VEGF‐C,VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours

Ramani P, Nash R, Radevsky L, Patel A, Luckett M & Rogers C
(2012) Histopathology
VEGF‐C, VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours Aims: More than 50% of neuroblastomas (NBs) present with haematogenous and/or lymphatic metastasis; however, little is known about the clinicopathological significance in NBs of the key lymphangiogenesis growth factors vascular endothelial growth factor (VEGF)‐C and VEGF‐D and the receptor VEGFR‐3. Methods and results: Ninety‐three NBs and nine ganglioneuromas (GNs) were immunostained for VEGF‐C, VEGF‐D and VEGFR‐3. VEGF‐C and VEGF‐D were present in 76% and 82% of the NBs, respectively. There was no significant difference in VEGF‐C expression between NBs and GNs. VEGF‐D expression was significantly higher in NBs compared with GNs and in MYCN‐amplified NBs. VEGFR‐3 tumoral cell expression (VEGFR‐3c), present in 48% of the NBs, was significantly higher in NBs from children ≥18 months at presentation and those belonging to a high‐risk group. VEGFR‐3 lymphovascular density was increased significantly in NBs compared with GNs and in NBs associated with adverse clinicopathological and biological factors. Lymphovascular invasion, assessed in VEGFR‐3‐stained vessels, was present in ～50% of NBs. Cox regression analyses demonstrated that VEGFR‐3c expression was associated with a significantly shorter event‐free survival and that its effect was independent of the important pathological variable, mitosis–karyorrhexis index. Conclusions: VEGF‐D and VEGFR‐3 up‐regulation support tumour progression in NB and VEGFR‐3c may provide a useful prognostic marker in NBs.     

Expression of VEGF‐C and VEGF‐D as Significant Markers for Assessment of Lymphangiogenesis and Lymph Node Metastasis in Non‐Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF)‐C and VEGF‐D induce lymphangiogenesis through activation of VEGF receptor 3 (VEGFR‐3) and have been implicated in tumor spread to the lymphatic system. Lymph node dissemination critically determines clinical outcome and therapeutic options of patients with non‐small cell lung cancer (NSCLC). However, the relationship of VEGF‐C, VEGF‐D, and lymph node metastasis in cancers, including NSCLC, is still controversial. To evaluate the relationship between lymphangiogenesis and lymph node metastasis, the expression of VEGF‐C and VEGF‐D in NSCLC tumors were detected by immunohistochemistry and quantitative real‐time polymerase chain reaction (QRT‐PCR). QRT‐PCR revealed that in marginal region VEGF‐C and VEGF‐D mRNA was significantly higher than in tumor center, and VEGF‐D mRNA was also higher than that in peritumoral lung tissue. Immunohistochemically, we observed the same heterogeneous expression of VEGF‐C and VEGF‐D proteins. The group with high expression of VEGF‐C and VEGF‐D in marginal region had a higher incidence of lymph node metastasis compared with the group with low expression. Furthermore, the group with high expression of VEGF‐D in marginal region had a higher incidence of lymphatic invasion. The group with high peritumoral lymphatic vessel density (LVD) had higher expression of VEGF‐C and VEGF‐D mRNA compared with the group with low peritumoral LVD. Our studies suggested that the expression of VEGF‐C and VEGF‐D at invasive edge was significantly associated with lymph node metastasis or lymphatic invasion in patients with NSCLC and may be involved in regulation of lymphangiogenesis and lymph node metastasis in NSCLC. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Impact of VEGF‐dependent tumour micro‐environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice

Fibronectin (FN) is an extracellular matrix cell‐adhesive glycoprotein. The alternative spliced isoform EDB‐FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro‐environments on EDB‐FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet‐FGF2 cells overexpressing fibroblast growth factor‐2 (FGF2) driven by the tetracycline‐responsive promoter were further transfected with a VEGF antisense cDNA, generating AS‐VEGF/Tet‐FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast‐growing, highly vascularized Tet‐FGF2 tumours. Down‐regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS‐VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro‐environment. Quantitative RT‐PCR analysis using human EDB‐FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up‐regulation of EDB‐FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki‐1 cells. However, in vivo down‐regulation of VEGF expression, as occurs in AS‐VEGF/Tet‐FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline‐treated Tet‐FGF2 tumour‐bearing animals, causes significant inhibition of EDB‐FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT‐PCR analysis. Accordingly, treatment of Tet‐FGF2 tumour‐bearing animals with the neutralizing anti‐murine VEGF receptor‐2 antibody DC101, or of Caki‐1 tumour‐bearing animals with the anti‐VEGF antibody bevacizumab, inhibited EDB‐FN expression in tumour grafts. EDB‐FN down‐regulation was paralleled by a decrease in vascularity, thus confirming EDB‐FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro‐environment, and in particular the VEGF/VEGFR‐2 system, plays a key role in modulating EDB‐FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB‐FN in combination with anti‐angiogenic and/or cytotoxic drugs. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

VEGF‐D promotes pulmonary oedema in hyperoxic acute lung injury

Leakage of fluid from blood vessels, leading to oedema, is a key feature of many diseases including hyperoxic acute lung injury (HALI), which can occur when patients are ventilated with high concentrations of oxygen (hyperoxia). The molecular mechanisms driving vascular leak and oedema in HALI are poorly understood. VEGF‐D is a protein that promotes blood vessel leak and oedema when overexpressed in tissues, but the role of endogenous VEGF‐D in pathological oedema was unknown. To address these issues, we exposed Vegfd‐deficient mice to hyperoxia. The resulting pulmonary oedema in Vegfd‐deficient mice was substantially reduced compared to wild‐type, as was the protein content of bronchoalveolar lavage fluid, consistent with reduced vascular leak. Vegf‐d and its receptor Vegfr‐3 were more highly expressed in lungs of hyperoxic, versus normoxic, wild‐type mice, indicating that components of the Vegf‐d signalling pathway are up‐regulated in hyperoxia. Importantly, VEGF‐D and its receptors were co‐localized on blood vessels in clinical samples of human lungs exposed to hyperoxia; hence, VEGF‐D may act directly on blood vessels to promote fluid leak. Our studies show that Vegf‐d promotes oedema in response to hyperoxia in mice and support the hypothesis that VEGF‐D signalling promotes vascular leak in human HALI. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

D2‐40‐positive lymphatic vessel invasion is not a poor prognostic factor in stage I lung adenocarcinoma

The present study investigates whether lymphatic vessel invasion (LVI) detected by D2‐40 staining is a prognostic factor for stage I adenocarcinoma of the lung. We retrospectively reviewed 124 patients who underwent complete resection for stage I adenocarcinoma of the lung from January 1983 to June 2003. LVI was microscopically evaluated using D2‐40 immunostaining. The median follow‐up was 71 months. The LVI positive rate was 37%. The 5‐year cancer‐specific survival rates of the D2‐40 positive LVI and negative groups were 88.8% and 84.3%, respectively (P = 0.630). The stage I lung adenocarcinoma patients who were determined to be LVI positive based on D2‐40 immunostaining did not have a significantly poorer prognosis than the LVI negative cases. Thus, lymphatic microinvasion may not be a prognostic indicator in early lung cancer, although advanced LVI does appear to correlate with survival. It is therefore unnecessary to use D2‐40 immunostaining to diagnose LVI in practical settings, and Hematoxylin‐Eosin and Elastica van Gieson staining should continue to be used to predict the prognosis of patients with stage I lung adenocarcinoma.     

Loss of p53 partially rescues embryonic development of Palb2 knockout mice but does not foster haploinsufficiency of Palb2 in tumour suppression

PALB2 interacts with BRCA1 and BRCA2 in supercomplexes involved in DNA repair via homologous recombination. Heterozygous germline mutations in PALB2 confer a moderate risk of breast cancer, while biallelic PALB2 mutations are linked to a severe form of Fanconi anaemia characterized by early childhood solid tumours and severe chromosomal instability. In contrast to BRCA1- or BRCA2-associated cancers, breast tumours in heterozygous PALB2 mutation carriers do not show loss of the wild-type allele, suggesting PALB2 might be haploinsufficient for tumour suppression. To study the role of PALB2 in development and tumourigenesis, we have generated Palb2(GT) mouse mutants using a gene trap approach. Whereas Palb2(GT/GT) homozygous mutant embryos died at mid-gestation due to massive apoptosis, Palb2(GT/+) heterozygous mice were viable and did not show any obvious abnormalities. Deletion of p53 alleviated the phenotype of Palb2(GT/GT) embryos, but did not rescue embryonic lethality. In addition, loss of p53 did not significantly collaborate with Palb2 heterozygosity in tumourigenesis in heterozygous or homozygous p53 knockout mice. Tumours arising in Palb2(GT/+) ;p53(+/-) or Palb2(GT/+) ;p53(-/-) compound mutant mice retained the wild-type Palb2 allele and did not display increased genomic instability.     

Optineurin deficiency in mice is associated with increased sensitivity to Salmonella but does not affect proinflammatory NF‐κB signaling

Optineurin (OPTN) is an evolutionary conserved and ubiquitously expressed ubiquitin‐binding protein that has been implicated in glaucoma, Paget bone disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. From in vitro studies, OPTN was shown to suppress TNF‐induced NF‐κB signaling and virus‐induced IRF signaling, and was identified as an autophagy receptor required for the clearance of cytosolic Salmonella upon infection. To assess the in vivo functions of OPTN in inflammation and infection, we generated OPTN‐deficient mice. OPTN knockout mice are born with normal Mendelian distribution and develop normally without any signs of spontaneous organ abnormality or inflammation. However, no differences in NF‐κB activation could be observed in OPTN knockout mice or fibroblasts derived from these mice upon TNF or LPS treatment. Primary bone marrow‐derived macrophages from OPTN‐deficient mice had slightly impaired IRF signaling and reduced IFN type I production in response to LPS or poly(I,C). Finally, OPTN‐deficient mice were more susceptible to infection with Salmonella, confirming in vivo the importance of OPTN in bacterial clearance.     

Poly (ADP‐ribose) polymerase‐1 deficiency does not affect ethylnitrosourea mutagenicity in liver and testis of lacZ transgenic mice

Poly (ADP‐ribose) polymerase‐1 (Parp1) has been implicated in DNA base excision repair, single‐ and double‐strand break repair pathways, as well as in cell death by apoptosis or necrosis. We used Parp1^?/? lacZ plasmid‐based transgenic mice to investigate whether Parp1 deficiency influences the in vivo mutagenic and clastogenic response to the alkylating agent N‐ethyl‐N‐Nitrosourea (ENU) in somatic and germ‐cell tissues. The comparison of the lacZ mutant frequencies (MFs) between Parp1^+/+ and Parp1^?/? mice showed that the ablation of Parp1 does not affect the spontaneous or ENU‐induced MFs in liver and testis. In addition, the spectrum of the ENU‐induced mutations was not dependent on the Parp1 status, given that similar spectra, consisting mostly of point mutations and a small fraction of deletions/insertions, wereobserved in organs of both Parp1^?/? and Parp1^+/+ mice. Sequencing of point mutations revealed a consistent significant increase in A:T → T:A base substitutions, typically induced by ENU. Overall, we observed that neither the frequency nor the spectrum of ENU‐induced mutations demonstrated a specificity that could be attributed to the Parp1 impairment in mice organs. The analysis of micronucleus frequency in peripheral blood reticulocytes showed that ENU was clastogenic in both Parp1^?/? and Parp1^+/+ mice and had a strong cytotoxic effect in Parp1^?/? mice only. The present data suggest that, at a whole‐organism level, Parp1‐independent repair mechanisms may be operative in the removal of ENU‐induced DNA lesions or that highly damaged cells may be preferentially committed to death when Parp1 is inactivated. Environ. Mol. Mutagen. 2010. © 2010 Wiley‐Liss, Inc.     

Surfactant protein D (SP‐D) deficiency is attenuated in humanised mice expressing the Met(11)Thr short nucleotide polymorphism of SP‐D: implications for surfactant metabolism in the lung

Surfactant protein D (SP-D) is part of the innate immune system involved in lung homeostasis. SP-D knockout mice show accumulations of foamy alveolar macrophages, alveolar lipoproteinosis and pulmonary emphysema. Three single nucleotide polymorphisms (SNPs) have been described in the coding sequence of the human SP-D gene SFTPD. Clinical studies showed that the SNP SFTPD with a nucleotide change from A to C resulting in a Met to Thr substitution at position 11 in the protein (Met(11)Thr), is relevant. This study set out to create a humanised mouse model of the Met(11)Thr SNP. Transgenic mice lines expressing either Met(11) or Thr(11) SP-D under the control of the ubiquitously expressed pROSA26 promoter in C57Bl/6 SP-D deficient mice (DKO) was created. Both Met(11) (142 ± 52 ng mL⁻¹) and Thr(11) (228 ± 76 ng mL⁻¹) mice lines expressed human SP-D at almost similar levels. According to the literature this was within the range of SP-D levels found in wildtype (WT) mice (253 ± 22 ng mL⁻¹). Met(11) or Thr(11) SP-D in serum from transgenic mice bound maltose in a calcium-dependent manner, and binding was inhibited in the presence of EDTA or maltose. Bronchoalveolar lavage showed for both transgenic mice lines complementation of the DKO phenotype by restoring cell counts, phospholipid levels and protein content back to WT levels. Cytospins of BAL pellet cells showed a resemblance to WT but both mice lines showed some foamy alveolar macrophages. The stereological analysis showed for none of the mice lines a complete abrogation of emphysematous alterations. However, both Met(11) and Thr(11) mice lines were partially reverted back to a WT phenotype when compared with DKO mice, indicating important effects on surfactant metabolism in vivo.     

Ccl2/Mcp‐1 blockade reduces glomerular and interstitial macrophages but does not ameliorate renal pathology in collagen4A3‐deficient mice with autosomal recessive Alport nephropathy

Lack of the α3 or α4 chain of type IV collagen (COL4) causes autosomal recessive Alport nephropathy in humans and mice that is characterized by progressive glomerulosclerosis and tubulointerstitial disease. Renal pathology is associated with chemokine‐mediated macrophage infiltrates but their contribution to the progression of Alport nephropathy is unclear. We found Ccl2 to be expressed in increasing amounts during the progression of nephropathy in Col4a3‐deficient mice; hence, we blocked Ccl2 with anti‐Ccl2 Spiegelmers, biostable L ‐enantiomeric RNA aptamers suitable for in vivo applications. Ccl2 blockade reduced the recruitment of ex vivo‐labelled macrophages into kidneys of Col4a3‐deficient mice. We therefore hypothesized that a prolonged course of Ccl2 blockade would reduce renal macrophage counts and prevent renal pathology in Col4a3‐deficient mice. Groups of Col4a3‐deficient mice received subcutaneous injections of either an anti‐mCcl2 Spiegelmer or non‐functional control Spiegelmer on alternate days, starting from day 21 or 42 of age. Glomerular and interstitial macrophage counts were found to be reduced with Ccl2 blockade by 50% and 30%, respectively. However, this was not associated with an improvement of glomerular pathology, interstitial pathology, or of overall survival of Col4a3‐deficient mice. We conclude that Ccl2 mediates the recruitment of glomerular and interstitial macrophages but this mechanism does not contribute to the progression of Alport nephropathy in Col4a3‐deficient mice. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Monoclonal antibody against macrophage colony‐stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus

This study's objective was to assess the effects of PD‐0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony‐stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD‐0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre‐ and post‐treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14⁺ CD16⁺ monocytes, urinary N‐terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater‐than‐dose‐proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14⁺ CD16⁺ cells, uNTX, ALT, AST and CK levels at most time‐points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between‐group differences were seen in CLASI. Patients receiving PD‐0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD‐0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end‐points.