Publications

<div>Abstract<p><b>Purpose:</b> Obesity promotes pancreatic and breast cancer progression via mechanisms that are poorly understood. Although obesity is associated with increased systemic levels of placental growth factor (PlGF), the role of PlGF in obesity-induced tumor progression is not known. PlGF and its receptor VEGFR-1 have been shown to modulate tumor angiogenesis and promote tumor-associated macrophage (TAM) recruitment and activity. Here, we hypothesized that increased activity of PlGF/VEGFR-1 signaling mediates obesity-induced tumor progression by augmenting tumor angiogenesis and TAM recruitment/activity.</p><p><b>Experimental Design:</b> We established diet-induced obese mouse models of wild-type C57BL/6, VEGFR-1 tyrosine kinase (TK)-null, or PlGF-null mice, and evaluated the role of PlGF/VEGFR-1 signaling in pancreatic and breast cancer mouse models and in human samples.</p><p><b>Results:</b> We found that obesity increased TAM infiltration, tumor growth, and metastasis in pancreatic cancers, without affecting vessel density. Ablation of VEGFR-1 signaling prevented obesity-induced tumor progression and shifted the tumor immune environment toward an antitumor phenotype. Similar findings were observed in a breast cancer model. Obesity was associated with increased systemic PlGF, but not VEGF-A or VEGF-B, in pancreatic and breast cancer patients and in various mouse models of these cancers. Ablation of PlGF phenocopied the effects of VEGFR-1-TK deletion on tumors in obese mice. PlGF/VEGFR-1-TK deletion prevented weight gain in mice fed a high-fat diet, but exacerbated hyperinsulinemia. Addition of metformin not only normalized insulin levels but also enhanced antitumor immunity.</p><p><b>Conclusions:</b> Targeting PlGF/VEGFR-1 signaling reprograms the tumor immune microenvironment and inhibits obesity-induced acceleration of tumor progression. <i>Clin Cancer Res; 22(12); 2993–3004. ©2016 AACR</i>.</p></div>

The healing of a fracture depends largely on the development of a new blood vessel network (angiogenesis) in the callus. During angiogenesis tip cells lead the developing sprout in response to extracellular signals, amongst which vascular endothelial growth factor (VEGF) is critical. In order to ensure a correct development of the vasculature, the balance between stalk and tip cell phenotypes must be tightly controlled, which is primarily achieved by the Dll4-Notch1 signaling pathway. This study presents a novel multiscale model of osteogenesis and sprouting angiogenesis, incorporating lateral inhibition of endothelial cells (further denoted MOSAIC model) through Dll4-Notch1 signaling, and applies it to fracture healing. The MOSAIC model correctly predicted the bone regeneration process and recapitulated many experimentally observed aspects of tip cell selection: the salt and pepper pattern seen for cell fates, an increased tip cell density due to the loss of Dll4 and an excessive number of tip cells in high VEGF environments. When VEGF concentration was even further increased, the MOSAIC model predicted the absence of a vascular network and fracture healing, thereby leading to a non-union, which is a direct consequence of the mutual inhibition of neighboring cells through Dll4-Notch1 signaling. This result was not retrieved for a more phenomenological model that only considers extracellular signals for tip cell migration, which illustrates the importance of implementing the actual signaling pathway rather than phenomenological rules. Finally, the MOSAIC model demonstrated the importance of a proper criterion for tip cell selection and the need for experimental data to further explore this. In conclusion, this study demonstrates that the MOSAIC model creates enhanced capabilities for investigating the influence of molecular mechanisms on angiogenesis and its relation to bone formation in a more mechanistic way and across different time and spatial scales.

Intracerebral-intraventricular hemorrhages (ICH/IVH) in very preterm neonates are responsible for high mortality and subsequent disabilities. In humans, tissue plasminogen activator (t-PA) initiates fibrinolysis and activates endoluminal-endothelial receptors; dysfunction of the t-PA inhibitor (PAI-1) results in recurrent hemorrhages. We used PAI-1 knockout (PAI-1) mice to examine the role of t-PA in age-dependent intracranial hemorrhages as a possible model of preterm ICH/IVH. Intracortical injection of 2 μL of phosphate-buffered saline produced a small traumatic injury and a high rate of hemorrhage in PAI-1 pups at postnatal day 3 (P3) or P5, whereas it had no effect in wild-type neonates. This resulted in white matter and cortical lesions, ventricle enlargement, hyperlocomotion, and altered cortical levels of serotonin and dopamine in the adult PAI mice. N-methyl-D-aspartate receptor blockers, plasmin- and matrix metalloproteinases inhibitors reduced hemorrhage and tissue lesions. In contrast to P3 to P5, no significant hemorrhages were induced in P10 PAI-1 pups and there were no behavioral or neurochemical alterations in adulthood. These data suggest that microvascular immaturity up to P5 in mice is a determinant factor required for t-PA-dependent vascular rupture. Neonatal PAI-1 mice could be a useful ICH/IVH model for studying the ontogenic window of vascular immaturity and vascular protection against later neurodisabilities.

<div>AbstractPurpose:<p>Placental growth factor (PlGF) and its receptor neuropilin 1 are elevated in malignant embryonal tumors and mediate tumor progression by promoting cell proliferation, survival, and metastasis. TB-403 is a blocking monoclonal antibody against PlGF that inhibits tumor growth and increases survival in orthotopic medulloblastoma models.</p>Patients and Methods:<p>We conducted a phase I, open-label, multicenter, dose-escalation study of TB-403 in pediatric subjects with relapsed or refractory cancers. The study involved four dose levels (20 mg/kg, 50 mg/kg, 100 mg/kg, 175 mg/kg) using a 3 + 3 dose-escalation scheme. Subjects received two doses of TB-403 (days 1 and 15) per cycle. After cycle 1, temozolomide or etoposide could be added. The primary objective was to determine the maximum tolerated dose (MTD) of TB-403 monotherapy during a dose-limiting toxicity assessment period. The secondary and exploratory objectives included efficacy, drug pharmacokinetics, and detection of pharmacodynamic biomarkers.</p>Results:<p>Fifteen subjects were treated in four dose levels. All subjects received two doses of TB-403 in cycle 1. Five serious treatment-emergent adverse events were reported in 3 subjects, but MTD was not reached. While no complete nor partial responses were observed, 7 of 11 relapsed subjects with medulloblastoma experienced stable disease, which persisted for more than 100 days in 4 of 7 subjects.</p>Conclusions:<p>TB-403 was safe and well tolerated at all dose levels. No MTD was reached. The results look encouraging and therefore warrant further evaluation of efficacy in pediatric subjects with medulloblastoma.</p></div>

<ns4:p>Background Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses. Methods We used a combination of <ns4:italic>in vivo</ns4:italic> and <ns4:italic>ex vivo</ns4:italic> models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes. Results We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter <ns4:italic>ex vivo</ns4:italic> neutrophil phenotypes or <ns4:italic>in vivo</ns4:italic> outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production. Conclusions PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target.</ns4:p>

Additional file 4: Table S3. Cryptic transcript promoter DNA methylation and expression levels. Shown are results from an analysis of combined single-cell methylome-and-transcriptome sequencing of colorectal cancer cells, as generated by Bian and colleagues [38].

Abstract The growth arrest‐specific gene 6 ( Gas6 ) plays a role in pro‐atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE −/− mice, but alters its plaque composition. Compared to Gas6 +/+ : ApoE −/− mice, initial and advanced plaques of Gas6 −/− : ApoE −/− mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFβ was increased. In addition, fewer macrophages were found in advanced plaques of Gas6 −/− : ApoE −/− mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

These are the models and molecular dynamics trajectories obtained for the paper Role of glutamine synthetase in angiogenesis beyond glutamine synthesis [G. Eelen et al. Nature 2018 vol. 561 (7721) pp. 63-69].<br>The PDB files (human.pdb and bacterial.pdb) contain representative structures extracted from the molecular dynamics simulations of the S. Typhimurium (bacterial.pdb) and Human Glutamine Synthetase (human.pdb). For the latter, models of the two most relevant binding modes of Palmitoyl-CoA are included in the file.<br><br>The trajectories included are the FINAL 450 ns of 2 fully atomistic molecular dynamics simulations of Glutamine Synthetase (GS) performed with GROMACS and the Amber ff14SB force field. The Human_GS.x is the trajectory of human GS (reference PDB 2OJW) with Palmitoyl-CoA. In this trajecory the palmitate tail points upwards (which was the most interesting orientation found). "Bacterial_GS" is the bacterial GS trajectory (reference starting PDB 1FPY) with Palmitoyl-CoA - palmitate tail points downwards. The trajectories are in GROMACS trr format and have been saved every 200ps.<b> </b>Water molecules and the initial 50 ns (relaxation) are not included in the files.

Abstract Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P≤0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6−/− mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P&amp;lt;0.001). Similarly, TNF-α was higher in Gas6−/− than in WT mice 1 hour after LPS (P&amp;lt;0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6−/− plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6−/− than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6−/− mice (P&amp;lt;0.05). LPS-induced mortality in Gas6 receptors Axl−/−, Tyro3−/− and Merkd was also enhanced when compared to WT mice (P&amp;lt;0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6−/− mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6−/− macrophages produced more TNF-α and IL-6 than WT macrophages. Cytokine release by Gas6−/− macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6−/− macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6−/− macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NFκB activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6−/− mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NFκB activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6−/− mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.