Publications

TNF has remarkable antitumor activities; however, therapeutic applications have not been possible because of the systemic and lethal proinflammatory effects induced by TNF. Both the antitumor and inflammatory effects of TNF are mediated by the TNF receptor p55 (p55TNFR) (encoded by the Tnfrsf1a gene). The antitumor effect stems from an induction of cell death in tumor endothelium, but the cell type that initiates the lethal inflammatory cascade has been unclear. Using conditional Tnfrsf1a knockout or reactivation mice, we found that the expression level of p55TNFR in intestinal epithelial cells (IECs) is a crucial determinant in TNF-induced lethal inflammation. Remarkably, tumor endothelium and IECs exhibited differential sensitivities to TNF when p55TNFR levels were reduced. Tumor-bearing Tnfrsf1a⁺⁺/⁻ or IEC-specific p55TNFR-deficient mice showed resistance to TNF-induced lethality, while the tumor endothelium remained fully responsive to TNF-induced apoptosis and tumors regressed. We demonstrate proof of principle for clinical application of this approach using neutralizing anti-human p55TNFR antibodies in human TNFRSF1A knockin mice. Our results uncover an important cellular basis of TNF toxicity and reveal that IEC-specific or systemic reduction of p55TNFR mitigates TNF toxicity without loss of antitumor efficacy.

Chronic hypoxia induces pulmonary vascular remodeling, leading to pulmonary hypertension, right ventricular hypertrophy, and heart failure. Heterozygous deficiency of hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates the cellular response to hypoxia by increasing expression of genes involved in erythropoiesis and angiogenesis, has been previously shown to delay hypoxia-induced pulmonary hypertension. HIF-2alpha is a homologue of HIF-1alpha and is abundantly expressed in the lung, but its role in pulmonary hypertension remains unknown. Therefore, we analyzed the pulmonary response of WT and viable heterozygous HIF-2alpha-deficient (Hif2alpha(+/-)) mice after exposure to 10% O(2) for 4 weeks. In contrast to WT mice, Hif2alpha(+/-) mice were fully protected against pulmonary hypertension and right ventricular hypertrophy, unveiling a critical role of HIF-2alpha in hypoxia-induced pulmonary vascular remodeling. Pulmonary expression levels of endothelin-1 and plasma catecholamine levels were increased threefold and 12-fold respectively in WT but not in Hif2alpha(+/-) mice after hypoxia, suggesting that HIF-2alpha-mediated upregulation of these vasoconstrictors contributes to the development of hypoxic pulmonary vascular remodeling.

uPAR enhances the internalization and thus the signaling downstream of a proangiogenic receptor.

Abstract Prolyl hydroxylase domain‐containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan‐hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells ( Phd1 f/ f Tie2:cre) protected mice from dextran sulphate sodium (DSS)‐induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2 f/+ Tie2:cre and Phd3 f/ f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell‐specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1 ‐deficient bone marrow‐derived macrophages towards an anti‐inflammatory M2 phenotype. These cells showed an attenuated nuclear factor‐κB‐dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin‐1β production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The thymus harbors an organ-typical dense network of branching and anastomosing blood vessels. To address the molecular basis for morphogenesis of this thymus-specific vascular pattern, we have inactivated a key vascular growth factor, VEGF-A, in thymus epithelial cells (TECs). Both Vegf-A alleles were deleted in TECs by a complementation strategy termed nude mouse [mutated in the transcription factor Foxn1 (forkhead box N1)] blastocyst complementation. Injection of Foxn1 +/+ ES cells into Foxn1 nu/nu blastocysts reconstituted a functional thymus. By dissecting thymus stromal cell subsets, we have defined, in addition to medullary TECs (mTECs) and cortical TECs (cTECs), another prominent stromal cell subset designated cortical mesenchymal cells (cMes). In chimeric thymi, mTECs and cTECs but not cMes were exclusively ES cell-derived. According to this distinct origin, the Vegf-A gene was deleted in mTECs and cTECs, whereas cMes still expressed Vegf-A . This genetic mosaic was associated with hypovascularization and disruption of the organ-typical network of vascular arcades. Thus, vascular growth factor production by TECs is required for normal thymus vascular architecture. These experiments provide insights into Foxn1 -dependent and Foxn1 -independent stromal cell development and demonstrate the value of this chimeric approach to analyzing gene function in thymus epithelium.

The accepted model of vessel branching distinguishes several endothelial cell fates. At the forefront of a vessel sprout, “tip cells” guide the sprouting vessel toward an angiogenic stimulus. Behind the tip, “stalk cells” proliferate to elongate the vessel branch and create a lumen. In mature vessels, endothelial cells acquire a streamlined shape to optimally conduct blood flow. For this purpose, endothelial cells switch to the “phalanx” cell fate, which is characterized by quiescent and nonproliferating cells aligned in a tight cobblestonelike layer. Vessel maturation also requires the recruitment of mural cells (ie, smooth muscle cells and pericytes). These cell fates are often altered in pathological conditions, most prominently during the formation of tumor vasculature. Given the essential role of hypoxia as the driving force for initiating angiogenesis, it is not surprising that the hypoxia-sensing machinery controls key steps in physiological and pathological angiogenesis.

Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest–specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo.

Mitotic spindle assembly is mediated by two processes: a centrosomal and a chromosomal pathway. RanGTP regulates the latter process by releasing microtubule-associated proteins from inhibitory complexes. NuSAP, a microtubule- and DNA-binding protein, is a target of RanGTP and promotes the formation of microtubules near chromosomes. However, the contribution of NuSAP to cell proliferation in vivo is unknown. Here, we demonstrate that the expression of NuSAP highly correlates with cell proliferation during embryogenesis and adult life, making it a reliable marker of proliferating cells. Additionally, we show that NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. As a result of sustained spindle checkpoint activity, the cells are unable to progress through mitosis, eventually leading to caspase activation and apoptotic cell death. Together, our findings demonstrate that NuSAP is essential for proliferation of embryonic cells and, simultaneously, they underscore the importance of chromatin-induced spindle assembly.

<title>Abstract</title> Conventional single-cell metabolomics approaches such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) generate biochemical snapshots, neglecting the inherent dynamic nature of metabolism. Here we describe a platform based on isotope tracing and MALDI-MSI that allows <italic>in-situ </italic>dynamic measurements of cell type-specific metabolism at single-cell resolution, and thus unravel cell metabolism within its tissue architecture. We applied different ¹³C-isotope-labeled nutrients on vibratome slices of fresh mouse kidney. MALDI-MSI at single-cell resolution (i.e. pixel size of 5 × 5 μm²) was then applied to detect metabolites and lipids from the harvested tissues. Following MALDI-MSI analysis, post-MSI-analyzed sections were stained and subsequently imaged using multiplexed immunofluorescence (IF) microscopy for cell-type identification. This platform allows to achieve single-cell resolution <italic>in situ </italic>and hence interpret cell type-specific metabolic dynamics in the context of structure and metabolism of neighboring cells.

Abstract Background Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo . Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGF δ/δ mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. Results Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGF δ/δ mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGF δ/δ mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGF δ/δ mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGF δ/δ mice, axon outgrowth is significantly reduced compared to wild-type littermates. Conclusions Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.