Publications

Abstract Background Plasmin system components are upregulated after partial hepatectomy, but their contribution to surgery-induced hepatic angiogenesis and regeneration is unclear. Liver regeneration and angiogenesis after partial hepatectomy were examined in mice lacking plasminogen or urokinase plasminogen activator (uPA). Methods Mice with a single-gene deletion of plasminogen or uPA were subjected to 70 per cent partial hepatectomy. Liver regeneration was measured as relative liver weight and cell proliferation index. Angiogenesis was quantified by determining hepatic microvessel density after staining for sinusoidal endothelial cells. Results The liver remnant weight was significantly reduced in mice lacking plasminogen or uPA compared with that in wild-type mice on days 2 and 7 after partial hepatectomy. This correlated with impaired cell proliferation. In wild-type mice, regeneration was accompanied by a significant increase in microvessel density after hepatectomy; this increase was impaired in plasminogen-deficient mice. Conclusion Plasminogen and uPA are essential for optimal liver regeneration. In addition, plasminogen appears to be a major determinant in regeneration-associated hepatic angiogenesis.

V ascular dysfunction is causally contributing to many dis- eases, including but not limited to cardiovascular disease, which is still the leading cause of death in the Western world.The endothelium that lines the inner wall of the blood vessels plays a critical role in the pathobiology of these illnesses.Particularly after ischemia or injury, the growth of new blood vessels, driven by endothelial expansion, is essential to maintain oxygen supply to the ischemic or injured tissue.Recent studies additionally suggest that the endothelium acts as a paracrine source for signals that determine tissue regeneration versus fibrosis after injury.Excessive vascularization, however, might also be unwanted, as in the case of cancer, neovascular eye diseases including diabetic retinopathy, atheroma growth, or the expansion of vasa vasorum, which leads to adverse vessel wall remodeling.Neovascularization is a tightly regulated and essential process that results in the formation of new blood vessels.Specific types of neovascularization include angiogenesis, the formation of new capillaries from existing capillaries, and arteriogenesis, the formation of new arteries from preexisting collaterals or de novo.Although endothelial cells (ECs) certainly are essential for both processes, the formation of functionally active vessels requires a complex molecular cross-talk of ECs with perivascular cells such as pericytes, smooth muscle cells, and macrophages.Simple in vitro models are best suited to examine specific aspects of particular processes involved in angiogenesis such as the biochemical interactions that regulate EC proliferation, motility, and apoptosis or lumen formation.However, in vivo models are required to understand the complex cellular interactions that enable the generation of functionally active and hierarchical blood vessel networks capable of providing an appropriate blood supply and paracrine stimuli to organs.In addition, the development of therapeutic strategies to either promote or inhibit vessel growth depends on reproducible measures and end points in experimental models that are relevant for the treatment of human diseases.Accordingly, the selection of an appropriate experimental model is critical to study specific aspects of the molecular and cellular mechanisms that are of physiological or pathological relevance.This scientific statement summarizes in vitro assays and in vivo models suitable for gaining insights into the basic mechanisms of vessel growth and provides an overview of models that are particularly useful for clinical translation. In Vitro AssaysConsiderable progress has been made over the past 20 years in the development of in vitro strategies to investigate the complex processes of vascular morphogenesis and maturation events in

Abstract Background There are no validated biomarkers to guide patient selection for treatment with the anti-VEGF antibody bevacizumab. We recently identified a locus, consisting of 4 SNPs in the VEGFR-1 TK domain, that correlated with progression-free (PFS) and overall survival (OS) of bevacizumab-treated pancreatic cancer patients in the AViTA phase III trial. The causal SNP underlying this association and its effect on VEGFR-1 expression or function are not yet known. Methods We conducted a fine-mapping study using data from the 1000 Genomes Project to identify the causal variant in this locus. The candidate SNP was functionally validated by studying effects on VEGFR-1 RNA (RT-PCR) or protein (ELISA) expression. Transcription and translation were measured using transient transfection and rabbit reticulocyte assays. Human umbilical vein endothelial cells (HUVECs) were isolated from healthy newborns. Association of causal SNPs was replicated in bevacizumab-treated renal cancer patients from the AVOREN phase III trial via Sequenom genotyping. Results Fine-mapping identified 48 common SNPs linked to one of the 4 SNPs in the VEGFR-1 locus. In silico functional prediction revealed that only one SNP (rs7993418) was located in exon 28. No other SNPs were located in exons or functionally important regions. Remarkably, rs7993418 is a synonymous SNP introducing a shift in codon usage (TAT to TAC codon) at an evolutionary conserved tyrosine (Tyr1213Tyr). In vitro transcription/translation of VEGFR-1 cDNAs carrying either the wildtype TAT or mutant TAC codon revealed that both constructs transcribe equal amounts of mRNA, whereas a 27% increased VEGFR-1 protein expression was found for TAC versus TAT-carrying constructs (P<0.001). Overexpression of both cDNAs in HEK293T cells demonstrated a similar increase in VEGFR-1 protein, but not mRNA expression for the TAC construct (P<0.001). Since transmembrane and soluble VEGFR-1 levels strongly correlate, we studied the effect of rs7993418 on sVEGFR-1 expression in human plasma. We found 18% and 19% increased median sVEGFR-1 levels in homozygous carriers of the TAC codon in two independent cohorts (P=0.006 and P=0.014). Primary HUVECs homozygous for the TAC codon also showed increased tmVEGFR-1 and sVEGFR-1 protein expression (P=0.049 and P=0.044), leading to increased VEGFR-1 downstream ERK1/2 signaling upon stimulation with the VEGFR-1 specific ligand PlGF (P<0.05). Finally, association of rs7993418 with bevacizumab treatment outcome was also replicated in AVOREN (per-allele HR=1.8; P=0.033). Conclusions The synonymous rs7993418 SNP (Tyr1213Tyr) is a functional SNP that potentially underlies the association of the VEGFR-1 locus with bevacizumab treatment outcome. In particular, rs7993418 causes a shift in codon usage, leading to increased VEGFR-1 expression and enhanced downstream signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-447. doi:1538-7445.AM2012-LB-447

The use of monoclonal antibodies targeting PlGF could thus offer a potential systemic treatment for patients who suffer from primary liver tumours.

<div>Abstract<p>Treatment of bone metastases is largely symptomatic and is still an unmet medical need. Current therapies mainly target the late phase of tumor-induced osteoclast activation and hereby inhibit further metastatic growth. This treatment method is, however, less effective in preventing initial tumor engraftment, a process that is supposed to depend on the bone microenvironment. We explored whether bone-derived placental growth factor (PlGF), a homologue of vascular endothelial growth factor-A, regulates osteolytic metastasis. Osteogenic cells secrete PlGF, the expression of which is enhanced by bone-metastasizing breast tumor cells. Selective neutralization of host-derived PlGF by anti-mouse PlGF (αPlGF) reduced the incidence, number, and size of bone metastases, and preserved bone mass. αPlGF did not affect metastatic tumor angiogenesis but inhibited osteoclast formation by preventing the upregulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand in osteogenic cells, as well as by blocking the autocrine osteoclastogenic activity of PlGF. αPlGF also reduced the engraftment of tumor cells in the bone and inhibited their interaction with matrix components in the metastatic niche. αPlGF therefore inhibits not only the progression of metastasis but also the settlement of tumor in the bone. These findings identify novel properties of PlGF and suggest that αPlGF might offer opportunities for adjuvant therapy of bone metastasis. Cancer Res; 70(16); 6537–47. ©2010 AACR.</p></div>

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.

<p>Overview of cancer-associated PPP2R4 exome mutations found in COSMIC and cBioportal cancer databases.</p>

<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.

VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF(120), VEGF(164), and VEGF(188) isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF(164) or only VEGF(188) (in VEGF(188/188) mice) was sufficient for metaphyseal development. VEGF(188/188) mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF(188) isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF(188/188) mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF(188) isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation.