Design and preclinical evaluation of a novel apelin-based PET radiotracer targeting APJ receptor for molecular imaging of angiogenesis.

APJ has been extensively described in the pathophysiology of angiogenesis and cell proliferation. The prognostic value of APJ overexpression in many diseases is now established. This study aimed to design a PET radiotracer that specifically binds to APJ. Apelin-F13A-NODAGA (AP747) was synthesized and radiolabeled with gallium-68 ([68Ga]Ga-AP747). Radiolabeling purity was excellent (> 95%) and stable up to 2 h. Affinity constant of [67Ga]Ga-AP747 was measured on APJ-overexpressing colon adenocarcinoma cells and was in nanomolar range. Specificity of [68Ga]Ga-AP747 for APJ was evaluated in vitro by autoradiography and in vivo by small animal PET/CT in both colon adenocarcinoma mouse model and Matrigel plug mouse model. Dynamic of [68Ga]Ga-AP747 PET/CT biodistributions was realized on healthy mice and pigs for two hours, and quantification of signal in organs showed a suitable pharmacokinetic profile for PET imaging, largely excreted by urinary route. Matrigel mice and hindlimb ischemic mice were submitted to a 21-day longitudinal follow-up with [68Ga]Ga-AP747 and [68Ga]Ga-RGD2 small animal PET/CT. [68Ga]Ga-AP747 PET signal in Matrigel was significantly more intense than that of [68Ga]Ga-RGD2. Revascularization of the ischemic hind limb was followed by LASER Doppler. In the hindlimb, [68Ga]Ga-AP747 PET signal was more than twice higher than that of [68Ga]Ga-RGD2 on day 7, and significantly superior over the 21-day follow-up. A significant, positive correlation was found between the [68Ga]Ga-AP747 PET signal on day 7 and late hindlimb perfusion on day 21. We developed a new PET radiotracer that specifically binds to APJ, [68Ga]Ga-AP747 that showed more efficient imaging properties than the most clinically advanced tracer of angiogenesis, [68Ga]Ga-RGD2.

Sublingual Atropine Administration as a Tool to Decrease Salivary Glands' PSMA-Ligand Uptake: A Preclinical Proof of Concept Study Using [68Ga]Ga-PSMA-11.

Prostate Specific Membrane Antigen (PSMA)-directed radionuclide therapy has gained an important role in the management of advanced castration-resistant prostate cancer. Although extremely promising, the prolongation in survival and amelioration of disease-related symptoms must be balanced against the direct toxicities of the treatment. Xerostomia is amongst the most common and debilitating of these, particularly when using an alpha emitter. It is therefore of main importance to develop new preventive strategies. This preclinical study has evaluated the effect of α-adrenergic and anticholinergic drugs on [99mTc]TcO4− Single Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) and [68Ga]Ga-PSMA-11 Positron Emission Tomography (PET/CT). Methods: The effects of phenylephrine, scopolamine, atropine, and ipratropium on salivary glands uptake were evaluated in non-tumor-bearing mice by [99mTc]TcO4− microSPECT/CT. The most efficient identified strategy was evaluated in non-tumor-bearing and xenografted mice by [68Ga]Ga-PSMA-11 PET/CT. Results: Scopolamine and atropine showed a significant decrease in the parotid glands' uptake on SPECT/CT whereas phenylephrine and ipratropium failed. Atropine premedication (sublingual route), which was the most effective strategy, also showed a drastic decrease of [68Ga]Ga-PSMA-11 salivary glands' uptake in both non-tumor-bearing mice (−51.6% for the parotids, p < 0.0001) and human prostate adenocarcinoma xenografted mice (−26.8% for the parotids, p < 0.0001). Conclusion: Premedication with a local administration of atropine could represent a simple, safe, and efficient approach for reducing salivary glands' uptake.

Tracking Radiolabeled Endothelial Microvesicles Predicts Their Therapeutic Efficacy: A Proof-of-Concept Study in Peripheral Ischemia Mouse Model Using SPECT/CT Imaging.

Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. METHODS: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. RESULTS: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. CONCLUSIONS: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection.

Involvement of Multiple Variants of Soluble CD146 in Systemic Sclerosis: Identification of a Novel Profibrotic Factor.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive fibrosis, immune dysfunction, and vascular damage, in which the expression of many growth factors is deregulated. CD146 was recently described as a major actor in SSc. Since CD146 also exists as a circulating soluble form (sCD146) that acts as a growth factor in numerous angiogenic- and inflammation-related pathologies, we sought to identify the mechanisms underlying the generation of sCD146 and to characterize the regulation and functions of the different variants identified in SSc. METHODS: We performed in vitro experiments, including RNA-Seq and antibody arrays, and in vivo experiments using animal models of bleomycin-induced SSc and hind limb ischemia. RESULTS: Multiple forms of sCD146, generated by both shedding and alternative splicing of the primary transcript, were discovered. The shed form of sCD146 was generated from the cleavage of both long and short membrane isoforms of CD146 through ADAM-10 and TACE metalloproteinases, respectively. In addition, 2 novel sCD146 splice variants, I5-13-sCD146 and I10-sCD146, were identified. Of interest, I5-13-sCD146 was significantly increased in the sera of SSc patients (P < 0.001; n = 117), in particular in patients with pulmonary fibrosis (P < 0.01; n = 112), whereas I10-sCD146 was decreased (P < 0.05; n = 117). Further experiments revealed that shed sCD146 and I10-sCD146 displayed proangiogenic activity through the focal adhesion kinase and protein kinase Cε signaling pathways, respectively, whereas I5-13-sCD146 displayed profibrotic effects through the Wnt-1/ß-catenin/WISP-1 pathway. CONCLUSION: Variants of sCD146, and in particular the novel I5-13-sCD146 splice variant, could constitute novel biomarkers and/or molecular targets for the diagnosis and treatment of SSc and other angiogenesis- or fibrosis-related disorders.

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 αvß3-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.

Early prediction of revascularisation by angiomotin-targeting positron emission tomography.

This study aimed to develop a PET imaging agent of angiomotin (AMOT) expression, a potential biomarker of functional tissue regeneration in post-ischaemic conditions. Methods: Hindlimb ischaemia was induced by ligature and resection of the right femoral artery in mice, and clinical score and limb perfusion were evaluated up to 30 days after surgery. AMOT expression was evaluated by histology and Western blot analysis. NODAGA-conjugates of AMOT ligand, sCD146, were designed, synthesised and radiolabelled with gallium-68. 68Ga-sCD146 microPET/CT imaging was performed from day 1 to day 30 after ischaemia. 68Ga-sCD146 specificity for AMOT was evaluated by autoradiography. Results: Immunohistochemistry showed a significant endothelial overexpression of AMOT from day 5 up to day 10 in the ischaemic hindlimb. 68Ga-sCD146 PET signal intensity correlated significantly with AMOT immunohistochemistry evaluation. 68Ga-sCD146 PET imaging showed a significant uptake in the ischaemic hindlimb from day 2 to day 15, peaking on day 5 (ipsi/contralateral ratio = 2.4 ± 1.3, P = 0.0005) and significantly decreased after pharmacological blocking (62.57 ± 11% decrease in PET signal P = 0.032). Finally, we observed a significant correlation between day 5 68Ga-sCD146 PET signal intensity and clinical recovery (day 28) or hindlimb perfusion recovery (day 30). Conclusions: This work reports for the first time an early and sustained increase in AMOT expression after hindlimb ischaemia in mice. We therefore developed an AMOT-targeting imaging agent, 68Ga-sCD146, and showed its specific uptake up to 21 days after ischaemic hindlimb using microPET imaging. Correlation of early post-ischaemic PET signal with both delayed perfusion recovery and clinical outcome allows us to postulate that 68Ga-sCD146 represents a promising radiotracer for tissue angiogenesis assessment.

Stem cell properties of peripheral blood endothelial progenitors are stimulated by soluble CD146 via miR-21: potential use in autologous cell therapy.

Cell-based therapies constitute a real hope for the treatment of ischaemic diseases. One of the sources of endothelial progenitors for autologous cell therapy is Endothelial Colony Forming Cells (ECFC) that can be isolated from peripheral blood. However, their use is limited by their low number in the bloodstream and the loss of their stem cell phenotype associated with the acquisition of a senescent phenotype in culture. We hypothesized that adding soluble CD146, a novel endothelial growth factor with angiogenic properties, during the isolation and growth procedures could improve their number and therapeutic potential. Soluble CD146 increased the number of isolated peripheral blood ECFC colonies and lowered their onset time. It prevented cellular senescence, induced a partial mesenchymal phenotype and maintained a stem cell phenotype by stimulating the expression of embryonic transcription factors. These different effects were mediated through the induction of mature miR-21. When injected in an animal model of hindlimb ischaemia, sCD146-primed ECFC isolated from 40 ml of blood from patients with peripheral arterial disease were able to generate new blood vessels and restore blood flow. Treatment with sCD146 could thus constitute a promising strategy to improve the use of autologous cells for the treatment of ischaemic diseases.

Portal vein stenosis preconditioning of living donor liver in swine: early mechanisms of liver regeneration and gain of hepatic functional mass.

To reduce the morbidity and mortality risk for the donor in living donor liver transplantation (LDLT), we previously identified 20% left portal vein (LPV) stenosis as an effective preconditioning method to induce cell proliferation in the contralateral lobe without downstream ipsilateral atrophy. In this study, we report the pathways involved in the first hours after preconditioning and investigate the changes in liver volume and function. Fourteen pigs were used this study. Five pigs were used to study the genetic, cellular and molecular mechanisms set up in the early hours following the establishment of our preconditioning. The remaining nine pigs were equally divided into three groups: sham-operated animals, 20% LPV stenosis, and 100% LPV stenosis. Volumetric scanning and 99 mTc-Mebrofenin hepatobiliary scintigraphy were performed before preconditioning and 14 days after to study morphological and functional changes in the liver. We demonstrated that liver regeneration triggered by 20% LPV stenosis in the contralateral lobe involves TNF-α, IL-6, and inducible nitric oxide synthase 2 by means of STAT3 and hepatocyte growth factor. We confirmed that our preconditioning was responsible for an increase in the total liver volume. Finally, we demonstrated that this volumetric gain was associated with an increase in hepatic functional capacity. NEW & NOTEWORTHY We describe a new preconditioning method for major hepatectomy that is applicable to hepatectomy for donation. We identified 20% left portal vein stenosis as effective preconditioning that is capable of inducing cell proliferation in the contralateral lobe without the downstream ipsilateral atrophy. In this study, we report the pathways involved in the first hours following preconditioning, and we confirm that 20% left portal vein stenosis is responsible for an increase in the functional capacity and total liver volume in a porcine model.

A novel anti-CD146 antibody specifically targets cancer cells by internalizing the molecule.

CD146 is an adhesion molecule present on many tumors (melanoma, kidney, pancreas, breast, ...). In addition, it has been shown to be expressed on vascular endothelial and smooth muscle cells. Generating an antibody able to specifically recognize CD146 in cancer cells (designated as tumor CD146), but not in normal cells, would thus be of major interest for targeting tumor CD146 without affecting the vascular system. We thus generated antibodies against the extracellular domain of the molecule produced in cancer cells and selected an antibody that specifically recognizes tumor CD146. This antibody (TsCD146 mAb) was able to detect CD146-positive tumors in human biopsies and in vivo, by PET imaging, in a murine xenograft model. In addition, TsCD146 mAb antibody was able to specifically detect CD146-positive cancer microparticles in the plasma of patients. TsCD146 mAb displayed also therapeutic effects since it was able to reduce the growth of human CD146-positive cancer cells xenografted in nude mice. This effect was due to a decrease in the proliferation and an increase in the apoptosis of CD146-positive cancer cells after TsCD146-mediated internalization of the cell surface CD146. Thus, TsCD146 mAb could be of major interest for diagnostic and therapeutic strategies against CD146-positive tumors in a context of personalized medicine.