Erik Samén

Vascular endothelial growth factor B controls endothelial fatty acid uptake

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb-/- mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved β-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Selenolthiol and dithiol C-terminal tetrapeptide motifs for one-step purification and labeling of recombinant proteins produced in E. coli

We have previously shown that a redox‐active selenocysteine‐containing tetrapeptide—Sel‐tag (Gly‐Cys‐Sec‐Gly)—can be used as a C‐terminal fusion motif for recombinant proteins produced in Escherichia coli. This Sel‐tag allows selenolate‐targeted one‐step purification, as well as fluorescent labeling or radiolabeling either with gamma emitters (75Se) or with positron‐emitting radionuclides (11C). Here we have analyzed four different redox‐active C‐terminal motifs, carrying either dithiol (Gly‐Cys‐Cys‐Gly or Ser‐Cys‐Cys‐Ser) or selenolthiol (Gly‐Cys‐Sec‐Gly or Ser‐Cys‐Sec‐Ser) motifs. Utilizing these different functional motifs with the same recombinant protein (Fel d 1), we were able to assess their relative reactivities and potential usefulness for biotechnological applications. We found that all four redox‐active tags could be utilized for efficient one‐step purification to provide pure protein from a crude bacterial lysate through reversible binding to phenylarsine oxide sepharose, with yields and purities comparable to those obtained for a His‐tagged protein purified by the more common approach with use of a Ni2+ column. For labeling with electrophilic fluorescent or radioactive compounds, however, the selenolthiol motifs were considerably more efficient than their dithiol counterparts. The results thus show that both the selenolthiol‐ and the dithiol‐containing tags can serve as efficient alternatives to His‐tags for protein purification, while the selenolthiol motifs offer additional and unique potential for Sec‐targeted labeling. It should therefore be possible to utilize these multifunctional tetrapeptide motifs to develop a wide range of novel biotechnological applications based on Sec targeting with electrophilic compounds.

HER2-Positive Tumors Imaged Within 1 Hour Using a Site-Specifically 11C-Labeled Sel-Tagged Affibody Molecule

A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. Methods: A HER2-binding Affibody molecule, ZHER2:342, was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either 11C or 68Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the 11C-labeled Affibody molecule. Results: Both the 11C- and 68Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4–5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the 11C-labeled protein, and its overall absorbed dose was considerably lower. 11C-ZHER2:342 showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. Conclusion: To our knowledge, the Sel-tagging technique is the first that enables site-specific 11C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, 11C-labeled Sel-tagged ZHER2:342 can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

Combining [11C]-AnxA5 PET Imaging with Serum Biomarkers for Improved Detection in Live Mice of Modest Cell Death in Human Solid Tumor Xenografts

Background In vivo imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model. Methodology/Principal Findings Modest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on 11C-labeled Sel-tagged Annexin A5 ([11C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified ex vivo for validation. Both PET imaging using [11C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [18F]-FDG uptake was not affected by this treatment in this tumor model. [11C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session. Conclusions/Significance The combined use of dynamic PET with [11C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors.

Metabolism of Epidermal Growth Factor Receptor Targeting Probe [11C]PD153035: Impact on Biodistribution and Tumor Uptake in Rats

Several tracers have been evaluated as probes for noninvasive epidermal growth factor receptor (EGFR) quantification with PET. One of the most promising candidates is the 11C-labeled analog of the EGFR tyrosine kinase inhibitor PD153035. However, previous in vitro studies indicated extensive metabolism of the tracer, which could be disadvantageous for the assessment of receptor density in vivo. The aim of this study was to investigate the in vivo metabolism of [11C]PD153035 to determine whether alterations in metabolite formation are accompanied by changes in biodistribution and tumor uptake. Methods: EGFR-overexpressing human epidermoid carcinoma xenografts in rats were used in all examinations of tumor uptake. Cytochrome P450 enzymes of subfamilies CYP2D and CYP3A were inhibited before intravenous injection of [11C]PD153035 into healthy and tumor-bearing male rats. Samples were taken from arterial blood and urine, and the occurrence of radioactive metabolites was assessed with radio–high-performance liquid chromatography. Dynamic PET examinations of healthy and tumor-bearing animals were performed. In 1 rat, the effect of local intraarterial administration was examined. Results: [11C]PD153035 labeled at position 6 was metabolized extensively in vivo in male rats, resulting in very low levels of the intact tracer in plasma only minutes after injection. The major identified radiolabeled metabolites found were the N-oxide and metabolites arising from O demethylation at position 7. They were reduced by inhibition of CYP2D and CYP3A enzymes. PET revealed enzyme activity–dependent changes in the radioactivity distribution in the liver and tumors. Local administration of [11C]PD153035 greatly increased radioactivity levels in the adjacent tumor compared with levels typically found after intravenous administration. The highest tumor-to-muscle ratio at 60 min after intravenous injection was found in the untreated animals, whereas the overall highest ratio was found in the tumor near the intraarterial administration site. Conclusion: We suggest that the metabolism of [11C]PD153035 should be taken into consideration when this tracer is used to quantify EGFR expression, as our results indicated that the distribution of radioactivity to EGFR-overexpressing tumors was affected by the rate of metabolism and the route of administration.

Visualization of angiogenesis during cancer development in the polyoma middle T breast cancer model: molecular imaging with (R)-[11C]PAQ

BackgroundVascular endothelial growth factor receptor 2 (VEGFR2) is a crucial mediator of tumour angiogenesis. High expression levels of the receptor have been correlated to poor prognosis in cancer patients. Reliable imaging biomarkers for stratifying patients for anti-angiogenic therapy could therefore be valuable for increasing treatment success rates. The aim of this study was to investigate the pharmacokinetics and angiogenesis imaging abilities of the VEGFR2-targeting positron emission tomography (PET) tracer (R)-[11C]PAQ.Methods(R)-[11C]PAQ was evaluated in the mouse mammary tumour virus-polyoma middle T (MMTV-PyMT) model of metastatic breast cancer. Mice at different stages of disease progression were imaged with (R)-[11C]PAQ PET, and results were compared to those obtained with [18 F]FDG PET and magnetic resonance imaging. (R)-[11C]PAQ uptake levels were also compared to ex vivo immunofluorescence analysis of tumour- and angiogenesis-specific biomarkers. Additional pharmacokinetic studies were performed in rat and mouse.ResultsA heterogeneous uptake of (R)-[11C]PAQ was observed in the tumorous mammary glands. Ex vivo analysis confirmed the co-localization of areas with high radioactivity uptake and areas with elevated levels of VEGFR2. In some animals, a high focal uptake was observed in the lungs. The lung uptake correlated to metastatic and angiogenic activity, but not to uptake of [18 F]FDG PET. The pharmacokinetic studies revealed a limited metabolism and excretion during the 1-h scan and a distribution of radioactivity mainly to the liver, kidneys and lungs. In rat, a high uptake was additionally observed in adrenal and parathyroid glands.ConclusionThe results indicate that (R)-[11C]PAQ is a promising imaging biomarker for visualization of angiogenesis, based on VEGFR2 expression, in primary tumours and during metastasis development.

Intra-arterial Administration of Placenta-Derived Decidual Stromal Cells to the Superior Mesenteric Artery in the Rabbit: Distribution of Cells, Feasibility, and Safety.

Selective administration of mesenchymal stromal cells to the mesenteric arteries is a potential technique to overcome pulmonary trapping and increase the density of transplanted cells in extensive mural inflammation of the intestine, such as in inflammatory bowel disease and graft-versus-host disease. We injected 5 × 106 111In-oxine-labeled human decidual stromal cells (DSCs) to the rabbit superior mesenteric artery (SMA) using clinical routine catheters guided by an angiographical system under sterile conditions. We used longitudinal single-photon emission tomography at 6 h and at 1, 2, and 5 days to assess trafficking and distribution of DSCs. We used digital subtraction angiography, computed tomography, and hematoxylin and eosin stainings to determine biodistribution of cells and to assess safety end points. We found that selective injection of human DSCs to the rabbit SMA does not result in acute embolic complications. Furthermore, we found that IV administration resulted in extensive retention of the radiolabeled DSCs in the lungs, corroborating previous studies on pulmonary trapping. In sharp contrast, selective injections to the SMA resulted in uptake distributed in the intestine supplied by the SMA and in the liver, indicating that this approach could significantly increase the fraction of injected DSCs reaching the target tissue.

Selective intra-arterial administration of 18F-FDG to the rat brain — effects on hemispheric uptake

IntroductionThe purpose of this study was to investigate the radioligand uptake and iodine contrast distribution in the intra- and extracranial circulation of the rat, after intra-arterial injections to the common carotid artery and different parts of the internal carotid artery.MethodsAll animal experiments were carried out in accordance with Karolinska Institutet’s guidelines and were approved by the local laboratory animal ethics committee. We used clinical neurointerventional systems to place microcatheters in the extra- or intracranial carotid artery of 15 Sprague–Dawley rats. Here, injection dynamics of iodine contrast was assessed using digital subtraction angiography. Maintaining the catheter position, the animals were placed in a micro PET and small-animal positron emission tomography (PET) was used to analyze injections [2-18F]-2-fluoro-2-deoxy-d-glucose (18F-FDG).ResultsMicrocatheters had to be placed in the intracranial carotid artery (iICA) for the infusate to distribute to the brain. Selective injection via the iICA resulted in a 9-fold higher uptake of 18F-FDG in the injected hemisphere (p < 0.005) compared to both intravenous and more proximal carotid artery injections. Furthermore, selective injection gave a dramatically improved contrast between the brain and extracranial tissue.ConclusionIntra-arterial injection increases the cerebral uptake of a radiotracer dramatically compared to systemic injection. This technique has potential applications for endovascular treatment of malignancies allowing intra-interventional modifications of injection strategy, based on information on tumor perfusion and risk to surrounding normal parenchyma. Furthermore the technique may increase diagnostic sensitivity and avoid problems due to peripheral pharmacological barriers and first passage metabolism of labile tracers.

Safety of Intra-Arterial Injection with Tumor-Activated T Cells to the Rabbit Brain Evaluated by MRI and SPECT/CT:

Glioblastoma multiforme (GBM) is the most common and most severe form of malignant gliomas. The prognosis is poor with current combinations of pharmaceutical, radiotherapy, and surgical therapy. A continuous search for new treatments has therefore been ongoing for many years. Therapy with tumor-infiltrating lymphocytes (TILs) is a clinically promising strategy to treat various cancers, including GBM. An endovascular intraarterial injection of TILs as a method of delivery may, instead of intravenous infusion, result in better retention of effector cells within the tumor. Prior to clinical trials of intra-arterial injections with any cells, preclinical safety data with special emphasis on embolic–ischemic events are necessary to obtain. We used native rabbits as a model for intra-arterial injections with routine clinical catheter material and a clinical angiography suite. We selectively infused a total dose of 20 million activated T cells at a cell concentration of 4,000 cells/ml over 8 min of injection time. The rabbits were evaluated for ischemic lesions by 9.4 T magnetic resonance imaging (MRI) (n = 6), and for tracking of injected cells, single-photon emission computed tomography/computed tomography (SPECT/CT) was used (n = 2). In this study, we show that we can selectively infuse activated T cells to a CNS volume of 3.5 cm3 (estimated from the volumetric MRI) without catastrophic embolic– ischemic adverse events. We had one adverse event with a limited basal ganglia infarction, probably due to catheter-induced mechanical occlusion of one of the lateral lenticulostriatal arteries. The cells pass through the native brain without leaving SPECT signals. The cells then, over the first hours, end up in the liver to a large extent and to a lesser degree by the spleen, pancreas, and kidneys. Virtually no uptake could be detected in the lungs. This indicates a difference in biodistribution as opposed to other cell types when infused intravenously.