Comparison of a New 68Ga-Radiolabelled PET Imaging Agent sCD146 and RGD Peptide for In Vivo Evaluation of Angiogenesis in Mouse Model of Myocardial Infarction

Ischemic vascular diseases are associated with elevated tissue expression of angiomotin (AMOT), a promising molecular target for PET imaging. On that basis, we developed an AMOT-targeting radiotracer, 68Ga-sCD146 and performed the first in vivo evaluation on a myocardial infarction mice model and then, compared AMOT expression and αvβ3-integrin expression with 68Ga-sCD146 and 68Ga-RGD2 imaging. After myocardial infarction (MI) induced by permanent ligation of the left anterior descending coronary artery, myocardial perfusion was evaluated by Doppler ultrasound and by 18F-FDG PET imaging. 68Ga-sCD146 and 68Ga-RGD2 PET imaging were performed. In myocardial infarction model, heart-to-muscle ratio of 68Ga-sCD146 imaging showed a significantly higher radiotracer uptake in the infarcted area of MI animals than in sham (* p = 0.04). Interestingly, we also observed significant correlations between 68Ga-sCD146 imaging and delayed residual perfusion assessed by 18F-FDG (* p = 0.04), with lowest tissue fibrosis assessed by histological staining (* p = 0.04) and with functional recovery assessed by ultrasound imaging (** p = 0.01). 68Ga-sCD146 demonstrated an increase in AMOT expression after MI. Altogether, significant correlations of early post-ischemic 68Ga-sCD146 uptake with late heart perfusion, lower tissue fibrosis and better functional recovery, make 68Ga-sCD146 a promising radiotracer for tissue angiogenesis assessment after MI.


Introduction
Myocardial infarction (MI) is a complex disease underlying different pathophysiological processes and issues. Early reperfusion therapies such as primary coronary intervention decrease mortality. Yet, functional recovery is often limited due to residual microcirculation dysfunction [1]. Boosted angiogenesis after acute MI is associated with favorable outcome in animal models as evidenced by better preserved heart function [2]. Post-MI angiogenesis processes are still subject to intensive research and many efforts are still committed to develop innovative pro-angiogenic therapeutic strategies based on pharmacological agents, cells [3], extracellular vesicles [4] or secretome [5]. In parallel with these developments, non-invasive clinical evaluation of the angiogenic process is critical for post-ischemic risk stratification, therapeutic eligibility, follow-up and management.
A non-invasive method assessing tissue angiogenic status could therefore offer a refined prognosis real-time monitoring of treatment efficacy. Positron emission tomography coupled with computed tomography (PET/CT) may represent an efficient and sensitive imaging modality to quantify angiogenesis, but the ideal molecular target and its associated radiotracer remain to be identified, developed and validated. Indeed, almost two decades after the first developments, PET imaging agents targeting integrins or molecules of the VEGF pathway still have not reached registration [6]. Several clinical studies in patients with MI and stroke showed an increased PET signal in the ischemic region in direct correlation with the phase and severity of the disease. However, these positive results must be confirmed in a larger number of patients in order to refine the prognostic power of these tracers and their use in the evaluation of pro-angiogenic therapies [7][8][9][10]. Efforts are needed to find innovative and relevant molecular targets.
Among them, we recently reported that angiomotin (AMOT) imaging was of interest for PET imaging of angiogenesis and tissue regeneration [11]. AMOT involvement in angiogenesis is widely described both in ischemic pathology [11], in post-hypoxia adaptation [12], in chronic intermittent hypoxia with re-oxygenation [13] and also during tumour vasculature growth promotion [14,15].
AMOT was first identified as an angiostatin-binding protein involved in the regulation of endothelial cell polarization, migration, proliferation and angiogenesis [16][17][18]. More recently, the soluble cluster of differentiation 146 (sCD146) was described as an endogenous AMOT ligand promoting angiogenic effects on endothelial populations [19]. sCD146 presented angiogenic properties in vitro and in vivo in hindlimb ischemia models [20] and tumour-bearing mouse models [21]. Furthermore, numerous reports highlighted that serum levels of sCD146 could be considered as a novel biomarker of heart failure patients [22,23].
Based on these observations, we recently developed an AMOT-targeting radiotracer, 68 Ga-sCD146 and proved its value as an angiogenesis PET imaging agent and as an early predictive tool to predict delayed revascularization in mouse hindlimb ischemia model [11]. The aim of this study was therefore to evaluate AMOT PET imaging using 68 Ga-sCD146 for post-ischemic angiogenesis evaluation and its predictive value for myocardial tissue recovery.

Animals
All procedures using animals were approved by the Institution's Animal Care and Use Committee (Project #2017040416045642 CE14 Aix-Marseille University) and were conducted according to the EU Directive 2010/63. Ten-week-old male CD1 mice (Janvier Labs) were housed in enriched cages placed in a temperature-and hygrometry-controlled room with daily monitoring and fed with water and commercial diet ad libitum.

Mouse Model of Myocardial Infarction
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection and following endotracheal intubation, were artificially ventilated. If necessary 1-2% isoflurane was added as maintenance anesthetic. For analgesia, buprenorphine. (0.1 mg/kg) was injected subcutaneously 30 min prior surgery. Following skin incision, lateral thoracotomy at the fourth intercostal space was performed by blunt dissection of the intercostal muscles. Under stereomicroscope control, the left anterior descending coronary artery was visualized and ligated (with 8.0 nonabsorbable silk suture) 2.0 mm below the left atrium, just above the bifurcation of the left diagonal arteries. Effective ligation of the coronary artery was confirmed by whitening of the LV affected region below the ligation site. The thoracic wall and skin incisions were then sutured with 6.0 non-absorbable and 4.0 absorbable silk sutures, respectively. Mice were then warmed for several minutes until recovery.

Ultrasound Imaging
In vivo heart structure and function of CD1 mice were evaluated using a high-frequency scanner (Vevo2100 VisualSonics, Bothell, WA, USA). Briefly, mice were anesthetized with 1-2% isoflurane inhalation and placed on a heated platform to maintain temperature during the analysis. Two-dimensional imaging was recorded with a 22-55 MHz transducer (MS550D) to capture long-and short-axis projections with guided M-Mode and B-Mode. Left parasternal long axis and left short axis view in M-mode. The left ventricular ejection fraction (VEF) is expressed as % and calculated by dividing the systolic ejection volume (TDV) and the telesystolic volume STV), commonly marked ESV) by the end-diastolic volume (TDV).

MicroPET/CT Imaging
Procedures for microPET/CT imaging experiments are summarized in Figure 1. Mi-croPET/CT acquisitions were performed on a NanoscanPET/CT camera (Mediso, Budapest, Hungary). For each PET tracer, radioactivity was injected intravenously in the retro-orbital sinus. Mice were maintained under 2% isoflurane anesthesia during acquisition. Static microPET imaging was performed 1 h after each radiotracer injection, during 20 min. Glucose metabolism was evaluated by 18 F-FDG imaging at early and later time after myocardial infarction. Indeed, while a normal 18 F-FDG uptake is characteristic of viable myocardium, a low uptake of 18 F-FDG indicates a hibernating myocardium and the absence of 18 F-FDG uptake indicates scar tissue.
Seven myocardial infarction mice and 4 sham mice, fasted for 4 h, were IV injected with 10 ± 3.5 MBq/50 µL of 18 F-FDG on days 16 and 30 post-ischemia. MicroPET images were acquired 1 h after injection.
Seven myocardial infarction mice and 4 sham mice were IV injected with 5.0 ± 2.4 MBq/100 µL of 68 Ga-sCD146 on days 15 and 22 post-MI. MicroPET images were acquired 1 h after injection.
Seven myocardial infarction mice and 4 sham mice were IV injected with 5.0 ± 3.2 MBq/100 µL of 68 Ga-RGD 2 on days 14 and 23 post-MI. MicroPET images were acquired 1 h after injection.
Semi-quantitative region-of-interest (ROI) analysis of the PET signal was performed on attenuation-and decay-corrected PET images using InterviewFusion software (Mediso) and tissue uptake values were expressed as a mean percentage of the injected dose per gram of tissue (%ID/g) ±SD for 18 F-FDG and as a mean heart-to-muscle signal ratio (H/M ischemic ) ± SD for 68 Ga-sCD146 and 68 Ga-RGD 2 . This imaging method only enables semi-quantitative analysis of the PET signal as neither dynamic imaging nor kinetic modeling study was carried out for true quantitative analysis.

Histological Sirius Red Staining
Forty-three days after myocardial infarction, hearts were collected and fixed in 4% PFA for 3 h and extensively washed in 1X PBS. Paraffin embedding was performed following dehydration using a graded ethanol series (50, 70, 90 and 100%), two xylene washes and three paraffin washes (Paraplast X-tra, Sigma P3808, Saint-Louis, MS, USA). Serial 13 µm sections were obtained and mounted on polylysine-treated slides. After dewaxing (xylene, 2 times) and rehydration in an ethanol series (100, 90, 70, 50% and H 2 O), paraffin sections were incubated in a 0.1% Sirius Red solution dissolved in saturated aqueous solution of picric acid for 1 h at room temperature. Subsequently, sections were washed 3 times in acidified water (0.5% acetic acid), dehydrated in ascending concentrations of ethanol (70%, 90% and 100%) and cleared in xylene. Sections were mounted in resinous medium (Entellan, Merck, Saint-Louis, MS, USA). Collagen and non-collagen components were redand orange-stained, respectively.

Statistical Analysis
Biodistribution data were analyzed using Prism software v9 (GraphPad, San Diego, CA, USA). Data were expressed as mean values ± SD. Earth to muscle ratio differences were analyzed using to Mann-Whitney test. Differences between sham and infarct hearts evaluated by immunohistochemistry and by ultrasound imaging were analyzed using parametric unpaired Student t-test. Gaussian distribution was assumed by a Shapiro-Wilk normality test. Analysis of correlation was realized with a Pearson two-tailed test. Differences were considered statistically significant when p < 0.05.
At the end of the experiment (day 43 post-surgery), myocardial fibrosis was evaluated on sham and infarcted hearts using histological Sirius Red staining analysis ( Figure 3C). Semi-quantitative analysis revealed that myocardial infarction resulted in a significant increase of myocardial fibrosis in ischemic hearts compared to sham hearts (Mean ischemic = 10.79 ± 4.52%, n = 7, Mean sham = 0.88 ± 0.07%, n = 4; p = 0.0061) at day 43 post-surgery ( Figure 3D). Semi-quantitative intra-animal comparison of both tracers, 68 Ga-sCD146 and 68 Ga-RGD 2 at early times, respectivly D14 and D15 after MI surgery. No significant difference was observed between 68 Ga-sCD146 and 68 Ga-RGD 2 PET uptake in the infarcted heart at this time (n = 7; p = 0.93). Semi-quantitative intra-animal comparison was also realized at a later time, respectively, D22 and D23 for 68 Ga-sCD146 and 68 Ga-RGD 2 . Significant differences was observed between both tracers 68 Ga-sCD146 and 68 Ga-RGD 2 PET uptake in the infarcted heart at this later time (n = 7; p = 0.01) (Figure 4). (E) Semi-quantitative intra-animal comparison of both tracers, 68 Ga-sCD146 and 68 Ga-RGD 2 at early times, respectively D14 and D15 after MI surgery (a). No significant difference was observed between 68 Ga-sCD146 and 68 Ga-RGD 2 PET uptake in the infarcted heart at this time (n = 7; p = 0.93). Semi-quantitative intra-animal comparison was also realized a later time, respectively D22 and D23 for 68 Ga-sCD146 and 68 Ga-RGD 2 (b). A significant difference was observed between 68 Ga-sCD146 and 68 Ga-RGD 2 PET uptakes in the infarcted hearts at this later time (n = 7; ** p = 0.01).

Early 68 Ga-sCD146 PET Signal Intensity on Day 15 Correlated with Delayed Residual Myocardial Perfusion
A significant positive correlation was observed between individual residual perfusion recovery evaluated by 18 F-FDG PET signal expressed as heart-to-muscle ratio on day 30 and 68 Ga-sCD146 PET signal intensity expressed as heart-to-muscle ratio ( Figure 5(Aa)) on day 15 post-ischemia (Pearson R 2 = 0.70; p = 0.04; n = 7). In contrast, no significant correlation was depicted between individual residual perfusion recovery evaluated by 18

Early 68 Ga-sCD146 PET Signal Intensity Was Inversely Correlated with Delayed Myocardial Fibrosis
Our results revealed a significant negative correlation between individual fibrosis quantification evaluated by histological Sirius red staining and expressed as percentage of fibrosis tissue on left ventricle on day 43 post-ischemia and 68 Ga-sCD146 PET signal intensity expressed as heart to muscles ratio ( Figure 5(Ba)) on day 15 post-ischemia (Pearson R 2 = −0.77; p = 0.04; n = 7). In contrast, no significant correlation was detected between individual fibrosis quantification 43 days post-myocardial infarction and 68 Ga-RGD 2 PET signal intensity expressed as heart to muscle ratio ( Figure 5(Bb)) on day 15 post-myocardial infarction (Pearson R 2 = −0.68; p = 0.06; n = 7).

Early 68 Ga-sCD146 PET Signal Correlated with Myocardial Functional Recovery
Most notably, a significant correlation was identified between individual heart functional recovery, explored using ultrasound imaging and expressed as a ratio between the VEF measured 43 and 22 days post-myocardial infarction and 68 Ga-sCD146 PET signal intensity expressed as heart-to-muscle ratio ( Figure 5(Ca)) on day 15 post-myocardial infarction (Pearson R 2 = 0.68; p = 0.01; n = 12).
All these correlations were also performed for 68 Ga-sCD146 and 68 Ga-RGD 2 late times, respectively D22 and D23 post IM. The results of these correlations are shown in supplemental Figure S1. We have also detailed the semi-quantitative results of each imaging method in Table S1 presented in additional data.

Early 18 F-FDG PET Signal Intensity Was Inversely Correlated with Delayed Myocardial Fibrosis and Wasn't Correlated with Myocardial Functional Recovery
A significant negative correlation was identified between individual fibrosis quantification expressed as percentage of fibrotic tissue on left ventricle on day 43 post-ischemia and 18 F-FDG PET signal intensity expressed as heart-to-muscle ratio ( Figure 5(Da)) on day 16 post-ischemia (Pearson R 2 = −0.83; p = 0.024; n = 10). In contrast, no significant correlation was detected between individual heart functional recovery explored using ultrasound imaging and expressed as a ratio between the VEF measured 43 and 22 days post-myocardial infarction and 18 F-FDG PET signal intensity expressed as heart-to-muscle ratio ( Figure 5(Db)) on day 16 post-ischemia (Pearson R 2 = −0.39; p = 0.25; n = 10).

Discussion
In this study, we demonstrated for the first time using microPET imaging an increase in AMOT expression after MI. We additionally observed that 68 Ga-sCD146 microPET imaging on day 15 post-ischemia was positively correlated with delayed residual myocardial perfusion, myocardial functional recovery and negatively correlated with delayed myocardial fibrosis. According to recent reports describing AMOT involvement in angiogenic processes [24], these results indicate that 68 Ga-sCD146 may be considered as a potentially sensitive tool to early predict recovery of recent myocardial injury.
MI affects millions of patients worldwide with increasing prevalence and is responsible for millions of deaths. MI is mainly due to rupture or erosion of vulnerable atherosclerotic plaque, inducing coronary artery occlusion and cell death in ischemic territory [25]. Despite advances in pharmacological and interventional therapies, only 10% of all MI patients survive with reduced left ventricular function, resulting in the development of adverse LV remodeling and heart failure [26][27][28]. Research and development of innovative therapies remain a major challenge for such high-risk patients. Wound healing after MI involves a robust angiogenic response as a key of therapeutics approach. The post-MI angiogenic mechanisms have been poorly described up to now but several studies showed a strong involvement of pre-existing ECs in the capillary formation in infarct region [29,30]. Endothelial cells (EC) emerged as a major therapeutic target for promoting angiogenesis and tissue regeneration after MI [2]. AMOT has been shown to promote EC migration and tube formation during angiogenesis [18]. AMOT is expressed on human endothelium and promotes angiogenesis by controlling directional migration [31]. A recent study reported the involvement of AMOT in EC migration through a key role in the mechanical signal's integration detected by the migrating endothelium-mediated the contractility of actomyosin, essential for EC migration [24]. In accordance with this, we already reported that AMOT mediated the beneficial effects of sCD146 on EC migration [19] and daily sCD146 injection in gastrocnemius induced a better revascularization and tissue regeneration in a hindlimb ischemia mouse model [20]. Moreover, many studies showed that sCD146 was a biomarker of endothelial damage, explaining the positive correlation between its plasma level and disease progression in chronic kidney failure and diabetic nephropathy [32]. High plasma levels of sCD146 were also found in acute coronary syndrome and reflected the severity of pulmonary congestion better than the brain natriuretic peptide [22]. Even if sCD146 was involved in angiogenesis [33], the sCD146/AMOT signaling pathway has not been clearly explored. We previously demonstrated that AMOT overexpression was early, intensively and sustainably upregulated in post-ischemic situation in ischemic hindlimb mouse model. In this previous report, we observed an overexpression of the p80 isoform of AMOT, an isoform involved in new vessel formation and EC migration and a later decrease of p130 isoform involved in blood vessel maturation and stabilization [11].
Altogether, the involvement of sCD146 and AMOT in angiogenesis and tissue regeneration in post-ischemic situations and the high AMOT expression in neovascular endothelial cells during angiogenesis makes AMOT an ideal target for imaging of the myocardial infarction healing process.
Thus, we recently developed and validated an AMOT-targeting, PET imaging agent named 68 Ga-sCD146, with an interesting early predictive value for delayed post-ischemic tissue recovery in hind limb ischemia model [11]. The preserved specificity for AMOT was confirmed by both ex vivo and in vivo experiments on the hind limb ischemia mouse model (respectively, by autoradiography on muscle sections and by microPET imaging with or without an excess of blocking peptide). In addition, a significant correlation between the intensity of 68 Ga-sCD146 PET signal and AMOT expression quantified by immunohistochemistry, validated the quantification of AMOT using 68 Ga-sCD146 PET imaging.
In this study, we applied 68 Ga-sCD146 microPET imaging to evaluate and quantify AMOT expression at different times post-MI and compared it to 68 Ga-RGD 2 , 18 F-FDG microPET imaging, ultrasound imaging and histological Sirius-red staining analysis.
The most reported radiotracers at the clinical stage for imaging angiogenesis are based on the RGD motif targeting the αvβ3 integrin. 68 Ga-RGD 2 PET imaging is still under clinical evaluation in many cardiovascular and oncologic diseases, but might suffer from high background noise and a low specificity for angiogenesis of the targeted αvβ3 integrin itself [34][35][36]. Despite multiple clinical evaluations, the RGD-derived tracers are still under evaluation and further studies will be needed to establish the clinical utility of αvβ3 integrin targeting [37]. In our MI mouse model, 68 Ga-sCD146 showed a significantly different microPET signal quantification on day 15 compared to sham mice, whereas 68 Ga-RGD 2 did not exhibit the least difference at this early time. In addition, analysis of 68 Ga-sCD146 PET images showed a high hepatic tracer uptake. In the context of cardiac uptake evaluation, this observation did not preclude signal analysis. A significant hepatic uptake is also systematically described with one of the gold standard radiotracers for cardiac perfusion scintigraphy, 99m Tc-MIBI [38,39]. Chemical structure optimizations must now be considered in order to increase the hydrophilicity of 68 Ga-sCD146, therefore improving pharmacokinetics via decreasing the hepatic retention, as already and successfully achieved with such as pegylated group addition or fluorinated RGD-derived radiotracers [40,41]. The superiority of 68 Ga-sCD146 microPET signal was supported by significant correlations between 68 Ga-sCD146 PET signal intensity at day 15 and delayed myocardial perfusion, myocardial recovery and lower myocardial fibrosis.

Conclusions
To conclude, this study demonstrated for the first time that AMOT was upregulated in post-MI mouse model and that AMOT expression could be considered as an informative key player in myocardial recovery processes. AMOT expression was successfully monitored by longitudinal 68 Ga-sCD146 PET imaging and that constituted an early predictive value for delayed myocardial recovery. Additionally, this innovative PET imaging approach would be an asset in the evaluation of experimental therapeutic strategies aimed at promoting post-MI regenerative processes.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cells10092305/s1. Figure S1: Correlation analysis, Table S1: Semi-quantitative analysis of PET imaging analysis, histological Sirius red intensity and ultrasound imaging expressed on Mean and Standard deviation for IM mice and Sham mice.

Conflicts of Interest:
The authors declare no conflict of interest.