Anu Autio

Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the ⁶⁸Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Nuclear imaging of inflammation: homing-associated molecules as targets.

The golden standard in nuclear medicine imaging of inflammation is the use of autologous radiolabeled leukocytes. Although their diagnostic accuracy is precise, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration (homing-associated molecules) could serve as targets for the development of imaging agents for inflammation. An excellent target would be a molecule that is absent or expressed at low levels in healthy tissues, but is present or upregulated at the sites of inflammation. In this paper, we will review the literature concerning the use of homing-associated molecules as imaging targets. We will especially concentrate on vascular adhesion protein-1 due to the promising results regarding its use as a target for the imaging of inflammation.

PET imaging of inflammation and adenocarcinoma xenografts using vascular adhesion protein 1 targeting peptide 68Ga-DOTAVAP-P1: comparison with 18F-FDG

PurposeThe aim of this study was to evaluate inflammation and tumour imaging with a vascular adhesion protein 1 (VAP-1) targeting peptide 68Ga-DOTAVAP-P1 in comparison with 18F-FDG.MethodsRats with both subcutaneous human pancreatic adenocarcinoma xenografts and turpentine oil-induced acute sterile inflammation were evaluated by dynamic positron emission tomography (PET) and by digital autoradiography of tissue cryosections. Subsequently, the autoradiographs were combined with histological and immunohistological analysis of the sections.Results68Ga-DOTAVAP-P1 delineated acute, sterile inflammation comparable with 18F-FDG. However, the tumour uptake of 68Ga-DOTAVAP-P1 was low in contrast to prominent 18F-FDG uptake. The standardised uptake values of inflammation and tumours by PET were 1.1 ± 0.4 (mean ± SEM) and 0.4 ± 0.1 for 68Ga-DOTAVAP-P1 and 2.0 ± 0.5 and 1.6 ± 0.8 for 18F-FDG, respectively. In addition, PET studies showed inflammation to muscle and tumour to muscle ratios of 5.1 ± 3.1 and 1.7 ± 0.3 for 68Ga-DOTAVAP-P1 and 6.2 ± 0.7 and 4.6 ± 2.2 for 18F-FDG, respectively. Immunohistochemistry revealed increased expression of luminal VAP-1 on the endothelium at the site of inflammation and low expression in the tumourConclusionThe 68Ga-DOTAVAP-P1 PET was able to visualise inflammation better than tumour, which was in accordance with the luminal expression of VAP-1 on vasculature in these experimental models.

Translating the concept of peptide labeling with 5-deoxy-5-[18F]fluororibose into preclinical practice: 18F-labeling of Siglec-9 peptide for PET imaging of inflammation.

Peptide glycosylation with 5-deoxy-5-[(18)F]fluororibose was translated into preclinical settings. The novel (18)F-labeled Siglec-9 peptide was produced using an automated synthesis procedure. The (18)F-labeled Siglec-9 peptide showed favorable binding in the animal model of inflammation in vivo.

USF1 deficiency activates brown adipose tissue and improves cardiometabolic health

Deficiency of USF1 protects against obesity, insulin resistance, and cardiovascular disease in mice and humans, and induces brown adipose tissue to burn triglycerides and glucose. Boosting metabolism with low USF1 Laurila et al. show that a deficiency of the transcription factor USF1 protects against obesity, insulin resistance, and cardiovascular disease. Even when fed a high-fat diet, USF1-deficient mice stayed lean and maintained a beneficial lipid profile with low triglycerides and high high-density lipoprotein (HDL) cholesterol. The mice had elevated energy expenditure because their brown adipose tissue was more active. In USF1-deficient mice, glucose and lipids were rapidly cleared from the circulation to be burned by brown fat. In humans, individuals with reduced USF1 expression also had higher plasma HDL cholesterol and lower triglycerides, and were more insulin-sensitive and less prone to hardening of the arteries. These findings identify USF1 as a potential therapeutic target for treating metabolic and cardiac diseases. USF1 (upstream stimulatory factor 1) is a transcription factor associated with familial combined hyperlipidemia and coronary artery disease in humans. However, whether USF1 is beneficial or detrimental to cardiometabolic health has not been addressed. By inactivating USF1 in mice, we demonstrate protection against diet-induced dyslipidemia, obesity, insulin resistance, hepatic steatosis, and atherosclerosis. The favorable plasma lipid profile, including increased high-density lipoprotein cholesterol and decreased triglycerides, was coupled with increased energy expenditure due to activation of brown adipose tissue (BAT). Usf1 inactivation directs triglycerides from the circulation to BAT for combustion via a lipoprotein lipase–dependent mechanism, thus enhancing plasma triglyceride clearance. Mice lacking Usf1 displayed increased BAT-facilitated, diet-induced thermogenesis with up-regulation of mitochondrial respiratory chain complexes, as well as increased BAT activity even at thermoneutrality and after BAT sympathectomy. A direct effect of USF1 on BAT activation was demonstrated by an amplified adrenergic response in brown adipocytes after Usf1 silencing, and by augmented norepinephrine-induced thermogenesis in mice lacking Usf1. In humans, individuals carrying SNP (single-nucleotide polymorphism) alleles that reduced USF1 mRNA expression also displayed a beneficial cardiometabolic profile, featuring improved insulin sensitivity, a favorable lipid profile, and reduced atherosclerosis. Our findings identify a new molecular link between lipid metabolism and energy expenditure, and point to the potential of USF1 as a therapeutic target for cardiometabolic disease.

Mini-PEG spacering of VAP-1-targeting 68Ga-DOTAVAP-P1 peptide improves PET imaging of inflammation.

BackgroundVascular adhesion protein-1 (VAP-1) is an adhesion molecule that plays a key role in recruiting leucocytes into sites of inflammation. We have previously shown that 68Gallium-labelled VAP-1-targeting peptide (68Ga-DOTAVAP-P1) is a positron emission tomography (PET) imaging agent, capable of visualising inflammation in rats, but disadvantaged by its short metabolic half-life and rapid clearance. We hypothesised that prolonging the metabolic half-life of 68Ga-DOTAVAP-P1 could further improve its imaging characteristics. In this study, we evaluated a new analogue of 68Ga-DOTAVAP-P1 modified with a mini-polyethylene glycol (PEG) spacer (68Ga-DOTAVAP-PEG-P1) for in vivo imaging of inflammation.MethodsWhole-body distribution kinetics and visualisation of inflammation in a rat model by the peptides 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were evaluated in vivo by dynamic PET imaging and ex vivo by measuring the radioactivity of excised tissues. In addition, plasma samples were analysed by radio-HPLC for the in vivo stability of the peptides.ResultsThe peptide with the mini-PEG spacer showed slower renal excretion but similar liver uptake as the original peptide. At 60 min after injection, the standardised uptake value of the inflammation site was 0.33 ± 0.07 for 68Ga-DOTAVAP-P1 and 0.53 ± 0.01 for 68Ga-DOTAVAP-PEG-P1 by PET. In addition, inflammation-to-muscle ratios were 6.7 ± 1.3 and 7.3 ± 2.1 for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1, respectively. The proportion of unchanged peptide in circulation at 60 min after injection was significantly higher for 68Ga-DOTAVAP-PEG-P1 (76%) than for 68Ga-DOTAVAP-P1 (19%).ConclusionThe eight-carbon mini-PEG spacer prolonged the metabolic half-life of the 68Ga-DOTAVAP-P1 peptide, leading to higher target-to-background ratios and improved in vivo PET imaging of inflammation.

Preliminary evaluation of novel 68Ga-DOTAVAP-PEG-P2 peptide targeting vascular adhesion protein-1.

Introduction:  Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent.

Preclinical Evaluation of a Radioiodinated Fully Human Antibody for In Vivo Imaging of Vascular Adhesion Protein-1–Positive Vasculature in Inflammation

Vascular adhesion protein-1 (VAP-1) is an endothelial glycoprotein mediating leukocyte trafficking from blood to sites of inflammation. BTT-1023 is a fully human monoclonal anti-VAP-1 antibody developed to treat inflammatory diseases. In this study, we preclinically evaluated radioiodinated BTT-1023 for inflammation imaging. Methods: Rabbits were intravenously injected with radioiodinated BTT-1023. Distribution and pharmacokinetics were assessed by PET/CT up to 72 h after injection. Human radiation dose estimates for 124I-BTT-1023 were extrapolated. Additionally, rabbits with chemically induced synovitis were imaged with 123I-BTT-1023 SPECT/CT. Results: Radioiodinated BTT-1023 cleared rapidly from blood circulation and distributed to liver and thyroid. Inflamed joints were delineated by SPECT/CT. The estimated human effective dose due to 124I-BTT-1023 was 0.55 mSv/MBq, if blockage of thyroid uptake is assumed. Conclusion: The radioiodinated BTT-1023 was able to detect mild inflammation in vivo. Clinical 124I-BTT-1023 PET studies with injected radioactivity of 0.5–0.7 MBq/kg may be justified.

68Ga-Chloride PET Reveals Human Pancreatic Adenocarcinoma Xenografts in Rats—Comparison with FDG

PurposeThe aim of the study was to compare 68Ga-chloride with 2-[18F]fluoro-2-deoxy-d-glucose (FDG) for the imaging of pancreatic xenografts.ProceduresRats with subcutaneous human pancreatic adenocarcinoma xenografts were evaluated in vivo by dynamic positron emission tomography (PET) and ex vivo by measuring radioactivity of excised tissues and by digital autoradiography of tumor cryosections.ResultsBoth tracers were capable of delineating all subcutaneous tumors from surrounding tissues by PET. The standardized uptake values of tumors by PET were 0.9 ± 0.3 (mean ± SD) for 68Ga-chloride (n = 13) and 1.8 ± 1.2 for FDG (n = 11). Ex vivo studies showed tumor-to-muscle ratio of 4.0 ± 0.3 for 68Ga-chloride (n = 4) and 7.9 ± 3.2 for FDG (n = 4).Conclusions68Ga-chloride delineated subcutaneously implanted pancreatic adenocarcinoma xenografts by PET, but the uptake was lower than FDG. Further studies to clarify the value of 68Ga-chloride for PET imaging of tumors are warranted.

Feasibility of experimental BT4C glioma models for somatostatin receptor 2-targeted therapies.

Abstract Somatostatin receptor subtype 2 (sstr2) is regarded as a potential target in malignant gliomas for new therapeutic approaches. Therefore, visualizing and quantifying tumor sstr2 expression in vivo would be highly relevant for the future development of sstr2-targeted therapies. The purpose of this study was to evaluate sstr2 status in experimental BT4C malignant gliomas. Methods. Rat BT4C malignant glioma cells were injected into BDIX rat brain or subcutaneously into nude mice. Tumor uptake of [68Ga]DOTA-(Tyr3)-Octreotide ([68Ga]DOTATOC), a somatostatin analog binding to sstr2, was studied by positron emission tomography/computed tomography (PET/CT). Additionally, subcutaneous tumor-bearing mice underwent PET imaging with 5-deoxy-5-[18F]fluororibose-NOC ([18F]FDR-NOC), a novel glycosylated peptide tracer also targeting sstr2. Ex vivo tissue radioactivity measurements, autoradiography and immunohistochemistry were performed to study sstr2 expression. Results. Increased tumor uptake of [68Ga]DOTATOC was detected at autoradiography with mean tumor-to-brain ratio of 68 ± 30 and tumor-to-muscle ratio of 9.2 ± 3.8 for rat glioma. High tumor-to-muscle ratios were also observed in subcutaneous tumor-bearing mice after injection with [68Ga]DOTATOC and [18F]FDR-NOC with both autoradiography (6.7 ± 1.5 and 4.3 ± 0.8, respectively) and tissue radioactivity measurements (6.5 ± 0.8 and 4.8 ± 0.6, respectively). Furthermore, sstr2 immunohistochemistry showed positive staining in both tumor models. However, surprisingly low tumor signal compromised PET imaging. Mean SUVmax for rat gliomas was 0.64 ± 0.28 from 30 to 60 min after [68Ga]DOTATOC injection. The majority of subcutaneous tumors were not visualized by [68Ga]DOTATOC or [18F]FDR-NOC PET. Conclusions. Experimental BT4C gliomas show high expression of sstr2. Weak signal in PET imaging, however, suggests only limited benefit of [68Ga]DOTATOC or [18F]FDR-NOC PET/CT in this tumor model for in vivo imaging of sstr2 status.