Regional Differences in Neuroinflammation-Associated Gene Expression in the Brain of Sporadic Creutzfeldt-Jakob Disease Patients.

Neuroinflammation is an essential part of neurodegeneration. Yet, the current understanding of neuroinflammation-associated molecular events in distinct brain regions of prion disease patients is insufficient to lay the ground for effective treatment strategies targeting this complex neuropathological process. To address this problem, we analyzed the expression of 800 neuroinflammation-associated genes to create a profile of biological processes taking place in the frontal cortex and cerebellum of patients who suffered from sporadic Creutzfeldt-Jakob disease. The analysis was performed using NanoString nCounter technology with human neuroinflammation panel+. The observed gene expression patterns were regionally and sub-regionally distinct, suggesting a variable neuroinflammatory response. Interestingly, the observed differences could not be explained by the molecular subtypes of sporadic Creutzfeldt-Jakob disease. Furthermore, analyses of canonical pathways and upstream regulators based on differentially expressed genes indicated an overlap between biological processes taking place in different brain regions. This suggests that even smaller-scale spatial data reflecting subtle changes in brain cells' functional heterogeneity and their immediate pathologic microenvironments are needed to explain the observed differential gene expression in a greater detail.

Molecular Characterization of the Danish Prion Diseases Cohort With Special Emphasis on Rare and Unique Cases.

The purpose of this study was to perform an updated reclassification of all definite prion disease cases with available fresh-frozen samples referred to the Danish Reference Center over the past 40 years, putting a special emphasis on the molecular characterization of novel disease subtypes. Investigation of the Danish prion diseases cohort revealed rare sporadic Creutzfeldt-Jakob disease cases with mixed subtypes and subtypes with previously uncharacterized white matter plaques, a new case of sporadic fatal insomnia, and 3 novel mutations, including 2 large octapeptide repeat insertions, and a point mutation in the prion protein gene. The evaluation of methionine and valine distribution at codon 129 among the prion disease patients in the cohort revealed the increased prevalence of methionine homozygotes compared to the general population. This observation was in line with the prevalence reported in other Caucasian prion disease cohort studies. Reclassification of the old prion diseases cohort revealed unique cases, the molecular characterization of which improves prion diseases classification, diagnostic accuracy, genetic counseling of affected families, and the understanding of disease biology.

Hypoxia-induced secretion of macrophage migration-inhibitory factor from MCF-7 breast cancer cells is regulated in a hypoxia-inducible factor-independent manner.

The cytokine MIF is over-expressed in tumors and is associated with tumor proliferation, angiogenesis and metastasis. Hypoxia, a hallmark feature of tumors, increases MIF expression from tumor cells. We examined the role of hypoxia-inducible transcription factors on MIF secretion from MCF-7 breast carcinoma cells. Secretion of MIF was induced by hypoxia after 24h but up-regulation of MIF mRNA was minimal. Inhibition of HIF-1alpha, HIF-2alpha, NF-kappaB and C/EBPbeta using siRNA had no effect on hypoxia-induced MIF secretion. However, inhibition of transcription and translation significantly decreased MIF production, suggesting that hypoxia-induced secretion of MIF in MCF-7 cells is via an alternative pathway.

Angiopoietin-4 inhibits angiogenesis and reduces interstitial fluid pressure.

Angiopoietins (Ang) are involved in the remodeling, maturation, and stabilization of the vascular network. Ang-4 was discovered more recently; thus, its effect on angiogenesis and its interplay with other angiogenic factors have not been equivocally established. The role of Ang-4 in angiogenesis was tested in Matrigel chambers implanted into the subcutaneous space of nude mice. Ang-4 inhibited the angiogenic response of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and GLC19 tumor cells. In Matrigel chambers with Ang-4-transfected cells, the mean response was significantly lower than that of mock cells. Subcutaneous tumor interstitial fluid pressure (IFP) was significantly lower in Ang-4-transfected GLC19 tumors than in mock-transfected tumors. IFP reduction in Ang-4-transfected tumors was comparable to the reduction seen after bevacizumab treatment. In vitro, we examined the effect of recombinant Ang-4 on endothelial cell migration in Boyden chambers. Human umbilical vein endothelial cell (HUVEC) migration induced by bFGF and VEGF was inhibited by Ang-4 to control levels. In conclusion, we show that rhAng-4, as well as transfection with Ang-4, inhibits angiogenesis induced by GLC19 tumor cells and that Ang-4 expression reduces elevated tumor IFP. In addition, we demonstrate that rhAng-4 inhibits HUVEC migration and growth factor-induced angiogenesis.

Augmenting tumor sensitivity to topotecan by transient hypoxia.

We examined how the effect of topotecan is modulated by transient hypoxia in three different tumor lines, Lewis lung carcinoma (LLC), U87 human glioblastoma and DMS273 human small cell lung cancer. Four groups of tumor bearing mice were treated with saline or a single dose of topotecan, immediately followed by 6-h or 72-h exposure to a hypoxic environment (10% O(2)) or normal air. Topotecan + hypoxia resulted in significantly greater suppression of tumor growth than normoxic topotecan or hypoxia alone. Correspondingly, the sensitivity of LLC cells to topotecan in a clonogenic survival assay was significantly higher under hypoxia. This effect of hypoxia was not a general phenomenon, since the tumor growth inhibitory effect of the alkylating agent cisplatin was not changed by hypoxic environment. In a parallel series of in vitro experiments, cell cultures were exposed to hypoxia (0.1% or 0.7% O(2)) in a hypoxic chamber or normoxia for 24 h. We found a dose-dependent downregulation of HIF-1alpha by topotecan (30-270 nM). The hypoxic upregulation of Glucose transporter-1 and VEGF secretion to the culture medium was inhibited by the addition of topotecan, while doses up to 270 nM had no effect on VEGF under normoxia. VEGF protein levels in tumors were also reduced by topotecan. These data show that the effect of topotecan is increased by transient hypoxia, and this may be a direct effect on the ability of cells to survive under hypoxia as well as an antiangiogenic effect, mediated through the HIF-1 inhibitory effect of topotecan.

Regulation of YKL-40 expression during genotoxic or microenvironmental stress in human glioblastoma cells.

YKL-40 is a 40 kDa secreted glycoprotein belonging to the family of 'mammalian chitinase-like proteins', but without chitinase activity. YKL-40 has a proliferative effect on fibroblasts, chondrocytes and synoviocytes, and chemotactic effect on endothelium and vascular smooth muscle cells. Elevated YKL-40 levels are found in serum of patients with diseases characterized by inflammation, fibrosis and tissue remodeling. Several studies have reported that high serum YKL-40 levels in patients with cancer are associated with poor prognosis. YKL-40 expression is strongly elevated in serum and biopsy material from glioblastomas patients. We investigated the expression of YKL-40 in three human malignant glioma cell lines exposed to different types of stress. Whereas a polymerase chain reaction transcript was detectable in all three cell lines, only U87 produced measurable amounts of YKL-40 protein. In U87, hypoxia and ionizing radiation induced a significant increase in YKL-40 after 24-48 h. The hypoxic induction of YKL-40 was independent of HIF1. Etoposide, ceramide, serum depletion and confluence all led to elevated YKL-40. Inhibition of p53 augmented the YKL-40 expression indicating that YKL-40 is attenuated by p53. In contrast, both basic fibroblast growth factor and tumor necrosing factor-alpha repressed YKL-40. These are the first data on regulation of YKL-40 in cancer cells. Diverse types of stress resulted in YKL-40 elevation, which strongly supports an involvement of YKL-40 in the malignant phenotype as a cellular survival factor in an adverse microenvironment.

Interactions between HIF-1 and Jab1: balancing apoptosis and adaptation. Outline of a working hypothesis.

When cells experience hypoxia, they either die by apoptosis or adapt to the hypoxic conditions by a series of compensatory mechanisms. Hypoxia inducible factor-1 (HIF-1) is a transcription factor involved in both processes, but the exact mechanisms regulating whether the cells survive (adapt) or perish by apoptosis are largely unknown. We hypothesize that the balancing between apoptosis and adaptation is governed by a triangular feedback system involving the alpha-subunit of HIF-1, p53, and jun activating binding protein 1 (Jab1). Jab1 and p53 bind competitively to the same domain on HIF-1alpha resulting in either stabilization or degradation of HIF-1alpha, respectively. Moreover, p53 is stabilized by binding to HIF-1alpha, whereas its interaction with Jab1 targets p53 for degradation. Thus as a consequence we propose that the ratio between p53 and Jab1 determine whether a hypoxic induction of HIF-1 results in apoptosis or adaptation, with Jab1 as the factor promoting adaptation. On this background we consider Jab1 an interesting molecular target for anticancer therapy.

Angiogenic synergy of bFGF and VEGF is antagonized by Angiopoietin-2 in a modified in vivo Matrigel assay.

A murine modification of the Matrigel chamber assay originally developed for use on rats is presented. This modified assay permits improved quantification due to subcutaneous Matrigel implants of constant shape and volume. We have quantitatively assessed the angiogenic potential of the growth factors basic fibroblast growth factor (bFGF), VEGF, and Angiopoietin-2 (Ang-2) with special emphasis on their mutual interactions. A reproducible dose-response relationship for bFGF was established for doses between 150 and 1000 ng per chamber, whereas VEGF did not display angiogenic activity on its own in the tested dose of up to 200 ng per chamber. Conversely, we found a strong synergistic action of bFGF and VEGF when combined in a 3:1 ratio. Two other combinations (ratios) with greater VEGF doses were also tested, but the synergistic effect was only observed when 50 ng of VEGF was added to 150 ng per chamber of bFGF. This synergistic effect of bFGF and VEGF was significantly reduced by further addition of 100 ng Angiopoietin-2. Inhibition of the response to bFGF and VEGF was confirmed by in vitro EC migration experiments, which, together with our in vivo results, indicates that Ang-2 may target chemotactic responses to bFGF and VEGF in vivo.

Differential regulation of VEGF, HIF1alpha and angiopoietin-1, -2 and -4 by hypoxia and ionizing radiation in human glioblastoma.

We examined how ionizing radiation (IR) delivered under either severe hypoxia (< 0.1% O2) or normoxia affects the expression of hypoxia inducible factor 1alpha (HIF-1alpha) and the angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins 1, 2 and 4 in U87 human glioblastoma cells. IR was delivered as single doses of 0, 2, 5, 10 and 20 Gy after 6-hr hypoxic incubation and in normoxic controls. Irradiation at any dose did not affect the cellular protein levels of any of the angiopoietins, whereas hypoxia led to increasing levels of both angiopoietin-4 and angiopoietin-2. Levels of angiopoietin-1 protein were unaltered throughout the observation period. A dose-dependent increase in levels of secreted VEGF in the medium occurred after IR at doses from 5-20 Gy. In hypoxic cells, 20 Gy IR induced an additional significant increase in VEGF relative to nonirradiated hypoxic control cells with elevated baseline VEGF levels induced by hypoxia. HIF-1alpha and glucose transporter-1 (Glut-1) were not correspondingly upregulated by IR. Blocking HIF-1alpha by antisense treatment induced a reduced baseline VEGF at normoxia, while the relative upregulation of VEGF by IR was unaffected. These data provide evidence that VEGF is upregulated by IR by mechanisms independent of HIF-1 transactivation.

Improved effect of an antiangiogenic tyrosine kinase inhibitor (SU5416) by combinations with fractionated radiotherapy or low molecular weight heparin.

The effect of combining SU5416 with fractionated radiotherapy or with low molecular weight (LMW) heparin (dalteparin) was studied in U87 human glioblastoma xenografts in nude mice. SU5416 is antiangiogenic by a specific inhibition of the vascular endothelial growth factor receptor 2 (VEGFR-2), and heparins are assumed to bind VEGF. Both SU5416 (100 mg/kg every second day in 5 days) and 3 Gyx5 produced moderate, yet significant, growth inhibition. Tumors treated with concomitant irradiation and short-term SU5416 maintained a lower growth rate during regrowth than the other treatment groups (P=.007). Dalteparin (1000 IE/kg subcutaneously once a day) had no growth-inhibitory effect on its own, but when this LMW heparin was added to the SU5416 schedule, a significantly enhanced growth inhibition was obtained. VEGF protein content in tumors was not significantly altered by SU5416, but a significant decrease in VEGF levels was found in tumors treated with concomitant dalteparin and SU5416 compared with controls (P=.03). We conclude that: 1) an additive growth-inhibitory effect is obtained by combining SU5416 and fractionated radiotherapy; and 2) LMW heparin (dalteparin), in combination with SU5416, decreases the level of VEGF in tumors and increases the growth-inhibitory effect of SU5416.