiangsu Institute of Nuclear Medicine, Nanjing, China). NOTA-MAL was purchased from CheMatech. 18F-labled fluoride was obtained using the HM-67 cyclotron (Sumitomo Heavy Industries, Ltd.) at the Jiangsu Institute of Nuclear Medicine by proton irradiation of 18O-enriched water. All other commercially obtained reagents were of analytical grade. For semi-preparative HPLC, an Xbridge C18 HPLC column (5 µm, 250×9 mm; Waters Corporation) and a Waters HPLC system with a Q1 Waters 2998 photodiode array detector (Waters Corporation) were used. The system was maintained at room temperature and the sample quantity was 1 ml. No internal standards were used. The flow rate was 5 ml/min. The linear gradient started at 95% A [0.1% trifluoroacetic acid (TFA) in water] and 5% B (0.1% TFA in acetonitrile) for 2 min, and increased to 35% A and 65% B at 32 min.

Preparation of NOTA-MAL-Cys40-exendin-4 analogs

The sequence of Cys40-exendin-4 is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Cys-NH2. Cys40-exendin-4 was conjugated with NOTA-MAL as previously described (19). Briefly, NOTA-MAL (2.16 mg, 3.2 µmol) was mixed with Cys40-exendin-4 (6.77 mg, 1.57 µmol) and dissolved in 6 ml 0.4 M ammonium acetate. Subsequently, 400 µl acetonitrile was added. Following stirring at 40°C overnight, the mixture was purified by semi-preparative HPLC, aforementioned. The desired product was collected and lyophilized.

Radiosynthesis of 18F-AlF-NOTA-MAL-Cys40-exendin-4

In a 2 ml centrifuge tube, 70 µg NOTA-MAL-Cys40-exendin-4 and 10 µl double-distilled water (ddH2O) were charged, and 10 µl glacial acetic acid (CH3COOH) was added. Cyclotron target water containing 18F (~50 µl containing 90 mCi) was added, followed by 280 µl acetonitrile (CH3CN) and 3 µl aluminum chloride (2 mM) in 0.5 M NaOAc (pH=4). After complete mixing, the resulting solution was incubated in oil at 100°C for 10 min. Following cooling, the reaction mixture was diluted with 15 ml ddH2O and eluted through a C18 column. The product was subsequently re-eluted with 20% aqueous ethanol containing 10 mM HCl. The elute was treated with 100 µl of 0.1 M NaOAc (pH 4) and concentrated to ~200 µl total volume under a stream of argon. The sample was diluted with PBS for animal injections.

Quality control of 18F-AlF-NOTA-MAL-Cys40-exendin-4

A sample of the elute (10 µl) was analyzed by reversed-phase HPLC on a C18 column (5 µm, 250×4.6 mm; Phenomenex) with linear gradient at 1 ml/min, as previously described (19). UV absorption and radioactivity were monitored by a Waters 2487 dual λ absorbance detector (Waters corporation) and a Radiomatic 610TR flow scintillation analyzer (PerkinElmer, Inc.), respectively. Mass spectra were obtained using a Q1 Waters LC-MS system (Waters corporation) featuring an Acquity UPLC system coupled with a Waters Q-Tof Premier high-resolution mass spectrometer (Waters Corporation). Positive mode was used with a vaporizer temperature of 300°C, a nebulizer pressure of 20 psi and a gas flow rate of 5 l/min. Positive ions were acquired in the multiple reaction monitoring mode (collision energy, 10 eV).

Animal model of myocardial ischemia and reperfusion

All animal studies were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Laboratory Animal Ethics Committee of The Fourth Military Medical University (Xi'an, China). A total of 36 adult male Sprague-Dawley rats (6–8 weeks old, 200–250 g) obtained from Laboratory Animal Center of the Fourth Military Medical University were used. The animals were housed under a 12:12-h light/dark cycle at 25°C and 50% relative humidity, with access to food and water ad libitum. Intraperitoneal injection of 3% pentobarbital sodium was conducted for anesthesia at a dose of 50 mg/kg initially with modifications according to the depth of anesthesia (20), and the animals were intubated for mechanical ventilation. MI/R models were established by ligation of the left anterior descending coronary artery 2–3 mm from the tip of the auricle with a 6-0 polypropylene suture. Reperfusion was achieved thirty minutes after the coronary artery occlusion by removal of the suture. In the sham-operated group, a suture was placed in the myocardium without ligation of the left coronary artery. The occlusion and reperfusion were confirmed by ST-segment elevation on an electrocardioscope and by 18F-fluorodeoxyglucose (FDG) imaging at post-surgery day 1 (see the ‘Micro-PET imaging’ section below for details).

Echocardiographic assessment of left ventricular (LV) contractility

Echocardiography was performed at day 1 post-MI/R. Gaseous anesthesia was conducted using 3% isoflurane, and the rats were placed on the scanning table. Echocardiographic images were obtained using a small-animal high-resolution imaging unit and a 30-MHz linear transducer (Vevo 770; FUJIFILM VisualSonics, Inc.). LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were measured using a parasternal short-axis view, and LV fractional shortening was calculated as [(LVEDD-LVESD)/LVEDD] ×100%. All measurements were averaged over three successive cardiac cycles.

Micro-PET imaging

PET scans and image analysis were performed using an Inveon micro-PET (Siemens Medical Solutions USA, Inc.). At 8 h, 1 day, 3 days, and 1 week post-MI/R, each rat was injected in the tail vein with 18.5 MBq (500 µCi) 18F-AlF-NOTA-MAL-Cys40-exendin-4 under 3% isoflurane anesthesia (n=6 rats/group). Ten-minute static PET scans were acquired 1 h post-injection. For the blocking experiment, 8 mg/kg of unlabeled Cys40-exendin-4 was pre-injected 10 min prior to the injection of labeled Cys40-exendin-4 at post-MI/R day 1 (n=6). The images were reconstructed using a three-dimensional ordered subsets expectation maximum algorithm, and no correction was applied for attenuation or scatter. For each scan, regions of interest (ROIs) were drawn around the ischemic myocardium, lung and surgical wounds using the vendor software ASI Pro 6.7.1.1 (Siemens AG) on whole-body coronal images. The radioactivity concentration (accumulation) within myocardium was obtained from mean pixel values within the multiple ROI volume, which was converted to MBq/ml/min using a conversion factor. The conversion to MBq/g/min assumed tissue density of 1 g/ml. The values were divided by the administered activity to obtain an image ROI-derived percentage of injected dose per gram (%ID/g). Following 18F-AlF-NOTA-MAL-Cys40-exendin-4 scanning, rats were kept on the scanning gantry for image co-registration for 1 h and injected with 18.5 MBq (500 µCi) of 18F-FDG. After 1 h, 10-min static scans were performed, and images were reconstructed and analyzed using the same procedure.

GLP-1R immunohistochemical staining

Heart samples from the rats were harvested after the rats were fully anesthetized and immediately placed into 10% formalin for fixation at room temperature for 24 h. Formalin-fixed samples were embedded in paraffin. Tissue sections (5-µm) were dried for 1 h at 90°C prior to deparaffinization in xylene, rehydrated by a series of descending ethanol concentrations and subsequently washed with PBS. Antigen retrieval was achieved by heat treatment at 95°C in a pressure cooker for 2 min in 10 mM citrate buffer (pH 6.5). Endogenous peroxidase was blocked at room temperature using 3% hydrogen peroxide for 20 min. The tissue section was incubated at 4°C overnight with a rabbit anti-rat polyclonal GLP-1R antibody (1:200; cat. no. ab188602; Abcam). After washing with PBS, the slide was incubated with a horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (1:50; cat. no. ab6721; Abcam) for 30 min at room temperature. Diaminobenzidine (Sangon Biotech Co., Ltd.) was used for staining according to the manufacturer's protocols, and the analysis was performed using an Olympus BX41 optical microscope (magnification, ×20; Olympus Corporation).

Western blot analysis of GLP-1R

The area of ischemia was defined as the region appearing pale. The corresponding areas of the heart were divided into different samples. The samples of each group (n=3) were homogenized, suspended in RIPA buffer (Sigma-Aldrich, Merck KGaA) and protease inhibitor cocktail (Roche Applied Science), and centrifuged at 14,000 × g for 10 min at 4°C. Protein concentration of the supernatant was measured by the bicinchoninic acid method; 50 µg of protein was separated by Tris-glycine 10% SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed with 5% skimmed milk and 0.1% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The membrane was incubated with rabbit anti-rat polyclonal GLP-1R primary antibody (1:500; cat. no. ab188602; Abcam) at 4°C overnight, washed with PBS, and incubated with HRP-labeled goat anti-rabbit IgG antibody (1:300; cat. no. ab6721; Abcam) at room temperature for 1 h. Chemiluminescence was performed with chemiluminescent HRP substrate using a SuperSignal West Pico Chemiluminescence Kit Detection System (Pierce, Thermo Fisher Scientific, Inc.). β-actin (1:500; cat. no. ab8227; Abcam) was used as internal control; each protein band was quantified using ImageJ software (v1.8.0, National Institutes of Health) and normalized to β-actin.

Statistical analysis

Data are expressed as mean ± SD. Statistical analysis was performed by one-way analysis of variance and a Student-Newman-Keuls multiple comparison post hoc test was performed to identify differences between groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Chemistry and radiochemistry

NOTA-MAL-Cys40-exendin-4 was synthesized by NOTA-MAL and Cys40-exendin-4. Following purification by semi-preparative HPLC, the yield of NOTA-MAL-Cys40-exendin-4 was 68%. Mass spectrometry results indicated a mass-to-charge ratio of 4,712.83 for MH+ (C205H314N56O68S2; calculated molecular weight, 4,713; Fig. 1). Radiosynthesis of 18F-AlF-NOTA-MAL-Cys40-exendin-4 was completed in 30 min, and the yield was 18.5±3.4% (no decay corrected) with a purity of >95%. The retention time of 18F-AlF-NOTA-MAL-Cys40-exendin-4 obtained by analytical HPLC was 23 min (Fig. 2).

Electrocardioscope monitoring during establishment of the MI/R models was used to confirm I/R conditions by ST-T segment change (Fig. 3A). At MI/R day 1, 18F-FDG PET scanning demonstrated a significant defect in the anterolateral wall of the LV (white arrow), whereas no uptake defect was observed in the sham-operated 1 day group (n=6; Fig. 3B). On the same day, M-mode high-resolution ultrasound was performed and revealed a decrease in the amplitude of the anterolateral wall of LV (Fig. 3C). Fractional shortening was decreased from 69±5.2 to 44±3.6% in rats following surgery; no compromised cardiac function was observed by M-mode high-resolution ultrasound in sham-operated animals (Fig. 3C).

Image colocalization

18F-FDG PET imaging was used to identify the area of ischemia. Co-registration of 18F-AlF-NOTA-MAL-Cys40-exendin-4 PET and 18F-FDG PET provided a visual method for anatomically localizing the high-radioactivity area of 18F-AlF-NOTA-MAL-Cys40-exendin-4 in hearts. To facilitate image co-registration, the rats were kept in the scanning gantry under anesthesia during tracer injection and imaging acquisition. Representative myocardial transaxial slices from one rat following in vivo PET imaging of 18F-AlF-NOTA-MAL-Cys40-exendin-4 and 18F-FDG are presented in Fig. 4. Compared with sham-operated rats, the hearts of MI/R day 1 rats exhibited decreased 18F-FDG uptake and increased 18F-AlF-NOTA-MAL-Cys40-exendin-4 uptake in the anterolateral wall of the LV. The area of ischemic myocardium was matched between 18F-FDG and 18F-AlF-NOTA-MAL-Cys40-exendin-4 uptake (Fig. 4).

Dynamic change in GLP-1R in PET imaging

Representative transaxial micro-PET images of 18F-AlF-NOTA-MAL-Cys40-exendin-4 at different time points following MI/R are presented in Fig. 5A. In the lung, the area of ischemia and surgery wound exhibited higher uptake of 18F-AlF-NOTA-MAL-Cys40-exendin-4 compared with surrounding tissues (Fig. 5A). Tracer uptake in the ROI of ischemic myocardium (white arrow) reached a peak (0.35±0.053 %ID/g) at 8 h post-MI/R, which was not observed in the sham-operated group (0.06±0.012 %ID/g; n=6; Fig. 5B). In addition, the radioactivity level remained high at post-MI/R day 1 (0.20±0.032 %ID/g; n=6) and decreased on day 3 (0.16±0.017 %ID/g; n=4; Fig. 5B). Minimal tracer uptake persisted at 1 week post-MI/R (0.08±0.006 %ID/g; n=6), but the levels were not significantly different (Fig. 5B).

To further test the GLP-1R binding specificity of 18F-AlF-NOTA-MAL-Cys40-exendin-4, unlabeled exendin-4 was used as a blocking agent. Tracer uptake was significantly lower compared with unblocked rats at post-MI/R day 1 (0.09±0.041 vs. 0.20±0.02 %ID/g, respectively; P<0.05; Fig. 5A and B).

Ischemic operation did not affect lung uptake of the radiotracer. The %ID/g of the lung remained high throughout the experiment (from 8 h to 1 week following MI/R) at 1.19±0.25 (n=6; Fig. 5C). 18F-AlF-NOTA-MAL-Cys40-exendin-4 accumulation within the surgical wound (yellow arrows) was 0.42±0.12 %ID/g at 8 h following MI/R (Fig. 5C).

Western blotting and immunohistochemical staining

To verify the direct relation between GLP-1R upregulation and high uptake of 18F-ALF-NOTA-MAL-Cys40-exendin-4, western blot analysis and immunohistochemical staining were preformed using a GLP-1R-specific antibody on myocardial samples from MI/R and sham-operated rats. GLP-1R protein level increased at 8 h post-MI/R compared with the sham-operated group (P<0.05; Fig. 6A), and the dynamic change observed later was consistent with that of tracer uptake (Fig. 5B). The immunostaining results revealed notably enhanced cardiomyocyte staining of GLP-1R at 8 h and 1 day post-MI/R compared with the sham-operated group. The staining density decreased with time and returned to control levels in 1 week (Fig. 6B).

Discussion

Reperfusion injury is a complex process that involves a series of signaling pathways (21). From extracellular ligands to intracellular signal cascades, receptors serve a key role in delivering signals. GLP-1 and its analogs have been reported to exert cardioprotective effects in several experimental models and preliminary clinical trials, but the underlying mechanism has yet to be studied (22,23). Therefore, an effective method for monitoring in vivo dynamic changes in GLP-1R expression may be of great value.

PET imaging is a promising method for receptor imaging for research use; however, the synthesis of radiotracers for PET requires consideration of biological and chemical issues. Several positron-emitting radionuclides have been used for PET imaging in the laboratory (64Cu, 68Ga, 18F, 11C), among which 18F stands out for its ideal imaging properties and may have the potential to be translated into clinical practice (14,24). The T1/2 of 18F (109.8 min) allows long-duration, multistep radiosynthesis, and the relatively low positron emission energy (0.64 MeV) provides a short tissue range, thus allowing generation of high-resolution images (25). However, combining 18F with target peptides has remained a challenge. In our previous study, 18F-FBEM-Cys40-exendin-4 was developed, which exhibited high and receptor-specific accumulation in INS-1 rat insulinoma models (17). However, the synthesis of 18F-FBEM-Cys40-exendin-4 requires a multistep procedure, specialized equipment and well-trained personnel, which may counteract the advantages that enable the tracer to translate into clinical practice.

In the present study, a novel GLP-1R specific radiotracer was synthesized by conjugating 18F-AlF to NOTA-MAL-Cys40-exendin-4; the entire process required only one step and was completed in 30 min, which was notably less compared with the time required to synthesize 18F-FBEM-Cys40-exendin-4 (17) and other GLP-1R-based radiotracers such as 18F-FBEM-EM3106B (26) and 18F-FBEM-Cys39-exendin-4 (27). The yield of 18F-AlF-NOTA-MAL-Cys40-exendin-4 was moderate at 18.5±3.4% (not decay corrected), with a purity of >95%. No HPLC purification was required, thus simplifying the synthesis process.

Longitudinal PET in a rat model of MI/R with 18F-AlF-NOTA-MAL-Cys40-exendin-4 was performed. Ischemic myocardium, lung and surgical wounds showed higher tracer accumulation compared with other tissues. Pre-injected unlabeled Cys40-exendin-4 significantly blocked tracer accumulation, which indicated that the accumulation of 18F-AlF-NOTA-MAL-Cys40-exendin-4 was GLP-1R-specific. Preliminary western blotting experiments demonstrated that GLP-1R expression was highest at 8 h following ischemia/reperfusion injury; therefore, 8 h was indicated as a critical time point. Compared with focal tracer uptake in ischemic myocardium at 8 h, 1 day, 3 days, and 1 week following the onset of ischemia, tracer uptake reached the highest level at 8 h post-MI/R; tracer accumulation then decreased over time. At 1 week post-MI/R, tracer uptake within ischemic myocardium was restored to the near-normal level. The performance of 18F-AlF-NOTA-MAL-Cys40-exendin-4 as a radiotracer in rat hearts was similar to that of 18F-FBEM-Cys40-exendin-4 (18). The results of western blotting and immunohistochemical staining were consistent with PET. Thus, a non-invasive method for molecular imaging was established to monitor dynamic changes in GLP-1R expression in ischemic myocardium in vivo. However, the experiments conducted in the present study offered only indirect evidence of association of the analog with GLP-1R, and the mechanism by which reperfusion induces GLP-1R upregulation remains unclear and requires further investigation.

Lung tissue exhibited higher tracer accumulation compared with ischemic myocardium at all time points owing to high levels of GLP-1R expression, which may compromise the value of 18F-AlF-NOTA-MAL-Cys40-exendin-4 in clinical use. However, considering that human lung tissue expresses significantly lower levels of GLP-1R compared with rat lung tissue, the ischemic myocardium-to-lung ratio of tracer uptake may be higher in humans (28). The analog may be eliminated through the lungs in a pharmacokinetic pathway, which requires further investigation. Surgical wounds exhibited increased tracer accumulation that could not be completely blocked with unlabeled Cys40-exendin-4. Thus, the increased accumulation of tracer in surgical wounds may have been due to increased vascular permeability caused by inflammatory reaction.

The results of the present preliminary evaluation of 18F-AlF-NOTA-MAL-Cys40-exendin-4 revealed a dynamic pattern of GLP-1R expression following MI/R in rats, which suggested that GLP-1 may exert its cardioprotective effects early following MI/R, when GLP-1R expression is relatively high. After 1 week, the therapeutic effect of GLP-1 may be compromised owing to low receptor expression. Consistent with the present study, previous studies demonstrated that the administration of GLP-1R agonists during the early stages of acute cardiac ischemia protected against MI/R injury (9,29).

In conclusion, 18F-AlF-NOTA-MAL-Cys40-exendin-4 was synthesized in a time-efficient and effective way. PET imaging using 18F-AlF-NOTA-MAL-Cys40-exendin-4 displayed high ischemic myocardium-to-background radioactivity ratios, and further analysis revealed dynamic changes in GLP-1R expression in rats following MI/R. The radiotracer offers a new approach to observe the dynamic changes of GLP-1R expression following MI/R injury, which may help clinicians make decisions regarding the exact time points of GLP-1R agonist administration during MI/R injury treatment.

Acknowledgements

Not applicable.

Funding

The present study was supported by The National Natural Science Foundation of China (grant no. 81570210).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XP established the myocardial ischemia/reperfusion rat models, performed positron emission tomography imaging and wrote the manuscript. QX synthesized the radiotracer and contributed to the acquisition of data. JC established the animal models. TW performed western blot analysis and immunohistochemical staining. MZ analyzed the data and revised the manuscript. HW and HG designed the experiments and critically revised the manuscript.

Ethics approval and consent to participate

All animal studies were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Laboratory Animal Ethics Committee of The Fourth Military Medical University (Xi'an, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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September 2019
Volume 20 Issue 3

Print ISSN: 1791-2997
Online ISSN:1791-3004

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APA
Pan, X., Xu, Q., Chen, J., Wang, T., Zhang, M., Wang, H., & Gao, H. (2019). Preliminary evaluation of 18F‑AlF‑NOTA‑MAL‑Cys40‑Exendin‑4 in rodent heart after myocardial ischemia and reperfusion. Molecular Medicine Reports, 20, 2276-2284. https://doi.org/10.3892/mmr.2019.10432
MLA
Pan, X., Xu, Q., Chen, J., Wang, T., Zhang, M., Wang, H., Gao, H."Preliminary evaluation of 18F‑AlF‑NOTA‑MAL‑Cys40‑Exendin‑4 in rodent heart after myocardial ischemia and reperfusion". Molecular Medicine Reports 20.3 (2019): 2276-2284.
Chicago
Pan, X., Xu, Q., Chen, J., Wang, T., Zhang, M., Wang, H., Gao, H."Preliminary evaluation of 18F‑AlF‑NOTA‑MAL‑Cys40‑Exendin‑4 in rodent heart after myocardial ischemia and reperfusion". Molecular Medicine Reports 20, no. 3 (2019): 2276-2284. https://doi.org/10.3892/mmr.2019.10432