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Introduction: We evaluated outcomes of closed incisional negative pressure therapy (ciNPT) on surgical site infection (SSI) rates in lower extremity bypass patients. We sought to determine whether or not the routine use of ciNPT is a cost-effective measure. Methods: During a period from May 2018 to August 2018, our institution transitioned to the routine use of ciNPT for re-vascularization procedures. We retrospectively reviewed our outcomes before and after the initiation of ciNPT. Group A included patients from September 2017 to April 2018 without ciNPT and Group B included patients from September 2018 to April 2019 with ciNPT. Chi-squared analysis was performed and the p value was set at

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long‐term exposure to concentrations of aluminium—in the form of aluminium chloride (AlCl3)—in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl3 form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl3. Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well‐established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens., What's new? Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives, and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. Here, the authors report that long‐term exposure to concentrations of aluminium in the range of those measured in the human breast enables normal murine mammary gland (NMuMG) epithelial cells to form tumors and metastasis in well‐established mouse cancer models. The observations indicate that aluminium salts could be environmental breast carcinogens.

Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspirants, and in the range of those recently measured in the human breast, results in loss of contact inhibition and anchorage-independent growth. These effects were preceded by an increase of DNA synthesis, DNA double strand breaks (DSBs), and senescence in proliferating cultures. AlCl(3) also induced DSBs and senescence in proliferating primary human mammary epithelial cells. In contrast, it had no similar effects on human keratinocytes or fibroblasts, and was not detectably mutagenic in bacteria. MCF-10A cells morphologically transformed by long-term exposure to AlCl(3) display strong upregulation of the p53/p21(Waf1) pathway, a key mediator of growth arrest and senescence. These results suggest that aluminium is not generically mutagenic, but similar to an activated oncogene, it induces proliferation stress, DSBs and senescence in normal mammary epithelial cells; and that long-term exposure to AlCl(3) generates and selects for cells able to bypass p53/p21(Waf1) -mediated cellular senescence. Our observations do not formally identify aluminium as a breast carcinogen, but challenge the safety ascribed to its widespread use in underarm cosmetics.

We screened maize (Zea mays) cDNAs for sequences similar to the single myb-like DNA-binding domain of known telomeric complex proteins. We identified, cloned, and sequenced five full-length cDNAs representing a novel gene family, and we describe the analysis of one of them, the gene Single myb histone 1 (Smh1). The Smh1 gene encodes a small, basic protein with a unique triple motif structure of (a) an N-terminal SANT/myb-like domain of the homeodomain-like superfamily of 3-helical-bundle-fold proteins, (b) a central region with homology to the conserved H1 globular domain found in the linker histones H1/H5, and (c) a coiled-coil domain near the C terminus. The Smh-type genes are plant specific and include a gene family in Arabidopsis and the PcMYB1 gene of parsley (Petroselinum crispum) but are distinct from those (AtTRP1, AtTBP1, and OsRTBP1) recently shown to encode in vitro telomere-repeat DNA-binding activity. The Smh1 gene is expressed in leaf tissue and maps to chromosome 8 (bin 8.05), with a duplicate locus on chromosome 3 (bin 3.09). A recombinant full-length SMH1, rSMH1, was found by band-shift assays to bind double-stranded oligonucleotide probes with at least two internal tandem copies of the maize telomere repeat, TTTAGGG. Point mutations in the telomere repeat residues reduced or abolished the binding, whereas rSMH1 bound nonspecifically to single-stranded DNA probes. The two DNA-binding motifs in SMH proteins may provide a link between sequence recognition and chromatin dynamics and may function at telomeres or other sites in the nucleus.

During meiotic prophase, telomeres actively attach themselves to the nuclear envelope and cluster in an arrangement called the bouquet. The bouquet is unique to meiosis, highly conserved, and thought to facilitate homologous chromosome synapsis. Analy sis of three-dimensional fluorescence in situ hybridization (3-D FISH) image data has been employed to characterize the bouquet in fixed pollen mother cells of maize (Zea mays L.). In order to examine the function of the bouquet further, several meiotic mutants were screened for telomeric defects using 3-D FISH as an assay. Two mutants, desynaptic (dy) and desynaptic1 (dsy1), were found to exhibit novel telomere-misplacement phenotypes. In both cases, the telomere-associated mutant phenotypes occurred prior to what was previously reported as the earliest affected stage. Three alleles of the desynaptic1 mutation (dsy1-1, dsy1-9101, and dsy1-9307) resulted in a partial bouquet phenotype at the zygotene stage of meiotic prophase. By contrast, dy nuclei contained apparently normal bouquets, but then resulted in a premature intranuclear localization of telomeres at the pachytene stage, when telomeres normally disperse but remain attached to the nuclear envelope. The dsy1 mutation is known to impair the fidelity and progression of homologous synapsis, whereas the dy mutation is known to reduce recombination rates. If the telomere misplacements are primary defects of these mutants, then these data would be consistent with the hypothesis that meiotic telomeres have at least two separable functions, one involving proper homologous chromosome synapsis at the bouquet stage and another involving post-bouquet cross-over control.

Oxygen tensions in the kidney are heterogeneous, and their changes presumably play an important role in renal physiologic and pathophysiologic processes. A family of hypoxia-inducible transcription factors (HIF) have been identified as mediators of transcriptional responses to hypoxia, which include the regulation of erythropoietin, metabolic adaptation, vascular tone, and neoangiogenesis. In vitro, the oxygen-regulated subunits HIF-1alpha and -2alpha are expressed in inverse relationship to oxygen tensions in every cell line investigated to date. The characteristics and functional significance of the HIF response in vivo are largely unknown. High-amplification immunohistochemical analyses were used to study the expression of HIF-1alpha and -2alpha in kidneys of rats exposed to systemic hypoxia bleeding anemia, functional anemia (0.1% carbon monoxide), renal ischemia, or cobaltous chloride (which is known to mimic hypoxia). These treatments led to marked nuclear accumulation of HIF-1alpha and -2alpha in different renal cell populations. HIF-1alpha was mainly induced in tubular cells, including proximal segments with exposure to anemia/carbon monoxide, in distal segments with cobaltous chloride treatment, and in connecting tubules and collecting ducts with all stimuli. Staining for HIF-1alpha colocalized with inducible expression of the target genes heme oxygenase-1 and glucose transporter-1. HIF-2alpha was not expressed in tubular cells but was expressed in endothelial cells of a small subset of glomeruli and in peritubular endothelial cells and fibroblasts. The kidney demonstrates a marked potential for upregulation of HIF, but accumulation of HIF-1alpha and HIF-2alpha is selective with respect to cell type, kidney zone, and experimental conditions, with the expression patterns partly matching known oxygen profiles. The expression of HIF-2alpha in peritubular fibroblasts suggests a role in erythropoietin regulation.

Mutations in the von Hippel-Lindau (VHL) gene are associated with hereditary and sporadic clear cell renal carcinoma. VHL acts in a ubiquitin ligase complex regulating hypoxia-inducible factor-1 (HIF-1), but the link between this function and cancer development is unclear. Here we show that in the kidneys of patients with VHL disease, HIF activation is an early event occurring in morphologically normal single cells within the renal tubules. In comparison, dysplastic lesions, cystic lesions, and tumors showed evidence of additional mechanisms that amplify HIF activation. Detection of cells with constitutive HIF activation identified a large number of previously unrecognized foci of VHL inactivation. In proximal tubules these were almost entirely unicellular, whereas multicellular foci were almost exclusively seen in the distal nephron.

Vascular endothelial growth factors (VEGFs) are endothelial cell-specific mitogens with potent angiogenic and vascular permeability-inducing properties. VEGF, VEGF-C, and VEGFRs -1, -2, and -3 were found to be expressed in post-pubertal (virgin) rodent mammary glands. VEGF was increased during pregnancy (5-fold) and lactation (15-19-fold). VEGF-C was moderately increased during pregnancy and lactation (2- and 3-fold respectively). VEGF levels were reduced by approximately 75% in cleared mouse mammary glands devoid of epithelial components, demonstrating that although the epithelial component is the major source of VEGF, approximately 25% is derived from stroma. This was confirmed by the findings (a) that VEGF transcripts were expressed predominantly in ductal and alveolar epithelial cells, and (b) that VEGF protein was localized to ductal epithelial cells as well as to the stromal compartment including vascular structures. VEGF was detected in human milk. Finally, transcripts for VEGFRs -2 and -3 were increased 2-3-fold during pregnancy, VEGFRs -1, -2 and -3 were increased 2-4-fold during lactation, and VEGFRs -2 and -3 were decreased by 20-50% during involution. These results point to a causal role for the VEGF ligand-receptor pairs in pregnancy-associated angiogenesis in the mammary gland, and suggest that they may also regulate vascular permeability during lactation.

Le facteur de croissance vasculaire endothelial (vascular endothelial growth factor (VEGF))-C est un nouveau membre de la famille des VEGFs, facteurs de croissance polypeptidiques qui jouent un role cle dans la physiologie et la pathologie de plusieurs aspects du systeme cardiovasculaire, y compris la vasculogenese, l’hematopoiese, l’angiogenese et la permeabilite vasculaire. Les signaux des VEGFs sont transmis dans les cellules endotheliales grâce a des recepteurs de type tyrosine kinase, les VEGFRs, dont on connait actuellement trois exemplaires, exprimes par les cellules endotheliales et les precurseurs hematopoietiques. Compare au premier VEGF decrit, le VEGF-A ; qui est un mitogene specifique des cellules endotheliales et facteur angiogenique de premier ordre, le VEGF-C semble avoir un role principal dans le developpement du systeme lymphatique. Ceci reflete le fait que, si VEGF-A est un ligand pour VEGFR-1 et VEGFR-2, VEGF-C exerce son activite biologique par l’intermediaire du recepteur-3, dont l’expression, au cours du developpement, va se restreindre aux vaisseaux lymphatiques et a certaines veines. Cependant, la capacite du VEGF-C de se lier aussi au VEGFR-2 et de l’activer est probablement responsable de son pouvoir d'induire une reponse angiogenique dans certains modeles, ce qui le rend interessant du point de vue des manipulations experimentales ou cliniques ayant pour but de bloquer ou de stimuler l’angiogenese. Dans cet article nous discutons brievement les connaissances actuelles sur l’activite biologique de VEGF-C, en mettant en evidence que celle-ci, comme dans le cas d’autres facteurs angiogeniques, est profondement influencee par l’activite d’autres regulateurs angiogeniques presents dans le microenvironnement des cellules endotheliales.

Department of Anaesthesiology, Pharmacology and Surgical Intensive Care, University Medical Center, University of Geneva, Switzerland. lucas@cmu.unige.chThe conditions under which tumor necrosis factor-alpha (TNF) induces apoptosis in primary microvascular endothelial cells (MVEC) were investigated. In the absence of sensitizing agents, TNF induced apoptosis after 3 days of incubation in confluent MVEC. In contrast, upon addition of the transcriptional inhibitor actinomycin D (Act. D), confluence was no longer required and apoptosis occurred already after 16 h. To assess the role of either TNF receptor (TNFR) type in apoptosis, MVEC isolated from mice genetically deficient in TNFR1 (Tnfr1o mice) or TNFR2 (Tnfr2o mice) were incubated with TNF in the presence or absence of Act. D. Under sensitized conditions, Tnfr2o MVEC were lysed like controls, whereas Tnfr1o MVEC were completely resistant, indicating an exclusive role for TNFR1. In contrast, in the absence of Act. D, confluent monolayers of wild-type cells were lysed by TNF, but both Tnfr1o and Tnfr2o MVEC were resistant to TNF-mediated toxicity, indicating a requirement for both TNFR types. Overexpression of the anti-apoptotic protein bcl-xL in MVEC led to a protection against the direct, but not the sensitized cytotoxicity of TNF. In conclusion, in pathophysiologically relevant conditions, both TNFR appear to be required for TNF-induced apoptosis in MVEC.

The vascular endothelial growth factor (VEGF) family has recently expanded by the identification and cloning of three additional members, namely VEGF-B, VEGF-C, and VEGF-D. In this study we demonstrate that VEGF-B binds selectively to VEGF receptor-1/Flt-1. This binding can be blocked by excess VEGF, indicating that the interaction sites on the receptor are at least partially overlapping. Mutating the putative VEGF receptor-1/Flt-1 binding determinants Asp 63 , Asp 64 , and Glu 67 to alanine residues in VEGF-B reduced the affinity to VEGF receptor-1 but did not abolish binding. Mutational analysis of conserved cysteines contributing to VEGF-B dimer formation suggest a structural conservation with VEGF and platelet-derived growth factor. Proteolytic processing of the 60-kDa VEGF-B 186 dimer results in a 34-kDa dimer containing the receptor-binding epitopes. The binding of VEGF-B to its receptor on endothelial cells leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor 1, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration.

We have previously reported the existence of a synergistic interaction between vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the induction of angiogenesis in vitro. Here we demonstrate that bFGF increases VEGF receptor-2 (VEGFR-2/Flk-1) expression: mRNA levels were increased by 4.5- to 8.0-fold and total protein by 2.0- to 3.5-fold, in bovine microvascular endothelial (BME), aortic endothelial (BAE), and transformed fetal aortic (GM7373) endothelial cells. VEGF itself did not affect VEGFR-2 expression, and neither bFGF nor VEGF altered expression of FGF receptor-1. We also show that synergism occurs at the level of proliferation when this is measured in a three-dimensional but not in a conventional two-dimensional assay. Differences in the level of VEGFR-2 expression were also observed when cells were grown on or within collagen gels under different conditions: mRNA levels were lowest under sparse conditions, increased 20- to 26-fold at confluence, and increased even further (57-fold) when cells were cultured in suspension in three-dimensional collagen gels. Finally, a synergistic increase was seen in the level of expression of urokinase and urokinase receptor mRNAs when cells were exposed to bFGF and VEGF for 4 days. These findings demonstrate that the level of VEGFR-2 expression can be modulated by environmental factors including cytokines and the geometry of the culture conditions and provide some insight into the mechanisms of synergism between bFGF and VEGF in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, both of which may be important for the function of islets of Langerhans. In this study, we have examined the expression of VEGF and its tyrosine kinase receptors (fit and flk-1) in isolated rat islets of Langerhans in vitro. When analyzed by in situ hybridization, islet tissue showed a significant 4.6-fold increase in VEGF mRNA expression over time in culture from 0 to 7 days. Islet tissue exposed to hypoxic/anoxic conditions for a period of 8 hr showed a 3.7-fold increase in VEGF mRNA when analyzed by Northern blot hybridization. Reverse transcriptase-polymerase chain reaction revealed the presence of both fit and flk.l in freshly isolated islets, and two VEGF isoforms, namely VEGF 120 and VEGF 164 . Three rodent β-cell lines derived from insulinomas (RINm5F-2A, INS-1, and MIN6) were also found to express VEGF by Northern blot hybridization. However, neither hypoxia/anoxia nor low (0.3 g/L)- or high (3.0 g/L)-glucose culture conditions modulated their expression of VEGF. VEGF derived from RINm5F-2A cells was bioactive in a three-dimensional in vitro model of angiogenesis, which assays for endothelial cell invasion and capillary morphogenesis. These findings demonstrate, first, that devascularization increases VEGF expression in isolated islet tissue, and they point to VEGF as a potentially important endogenous angiogenic stimulus for subsequent revascularization in vivo. Second, our observations raise the possibility that survival of transplanted islets may be improved by increasing VEGF expression before transplantation.

We have previously reported that culture medium conditioned by human SK-Hep1 hepatoma cells or mouse S180 sarcoma cells induces in vitro angiogenesis and stimulates production of urokinase plasminogen activator (uPA) in vascular endothelial cells. These activities are mediated by a 3.5-10 kDa, heparin-binding peptide that upregulates endothelial cell expression of basic fibroblast growth factor (bFGF; Peverali et al., 1994, J. Cell. Physiol. 161:1-14.) We now report that SK-Hep 1 or S180 cell-conditioned medium rapidly induces a 4- to 5-fold increase in cell-bound uPA activity and in the high-affinity binding of 125I-prouPA to vascular endothelial cells. Ligand blotting and purification experiments show an equivalent increase in the synthesis of a cell surface protein corresponding to the endothelial cell uPA receptor (uPAR) on the basis of M, (45-50 kDa) and sensitivity to phosphatidylinositol-specific phospholipase C (PI-PLC). The tumor cell-conditioned media also upregulate uPAR mRNA levels in endothelial cells. Thus, the increase in uPA binding capacity of endothelial cells is mediated by an increased expression of uPAR. The uPAR-inducing activity of SK-Hep 1 or S180 cell-conditioned medium is not neutralized by antibodies to bFGF, and is associated with a peptide that has a M, higher than 10 kDa and no affinity for heparin. Therefore, it appears to be distinct from the bFGF/uPA-inducing factor secreted by the same cells, and from other heparin-binding cytokines that upregulate uPAR expression in endothelial cells.

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.

Extracellular proteolysis is required for matrix degradation and the regulation of cytokine activity during angiogenesis, and this is dependent on a cohort of proteases and protease inhibitors produced by endothelial and nonendothelial cells. The plasminogen activator (PA)/plasmin system has been extensively investigated in these processes, and descriptive studies have demonstrated that urokinase-type PA (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) are expressed by endothelial cells during angiogenesis in vivo. In vitro studies have led to the notion that normal capillary morphogenesis is dependent on a protease-antiprotease equilibrium. These findings are discussed in the context of recent observations on uPA-, uPAR-, PAI-1 and plaminogen-deficient mice, in which developmental and physiological angiogenesis appear to occur normally. This has led to a reevaluation of the role of the PA/plasmin system during angiogenesis. In particular, these observations raise the possibility that the role of this system may be limited to situations in which endothelial cells encounter and must degrade fibrin in order to form new capillary sprouts.

Cellular responses to oxygen are increasingly recognized as critical in normal development and physiology, and are implicated in pathological processes. Many of these responses are mediated by the transcription factors HIF-1 and HIF-2. Their regulation occurs through oxygen-dependent proteolysis of the alpha subunits HIF-1alpha and HIF-2alpha, respectively. Both are stabilized in cell lines exposed to hypoxia, and recently HIF-1alpha was reported to be widely expressed in vivo. In contrast, regulation and sites of HIF-2alpha expression in vivo are unknown, although a specific role in endothelium was suggested. We therefore analyzed HIF-2alpha expression in control and hypoxic rats. Although HIF-2alpha was not detectable under baseline conditions, marked hypoxic induction occurred in all organs investigated, including brain, heart, lung, kidney, liver, pancreas, and intestine. Time course and amplitude of induction varied between organs. Immunohistochemistry revealed nuclear accumulation in distinct cell populations of each tissue, which were exclusively non-parenchymal in some organs (kidney, pancreas, and brain), predominantly parenchymal in others (liver and intestine) or equally distributed (myocardium). These data indicate that HIF-2 plays an important role in the transcriptional response to hypoxia in vivo, which is not confined to the vasculature and is complementary to rather than redundant with HIF-1.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

The establishment and maintenance of a vascular supply is an absolute requirement for the growth of normal and neoplastic tissues, and as might be predicted, the cardiovascular system is the first organ system to develop and to become functional during embryogenesis. Both during development and in postnatal life, all new blood vessels begin as simple endothelial-lined tubes. Some become capillaries after developing an intimate association with pericytes, whereas others develop into vessels of larger diameter (arteries and veins) and acquire a variable number of concentrically disposed smooth-muscle cell layers. Traditionally, the formation of new blood vessels has been ascribed to two interrelated but separable processes, vasculogenesis, and angiogenesis (Fig. 1).

Mutations in the von Hippel-Lindau (VHL) gene are associated with hereditary and sporadic clear cell renal carcinoma. VHL acts in a ubiquitin ligase complex regulating hypoxia-inducible factor-1 (HIF-1), but the link between this function and cancer development is unclear. Here we show that in the kidneys of patients with VHL disease, HIF activation is an early event occurring in morphologically normal single cells within the renal tubules. In comparison, dysplastic lesions, cystic lesions, and tumors showed evidence of additional mechanisms that amplify HIF activation. Detection of cells with constitutive HIF activation identified a large number of previously unrecognized foci of VHL inactivation. In proximal tubules these were almost entirely unicellular, whereas multicellular foci were almost exclusively seen in the distal nephron.

The pivotal role of vascular endothelial growth factor (VEGF-A) in the regulation of angiogenesis, in particular in the onset and maintenance of tumor angiogenesis, has been demonstrated repeatedly in experimental model systems and, more recently, in clinical trials. Experimental evidence has also suggested that up-regulated expression of VEGF-A may cooperate with other genetic or epigenetic changes to induce or accelerate tumor progression to invasive and metastatic cancers. Here we report the generation of transgenic mouse lines that express human VEGF-A165 under the control of the rat insulin promoter in the beta cells of pancreatic islets of Langerhans (Rip1VEGF-A). These mice do not exhibit detectable changes in islet development, vascularization, or physiology. Intercrosses of these mice with a transgenic mouse model of pancreatic beta cell carcinogenesis (Rip1Tag2) result in an earlier onset of tumor angiogenesis and with it accelerated tumor growth and mortality. The transition from benign tumors (adenoma) to malignant tumors (carcinoma) is modestly accelerated; however, tumor metastases are not observed. Our findings indicate that in beta-cell tumorigenesis, overexpression of VEGF-A165 accelerates the onset of tumor angiogenesis and with it tumor progression but is not sufficient to induce tumor metastasis.

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.

Angiopoietins are ligands for the endothelial cell tyrosine kinase receptor Tie-2. Ang-1, the major physiological activator of Tie-2, promotes blood vessel maturation and stability. Ang-2 counteracts this effect by competitively inhibiting the binding of Ang-1 to Tie-2. Using a combined RNase protection/semiquantitative reverse transcriptase-polymerase chain reaction approach, we demonstrate that hypoxia up-regulates Ang-2 mRNA levels by up to 3.3-fold in two human endothelial cell lines. In bovine microvascular endothelial (BME) cells, the flavoprotein oxidoreductase inhibitor diphenylene iodonium (DPI) and the related compound iodonium diphenyl mimic induction of Ang-2 but not vascular endothelial growth factor (VEGF) by hypoxia; in combination with hypoxia, DPI further increases Ang-2 expression but has no effect on the induction of VEGF by hypoxia. Neither Ang-2 or VEGF was increased by cyanide or rotenone, suggesting that failure in mitochondrial electron transport is not involved in the oxygen-sensing system that controls their expression. In ischemic rat dorsal skin flaps or in the brain of rats maintained for 12 hours under conditions of hypoxia, Ang-2 mRNA was up-regulated 7.5- or 17.6- fold, respectively. VEGF was concomitantly increased, whereas expression of Ang-1, Tie-2, and the related receptor Tie-1 was unaltered. In situ hybridization localized Ang-2 mRNA to endothelial cells in hypoxic skin. These findings 1) show that up-regulation of Ang-2 by hypoxia occurs widely in endothelial cells in vitro and in vivo; 2) suggest that induction of Ang-2, but not VEGF, by hypoxia in BME cells is controlled by a flavoprotein oxidoreductase that is sensitive to iodonium compounds; and 3) point to Ang-2 and VEGF as independently regulated and selective effectors of hypoxia-induced vascular sprouting.

Abstract —Angiopoietin-2 (Ang2) is a ligand for the endothelial cell tyrosine kinase receptor Tie2 and counteracts blood vessel maturation/stability mediated by angiopoietin-1 (Ang1), the other known ligand of Tie2. Using degenerate oligonucleotides and reverse transcriptase–polymerase chain reaction, we have screened bovine microvascular endothelial (BME), aortic, lymphatic, pulmonary artery, and transformed fetal aortic endothelial cells, as well as rat smooth muscle cells for Ang1 and Ang2 expression. Except for high Ang2 mRNA levels found in BME cells, none of the endothelial cell types studied expressed appreciable levels of Ang1 or Ang2 mRNAs, whereas smooth muscle cells expressed both Ang1 and Ang2. BME cell Ang2 mRNA levels were increased by vascular endothelial growth factor (1.9- to 2.9-fold), basic fibroblast growth factor (1.6- to 2-fold), both cytokines in combination (2.9- to 4-fold), and hypoxia (3.1- to 5.6-fold) and were decreased by Ang1 (31% to 70%) or transforming growth factor-β 1 (64% to 81%). Ang2 also decreased (60% to 82%) BME cell Ang2 mRNA. mRNA levels for the Tie1 or Tie2 receptors were only slightly modulated under the conditions described above. These findings suggest that the angiogenic effect of a number of regulators may be achieved in part through the regulation of an autocrine loop of Ang2 activity in microvascular endothelial cells.

Induction of in vitro angiogenesis and upregulation of urokinase- and tissue type-plasminogen activator (uPA, tPA) expression are two hallmarks of vascular endothelial growth factor (VEGF) activity on cultured endothelial cells. We report here that neutralizing antibodies to basic fibroblast growth factor (bFGF) inhibit VEGF-induced in vitro angiogenesis in bovine microvascular endothelial (BME) cells. Analysis of VEGF receptor-2 (VEGFR-2) expression revealed no alteration in VEGFR-2 mRNA or total protein in anti-bFGF antibody-treated BME or bovine aortic endothelial (BAE) cells. Ethidium bromide/agarose gel electrophoresis on the cytosolic fraction of BME cells revealed a basal level of fragmented DNA that was increased by anti-bFGF antibodies to an extent not exceeding that observed in parallel cultures incubated with concentrations of transforming growth factor-ss1 that increase VEGF-induced in vitro angiogenesis. In both BME and BAE cells, antibodies to bFGF also decreased basal levels of cell-associated uPA activity, and completely blocked the VEGF-mediated increase in uPA and tPA expression observed in parallel cultures incubated with VEGF alone. In contrast, PA inhibitor-1 expression was strongly upregulated in BME and BAE cells incubated with antibodies to bFGF, either alone or in combination with VEGF. These findings demonstrate that: (1) VEGF-induced in vitro angiogenesis and PA expression are dependent on endogenous bFGF, (2) that this phenomenon is not mediated by a decrease in VEGFR-2 expression and that apoptosis does not necessarily correlate with inhibition of invasion, and (3) that inhibition of endogenous bFGF in VEGF-treated cells results in a net antiproteolytic (and possibly also anti-adherent) effect, which could account in part for the inhibitory effect of the anti-bFGF antibodies. These findings point to a novel and unsuspected role for endogenous bFGF in regulating VEGF-induced in vitro angiogenesis.

The establishment of a vascular supply is an absolute requirement for the growth of normal and neoplastic tissues and, as might be expected, the cardiovascular system is the first organ system to develop and to become functional during embryogenesis. It has been known since the beginning of this century (Evans 1909) that, both during development and in postnatal life, all blood vessels begin as simple endothelial-lined capillaries. Although some remain as capillaries, many of these newly formed vessels develop into larger vessels, in part through the addition of a variable number of concentrically disposed smooth muscle cell layers. The heterogeneity and variable complexity of blood vessels thus generated serves to ensure structural and functional diversity in the vascular tree.

not Available.

SINCE the development of soft-walled traps suitable for the capture of small to medium-sized macropodids (Kinnear et al. 1988; Pollock and Montague 1991), traps of similar design have been used to capture swamp wallabies (Wallabia bicolor) by a number of workers (Wood 2002; Ben-Ami 2005; Paplinska 2005; B Parker, pers. comm.). Although immobilising drugs delivered by syringe darts have also been used to capture W. bicolor successfully (Troy et al. 1992), this species is difficult to dart relative to other similar sized wallabies (Wood 2002), and once darted can be hard to find within its often densely vegetated habitat (Pollock and Montague 1991). The difficulty of locating drugged animals in dense vegetation or steep terrain also makes the use of bait laced with diazepam or alpha chloralose (e.g., Norbury et al. 1994) impractical. Other capture techniques, such as stunning and netting (e.g., Taggart et al. 2003) are also rendered ineffective due to the habitat selected by this species.

Extracts from HeLa cell nuclei assemble RNAs containing the adenovirus type 2 L3 polyadenylation site into a number of rapidly sedimenting heterodisperse complexes. Briefly treating reaction mixtures prior to sedimentation with heparin reveals a core 25S assembly formed with substrate RNA but not an inactive RNA containing a U----C mutation in the AAUAAA hexanucleotide sequence. The requirements for assembly of this heparin-stable core complex parallel those for cleavage and polyadenylation in vitro, including a functional hexanucleotide, ATP, and a uridylate-rich tract downstream of the cleavage site. The AAUAAA and a downstream U-rich element are resistant in the assembly to attack by RNase H. The poly(A) site between the two protected elements is accessible, but is attacked more slowly than in naked RNA, suggesting that a specific factor or secondary structure is located nearby. The presence of a factor bound to the AAUAAA in the complex is independently demonstrated by immunoprecipitation of a specific T1 oligonucleotide containing the element from the 25S fraction. Precipitation of this fragment from reaction mixtures is blocked by the U----C mutation. However, neither ATP nor the downstream sequence element is required for binding of this factor in the nuclear extract, suggesting that recognition of the AAUAAA is an initial event in complex assembly.

Fetal thymus organ culture was used to study the expression and function of antigen-specific, major histocompatibility complex-restricted receptors on thymocytes. Receptor gene rearrangement and expression occurred de novo in organ culture indicating that these events are induced in the thymus itself, presumably in response to thymus-derived stimuli. During organ culture a population of immature thymocytes expressing low levels of receptors developed first, and then diminished as mature thymocytes with high levels of receptor expression appeared. Continuous culture with antireceptor antibody modulated receptor from the surfaces of immature thymocytes, but did not prevent their appearance or accumulation. By contrast, appearance of receptor-bearing mature thymocytes was prevented in the presence of antireceptor antibody. These results indicate that the receptor is not essential for the generation of immature thymocytes but is involved in the selection or maintenance of mature cells from this pool.